ASYMMETRIC CONJUGATE COMPOUNDS
20190144443 ยท 2019-05-16
Inventors
- Paul Joseph Mark Jackson (Hertfordshire, GB)
- David Edwin Thurston (Hertfordshire, GB)
- Khondaker Mirazur Rahman (Hertfordshire, GB)
Cpc classification
A61K47/6803
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
The invention relates to compound of formula (I):
A-X.sub.1-L-X.sub.2B (I)
and salts, solvates and tautomers thereof, which are useful as medicaments, in particular as anti-proliferative agents and for use as a drug in an antibody-drug conjugate; wherein A is a group selected from:
##STR00001##
X.sub.1 and X.sub.2 are independently selected from O, S, NR.sub.28, CR.sub.28R.sub.29, CR.sub.28R.sub.29O, C(?O), C(?O)NR.sub.28, NR.sub.28C(?O), C(O)R.sup.AC(O)NH, C(O)R.sup.ANHC(O), C(O)NHR.sup.AC(O), NHC(O)R.sup.AC(O), NHC(O)R.sup.AC(O)NH, NHC(O)R.sup.ANHC(O), C(O)NHR.sup.ANHC(O), C(O)NHR.sup.AC(O)NH, OC(O) and C(O)O or is absent;
L is selected from an amino acid, a peptide chain having from 2 to 12 amino acids, a paraformaldehyde chain (OCH.sub.2).sub.1-24, a polyethylene glycol chain (OCH.sub.2CH.sub.2).sub.1-12 and (CH.sub.2).sub.mY.sup.6(CH.sub.2).sub.n wherein Y.sup.6 is selected from (CH.sub.2).sub.z and a group (L1) a group (L1) that is selected from arylene, monocyclic heteroarylene, monocyclic cycloalkylene, monocyclic cycloalkenylene and monocyclic heterocyclylene groups optionally substituted with up to three optional substituent groups; and
B is a polycyclic group selected from:
##STR00002##
Claims
1. A method of treatment of a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically effective amount of an antibody-drug conjugate, wherein the drug is a compound of formula (I):
A-X.sub.1-L-X.sub.2B (I) and salts, solvates and tautomers thereof, wherein; A is a group selected from: ##STR00322## h is 0 or 1; R.sub.1 is selected from H and halogen; either R.sub.2 is selected from CH.sub.2-halogen, C.sub.1-6 alkyl and H, and R.sub.3 is H; or R.sub.2 and R.sub.3 together with the carbon atoms to which they are attached form a cyclopropyl ring; p is 0 or 1; and when p is 1 then Y is CR.sub.7, Y.sup.2 is CR.sub.6, Y.sup.3 is CR.sub.5 and Y.sup.4 is CR.sub.4; and for (A1) and (A2) when p is 0 either (a) Y is selected from NR.sub.19, O and S; Y.sup.2 is selected from CR.sub.6 and N; and Y.sup.3 is CR.sub.5; or (b) Y.sup.3 is selected from NR.sub.19, O and S; Y.sup.2 is selected from CR.sub.6 and N; and Y is CR.sub.7; and for (A3) when p is 0, Y is selected from NR.sub.9, O and S; and Y.sup.2 is selected from CR.sub.6 and N; R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are each independently selected from H and R.sub.20, or one of R.sub.4 and R.sub.5, or R.sub.5 and R.sub.6, or R.sub.6 and R.sub.7 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups; R.sub.8 is selected from selected from H, nitrogen protecting groups and R.sub.20; X.sub.3 is selected from C?O, COH and CR; or Y.sup.5 is selected from C?O, COH, CNH.sub.2 and CR; with the carbon forming part of the ring; and when X.sub.3 or Y.sup.5 is C?O then represents an ?,?-unsaturated double bond conjugated with the C?O; and when X.sub.3 is COH or CR or Y.sup.5 is COH, CN.sub.H or CR then
represents the double bonds of an aromatic 6-membered ring and R.sub.3 is absent; wherein R is a prodrug moiety containing carbonyl, carbamoyl, glycosyl, O-amino, O-acylamino, para-aminobenzyl ether, peptidyl or phosphate groups; X.sub.1 is selected from O, S, NR.sub.21, CR.sub.21R.sub.22, CR.sub.21R.sub.22O, C(?O), C(?O)NR.sub.21, NR.sub.21C(?O), C(O)R.sup.AC(O)NH, C(O)R.sup.ANHC(O), C(O)NHR.sup.AC(O), NHC(O)R.sup.AC(O), NHC(O)R.sup.AC(O)NH, NHC(O)R.sup.ANHC(O), C(O)NHR.sup.ANHC(O), C(O)NHR.sup.AC(O)NH, OC(O) and C(O)O or is absent; L is selected from an amino acid, a peptide chain having from 2 to 12 amino acids, a paraformaldehyde chain (OCH.sub.2).sub.1-24, a polyethylene glycol chain (OCH.sub.2CH.sub.2).sub.1-12 and (CH.sub.2).sub.mY.sup.6(CH.sub.2).sub.n wherein m is an integer selected from 0 to 12, n is an integer selected from 0 to 12, and Y.sup.6 is selected from (CH.sub.2).sub.z and a group (L1) that is selected from arylene, monocyclic heteroarylene, monocyclic cycloalkylene, monocyclic cycloalkenylene and monocyclic heterocyclylene groups optionally substituted with up to three independently selected optional R.sub.20 groups; z is an integer selected from 1 to 5; X.sub.2 is selected from O, S, NR.sub.23, CR.sub.23R.sub.24, CR.sub.23R.sub.24O, C(?O), C(?O)NR.sub.23, NR.sub.24C(?O), C(O)R.sup.AC(O)NH, C(O)R.sup.ANHC(O), C(O)NHR.sup.AC(O), NHC(O)R.sup.AC(O), NHC(O)R.sup.AC(O)NH, NHC(O)R.sup.ANHC(O), C(O)NHR.sup.ANHC(O), C(O)NHR.sup.AC(O)NH, OC(O) and C(O)O or is absent; B is a polycyclic group selected from: ##STR00323## the dotted lines indicate the optional presence of one or more double bonds; q is 0 or 1; and R.sub.9 and R.sub.10 are selected such that either: (i) R.sub.9 and R.sub.10 together form a double bond; (ii) R.sub.9 is H and R.sub.10 is OH; (iii) R.sub.9 is H and R.sub.10 is OC.sub.1-6 alkyl; (iv) R.sub.9 is selected from SO.sub.3H, nitrogen protecting groups and R.sub.20; and R.sub.10 is H; or (v) R.sub.9 is H or C.sub.1-6 alkyl, and R.sub.10 is oxo or H; R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are independently selected from H, R.sub.20, R.sub.25, ?CH.sub.2, ?CH(CH.sub.2).sub.sCH.sub.3, ?CH(CH.sub.2).sub.sR.sub.25, ?O, (CH.sub.2)OR.sub.25, (CH.sub.2).sub.sCO.sub.2R.sub.25, (CH.sub.2).sub.sNR.sub.25R.sub.26, O(CH.sub.2).sub.tNR.sub.25R.sub.26, NHC(O)R.sub.25, O(CH.sub.2).sub.tNHC(O)R.sub.25, O(CH.sub.2).sub.tC(O)NHR.sub.25, (CH.sub.2).sub.sSO.sub.2R.sub.25, OSO.sub.2R.sub.25, (CH.sub.2).sub.sC(O)R.sub.25 and (CH.sub.2).sub.sC(O)NR.sub.25R.sub.26; or one of R.sub.11 and R.sub.12, R.sub.12 and R.sub.13, or R.sub.13 and R.sub.14 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups; each s is an integer independently selected from 0 to 6; each t is an integer independently selected from 1 to 6; R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are independently selected from H and R.sub.20; each R.sub.20 is independently selected from (CH.sub.2).sub.jOH, C.sub.1-6 alkyl, OC.sub.1-6 alkyl, OCH.sub.2Ph, (CH.sub.2).sub.jCO.sub.2R.sub.27, O(CH.sub.2).sub.kNR.sub.27R.sub.28, (CH.sub.2).sub.jNR.sub.27R.sub.28, C(?O)NH(CH.sub.2).sub.kNR.sub.27R.sub.28, C(?O)NHC.sub.6H.sub.4(CH.sub.2).sub.jR.sub.27 and C(?O)NH(CH.sub.2).sub.kC(?NH)NR.sub.27R.sub.28; each j is an integer independently selected from 0 to 6; each k is an integer independently selected from 1 to 6; each R.sub.19, R.sub.21, R.sub.22, R.sub.23, R.sub.24, R.sub.26, R.sub.27 and R.sub.28 is independently selected from H and C.sub.1-6 alkyl; and each R.sub.25 is independently selected from H, C.sub.1-12 alkyl, C.sub.5-9 heteroaryl, C.sub.6-15 heteroarylalkyl, phenyl and C.sub.7-12 aralkyl groups; wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three independently selected optional R.sub.20 groups; each R.sup.A is independently selected from: NR.sup.B-T.sup.1-NR.sup.C where R.sup.B and R.sup.c are each independently selected from H and C.sub.1-8 alkyl, or together R.sup.B and R.sup.c join to form a ring and together are (CH.sub.2).sub.2-3, where T.sup.1 is selected from C(O), C(O)(CH.sub.2).sub.0-50C(O).sup.?, C(O)PhC(O) where Ph is 1,3- or 1,4-phenylene; -het- wherein het is a mono-, bi-, or tricyclic heteroarylene of 5 to 12 members, containing one, two, or three heteroatoms independently selected from O, N, S, P and B, wherein het is optionally substituted up to three independently selected optional R.sub.20 groups; X.sup.A-T.sup.2-X.sup.A, where T.sup.2 is: ##STR00324## wherein each X.sup.A is independently selected from a bond, NH, N(C.sub.1-8 alkyl)-, O and S, each R.sup.D, R.sup.E, R.sup.F, and R.sup.G are each independently H or R.sub.20, or R.sup.D and R.sup.E form a ring system, or R.sup.F and R.sup.G form a ring system, or both R.sup.D and R.sup.E, and R.sup.F and R.sup.G independently form ring systems, where said ring systems are independently selected from C.sub.1-C.sub.10 heterocyclyl or C.sub.3-C.sub.8 carbocyclycl, or R.sup.D, R.sup.E, R.sup.F, and R.sup.G are each bonds to different carbons on D, wherein f and g are each independently an integer from 0 to 50 and w is an integer from 1 to 50, and wherein D is a bond or is selected from the group consisting of S, C.sub.1-C.sub.8 alkylene-, C.sub.6-C.sub.14 arylene-, C.sub.6-C.sub.14 heteroarylene-, C.sub.1-C.sub.8 heteroalkylene-, C.sub.7-C.sub.22 aralkylene, C.sub.1-C.sub.10 heterocyclo and C.sub.3-C.sub.8 carbocyclo, where said C.sub.1-C.sub.8 alkylene-, C.sub.6-C.sub.14 arylene-, C.sub.6-C.sub.14 heteroarylene-, C.sub.1-C.sub.8 heteroalkylene-, C.sub.7-C.sub.22 aralkylene, C.sub.1-C.sub.10 heterocyclo and C.sub.3-C.sub.8 carbocyclo are optionally substituted up to three independently selected optional R.sub.20 groups; with the proviso that when the compound is: ##STR00325## that at least one of R.sub.11, R.sub.12 and R.sub.13 is independently selected from C.sub.5-9 heteroaryl, C.sub.6-15 heteroarylalkyl, phenyl and C.sub.7-12 aralkyl groups and these groups are optionally substituted with up to three independently selected optional R.sub.20 groups, or that one of R.sub.11 and R.sub.12 or R.sub.12 and R.sub.13, or R.sub.13 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups; with the proviso that R.sub.5 and R.sub.6 are each independently selected from H and R.sub.20 when B, q and A are selected as (B1), O and (A4) respectively; with the proviso that when R.sub.2 is C.sub.1-6 alkyl or H, that R.sub.9 and R.sub.10 are selected from options (i), (ii), (iii) or (iv); and with the proviso that when (v) R.sub.9 is H or C.sub.1-6 alkyl, and R.sub.10 is oxo or H; then either R.sub.2 is CH.sub.2-halogen and R.sub.3 is H; or R.sub.2 and R.sub.3 together with the carbon atoms to which they are attached form a cyclopropyl ring.
2. A method of treatment according to claim 1, wherein one of R.sub.11 and R.sub.12, R.sub.12 and R.sub.13, or R.sub.13 and R.sub.14 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups.
3. A compound of formula (I):
A-X.sub.1-L-X.sub.2B (I) and salts, solvates and tautomers thereof, wherein; A is a group selected from: ##STR00326## h is 0 or 1; R.sub.1 is selected from H and halogen; either R.sub.2 is selected from CH.sub.2-halogen, C.sub.1-6 alkyl and H, and R.sub.3 is H; or R.sub.2 and R.sub.3 together with the carbon atoms to which they are attached form a cyclopropyl ring; p is 0 or 1; and when p is 1 then Y is CR.sub.7, Y.sup.2 is CR.sub.6, Y.sup.3 is CR.sub.5 and Y.sup.4 is CR.sub.4; and for (A1) and (A2) when p is 0 either (a) Y is selected from NR.sub.19, O and S; Y.sup.2 is selected from CR.sub.6 and N; and Y.sup.3 is CR.sub.5; or (b) Y.sup.3 is selected from NR.sub.19, O and S; Y.sup.2 is selected from CR.sub.6 and N; and Y is CR.sub.7; and for (A3) when p is 0, Y is selected from NR.sub.19, O and S; and Y.sup.2 is selected from CR.sub.6 and N; R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are each independently selected from H and R.sub.20, or one of R.sub.4 and R.sub.5, or R.sub.5 and R.sub.6, or R.sub.6 and R.sub.7 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups; R.sub.8 is selected from selected from H, nitrogen protecting groups and R.sub.20; X.sub.3 is selected from C?O, COH and CR; or Y.sup.5 is selected from C?O, COH, CNH.sub.2 and CR; with the carbon forming part of the ring; and when X.sub.3 or Y.sup.5 is C?O then represents an ?,?-unsaturated double bond conjugated with the C?O; and when X.sub.3 is COH or CR; or Y.sup.5 is COH, CNH.sub.2 or CR then
represents the double bonds of an aromatic 6-membered ring and R.sub.3 is absent; wherein R is a prodrug moiety containing carbonyl, carbamoyl, glycosyl, O-amino, O-acylamino, para-aminobenzyl ether, peptidyl or phosphate groups X.sub.1 is selected from O, S, NR.sub.21, CR.sub.21R.sub.22, CR.sub.21R.sub.22O, C(?O), C(?O)NR.sub.21, NR.sub.21C(?O), C(O)R.sup.AC(O)NH, C(O)R.sup.ANHC(O), C(O)NHR.sup.AC(O), NHC(O)R.sup.AC(O), NHC(O)R.sup.AC(O)NH, NHC(O)R.sup.ANHC(O), C(O)NHR.sup.ANHC(O), C(O)NHR.sup.AC(O)NH, OC(O) and C(O)O or is absent; L is selected from an amino acid, a peptide chain having from 2 to 12 amino acids, a paraformaldehyde chain (OCH.sub.2).sub.1-24, a polyethylene glycol chain (OCH.sub.2CH.sub.2).sub.1-12 and (CH.sub.2).sub.mY.sup.6(CH.sub.2).sub.n wherein m is an integer selected from 0 to 12, n is an integer selected from 0 to 12, and Y.sup.6 is selected from (CH.sub.2).sub.z and a group (L1) that is selected from arylene, monocyclic heteroarylene, monocyclic cycloalkylene, monocyclic cycloalkenylene and monocyclic heterocyclylene groups optionally substituted with up to three independently selected optional R.sub.20 groups; z is an integer selected from 1 to 5; X.sub.2 is selected from O, S, NR.sub.23, CR.sub.23R.sub.24, CR.sub.23R.sub.24O, C(?O), C(?O)NR.sub.23, NR.sub.24C(?O), C(O)R.sup.AC(O)NH, C(O)R.sup.ANHC(O), C(O)NHR.sup.AC(O), NHC(O)R.sup.AC(O), NHC(O)R.sup.AC(O)NH, NHC(O)R.sup.ANHC(O), C(O)NHR.sup.ANHC(O), C(O)NHR.sup.AC(O)NH, OC(O) and C(O)O or is absent; B is a polycyclic group selected from: ##STR00327## the dotted lines indicate the optional presence of one or more double bonds; q is 0 or 1; and R.sub.9 and R.sub.10 are selected such that either: (i) R.sub.9 and R.sub.10 together form a double bond; (ii) R.sub.9 is H and R.sub.10 is OH; (iii) R.sub.9 is H and R.sub.10 is OC.sub.1-6 alkyl; (iv) R.sub.9 is selected from SO.sub.3H, nitrogen protecting groups and R.sub.20; and R.sub.10 is H; or (v) R.sub.9 is H or C.sub.1-6 alkyl, and R.sub.10 is oxo or H R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are independently selected from H, R.sub.20, R.sub.25, ?CH.sub.2, ?CH(CH.sub.2).sub.sCH.sub.3, ?CH(CH.sub.2).sub.sR.sub.25, ?O, (CH.sub.2).sub.sOR.sub.25, (CH.sub.2).sub.sCO.sub.2R.sub.25, (CH.sub.2).sub.sNR.sub.25R.sub.26, O(CH.sub.2).sub.tNR.sub.25R.sub.26, NHC(O)R.sub.25, O(CH.sub.2).sub.tNHC(O)R.sub.25, O(CH.sub.2).sub.tC(O)NHR.sub.25, (CH.sub.2).sub.sSO.sub.2R.sub.25, OSO.sub.2R.sub.25, (CH.sub.2)SC(O)R.sub.25 and (CH.sub.2).sub.1C(O)NR.sub.25R.sub.26; or one of R.sub.11 and R.sub.12, R.sub.12 and R.sub.13, or R.sub.13 and R.sub.14 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups; each s is an integer independently selected from 0 to 6; each t is an integer independently selected from 1 to 6; R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are independently selected from H and R.sub.20; each R.sub.20 is independently selected from (CH.sub.2).sub.jOH, C.sub.1-6 alkyl, OC.sub.1-6 alkyl, OCH.sub.2Ph, (CH.sub.2).sub.jCO.sub.2R.sub.27, O(CH.sub.2).sub.kNR.sub.27R.sub.28, (CH.sub.2).sub.jNR.sub.27R.sub.28, C(?O)NH(CH.sub.2).sub.kNR.sub.27R.sub.28; C(?O)NHC.sub.6H.sub.4(CH.sub.2).sub.jR.sub.27 and C(?O)NH(CH.sub.2).sub.kC(?NH)NR.sub.27R.sub.28; each j is an integer independently selected from 0 to 6; each k is an integer independently selected from 1 to 6; each R.sub.19, R.sub.21, R.sub.22, R.sub.23, R.sub.24, R.sub.26, R.sub.27 and R.sub.28 is independently selected from H and C.sub.1-6 alkyl; and each R.sub.25 is independently selected from H, C.sub.1-12 alkyl, C.sub.5-9 heteroaryl, C.sub.6-15 heteroarylalkyl, phenyl and C.sub.7-12 aralkyl groups; wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three independently selected optional R.sub.20 groups; each R.sup.A is independently selected from: NR.sup.B-T.sup.1-NR.sup.C where R.sup.B and R.sup.c are each independently selected from H or C.sub.1-8 alkyl, or together R.sup.B and R.sup.c join to form a ring and together are (CH.sub.2).sub.2-3, where T.sup.1 is selected from C(O), C(O)(CH.sub.2).sub.0-50C(O).sup.?, C(O)PhC(O) where Ph is 1,3- or 1,4-phenylene; -het- wherein het is a mono-, bi-, or tricyclic heteroarylene of 5 to 12 members, containing one, two, or three heteroatoms independently selected from O, N, S, P and B, wherein het is optionally substituted up to three independently selected optional R.sub.20 groups; X.sup.A-T.sup.2-X.sup.A, where T.sup.2 is: ##STR00328## wherein each X.sup.A is independently selected from a bond, NH, N(C.sub.1-8 alkyl)-, O and S, each R.sup.D, R.sup.E, R.sup.F, and R.sup.G are each independently H or R.sub.20, or R.sup.D and R.sup.E form a ring system, or R.sup.F and R.sup.G form a ring system, or both R.sup.D and R.sup.E, and R.sup.F and R.sup.G independently form ring systems, where said ring systems are independently selected from C.sub.1-C.sub.10 heterocyclyl or C.sub.3-C.sub.8 carbocyclycl, or R.sup.D, R.sup.E, R.sup.E, and R.sup.G are each bonds to different carbons on D, wherein f and g are each independently an integer from 0 to 50 and w is an integer from 1 to 50, and wherein D is a bond or is selected from the group consisting of S, C.sub.1-C.sub.8 alkylene-, C.sub.6-C.sub.14 arylene-, C.sub.6-C.sub.14 heteroarylene-, C.sub.1-C.sub.8 heteroalkylene-, C.sub.7-C.sub.22 aralkylene, C.sub.1-C.sub.10 heterocyclo and C.sub.3-C.sub.8 carbocyclo, where said C.sub.1-C.sub.8 alkylene-, C.sub.6-C.sub.14 arylene-, C.sub.6-C.sub.14 heteroarylene-, C.sub.1-C.sub.8 heteroalkylene-, C.sub.7-C.sub.22 aralkylene, C.sub.1-C.sub.10 heterocyclo and C.sub.3-C.sub.8 carbocyclo are optionally substituted up to three independently selected optional R.sub.20 groups; with the proviso that when R.sub.2 is C.sub.1-6 alkyl or H, that R.sub.9 and R.sub.10 are selected from options (i), (ii), (iii) or (iv); and with the proviso that when (v) R.sub.9 is H or C.sub.1-6 alkyl, and R.sub.10 is oxo or H; then either R.sub.2 is CH.sub.2-halogen and R.sub.3 is H; or R.sub.2 and R.sub.3 together with the carbon atoms to which they are attached form a cyclopropyl ring.
4. A compound of formula (I) and salts, solvates and tautomers thereof according to claim 3, wherein A is (A1).
5. A compound of formula (I) according to claim 3, wherein the compound has the formula (III): ##STR00329## and salts, solvates and tautomers thereof.
6. A compound of formula (I) according to claim 3, wherein the compound has the formula (VI): ##STR00330## and salts, solvates and tautomers thereof.
7. A compound of formula (I) according to claim 3, wherein the compound has the formula (IX): ##STR00331## and salts, solvates and tautomers thereof.
8. A compound of formula (I) according to claim 3, wherein the compound has the formula (XII): ##STR00332## and salts, solvates and tautomers thereof.
9. A compound of formula (I) according to claim 3, wherein the compound has the formula (XIV): ##STR00333## and salts, solvates and tautomers thereof.
10. A compound of formula (I) and salts, solvates and tautomers thereof according to claim 3, wherein one of R.sub.11 and R.sub.12, R.sub.12 and R.sub.13, or R.sub.13 and R.sub.14 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three independently selected optional R.sub.20 groups.
11. A compound of formula (I) and salts, solvates and tautomers thereof according to claim 3, wherein X.sub.1 is selected from C(?O) and NHC(?O).
12. A compound of formula (I) and salts, solvates and tautomers thereof according to claim 3, wherein X.sub.2 is selected from O and CH.sub.2, or is absent.
13. A compound of formula (I) and salts, solvates and tautomers thereof according to claim 3, wherein L is selected from (CH.sub.2).sub.m(CH.sub.2).sub.z(CH.sub.2).sub.n, ##STR00334## and R.sub.29, R.sub.30 and R.sub.31 are each independently selected from H and R.sub.20.
14. A compound of formula (I) and salts, solvates and tautomers thereof according to claim 3, wherein L is (CH.sub.2).sub.3.
15. A pharmaceutical composition comprising a compound of formula (I) and salts, solvates and tautomers thereof of claim 3 and a pharmaceutically acceptable carrier or diluent.
16. A method of treatment of a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically effective amount of a compound of formula (I) and salts, solvates and tautomers thereof of claim 3.
17. A method of treatment according to claim 16, wherein the proliferative disease is selected from bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, oesophageal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0953] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:
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EXAMPLES
[0984] General Remarks
[0985] Unless otherwise stated, all reagents and synthetic building blocks and reagents were purchased from standard commercial suppliers, such as Maybridge Chemicals (UK), Fluorochem (USA), ChemShuttle Inc (USA), Merck KGaA, (Germany), VWR Ltd., Avantor Inc., (USA), Fischer Scientific, Inc. (USA), and Sigma-Aldrich (UK) and used as purchased. 3-(Bromomethyl)-benzeneacetic acid methyl ester was purchased from Beta Pharma Scientific Inc. (USA). Methyl 3-(bromomethyl)-1-benzothiophene-2-carboxylate was purchased from Enamine Ltd. (Ukraine). Methyl (2S)-piperidinecarboxylate hydrochloride and methyl 2-[6-(chloromethyl)-2-pyridyl]acetate hydrochloride were purchased from Apollo Scientific Ltd. (UK). Methyl 8-bromooctanoate was purchased from Combi-Blocks, Inc. (USA). (S)-(+)-2-Indolinemethanol was purchased from Carbosynth Ltd. (UK). N-Boc O-Bn-(S)-seco-CBIN-Boc O-Bn-(S)-seco-CBI, Alloc-Val-Ala-OH and Alloc-Val-Ala-PAB-PNP were purchased from YProTech (UK) and SYNthesis Med Chem (UK). Solvents were purchased from Sigma-Aldrich (UK) and Fisher Scientific (UK). Anhydrous reactions were carried out in pre-oven-dried glassware under an inert atmosphere of nitrogen or argon. Anhydrous solvents were used as purchased without further drying. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F.sub.254), and flash column chromatography was carried out either manually, using silica gel (Merck 9385, 230-400 mesh ASTM, 40-63 ?M) (whilst monitoring by thin layer chromatography: UV (254 nm) and an aqueous alkaline solution of potassium permanganate as stain), or using a Grace Reveleris? X2 automated Flash Chromatography System, or using a Biotage Isolera Dalton 2000 (automated mass-directed flash chromatography system). All Nuclear Magnetic Resonance (NMR) spectra were obtained at room temperature using a Bruker DPX400 or a Varian Mercury Vx Agilent 400 MHz spectrometer, for which chemical shifts are expressed in ppm relative to the solvent and coupling constants are expressed in Hz. Microwave reactions were carried out on an Anton Paar Monowave 300 microwave synthesis reactor, or a Biotage Initiator+microwave synthesizer. High Resolution Mass Spectrometry (HRMS) was performed on a Thermo Scientific-Exactive HCD Orbitrap Mass Spectrometer. Yields refer to isolated material (homogeneous by TLC or NMR) unless otherwise stated and names are assigned according to IUPAC nomenclature.
[0986] Liquid Chromatography Mass Spectroscopy (LCMS) analysis Methods A-C were performed on a Waters Alliance 2695 with water (A) and acetonitrile (B) comprising the mobile phases. Formic acid (0.1%) was added to both acetonitrile and water to ensure acidic conditions throughout the analysis. Function type: Diode array (535 scans). Column type: Monolithic C18 50?4.60 mm. Mass spectrometry data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (? C.), 100; Desolvation Temperature (? C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. LCMS gradient conditions are described as follows.
[0987] Method A (10 min): from 95% A/5% B to 50% B over 3 min. Then from 50% B to 80% B over 2 min. Then from 80% B to 95% B over 1.5 min and held constant for 1.5 min. This was then reduced to 5% B over 0.2 min and maintained to 5% B for 1.8 min. The flow rate was 0.5 mL/min, 200 ?L was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-400 nm.
[0988] Method B (5 min): from 95% A/5% B to 90% B over 3 min. Then from 90% B to 95% B over 0.5 min and held constant for 1 min. This was then reduced to 5% B over 0.5 min. The flow rate was 1.0 mL/min, 100 ?L was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm.
[0989] Method C (5 min): from 95% A/5% B, which was increased to 90% B over 3 min and to 95% B over a further 0.5 min. The gradient was then held at 95% B for 1 min and then returned to 5% B over 0.5 min. The total duration of the run was 5 minutes and the solvent flow rate was 1 mL/min, 100 ?L was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm.
[0990] Liquid Chromatography Mass Spectrometry (LCMS) analysis Methods D-G were performed on a Shimadzu LC-20AD series, Binary Pump, Diode Array Detector. Column type: Agilent Poroshell 120 EC-C18, 2.7 ?m, 4.6?50 mm. Mobile phase: A: 0.05% formic acid in water (v/v); B: 0.05% formic acid in acetonitrile (v/v). Flow Rate: 1 mL/min at 25? C. Detector: 214 nm, 254 nm. Gradient stop time: 5 min. MS: 2020, Quadrupole LC/MS, Ion Source: API-ESI, TIC: 100-1300 m/z, Drying gas flow: 15 L/min, Nebulizer pressure: 1.5 L/min, Drying gas temperature: 250? C., Vcap: 4500V. Sample preparation: samples were dissolved in methanol at 1-10 ?g/mL, then filtered through a 0.22 ?m filter membrane. Injection volume: 1-10 ?L. Gradient conditions are described as follows.
[0991] Method D (5 min): 20% A/80% B for 0.5 min, which was increased to 100% B over 3.5 min, then held at 100% B for 0.5 min. This was then returned to 20% A/80% B for 0.5 min.
[0992] Method E (5 min): 50% A/50% B for 0.5 min, which was increased to 100% B over 3.5 min, then held at 100% B for 0.5 min. This was then returned to 50% A/50% B for 0.5 min.
[0993] Method F (5 min): 85% A/15% B for 0.5 min, which was increased to 100% B over 3.5 min, then held at 100% B for 0.5 min. This was then returned to 85% A/15% B for 0.5 min.
[0994] Method G (5 min): 97% A/3% B for 0.5 min, which was increased to 30% A/70% B over 3.5 min, then to 100% B over 0.5 min. This was then returned to 97% A/3% B for 0.5 min.
[0995] Optical rotations were measured on a SGWzz-1 automatic Polarimeter (Shanghai Shen Guang Instrument Co., Ltd.
Example 1: Molecular Modeling
[0996] Methodology
[0997] Ligand Preparation
[0998] Each ligand used in the study was built using ChemBioOffice, and was energy-minimized using the MMFF94 (23) force-field. Ligand structures were then imported into AMBER (v11) (24) software, AMBER modules were loaded, and antechamber was used to convert the structures to mol2 files with the application of Gasteiger charges. Further missing parameters were then generated using parmchk, which uses the gaff.dat force-field to facilitate this process.
[0999] DNA Preparation
[1000] DNA was built in every instance using the nuc module of AMBER. The gaff and DNA optimised parm99bsco (25) force-fields and modified DNA library were loaded for DNA. Parmbsco considers ?/? bond rotations of nucleic acids, reducing fraying of bases over long-scale MD simulations (25).
[1001] Ligand:DNA Adduct Simulation
[1002] The AMBER module xleap was used to make initial approximate docking alignments of ligands into the minor groove of DNA (with the appropriate sequence), prior to subsequent energy minimization. The placement was done such that the N10 of the PBD was within 2 ? of the intended exocyclic amino group of the reacting guanine. This was undertaken as the PBD is thought to form a reaction-mediating H-bond with the DNA, which in turn pulls the molecule into the minor groove, and non-covalent simulations allowed the investigation of initial ligand: DNA contacts.
[1003] A similar process was undertaken for covalently bound simulations where the ligand was first docked in the DNA minor groove of each individual sequence, and covalent bonds were then created. The CBI was first covalently bound to N3 of the appropriate adenine (parameters for covalent attachment derived in-house), and simulated mono-covalently bound in order to investigate DNA span of unsymmetrical dimers. A third set of simulations was also undertaken where the covalent bond was created between both the N3 of an adenine and the cyclopropane of the CBI and between the exocyclic amine of guanine and the N10-C11 imine of the PBD using the AMBER module xleap. C11S stereochemistry was maintained in every case at the binding interface of the PBD. Parameters for the covalent attachment of the PBD to DNA were created using parameters derived previously through molecular mechanics calculations (26).
[1004] Each adduct was then minimized in a stepwise manner to facilitate accommodation in the minor groove. In this procedure, positional restraints are initially used on the DNA atoms to keep their positions fixed and the ligands are then energy minimized alone, followed by full minimization of the system (without restraints) to ensure ligands are accommodated deep in the minor groove.
[1005] Production simulations were undertaken in implicit solvent, where the Generalised Born solvation method was used, which is equivalent to the Poisson Boltzmann method, but includes a surface area term to enable accuracy in the simulation of macromolecules (27). A term to allow for monovalent electrostatic ion screening was also employed to simulate the effect of Na.sup.+ ions in the surrounding environment.
[1006] The choice of simulation time is important, and is generally judged upon availability of hardware and time required for the simulation to equilibrate to a degree where potential energy can be considered stable over time. A valuable indicator of this is obtained through plotting conformational variability of the ligand:DNA adduct over time. This is achieved by comparing the coordinates of each frame with the first frame, finding the best RMS fit in each case. In the case of DNA macromolecule simulation (and particularly PBD:DNA adduct simulation), a simulation time of 10 ns in duration in implicit solvent is sufficient to represent the ligand:DNA complex, as simulations of this type are well established in literature (28, 29, 30) using the protocol outlined. Example RMSD graphs of simulations contained within this study are provided in Supporting Information, proving simulations went to equilibrium as expected. RMSD calculations were also conducted between the lowest energy snapshot of the MD simulations (derived through a ptraj script) and the ligand:DNA adduct structure post full minimization of the system, which provided a numeric measurement of DNA disorder.
[1007] Discussion
[1008] The novel hybrid molecules described herein have been designed through molecular modeling to ensure that they (a) fit snugly in the DNA minor groove, and (b) the two alkylating moieties are in the appropriate positions to cross-link GC and AT base pairs (
[1009] In the case of the PBD-Phenyl-CBI molecule (
[1010] Furthermore, RMSD calculations were also conducted between the lowest energy snapshot of the MD simulations (derived through a ptraj script) and the ligand:DNA adduct structure post full minimization of the system, which provided a numeric measurement of DNA disorder. In the case of the known PBD-CBI hybrid 27eS, DNA disorder was calculated to be 1.18, whereas slightly less disorder (0.80) was observed in simulations of the PBD-Phenyl-CBI hybrid, again suggesting similar potency as degree of DNA distortion is correlated with DNA-binding ability. Base-pairing was maintained throughout simulation, suggesting stable adducts were generated.
[1011] Examples of PDD-CBI molecules connected via the same linkage as the PBD-CBI molecule 27eS also suggest strong affinity with the DNA minor groove, inferring similar potency. For example, the PDD-CBI molecule containing a pentamethylene linker possesses a free energy of binding value of ?40.52 kcal/mol and an RMSD value of 1.02, which suggests strong DNA-binding ability and little distortion of DNA.
[1012] Simulations of the C1-linked PBD-CBI dimer containing a phenyl linker (suggest similar interaction with the minor groove (
[1013] Furthermore, little distortion of the minor groove was evident in simulations (RMSD of 1.06), and base pairing was maintained, suggesting strong interactivity with the minor groove floor.
[1014] The PBD-CBI conjugate linked via C7 on the A-ring of the CBI also exhibits extensive interactions with the minor groove floor. The molecule fits snugly in the minor groove (in a similar manner to the PBD dimers), causing little DNA distortion. The imine of the PBD is ideally located to alkylate DNA (in this instance G26 on the reverse strand), and the CBI is also ideally situated to alkylate an adenine base four base pairs away (i.e., A11). As such, the PBD-CBI conjugate spans five base-pairs (5-C(G)ATTA-3), and in the example snapshot (
[1015] Surprisingly, dimers linked via the C8 of the CBI and C8 of the PDD/PBD also suggest comparable binding affinity and accommodation in the minor groove of DNA to those linked via C7.
##STR00093##
[1016] Free energy of binding calculations suggest an affinity of ?37.72 kcal/mol for the DNA sequence, which is slightly less favourable than other molecules simulated (Table 1). However, both PBD/PDD and CBI are located at precise orientations in non-covalent simulations, suggesting that alkylation of the guanine (by PBD/PDD) and adenine (by the CBI) would readily occur. The example illustrated below (
TABLE-US-00012 TABLE1 RMSD Calculations FreeEnergy (generatedfrom) Base-pairing DNASequence(Spanof ofBinding covalently maintained Asymmetric moleucleinnon-covalent Calculations bound during molecule simulationsinbold) (kcal/mol) simulations) simulation PBD-CBI(27eS) 5-GCTATAACATTATATAC-3 ?40.73 1.18 Y PBD/PDD- 5-GCTATAACATTATATAC-3 ?41.55 0.80 Y Phenyl-CBI PDD-CBI 5-GCTATAACATTATATAC-3 ?40.52 1.02 Y C.sub.1PBD-Phenyl- 5-GCTATAACATTATATAC-3 ?43.22 1.06 Y CBI PBD/PDD-CBI 5-GCTATAACATTATATAC-3 ?41.56 0.64 Y (C.sub.7-linked) PBD/PDD-CBI 5-GCTATAACATTATATAC-3 ?37.72 0.78 Y (C8-linked) Free energy of binding, RMSD calculations and degree of base-pair maintenance of PBD-CBI and PDD-CBI molecules in cross-linked DNA sequences (span of molecule highlighted in red).
[1017] General Synthetic Scheme for PDD Precursor
##STR00094## ##STR00095##
Example 2: Methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (1)
[1018] ##STR00096##
[1019] A mixture of vanillin (20.0 g, 131 mmol), methyl 4-bromobutanoate (17.5 mL, 139 mmol) and potassium carbonate (27.2 g, 197 mmol) in N,N-dimethylformamide (100 mL) was stirred at room temperature for 18 h. The reaction mixture was diluted with water (500 mL) and the title compound (30.2 g, 91%) was obtained by filtration as a white solid. The product was carried through to the next step without any further purification.
[1020] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.84 (s, 1H), 7.46-7.37 (m, 2H), 6.98 (d, J=8.2 Hz, 1H), 4.16 (t, J=6.3 Hz, 2H), 3.91 (s, 3H), 3.69 (s, 3H), 2.56 (t, J=7.2 Hz, 2H), 2.20 (quin, J=6.7 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 190.9, 173.4, 153.8, 149.9, 130.1, 126.8, 111.6, 109.2, 67.8, 56.0, 51.7, 30.3, 24.2; MS M/Z (EIMS)=271.9 (M+Na).sup.+, 253 (M+H).sup.+; LCMS (Method A): t.sub.R=6.48 min.
Example 3: Methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (2)
[1021] ##STR00097##
[1022] To a stirring solution of potassium nitrate (10.0 g, 98.9 mmol) in TFA (50 mL) at 0? C. was added dropwise a solution of methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (1) (20.0 g, 79.2 mmol) in TFA (50 mL). The reaction mixture was stirred at room temperature for 1 h. It was then concentrated in vacuo and diluted with ethyl acetate (400 mL). The organic layer was sequentially washed with brine (3?100 mL) and a saturated aqueous solution of sodium hydrogen carbonate (2?80 mL), dried over sodium sulfate, filtered and concentrated to give the title compound (23.5 g, 100%) as a yellow solid. The product was carried through to the next step without any further purification.
[1023] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 10.42 (s, 1H), 7.60 (s, 1H), 7.39 (s, 1H), 4.21 (t, J=6.3 Hz, 2H), 3.98 (s, 3H), 3.70 (s, 3H), 2.61-2.53 (m, 2H), 2.22 (quin, J=6.6 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 187.8, 173.2, 153.5, 151.7, 143.8, 125.5, 109.9, 18.1, 68.6, 56.6, 51.8, 30.2, 24.1; MS M/Z (EIMS)=298 (M+H), 296.1 (M?H).sup.?; LCMS (Method A): t.sub.R=6.97 min.
Example 4: 5-Methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (3)
[1024] ##STR00098##
[1025] To a solution of methyl 4-4-formyl-2-met oxy-5-nitropenoxy)butanoate (2) (23.0 g, 77.4 mmol) in acetone (600 mL) was quickly added a hot (70? C.) solution of potassium permanganate (46.0 g, 291 mmol) in water (400 mL). The reaction mixture was stirred at 70? C. for 3 h. The reaction mixture was cooled to room temperature and passed through celite. The cake of celite was washed with hot water (200 mL). A solution of sodium bisulfite in HCl (1 M, 200 mL) was added to the filtrate which was extracted with dichloromethane (2?400 mL). The combined organic extracts were then was dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 50%), to give the title compound (17.0 g, 70%) as a pale yellow solid.
[1026] .sup.1H NMR (400 MHz, MeOD) ? 7.47 (s, 1H), 7.25 (s, 1H), 4.13 (t, J=6.2 Hz, 2H), 3.94 (s, 3H), 3.68 (s, 3H), 2.54 (t, J=7.2 Hz, 2H), 2.17-2.06 (m, 2H); .sup.13C NMR (100 MHz, MeOD) ? 175.3, 168.6, 153.8, 151.3, 143.1, 122.8, 112.4, 109.2, 69.6, 57.0, 52.2, 31.2, 25.5; MS M/Z (EIMS)=314 (M+H).sup.+, 311.9 (M?H).sup.?; LCMS (Method A): t.sub.R=6.22 min.
Example 5: Methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)butanoate (4)
[1027] ##STR00099##
[1028] A mixture of 5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (3) (8.0 g, 25.5 mmol), oxalyl chloride (6.6 mL, 77.0 mmol) and anhydrous N,N-dimethyl-formamide (2 drops) in anhydrous dichloromethane (100 mL) was stirred at room temperature for 1 h. Anhydrous toluene (20 mL) was added to the reaction mixture which was then concentrated in vacuo. A solution of the resulting residue in anhydrous dichloromethane
[1029] (10 mL) was added dropwise to a solution of (S)-piperidin-2-ylmethanol (3.8 g, 33.4 mmol) and triethylamine (10.7 mL, 77.0 mmol) in anhydrous dichloromethane (90 mL) at ?10? C. The reaction mixture was stirred at room temperature for 2 h and then washed with hydrochloric acid (1 M, 50 mL) and brine (50 mL), dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with methanol/dichloro-methane (from 0% to 5%), to give the title compound (9.2 g, 73%) as a yellow oil.
[1030] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.68-7.64 (m, 1H), 6.77-6.70 (m, 1H), 4.16-4.07 (m, 3H), 3.93-3.89 (m, 3H), 3.83 (s, 1H), 3.67 (s, 3H), 3.15 (d, J=1.4 Hz, 1H), 3.11 (s, 1H), 2.78 (s, 1H), 2.56-2.50 (m, 3H), 2.21-2.12 (m, 4H), 1.74-1.55 (m, 4H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 173.3, 168.1, 154.6, 148.2, 137.4, 127.6, 111.4, 108.3, 68.3, 60.6, 56.7, 53.5, 51.7, 43.3, 38.0, 34.9, 30.3, 24.1, 19.7; MS M/Z (EIMS)=411.0 (M+H).sup.+; LCMS (Method A): t.sub.R=6.28 min.
Example 6: Methyl (S)-4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (5)
[1031] ##STR00100##
[1032] To a solution of methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)butanoate (4) (9.2 g, 22.4 mmol) in ethanol (40 mL) and ethyl acetate (10 mL) was added palladium on activated charcoal (10% wt. basis) (920 mg). The reaction mixture was hydrogenated at 35 psi for 3 h in a Parr apparatus. The reaction mixture was filtered through celite and the resulting cake was washed with ethyl acetate. The filtrate was concentrated in vacuo to give the title compound (9.0 g, 90%) as a pink solid. The product was carried through to the next step without any further purification.
[1033] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.69 (s, 1H), 6.27-6.18 (m, 1H), 4.03-3.94 (m, 3H), 3.94-3.82 (m, 3H), 3.81-3.76 (m, 1H), 3.74 (s, 3H), 3.73-3.68 (m, 1H), 3.67-3.65 (m, 3H), 3.56 (d, J=4.8 Hz, 1H), 3.03 (s, 1H), 2.51 (t, J=7.2 Hz, 2H), 2.11 (quin, J=6.7 Hz, 2H), 1.68-1.59 (m, 4H), 1.55-1.40 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 173.6, 171.2, 150.3, 141.8, 141.1, 113.2, 112.3, 102.4, 67.5, 60.8, 60.4, 56.8, 51.6, 30.4, 25.8, 24.3, 21.0, 19.9, 14.2; MS M/Z (EIMS)=381.0 (M+H).sup.+; LCMS (Method A): t.sub.R=5.52 min.
Example 7: Methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxyl-methyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (6)
[1034] ##STR00101##
[1035] To a solution of methyl (S)-4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (5) (9.0 g, 23.7 mmol) and pyridine (4.4 mL, 54.4 mmol) in anhydrous dichloromethane (100 mL) at ?10? C. was added dropwise a solution of allylchloroformate (2.6 mL, 24.8 mmol) in anhydrous dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was sequentially washed with a saturated aqueous solution of copper (II) sulfate (80 mL), water (80 mL) and a saturated aqueous solution of sodium hydrogen carbonate (80 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue (2.0 g out of the 11.0 g crude) was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 1%), to give the title compound (930 mg, 47% based on the amount purified) as a yellow oil.
[1036] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.30 (br s, 1H), 7.63 (br s, 1H), 6.76 (br s, 1H), 5.92 (ddt, J=17.2, 10.6, 5.4, 5.4 Hz, 1H), 5.37-5.28 (m, 1H), 5.20 (dq, J=10.4, 1.3 Hz, 1H), 4.65-4.56 (m, 2H), 4.06 (t, J=6.2 Hz, 2H), 3.94-3.82 (m, 1H), 3.79 (s, 3H), 3.66 (s, 3H), 3.62-3.54 (m, 1H), 3.40 (br s, 1H), 3.10-2.88 (m, 1H), 2.52 (t, J=7.4 Hz, 2H), 2.22-2.04 (m, 3H), 1.64 (br s, 4H), 1.56-1.31 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 173.5, 170.6, 153.9, 149.7, 144.8, 132.6, 130.1, 117.6, 116.9, 110.8, 107.1, 106.0, 67.7, 65.6, 60.7, 56.3, 53.5, 51.6, 43.1, 30.5, 25.7, 24.4, 19.7; MS M/Z (EIMS)=465.1 (M+H).sup.+; LCMS (Method A): t.sub.R=6.47 min.
Example 8: Allyl (6S,6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (7)
[1037] ##STR00102##
[1038] To a solution of methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)-piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (6) (930 mg, 2.0 mmol) in dichloromethane (45 mL) was added 2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) (32 mg, 0.20 mmol) and (diacetoxyiodo)-benzene (773 mg, 2.4 mmol). The reaction mixture was stirred at room temperature for 16 h, and was then sequentially washed with a saturated aqueous solution of sodium metabisulfite (20 mL), a saturated aqueous solution of sodium hydrogen carbonate (20 mL), water (20 mL) and brine (20 mL). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 5%), to give the title compound (825 mg, 89%) as a cream solid.
[1039] MS M/Z (EIMS)=462.9 (M+H).sup.+; LCMS (Method A): t.sub.R=6.30 min.
Example 9: Allyl (6S6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)ox)-6,6a7,8,9,10-hexahydrobenzoelpyrido-[1,2-a][1,4]diazepine-5(12H)-carboxylate (8)
[1040] ##STR00103##
[1041] A mixture of allyl (6S,6aS)-6-hydroxy-2-methoxy-3-4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,1-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (7) (825 mg, 1.8 mmol), 3,4-dihydro-2H-pyran (1.7 mL, 18.2 mmol) and p-toluenesulfonic acid monohydrate (pTSA) (8.5 mg, 1% w/w) in ethyl acetate (12 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with ethyl acetate (50 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 2%), to give the title compound (820 mg, 84%) as a cream solid.
[1042] MS M/Z (EIMS)=546.7 (M+H).sup.+; LCMS (Method A): t.sub.R=7.70 min.
Example 10: 4-(((6S,6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9)
[1043] ##STR00104##
[1044] To a solution of allyl (6S,6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (8) (770 mg, 1.4 mmol) in 1,4-dioxane (10 mL) was added a 0.5 M aqueous solution of sodium hydroxide (10 mL, 5.0 mmol). The reaction mixture was stirred at room temperature for 2 h and was then concentrated in vacuo, after which water (20 mL) was added and the aqueous layer was acidified to pH=1 with an aqueous 1 M citric acid solution (5 mL). The aqueous layer was then extracted with ethyl acetate (2?50 mL). The combined organic extracts were then washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated to give the title compound (700 mg, 93%) as a yellow oil. The product was carried through to the next step without any further purification.
[1045] MS M/Z (EIMS)=532.9 (M+H).sup.+; LCMS (Method A): t.sub.R=6.98 min.
[1046] Reaction Scheme for Preparing Compound (11)
##STR00105##
Example 11: tert-Butyl (S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-H-benzo[e]indole-3-carboxylate (10)
[1047] ##STR00106##
[1048] A solution of tert-butyl (S)-5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]-indole-3-carboxylate (460 mg, 1.09 mmol) in tetrahydrofuran (10 mL) was charged with palladium on activated charcoal (10 wt. % basis) (230 mg) and a solution of ammonium formate (547 mg, 8.68 mmol) in water (2 mL) and then heated to 35? C. under an inert atmosphere of argon. After 1 h, the mixture was allowed to cool and filtered through a pad of celite, which was then washed with ethyl acetate. After extracting the filtrate with ethyl acetate (2?50 mL), the combined organic extracts were dried over magnesium sulfate and concentrated in vacuo. The residue was then purified by recrystallisation (ethyl acetate/hexane) to give the title compound (244 mg, 67%) as a white solid.
[1049] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.23 (d, J=8.3 Hz, 1H), 7.86 (br s, 1H), 7.63 (d, J=8.3, 1H), 7.50 (ddt, J=6.8, 1.4, 1.3 Hz, 1H), 7.38-7.33 (m, 1H), 4.27 (d, J=11.5 Hz, 1H), 4.17-4.09 (m, 1H), 3.95 (tt, J=10.0, 2.8 Hz, 2H), 3.43 (t, J=11.5 Hz, 1H), 1.64 (s, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 154.1, 153.2, 141.1, 130.4, 127.6, 123.7, 122.9, 121.8, 121.7, 114.3, 99.2, 81.9, 53.2, 46.5, 41.8, 28.6; MS M/Z (ES?)=332 (M?1).sup.?; LCMS (Method C): t.sub.R=3.73 min.
Example 12: (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11)
[1050] ##STR00107##
[1051] A solution of tert-butyl (S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]-indole-3-carboxylate (10) (30 mg, 0.090 mmol) in hydrochloric acid (4 M in 1,4-dioxane) was stirred at room temperature under argon. Progress was monitored by LCMS and after approximately 1 h, the reaction mixture was concentrated in vacuo to give the title compound (24 mg, quant.) as a pale green crystalline solid (unstable), which was used immediately in the subsequent step without further purification.
[1052] MS M/Z (EIMS)=234 (M+H).sup.+; LCMS (Method C): t.sub.R=2.62 min.
Example 13: Synthesis of an Asymmetric Conjugate Compound (13)
[1053] ##STR00108##
[1054] Deprotection of (S)-tert-butyl 1-(chloromethyl)-5-hydroxy-1H-benzo[e]indole-3(2H)-carboxylate (10) [Sigma-Aldrich] is carried out under acid catalysis to provide the crude hydrochloride salt (11) which is then coupled with the protected PDD compound (9) using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-(dimethylamino)pyridine or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in N,N-dimethylacetamide to give the protected asymmetric conjugate compound (12). Deprotection of (12) along with imine formation is achieved by reacting
[1055] (12) with tetrakis(triphenylphosphine)palladium(0) in the presence of triphenyl-phosphine in pyrrolidine and dichloromethane results in (13).
[1056] Analogous compounds comprising a PBD unit can be prepared using a protected PBD compound that is equivalent to (9). The synthesis of such protected PBD compounds is disclosed in WO2007/039752 and WO 2013/164593.
Example 14: Allyl (6S,6aS)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (12)
[1057] ##STR00109##
[1058] A solution of 4-(((6S,6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (97.0 mg, 0.183 mmol) in N,N-dimethylacetamide (2.5 mL) was charged with (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (49.0 mg, 0.183 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (70.0 mg, 0.365 mmol) and stirred at room temperature under argon for 18 h. The reaction mixture was subsequently quenched with a saturated aqueous solution of sodium hydrogen carbonate, then taken up into ethyl acetate, separated and extracted with ethyl acetate (2?50 mL). The combined organic extracts were then washed with brine (50 mL), dried over magnesium sulfate and concentrated in vacuo. Column chromatography (silica), eluting with ethyl acetate/hexane (from 25% to 100%) afforded the title compound
[1059] (18 mg, 14%) as a pale green oil.
[1060] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.28 (d, J=8.2 Hz, 1H), 8.11 (br s, 1H), 7.65 (d, J=8.2 Hz, 1H), 7.50 (t, J=7.6 Hz, 1H), 7.36 (t, J=7.3 Hz, 1H), 7.28 (s, 1H), 7.18 (br s, 1H), 6.18 (d, J=9.3 Hz, 1H), 5.78-5.64 (m, 1H), 5.13-4.96 (m, 2H), 4.54 (d, J=11.2 Hz, 1H), 4.39 (br s, 1H), 4.26 (d, J=70.6 Hz, 2H), 4.18 (d, J=9.4 Hz, 1H), 4.02 (d, J=8.3 Hz, 1H), 3.95 (d, J=8.3 Hz, 1H), 3.89 (s, 3H), 3.86 (br s, 1H), 3.82 (d, J=90.1 Hz, 1H), 3.74-3.65 (m, 1H), 3.56-3.44 (m, 2H), 3.39 (t, J=10.7 Hz, 2H), 2.81-2.70 (m, 1H), 2.68-2.62 (m, 1H), 2.39-2.23 (m, 3H), 1.85-1.74 (m, 2H), 1.71-1.63 (m, 4H), 1.54-1.38 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.1, 169.4, 155.0, 149.1, 141.4, 138.7, 135.1, 130.0, 129.3, 127.6, 124.0, 123.3, 122.7, 122.6, 121.9, 118.8, 117.3, 114.3, 113.2, 110.5, 100.3, 94.0, 83.8, 66.7, 63.8, 56.1, 55.7, 53.2, 46.4, 45.4, 42.3, 38.8, 38.1, 35.5, 30.4, 25.2, 23.3, 22.9, 21.4, 18.2; MS M/Z (EIMS)=770 (M+Na).sup.+; MS M/Z (ES?)=746 (M?1).sup.?; LCMS (Method C): t.sub.R=3.77 min.
Example 15: (S)-3-(4-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-12(6aH)-one (13)
[1061] ##STR00110##
[1062] A solution of allyl (6S,6aS)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (12) (17.5 mg, 0.023 mmol) in dichloromethane (1 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (1 mg) and pyrrolidine (10 ?L) and then stirred at room temperature under argon. After approximately 1 min, the resulting mixture was concentrated in vacuo and immediately purified by column chromateography, eluting with methanol/ethyl acetate (from 0% to 10%), to give the title compound (2.5 mg, 19%) as a yellow oil.
[1063] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.27 (br s, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.21 (s, 1H), 7.88 (d, J=5.7 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.51 (t, J=7.7 Hz, 1H), 7.41 (s, 1H), 7.38 (t, J=7.7 Hz, 1H), 6.85 (s, 1H), 4.36-4.19 (m, 4H), 4.06-4.00 (m, 1H), 3.96-3.91 (m, 2H), 3.84 (s, 3H), 3.72 (dd, J=10.0, 4.4 Hz 1H), 3.40 (dd, J=10.8, 4.8 Hz, 1H), 3.23 (ddd, J=14.1, 10.8, 4.0 Hz, 1H), 2.90-2.86 (m, 1H), 2.80-2.76 (m, 1H), 2.47-2.34 (m, 2H), 1.89-1.62 (m, 6H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 170.6, 166.8, 164.0, 160.3, 150.8, 147.9, 142.4, 140.3, 130.4, 127.2, 123.2, 122.7, 122.4, 121.3, 114.4, 113.5, 111.7, 110.0, 100.4, 67.8, 59.6, 55.4, 52.9, 49.6, 47.0, 41.7, 39.1, 31.7, 24.2, 22.9, 18.2; MS M/Z (EIMS)=562 (M+H).sup.+; LCMS (Method C): t.sub.R=3.28 min, LCMS (Method A): t.sub.R=6.85 min; HRMS (ESI) calculated for: [C.sub.31H.sub.33ClN.sub.3O.sub.5].sup.+: 562.2103, found: 562.2098.
[1064] General Reaction Scheme for Asymmetric Conjugate Compound (24)
##STR00111## ##STR00112##
Example 16: Ethyl 6-(4-formyl-2-methoxyphenoxy)hexanoate (14)
[1065] ##STR00113##
[1066] A solution of vanillin (6.50 g, 42.7 mmol), ethyl 6-bromohexanoate (8.00 mL, 45.0 mmol) and potassium carbonate (8.70 g, 63.0 mmol) in N,N-dimethylformamide (500 mL) was stirred at room temperature for 18 h. The reaction mixture was then diluted with water
[1067] (100 mL), separated and extracted with ethyl acetate (120 mL). The combined organic extracts were sequentially washed with water (100 mL), brine (100 mL), dried over magnesium sulfate, filtered and concentrated to give the title compound (12.5 g, 99%) as a yellow oil, which was carried through to the next step without any further purification.
[1068] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.84 (s, 1H), 7.43 (dd, J=8.1, 1.9 Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 6.96 (d, J=8.1 Hz, 1H), 4.08-4.15 (m, 4H), 3.92 (s, 3H), 2.34 (t, J=7.5 Hz, 2H), 1.87-1.94 (m, 2H), 1.68-1.75 (m, 2H), 1.49-1.56 (m, 2H), 1.25 (t, J=7.2 Hz, 3H); MS M/Z (EIMS)=317 (M+Na).sup.+; LCMS (Method B): t.sub.R=3.82 min.
Example 17: Ethyl 6-(4-formyl-2-methoxy-5-nitrophenoxy)hexanoate (15)
[1069] ##STR00114##
[1070] A solution of potassium nitrate (5.40 g, 53.0 mmol) in trifluoroacetic acid (25 mL) at room temperature was charged slowly with a solution of ethyl 6-(4-formyl-2-methoxy-phenoxy)hexanoate (14) (12.5, 42.0 mmol) in trifluoroacetic acid (25 mL). The reaction mixture was stirred for 1 h and then concentrated in vacuo, after which the resulting residue was dissolved in ethyl acetate (200 mL). This was then washed with brine (3?50 mL) followed by a saturated aqueous solution of sodium hydrogen carbonate (2?40 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo to give the title compound (14.4 g, 99%) as a yellow solid. This was carried through to the next step without any further purification.
[1071] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 10.43 (s, 1H) 7.58 (s, 1H), 7.40 (s, 1H), 4.10-4.16 (m, 4H), 4.00 (s, 3H), 2.35 (t, J=7.4 Hz, 2H), 1.84-1.96 (m, 2H), 1.69-1.76 (m, 2H), 1.50-1.58 (m, 2H), 1.25 (t, J=7.2 Hz, 3H); MS M/Z (EIMS)=340 (M+H).sup.+; LCMS (Method B): t.sub.R=4.02 min.
Example 18: 4-((6-Ethoxy-6-oxohexyl)oxy)-5-methoxy-2-nitrobenzoic acid (16)
[1072] ##STR00115##
[1073] A solution of ethyl 6-(4-formyl-2-methoxy-5-nitrophenoxy)hexanoate (15) (7.80 g, 23.0 mmol) in acetone (200 mL) was charged with a hot (70? C.) solution of potassium permanganate (13.6 g, 86.0 mmol) in water (100 ml). The resulting mixture was stirred at 70? C. for 4 h and then cooled to room temperature and filtered through a pad of celite. The filter cake was subsequently washed with hot water (100 mL). A solution of sodium bisulfite in hydrochloric acid (1 M, 100 mL) was added to the filtrate, which was then extracted with dichloromethane (2?200 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo to give the title compound (5.0 g, 61%) as a yellow solid which was used in the next step without further purification.
[1074] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.34 (s, 1H), 7.14 (s, 1H), 3.96-4.03 (m, 4H), 3.84 (s, 3H), 2.24 (t, J=7.4 Hz, 2H), 1.70-1.77 (m, 2H), 1.55-1.62 (m, 2H), 1.39-1.45 (m, 2H), 1.13 (t, J=7.1 Hz, 3H); MS M/Z (EIMS)=354 (M?H).sup.+; LCMS (Method B): t.sub.R=3.63 min.
Example 19: Ethyl (S)-6-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)hexanoate (17)
[1075] ##STR00116##
[1076] A solution of 4-((6-ethoxy-6-oxohexyl)oxy)-5-methoxy-2-nitrobenzoic acid (16) (2.00 g, 5.60 mmol) in dichloromethane (40 mL) was charged with trimethylamine (4.70 mL, 33.8 mmol) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (2.20 g, 5.90 mmol) and the resulting mixture was stirred for 2 h at room temperature. A solution of (S)-piperidin-2-ylmethanol (647 mg, 5.63 mmol) in dichloromethane (10 mL) was then added and the resulting mixture was stirred for 16 h at room temperature. The reaction was quenched with a saturated aqueous solution of sodium hydrogen carbonate (40 mL), the phases were separated and the aqueous layer was further extracted with dichloromethane (20 mL). The combined organic extracts were washed with brine (40 mL), dried over magnesium sulfate, filtered and concentrated to give an amber oil. Purification was carried out by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 100%), to give the title compound (1.20 g, 48%) as a colourless oil.
[1077] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.63-7.60 (m, 1H), 6.77-6.75 (m, 1H), 4.13-4.02 (m, 4H), 3.93 (s, 3H), 3.78-3.70 (m, 1H), 3.68-3.39 (m, 1H), 3.18-3.11 (m, 3H), 2.32 (t, J=7.6 Hz, 2H), 1.91-1.83 (m, 2H), 1.72-1.39 (m, 11H), 1.26 (t, J=7.1 Hz, 3H); MS M/Z (EIMS)=453 (M+H).sup.+; LCMS (Method B): t.sub.R=3.63 min.
Example 20: Ethyl (S)-6-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate (18)
[1078] ##STR00117##
[1079] A solution of ethyl (S)-6-(4-2-hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy) hexanoate (17) (1.20 g, 2.70 mmol) in methanol (20 mL) was charged with Raney?-Nickel (slurry in H.sub.2O) (120 mg). The resulting mixture was hydrogenated at 4 atm for 1.5 h in a Parr apparatus, then filtered through a pad of celite and concentrated in vacuo to give the title compound (991 mg, 87%) as a grey oil that solidifies upon standing. The resulting material was carried through to the next step without further purification.
[1080] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.69 (s, 1H), 6.32 (s, 1H), 4.13 (m, 4H), 3.98 (t, J=6.5 Hz, 2H), 3.79 (s, 3H), 3.67-3.57 (m, 1H), 3.22-3.19 (m, 4H), 2.87 (s, 2H), 2.36-2.32 (m, 2H), 1.89-1.82 (m, 2H), 1.73-1.65 (m, 6H), 1.55-1.47 (m, 3H), 1.27 (t, J=7.1 Hz, 3H); MS M/Z (EIMS)=423 (M+H).sup.+; LCMS (Method B): t.sub.R=3.23 min.
Example 21: Ethyl (S)-6-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxy-methyl)piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate (o)
[1081] ##STR00118##
[1082] A solution of ethyl (S)-6-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy) hexanoate (18) (1.23 g, 2.91 mmol) and anhydrous pyridine (542 ?L, 6.69 mmol) in anhydrous dichloromethane (20 mL), at ?10? C., was charged with a solution of allyl chloroformate (278 ?L, 2.62 mmol) in dichloromethane (12 mL), dropwise. The resulting reaction mixture was stirred at room temperature for 0.5 h, quenched with a saturated aqueous solution of copper (II) sulfate (25 mL), diluted with dichloromethane
[1083] (10 mL), separated and successively washed with water (20 mL), a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (20 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo to give the title compound (588 mg, 40%) as an orange oil. The resulting material was carried through to the next step without further purification.
[1084] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.23 (br s, 1H), 7.70 (br s, 1H), 6.78 (s, 1H), 6.00-5.90 (m, 1H), 5.38-5.33 (m, 1H), 5.24 (dd, J=10.4, 1.3 Hz, 1H), 4.63 (m, 2H), 4.12 (q, J=7.1 Hz, 2H) 4.05 (t, J=6.6 Hz, 2H), 3.83 (s, 3H), 3.72-3.64 (m, 1H), 3.12-3.02 (m, 1H), 2.33 (t, J=7.6 Hz, 2H), 1.91-1.84 (m, 2H), 1.74-1.67 (m, 10H), 1.66-1.54 (m, 4H), 1.26 (t, J=7.1 Hz, 3H); MS M/Z (EIMS)=507 (M+H).sup.+; LCMS (Method B): t.sub.R=3.70 min.
Example 22: Allyl (6S,6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(1aH)-carboxylate (20)
[1085] ##STR00119##
[1086] A solution of ethyl (S)-6-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)-piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate (19) (1.70 g, 3.40 mmol) in dichloromethane (80 mL) was charged with 2,2,6,6-tetramethyl-1-piperidinyloxy (53 mg, 0.30 mmol) and (diacetoxyiodo)benzene (1.30 g, 4.00 mmol). The resulting mixture was stirred at room temperature for 16 h and was then cooled in an ice bath and quenched with a saturated aqueous solution of sodium metabisulfite (35 mL). The mixture was then diluted with dichloromethane (30 mL), separated and sequentially washed with a saturated aqueous solution of sodium hydrogen carbonate (30 mL), water (30 mL) and brine (30 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. Purification was carried out by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 80%) to give the desired compound (1.10 g, 66%) as a colourless oil.
[1087] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.72-7.70 (m, 1H), 7.13-7.09 (m, 1H), 5.98-5.08 (m, 1H), 5.38-5.25 (m, 1H), 5.19-5.14 (m, 2H), 4.72-4.63 (m, 2H), 4.50-4.35 (m, 1H), 4.13 (q, J=7.1 Hz, 2H), 4.08-4.03 (m, 1H), 4.01-3.96 (m, 2H), 3.91 (s, 3H), 3.83-3.81 (m, 1H), 3.53-3.45 (m, 1H), 3.10-3.03 (m, 1H), 2.33 (t, J=7.6 Hz, 2H), 1.90-1.83 (m, 2H), 1.74-1.62 (m, 10H), 1.53-1.48 (m, 2H); MS M/Z (EIMS)=505 (M+H).sup.+; LCMS (Method B): t.sub.R=3.57 min.
Example 2: Allyl (6S,6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4a]diazepine-5(12H)-carboxylate (21)
[1088] ##STR00120##
[1089] A solution of allyl (6S,6aS)-3-(6-ethoxy-6-oxohexyl)oxy)-6-hydro-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (20) (1.10 g, 2.20 mmol) in dichloromethane (50 mL) was charged with 3,4-dihydro-2H-pyran (2.00 mL, 22.4 mmol) and p-toluenesulfonic acid monohydrate (113 mg, 1% w/w). The resulting mixture was stirred at room temperature for 4 h. The reaction mixture was then diluted with dichloromethane (50 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate (50 mL) and brine (50 mL). The organic extract was then dried over magnesium sulfate and concentrated. Purification by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 70%), gave the title compound (863 mg, 66%) as a yellow oil.
[1090] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.16 (m, 1H), 6.50 (s, 1H), 6.10 (m, 1H), 5.81-5.76 (m, 1H), 5.14-5.03 (m, 2H), 4.69-4.57 (m, 2H), 4.47-4.37 (m, 1H), 4.34-4.26 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 4.01-3.94 (m, 3H), 3.90 (s, 3H), 3.68-3.62 (m, 1H), 3.68-3.46 (m, 2H), 3.12-3.03 (m, 1H), 2.33 (t, J=7.4 Hz, 2H), 1.89-1.66 (m, 11H), 1.57-1.47 (m, 6H), 1.25 (t, J=7.1 Hz, 3H); MS M/Z (EIMS)=589 (M+H).sup.+; LCMS (Method B): t.sub.R=4.32 min.
Example 24: 6-(((6S,6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (22)
[1091] ##STR00121##
[1092] A solution of allyl (6S,6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (21) (200 mg, 0.340 mmol) in 1,4-dioxane (3 ml) was charged with an aqueous solution of sodium hydroxide (0.5 M, 1.20 mL). The reaction mixture was stirred at room temperature for 2 h and was then concentrated in vacuo, after which water (6 ml) was added and the aqueous layer was then acidified to pH=1 with citric acid (1 M). The aqueous layer was then extracted with ethyl acetate (2?40 mL) and the combined organic extracts were then washed with brine (40 ml), dried over sodium sulfate and concentrated to give the title compound as a yellow oil (181 mg, 95%) which was used in the subsequent step without further purification.
[1093] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.18 (s, 1H), 6.19 (s, 1H), 6.18-5.99 (m, 1H), 5.81-5.71 (m, 1H), 5.12-5.02 (m, 2H), 4.67-4.51 (m, 1H), 4.48-4.36 (m, 1H), 4.31-4.23 (m, 1H), 4.00-3.88 (m, 7H), 3.66-3.46 (m, 2H), 3.12-3.02 (m, 1H), 2.36 (t, J=7.4 Hz, 2H), 1.81-1.79 (m, 2H), 1.75-1.65 (m, 10H), 1.55-1.49 (m, 7H); MS M/Z (EIMS)=561 (M+H).sup.+; LCMS (Method B): t.sub.R=3.78 min.
Example 25: Allyl (6S,6aS)-3-((6-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)ox)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (21)
[1094] ##STR00122##
[1095] A solution of 6-(((6S,6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (22) (37 mg, 0.066 mmol) in N,N-dimethylacetamide (1.0 mL) was charged with (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11)
[1096] (18 mg, 0.066 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (26.0 mg, 0.133 mmol) and stirred at room temperature under argon for 18 h. The reaction mixture was subsequently quenched with a saturated aqueous solution of sodium hydrogen carbonate, then taken up into ethyl acetate, separated and extracted with ethyl acetate (2?50 mL). The combined organic extracts were then washed with brine (50 mL), dried over magnesium sulfate and concentrated in vacuo. Column chromatography (silica), eluting with ethyl acetate/hexane (from 10% to 100%) followed by methanol
[1097] (100%) afforded the title compound (11.4 mg, 22%) as a yellow oil.
[1098] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.34 (d, J=7.2 Hz, 1H), 8.29 (d, J=8.2 Hz, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.54-7.48 (m, 1H), 7.39-7.33 (m, 1H), 7.18 (s, 1H), 6.58 (s, 1H), 6.19 (d, J=10.0 Hz, 1H), 6.01 (d, J=10.0 Hz, 1H), 5.81-5.66 (m, 1H), 5.17-4.99 (m, 3H), 4.68-4.42 (m, 2H), 4.35-4.24 (m, 3H), 4.09-4.01 (m, 3H), 3.88 (s, 3H), 3.85-3.80 (m, 1H), 3.67-3.60 (m, 1H), 3.52-3.46 (m, 1H), 3.42 (t, J=11 Hz, 1H), 3.13-3.02 (m, 1H), 2.74-2.55 (m, 2H), 2.01-1.88 (m, 6H), 1.82-1.61 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.2, 163.7, 156.3, 155.1, 149.3, 146.6, 141.2, 131.6, 130.0, 127.6, 130.0, 127.6, 125.4, 123.9, 123.5, 122.7, 122.0, 117.9, 116., 114.6, 110.5, 108.0, 106.4, 100.4, 94.7, 69.1, 66.8, 63.0, 60.4, 56.1, 55.9, 52.3, 46.9, 46.3, 42.3, 35.7, 31.9, 29.7, 29.4, 25.5, 25.4, 25.2, 23.1, 22.7; MS M/Z (ES?)=774 (M?1).sup.?; MS M/Z (EIMS)=798 (M+Na).sup.+; LCMS (Method C): t.sub.R=3.93 min.
Example 26: (S)-3-((6-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo-[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (24)
[1099] ##STR00123##
Experiment (i)
[1100] A solution of allyl (6S,6aS)-3-((6-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate
[1101] (23) (11 mg, 0.014 mmol) in dichloromethane (1 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (1 mg) and pyrrolidine (10 ?L) and then stirred at room temperature under argon. After approximately 1 min, the resulting mixture was concentrated in vacuo and immediately purified by column chromatography, eluting with ethyl acetate/hexane (from 50% to 100%) then with methanol/ethyl acetate (from 0% to 100%), to give the title compound (0.6 mg, 7.5%) as a yellow oil.
[1102] MS M/Z (EIMS)=590 (M+H).sup.+; LCMS (Method C): t.sub.R=3.80 min.
Experiment (ii)
[1103] A solution of allyl (6S,6aS)-3-((6-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate
[1104] (23) (27 mg, 0.035 mmol) in dichloromethane (4 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (4 mg) and pyrrolidine (4 ?L) and then stirred at room temperature under argon. After approximately 1 min, the resulting mixture was concentrated in vacuo and immediately purified by column chromatography, eluting with ethyl acetate/hexane (from 50% to 100%), to give the title compound (8 mg, 38%) as a yellow oil.
[1105] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.30 (br s, 1H), 8.21 (d, J=8.2 Hz, 1H), 8.13 (s, 1H), 7.97 (d, J=5.5 Hz, 1H), 7.80 (d, J=8.6 Hz, 1H), 7.74-7.67 (m, 1H), 7.63-7.49 (m, 1H), 7.33 (s, 1H), 6.78 (s, 1H), 4.39-4.30 (m, 2H), 4.19-4.11 (m, 3H), 4.10-4.05 (m, 1H), 4.01 (dd, J=11.0, 3.5 Hz, 1H), 3.85 (s, 3H), 3.79-3.68 (m, 2H), 3.16 (td, J=11.3, 3.1 Hz, 1H), 2.70-2.56 (m, 2H), 2.18-2.10 (m, 1H), 2.02-1.95 (m, 1H), 1.94-1.76 (m, 6H), 1.70-1.60 (m, 4H); MS (ES+): m/z=590 (M+H).sup.+; LCMS (Method C): t.sub.R=3.80 min, LCMS (Method A): t.sub.R=7.20 min.
Example 27: tert-Butyl (8bR,9aS)-4-oxo-9,9a-dihydro-1H-benzo[e]cyclopropa[c]indole-2(4H)-carboxylate (25)
[1106] ##STR00124##
[1107] A solution of tert-butyl (S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]-indole-3-carboxylate (10) (20 mg, 0.060 mmol) in anhydrous N,N-dimethylacetamide (1.0 mL) was cooled to 0? C. and charged with potassium carbonate (58.0 mg, 0.419 mmol) and stirred at this temperature for 25 min. The reaction mixture was then quenched (cold) with a saturated aqueous solution of sodium hydrogen carbonate and the resulting slurry extracted twice with ethyl acetate. The combined organic extracts were then dried over magnesium sulfate and concentrated in vacuo before purification was enacted by column chromatography (silica), eluting with ethyl acetate/hexane (25%, isocratic) to give the title compound (14 mg, 79%) as a yellow solid.
[1108] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.22 (dd, J=7.9, 1.1 Hz, 1H), 7.49 (dd, J=7.7, 1.4 Hz, 1H), 7.39 (dt, J=7.6, 1.2 Hz, 1H), 6.86 (dd, J=7.8, 0.6 Hz, 1H), 6.82 (br s, 1H), 4.04-3.96 (m, 2H), 2.79-2.73 (m, 1H), 1.62 (dd, J=7.7, 4.4 Hz, 1H), 1.57 (s, 9H), 1.47 (t, J=4.7 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 186.1, 159.7, 151.7, 140.2, 132.7, 131.8, 126.9, 126.5, 120.9, 108.7, 83.5, 52.9, 33.5, 28.2, 23.4, 14.1; MS M/Z (EIMS)=298 (M+H).sup.+; LCMS (Method C): t.sub.R=3.37 min.
[1109] General Synthetic Scheme to Prepare an A Group Precursor
##STR00125## ##STR00126## ##STR00127##
[1110] In step a, an aryl aldehyde is reacted with the phosphonate ester to produce an alkene compound. The tert-butyl protecting group is removed in step b to provide a carboxylic acid. In step c, this carboxylic acid is coupled with acetic anhydride to yield a naphthalene derivative. The acetyl group is removed in step d to produce the alcohol. In step d, the alcohol group is protected with a benzyl group. The ethyl ester is then removed in step f and the carboxylic acid is reacted with diphenylphosphoryl azide to produce an acyl azide that undergoes a Curtis rearrangement in the presence of tert-butanol to provide the tert-butyl carbamate in step g. The naphthalene ring is iodinated in step h to provide an aryl iodide derivative. In step i, the aryl carbamate is coupled with allyl chloride compound at the carbamate nitrogen. The aryl nitro group is reduced to the aryl amine in step l, and this aryl amine is protected with a Fmoc protecting group in step m. Radical cyclisation is carried out in step n to preferentially provide the 5-membered ring exo cyclisation product. Removal of the Boc protecting group provides the A group precursor.
[1111] Further methods and experimental procedures for making A-rings and A-ring precursors have been disclosed by Jia and Lown (31) and by Elgersma et al. (32).
Example 28: (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11)
[1112] ##STR00128##
[1113] A solution of tert-butyl (S)-5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (100 mg, 0.236 mmol) in anhydrous dichloromethane (3 mL) was charged with boron trichloride (1 M solution in dichloromethane, 708 ?L, 0.708 mmol), in a dropwise manner via syringe, at room temperature and under an inert atmosphere of argon. The resulting orange solution was stirred for 5 min before being quenched by cautious addition of methanol (5 mL), then concentrated in vacuo, charged again with methanol (5 mL) and re-concentrated to give the title compound (55 mg, quant.) as a pale green crystalline solid (unstable), which was used immediately in the subsequent step without further purification.
[1114] MS (ES+): m/z=234 (M+H).sup.+; LCMS (Method C): t.sub.R=2.62 min.
Example 29: tert-Butyl (S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (26)
[1115] ##STR00129##
[1116] A solution of tert-butyl (S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (10) (50 mg, 0.15 mmol) in dichloromethane (5 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (89 mg, 0.45 mmol), 4-(dimethylamino)pyridine (20 mg, 0.17 mmol) and triethylamine (73 ?L, 0.52 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently washed with water (2?10 mL), dried over magnesium sulfate and concentrated in vacuo, to give the title compound (59 mg, 86%) as a yellow solid.
[1117] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.05 (br s, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.68 (d, J=8.3 Hz, 1H), 7.51-7.45 (m, 1H), 7.38-7.33 (m, 1H), 4.28-4.21 (br, 1H), 4.15-4.07 (m, 1H), 4.03-3.96 (m, 1H), 3.94-3.88 (m, 1H), 3.72 (t, J=4.9 Hz, 2H), 3.68-3.60 (m, 2H), 3.45 (t, J=10.8 Hz, 1H), 2.61-2.50 (m, 4H), 2.31 (s, 3H), 1.57 (s, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 153.4, 152.4, 148.4, 148.3, 130.2, 127.6, 124.2, 124.1, 122.6, 122.3, 120.1, 109.3, 81.2, 54.6, 54.2, 48.5, 46.3, 46.1, 45.8, 28.4; MS (ES+): m/z=460 (M+H).sup.+; LCMS (Method C): t.sub.R=3.00 min.
[1118] Reaction Scheme for Preparing Compound (29)
##STR00130##
Example 30: (S)-5-(Benzyloxy)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole hydrochloride (27)
[1119] ##STR00131##
[1120] A solution of tert-butyl (S)-5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (100 mg, 0.236 mmol) in 1,4-dioxane (1 mL) was charged with hydrochloric acid (4 M in 1,4-dioxane) (2 mL) dropwise and stirred at room temperature for 2 h, whereupon it was concentrated in vacuo to give the title compound
[1121] (85 mg, quant.) as a green solid (unstable), which was used immediately in the subsequent step without further purification.
[1122] MS (ES+): m/z=324 (M+H).sup.+; LCMS (Method C): t.sub.R=3.77 min.
Example 31: (S)-1-(5-(Benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo-[e]indol-3-yl)ethan-1-one (6)
[1123] ##STR00132##
[1124] A solution of (S)-5-(benzyloxy)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole hydrochloride (27) (85 mg, 0.24 mmol) in N,N-dimethylacetamide (1 mL) was charged with acetic acid (100 ?L), and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (45 mg, 0.24 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently quenched with a saturated aqueous solution of sodium hydrogen carbonate, and extracted with ethyl acetate (2?50 mL). The combined organic extracts were then washed with brine (50 mL), dried over magnesium sulfate and concentrated in vacuo. Column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 20%) afforded the title compound (53 mg, 62%) as a white solid.
[1125] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.34 (d, J=8.4 Hz, 1H), 8.18 (s, 1H), 7.67 (d, J=8.3 Hz, 1H), 7.58-7.53 (m, 3H), 7.46-7.41 (m, 2H), 7.40-7.35 (m, 2H), 5.30 (dd, J=11.8, 2.0 Hz, 2H), 4.28 (br, 1H), 4.08-4.01 (m, 1H), 3.98 (dd, J=11.2, 3.0 Hz, 1H), 3.80-3.63 (m, 1H), 3.47-3.41 (m, 1H), 2.33 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.1, 155.9, 141.7, 136.8, 129.8, 128.6, 128.0, 127.6, 123.7, 123.7, 123.3, 122.0, 115.2, 110.3, 98.0, 70.4, 54.0, 46.2, 42.4, 22.7; MS (ES+): m/z=366 (M+H).sup.+; LCMS (Method C): t.sub.R=4.38 min.
Example 32: (S)-1-(1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)ethan-1-one (7)
[1126] ##STR00133##
[1127] A solution of (S)-1-(5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-3-yl)ethan-1-one (28) (24 mg, 0.66 mmol) in tetrahydrofuran (1 mL) was charged with ammonium formate (25% aqueous solution) (132 ?L, 0.53 mmol) and palladium on activated charcoal (10 wt. % basis) (2 mg) and then heated to 35? C. under an inert atmosphere of argon. After 3 h, the mixture was allowed to cool and filtered through a pad of celite, washed with acetone and then concentrated in vacuo. After diluting in ethyl acetate and washing with water (50 mL) followed by brine (50 mL), the organic extract was dried over magnesium sulfate and concentrated in vacuo to give the title compound (6.3 mg, 35%) as a green solid.
[1128] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.47 (s, 1H), 8.32 (d, J=8.1 Hz, 1H), 7.69 (d, J=8.3 Hz, 1H), 7.48 (td, J=7.6, 1.3 Hz, 1H), 7.38 (m, 1H), 4.24-4.16 (m, 1H), 3.80-3.69 (m, 4H), 2.38 (s, 3H); MS (ES+): m/z=276 (M+H).sup.+; LCMS (Method C): t.sub.R=3.18 min.
Example 33: Ethyl 2-(3-(bromomethyl)phenyl)acetate (30)
[1129] ##STR00134##
[1130] A mixture of N-bromosuccinimide (12.5 g, 71.2 mmol), azobisisobutyronitrile (366 mg, 2.30 mmol) and ethyl m-tolylacetate (10 mL, 56.6 mmol) in carbon tetrachloride (60 mL) was stirred at reflux for 3 h. The reaction mixture was then allowed to cool to room temperature, filtered, and the filtrate concentrated in vacuo. Purification by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 9%) gave the title compound (6.7 g, 46%) as a colourless oil.
[1131] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.34-7.23 (m, 4H), 4.50 (s, 2H), 4.22-4.13 (m, 2H), 3.63 (s, 2H), 1.29 (t, J=8.2 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) 3171.3, 138.1, 134.7, 129.9, 129.3, 129.0, 128.7, 60.9, 41.1, 33.3, 14.2; MS (ES+): m/z=258 (M+H).sup.+; LCMS (Method B): t.sub.R=3.90 min.
[1132] Reaction Scheme for Preparing Compound (42)
##STR00135## ##STR00136## ##STR00137##
Example 34: 4-(Benzloxy)-3-methoxybenzaldehyde (31)
[1133] ##STR00138##
[1134] Method (i)A mixture of vanillin (15.0 g, 99 mmol), benzyl bromide, (12.9 mL, 109 mmol) and potassium carbonate (6.7 g, 0.49 mmol) in acetone (225 mL) was stirred at room temperature for 18 h. The reaction was diluted with water (200 mL) and extracted with ethyl acetate (2?200 mL). The combined organics were then washed with water (100 mL) and brine (100 mL), dried over magnesium sulfate, filtered and concentrated to give the title compound (12.5 g, 62%) as a pale yellow solid. The product was carried through to the next step without further purification.
[1135] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.84 (s, 1H), 7.45-7.31 (m, 7H), 6.98 (d, J=8.2 Hz, 1H), 5.24 (s, 2H), 3.94 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 190.9, 153.6, 150.1, 136.0, 1300.3, 128.7, 128.2, 127.2, 126.6, 112.4, 109.4, 70.9, 56.0; MS (ES+): m/z=243 (M+H).sup.+, MS (ES?): m/z=241 (M?1).sup.?; LCMS (Method B): t.sub.R=3.82 min; LCMS (Method A): t.sub.R=7.53 min.
[1136] Method (ii)A mixture of compound vanillin (200 g, 1.31 mol), benzyl bromide (236 g, 1.38 mol) and potassium carbonate (545 g, 3.94 mol) in methanol (1.20 L) was refluxed for 5 h. The reaction mixture was filtered, and the filtrate evaporated under reduced pressure to afford the title compound (271 g, 85%) as a pale yellow solid.
[1137] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.83 (s, 1H), 7.47-7.35 (m, 6H), 7.33 (d, J=7.2 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 5.24 (s, 2H), 3.94 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 191.0, 153.6, 150.1, 136.0, 130.3, 128.7, 128.2, 127.2, 126.6, 112.3, 109.3, 70.9, 56.1; MS (ES+): m/z=243 (M+H).sup.+; LCMS (Method A): t.sub.R=7.53 min.
Example 35: 4-(Benzyloxy)-5-methoxy-2-nitrobenzaldehyde (32)
[1138] ##STR00139##
[1139] Method (i)A solution of potassium nitrate (5.4 g, 53 mmol) in trifluoroacetic acid (25 mL) was added dropwise to a solution of 4-(benzyloxy)-3-methoxybenzaldehyde (31) (12.5 g, 42 mmol) in trifluoroacetic acid (25 mL) at room temperature. The reaction mixture was stirred for 1 h. It was then concentrated in vacuo and the residue was dissolved in ethyl acetate (200 mL). The organic layer was successively washed with brine (3?50 mL) and a saturated aqueous solution of sodium hydrogen carbonate (2?40 mL), dried over magnesium sulfate, filtered and concentrated to give the title compound (14.4 g, 100%) as a yellow solid. The product was used in the next step without further purification.
[1140] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 10.21 (s, 1H), 7.84 (s, 1H), 7.50-7.38 (m, 6H), 5.33 (s, 2H), 3.96 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 188.6, 152.8, 150.8, 135.6, 128.6, 128.5, 128.3, 128.1, 124.9, 110.2, 108.7, 70.7, 56.5; MS (ES+): m/z=288 (M+H).sup.+, MS (ES?): m/z=286 (M?1).sup.?; LCMS (Method B): t.sub.R=3.98 min, LCMS (Method A): t.sub.R=7.67 min.
[1141] Method (ii) A solution of 4-(benzyloxy)-3-methoxybenzaldehyde (31) (130 g, 537 mmol) in trifluoroacetic acid (600 mL) was charged with a solution of potassium nitrate (65 g, 644 mmol), in trifluoroacetic acid (600 mL) dropwise at 0? C. The reaction mixture was stirred for 1 h and then diluted with water (2.40 L). The resulting precipitate was filtered and washed with cold water (500 mL?2) to afford the title compound (125 g, 81%) as a yellow solid.
[1142] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 10.43 (s, 1H), 7.67 (s, 1H), 7.46-7.30 (m, 6H), 5.27 (s, 2H), 4.02 (S, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 187.8, 153.7, 151.4, 134.85, 129.0, 128.9, 128.7, 127.6, 125.7, 110.0, 108.9, 71.6, 56.7; MS (ES?): m/z=286 (M?H).sup.?; LCMS (Method A): t.sub.R=7.87 min.
Example 36: 4-(Benzyloxy)-5-methoxy-2-nitrobenzoic acid (33)
[1143] ##STR00140##
[1144] A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (32) (8.0 g, 28 mmol) in acetone (300 mL) was quickly charged with a hot (70? C.) solution of potassium permanganate (16.5 g, 104 mmol) in water (150 ml). The mixture was then stirred at 70? C. for 4 h. The reaction mixture was then allowed to cool to room temperature and passed through a pad of celite, which was then washed with hot water (120 mL). A solution of sodium bisulfite in hydrochloric acid (1 M, 120 mL) was added to the filtrate, which was then extracted with dichloromethane (2?200 mL). The combined organic extracts were subsequently dried over sodium sulfate, filtered and concentrated to give the title compound (6.7 g, 79%) as a yellow solid, which was used in the subsequent step without further purification.
[1145] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.55 (s, 1H), 7.49-7.37 (m, 6H), 5.17 (s, 2H), 4.99 (br s, 1H), 3.93 (s, 3H); .sup.13C NMR (100 MHz, MeOD) ?168.6, 154.1, 151.0, 142.9, 137.3, 129.7, 129.4, 129.0, 123.2, 112.5, 110.0, 72.3, 57.1; MS (ES+): m/z=302 (M+H).sup.+, MS (ES?): m/z=302 (M?1).sup.?; LCMS (Method B): t.sub.R=3.62 min, LCMS (Method A): t.sub.R=7.02 min.
Example 37: (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxy-methyl)piperidin-1-yl)methanone (34)
[1146] ##STR00141##
[1147] A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (33) (1.00 g, 3.30 mmol) and oxalyl chloride (0.84 mL, 9.90 mmol) in anhydrous dichloromethane (10 mL) was charged with N,N-dimethylformamide (drops) at 0? C. The resulting mixture was stirred for 2 h at room temperature, and then concentrated in vacuo. Anhydrous toluene was then charged to the resulting residue and the mixture concentrated again. After re-solubilising in anhydrous dichloromethane (10 mL), the resulting solution was then added dropwise to a solution of (S)-piperidin-2-ylmethanol (494 mg, 4.30 mmol) and triethylamine (1.4 mL, 9.9 mmol) in anhydrous dichloromethane (10 mL). The resulting mixture was stirred for 16 h at room temperature. The reaction was quenched with hydrochloric acid (1 M, 20 mL), the phases were separated and the organic extract was washed with brine (15 mL), dried over sodium sulfate, filtered and concentrated in vacuo. Purification was carried out by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 500%), to give the title compound (974 mg, 74%) as an amber oil.
[1148] .sup.1H NMR (400 MHz, CDCl.sub.3) 37.76 (s, 1H), 7.44-7.38 (m, 5H), 6.83 (s, 1H), 5.20 (s, 2H), 4.37 (br s, 1H), 3.98 (s, 3H), 3.94-3.78 (m, 4H), 3.16 (m, 2H), 2.19-1.83 (m, 5H); MS (ES+): m/z=401 (M+H).sup.+; LCMS (Method B): t.sub.R=3.60 min.
Example 38: (S)-(2-Amino-4-(benzyloxy)-5-methoxyphenyl)(2-(hydroxy-methyl)piperidin-1-yl)methanone (35)
[1149] ##STR00142##
[1150] A solution of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-piperidin-1-yl)methanone (34) (1.67 g, 4.18 mmol) in methanol (60 mL) and water (60 mL) was sequentially charged with activated charcoal (2.26 g, 188 mmol), iron(III) chloride hexahydrate (678 mg, 2.51 mmol) and hydrazine monohydrate (2.51 mL, 50.2 mmol) under an inert atmosphere of nitrogen. The reaction mixture was then heated to reflux for 16 h, before cooling to room temperature, filtering through a pad of celite and concentrating in vacuo. After extracting with ethyl acetate (2?80 mL), the organic extracts were combined, washed with brine (100 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography (silica), eluting with ethyl acetate/hexane (from 0 to 100%) gave the title compound (991 mg, 82%) as a yellow oil that solidifies upon standing.
[1151] MS (ES+): m/z=371 (M+H).sup.+, MS (ES?): m/z=369 (M?1).sup.?; LCMS (Method B): t.sub.R=3.22 min.
Example 39: Allyl (S)-(5-(benzyloxy)-2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxyphenyl)carbamate (36)
[1152] ##STR00143##
[1153] A solution of (S)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(hydroxymethyl)-piperidin-1-yl)methanone (35) (942 mg, 2.54 mmol) and pyridine (473 ?L, 5.48 mmol) in anhydrous dichloromethane (10 mL) at ?10? C., was slowly charged with a solution of allylchloroformate (243 ?L, 2.29 mmol) in dichloromethane (10 mL). The resulting mixture was stirred at room temperature for 0.5 h, before diluting with dichloro-methane (10 mL) and extracting with a saturated aqueous solution of copper (II) sulfate (25 mL). The organic phase was then washed successively with water (20 mL), a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (20 mL), then dried over sodium sulfate, filtered and concentrated in vacuo. Purification by column chromatography (silica), eluting with ethyl acetate/hexane (from 0 to 100%) gave the title compound (789 mg, 68%) as a colourless oil that solidifies upon standing.
[1154] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.30 (br s, 1H), 7.80 (br s, 1H), 7.49-7.47 (m, 2H) 7.40-7.30 (m, 3H) 6.82 (brs, 1H), 6.00-5.91 (m, 1H), 5.39-5.34 (m, 1H), 5.24 (dd, J=10.4, 1.3 Hz, 1H), 5.16 (s, 2H), 4.64 (dd, J=5.4, 1.3 Hz, 2H), 3.98-3.90 (m, 1H), 3.85 (s, 3H), 3.71-3.57 (m, 2H), 3.25-2.98 (m, 2H), 1.79-1.63 (m, 4H), 1.58-1.44 (m, 2H); MS (ES+): m/z=455 (M+H).sup.+, MS (ES?): m/z=453 (M?1).sup.?; LCMS (Method B): t.sub.R=3.72 min.
Example 40: Allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (37)
[1155] ##STR00144##
[1156] A solution of allyl (S)-(5-(benzyloxy)-2-(2-(hydroxymethyl)piperidine-1-carbonyl-4-methoxyphenyl)carbamate (36) (789 mg, 1.74 mmol) in dichloromethane (20 mL) was charged with 2,2,6,6-tetramethyl-1-piperidinyloxy (28 mg, 0.17 mmol) and (diacetoxyiodo)benzene (672 mg, 2.10 mmol). The reaction mixture was stirred at room temperature for 16 h and then placed in an ice bath before quenching with a saturated aqueous solution of sodium metabisulfite (15 mL). After extracting with dichloro-methane
[1157] (20 mL), the organic layer was sequentially washed with a saturated aqueous solution of sodium hydrogen carbonate (20 mL), water (20 mL) and brine (20 mL), then dried over sodium sulfate, filtered and concentrated in vacuo. Purification was carried out by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 100%) to give the title compound (347 mg, 44%) as a colourless oil.
[1158] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.43-7.29 (m, 5H), 7.20 (s, 1H), 6.69 (br s, 1H), 5.90 (d, J=10.3 Hz, 1H), 5.29 (s, 2H), 5.17-5.05 (m, 4H), 4.50 (br s, 1H), 4.44 (br s, 1H), 4.41-4.31 (m, 1H), 3.92 (s, 3H), 3.48 (ddd, J=10.2, 6.0, 3.8 Hz, 1H), 3.11-3.00 (m, 1H), 2.07-1.99 (m, 1H), 1.82-1.55 (m, 5H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 207.1, 168.9, 156.1, 150.0, 149.2, 136.3, 131.9, 128.6, 128.1, 127.3, 125.6, 118.0, 114.2, 110.8, 82.4, 71.1, 66.7, 56.2, 55.3, 38.7, 30.9, 23.2, 23.0; MS (ES+): m/z=453 (M+H).sup.+, MS (ES?): m/z=451 (M?1).sup.?; LCMS (Method B): t.sub.R=3.53 min.
Example 41: Allyl (6aS)-3,6-dihydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzoelpyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (38)
[1159] ##STR00145##
[1160] A solution of allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (37) (861 mg, 1.90 mmol) in anhydrous dichloromethane (30 mL) was cooled to ?78? C. and slowly charged with boron trichloride (1 M in toluene) (3.81 mL, 3.80 mmol). The resulting mixture was stirred at the same temperature for 30 min and subsequently quenched by cautious addition of water (5 mL). After diluting with dichloromethane (50 mL) and separating, the organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (407 mg, 59%) as an orange solid, which was used in the subsequent step without further purification.
[1161] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.18 (s, 1H) 6.75 (s, 1H) 6.30 (br s, 1H) 5.95-5.92 (m, 1H) 5.83-5.77 (m, 1H), 5.18-5.13 (m, 2H), 4.66-4.62 (m, 1H), 4.50-4.47 (m, 1H), 4.37-4.32 (m, 1H), 3.92 (s, 3H), 3.50-3.45 (m, 1H), 3.10-3.03 (m, 1H), 2.08-2.03 (m, 1H), 1.82-1.61 (m, 6H); MS (ES+): m/z=363 (M+H).sup.+; LCMS (Method B): t.sub.R=2.78 min.
Example 42: Allyl (6aS)-3-((3-(2-ethoxy-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (39)
[1162] ##STR00146##
[1163] A solution of allyl (6aS)-3,6-dihydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (38) (492 mg, 1.36 mmol) and ethyl 2-(3-(bromomethyl)phenyl)acetate (30) (492 mg, 1.36 mmol) in N,N-dimethyl-formamide (100 mL) was charged with potassium carbonate (282 mg, 2.04 mmol). After stirring at room temperature for 16 h, water (10 mL) was added and the mixture extracted with ethyl acetate (2?15 mL). The combined organic extracts were then washed with an aqueous solution of lithium chloride (1 M, 2?10 mL), dried over sodium sulfate, filtered and concentrated in vacuo. Purification was carried out by column chromatography (silica), eluting with ethyl acetate/hexane (from 0% to 100%) to give the title compound
[1164] (385 mg, 52%) as a white solid.
[1165] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.36-7.33 (m, 4H) 7.23-7.22 (m, 1H), 7.20 (s, 1H), 6.70 (br s, 1H), 5.90 (d, J=10.4 Hz, 1H), 5.72-5.70 (m, 1H), 5.14-5.12 (m, 3H), 4.54-4.36 (m, 3H), 4.15 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 3.62 (s, 2H), 3.50-3.45 (m, 1H), 3.07-3.04 (m, 1H), 2.06-2.02 (m, 1H), 1.81-1.64 (m, 5H), 1.26 (t, J=7.2 Hz, 3H); MS (ES+): m/z=539 (M+H).sup.+; LCMS (Method B): t.sub.R=3.68 min.
Example 43: 2-(3-((((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-2-methoxy-1-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)-oxy)methyl)phenyl)acetic acid (40)
[1166] ##STR00147##
[1167] A solution of ally (6aS)-3-((3-2-ethoxy-2-oxoetyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (39) (273 mg, 0.51 mmol) in 1,4-dioxane (2 mL) was treated with an aqueous solution of sodium hydroxide (1 M, 2 mL). The reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The resulting residue was dissolved in water (5 mL), acidified with acetic acid to pH=1, and extracted with ethyl acetate (2?5 mL). The combined organics were then washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (206 mg, 80%) as a white solid, which was used in subsequent steps with no further purification.
[1168] .sup.1H NMR (400 MHz, CDCl.sub.3) 37.28-7.23 (m, 3H), 7.20-7.14 (m, 2H), 7.10 (s, 1H), 6.62 (br s, 1H), 5.81 (d, J=10.4 Hz, 1H), 5.65-5.57 (m, 1H), 5.05-5.01 (m, 4H), 4.46-4.25 (m, 3H), 3.81 (s, 3H), 3.55 (s, 2H), 3.43-3.37 (m, 1H), 3.01-2.94 (m, 1H), 1.98-1.93 (m, 1H), 1.72-1.51 (m, 5H); MS (ES+): m/z=511 (M+H).sup.+; LCMS (Method B): t.sub.R=3.22 min.
Example 44: Allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (41)
[1169] ##STR00148##
[1170] A solution of 2-(3-((((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-2-methoxy-12-oxo-5, 6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)-phenyl)acetic acid (40) (44 mg, 0.087 mmol) in N,N-dimethylacetamide (1.0 mL) was charged with (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (2)
[1171] (20 mg, 0.087 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (50.0 mg, 0.261 mmol) and stirred at room temperature under argon for 18 h. The reaction mixture was subsequently quenched with a saturated aqueous solution of sodium hydrogen carbonate, and extracted with ethyl acetate (2?50 mL). The combined organic extracts were then washed with brine (50 mL), dried over magnesium sulfate and concentrated in vacuo. Column chromatography (silica), eluting with ethyl acetate/hexane (from 50% to 100%) afforded the title compound (44 mg, 71%) as a yellow oil.
[1172] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.29-8.13 (m, 2H), 7.63 (t, J=9.2 Hz, 1H), 7.54-7.47 (m, 1H), 7.45-7.29 (m, 4H), 7.20 (br s, 1H), 7.15 (d, J=4.2 Hz, 1H), 6.78 (s, 1H), 5.99 (dd, J=14.7, 10.0 Hz, 1H), 5.74-5.61 (m, 1H), 5.41-5.24 (m, 1H), 5.11-4.99 (m, 3H), 4.60-4.50 (m, 1H), 4.49-4.41 (m, 1H), 4.37-4.20 (m, 3H), 4.05-3.93 (m, 2H), 3.91 (s, 3H), 3.38 (t, J=10.7 Hz, 1H), 3.32-3.22 (m, 1H), 3.08-2.93 (m, 3H), 1.98-1.83 (m, 1H), 1.79-1.69 (m, 2H), 1.68-1.60 (m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 170.3, 169.3, 156.0, 155.0, 149.4, 144.8, 141.2, 141.1, 136.7, 133.8, 131.7, 130.0, 129.4, 129.2, 127.7, 126.4, 126.0, 123.8, 123.6, 122.7, 122.6, 122.1, 117.6, 117.5, 110.3, 100.6, 100.4, 82.3, 71.2, 70.0, 66.7, 56.1, 53.3, 46.6, 46.2, 42.3, 38.9, 31.9, 29.3, 23.0; MS (ES?): m/z=724 (M?1).sup.?, MS (ES+): m/z=748 (M+Na).sup.+; LCMS (Method C): t.sub.R=3.52 min.
Example 4f: (S)-3-((3-(2-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (42)
[1173] ##STR00149##
[1174] A solution of allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (41) (44 mg, 0.061 mmol) in dichloromethane (1 mL) was charged with tetrakis(triphenyl-phosphine)palladium(0) (1 mg) and pyrrolidine (10 ?L) and then stirred at room temperature under argon. After approximately 5 min, the resulting mixture was concentrated in vacuo and immediately purified by column chromatography, eluting with acetone/dichloromethane (from 30% to 100%) then with methanol/acetone (10%, isocratic), to give the title compound (19 mg, 500%) as a yellow oil.
[1175] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.34 (br s, 1H), 8.21 (d, J=8.6 Hz, 1H), 8.10 (br s, 1H), 7.92 (d, J=5.9 Hz, 1H), 7.80 (d, J=7.8 Hz, 1H), 7.75-7.67 (m, 1H), 7.65-7.59 (m, 1H), 7.57-7.50 (m, 2H), 7.40 (d, J=7.0 Hz, 1H), 7.38-7.34 (m, 2H), 6.84 (br, 1H), 5.26-5.12 (m, 1H), 5.13-5.06 (m, 1H), 4.45-4.39 (m, 2H), 4.37-4.31 (m, 2H), 4.14-4.09 (m, 2H), 4.00-3.93 (m, 2H), 3.82 (s, 3H), 3.73-3.70 (s, 1H), 3.67-3.60 (m, 1H), 2.50 (t, J=7.4 Hz, 1H), 2.32 (dt, J=7.4, 2.0 Hz, 1H), 1.82-1.77 (m, 2H), 1.64-1.55 (br, 2H); MS (ES+): m/z=624 (M+H).sup.+; LCMS (Method C): t.sub.R=4.02 min, LCMS (Method A): t.sub.R=7.60 min.
##STR00150## ##STR00151## ##STR00152##
[1176] Reaction Scheme for Preparing Compound (56)
Example 46: Methyl (S)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (43)
[1177] ##STR00153##
[1178] A mixture of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (33) (2.0 g, 6.6 mmol), oxalyl chloride (1.70 mL, 19.8 mmol) and anhydrous N,N-dimethylformamide (2 drops) in anhydrous dichloromethane (40 mL) was stirred at room temperature for 3 h. Anhydrous toluene (8 mL) was added to the reaction mixture which was then concentrated in vacuo. A solution of the resulting residue in anhydrous dichloro-methane (10 mL) was added dropwise to a solution of methyl (S)-1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (1.65 g, 7.26 mmol) and triethylamine (2.0 mL, 14.5 mmol) in anhydrous dichloromethane (30 mL), at ?10? C. The reaction mixture was stirred at room temperature for 2 h and then washed with hydrochloric acid (1 M, 20 mL) and brine (20 mL), dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloro-methane (from 0% to 30%), to give the title compound (2.5 g, 79%) as a yellow oil.
[1179] .sup.1H NMR (400 MHz, CDCl.sub.3) 37.49-7.42 (m, 6H), 7.24-7.19 (m, 5H), 5.25 (s, 2H), 4.64-4.60 (m, 1H), 4.38-4.26 (m, 2H), 3.93 (s, 3H), 3.58 (s, 3H), 3.33-3.23 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 170.8, 170.3, 154.6, 148.4, 135.3, 133.5, 130.5, 130.1, 128.9, 128.8, 128.6, 128.4, 127.7, 127.4, 126.7, 109.3, 109.1, 71.4, 56.8, 52.6, 31.8, 31.0, 30.5; MS (ES+): m/z=477 (M+H).sup.+; LCMS (Method B): t.sub.R=4.10 min.
Example 47: (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(3-(hydroxy-methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (44)
[1180] ##STR00154##
[1181] A solution of methyl (S)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-1,2,3,4-tetra hydroisoquinoline-3-carboxylate (43) (2.4 g, 5.0 mmol) in anhydrous tetrahydrofuran (48 mL) was charged with a solution of lithium borohydride (2 M in tetrahydrofuran, 3.8 mL, 8.7 mmol) at 0? C. The reaction mixture was stirred at room temperature for 3 hours. Water (150 mL) was added dropwise at 0? C. and the reaction mixture was then extracted with ethyl acetate (2?100 mL). The combined organic extracts were then concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 30%), to give the title compound (2.2 g, 97%) as creamy oil.
[1182] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.42-7.39 (m, 4H), 7.36-7.34 (m, 5H), 7.30 (s, 1H), 7.29 (s, 1H), 5.17 (s, 2H), 4.62 (s, 1H), 4.36-4.25 (m, 1H), 4.23-4.16 (m, 2H), 3.87 (s, 3H), 3.70-3.63 (m, 1H), 3.58-3.50 (m, 1H), 3.05-2.97 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 168.2, 150.2, 148.3, 133.7, 128.9, 128.9, 128.8, 128.6, 127.7, 127.6, 127.5, 127.0, 126.5, 114.4, 110.6, 108.9, 103.9, 91.6, 71.4, 65.4, 54.4, 33.3; MS (ES+): m/z=449 (M+H).sup.+; LCMS (Method B): t.sub.R=3.78 min.
Example 48: (S)-(2-Amino-4-(benzyloxy)-5-methoxyphenyl)(3-(hydroxy-methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (45)
[1183] ##STR00155##
[1184] A solution of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(3-(hydroxymethyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (44) (2.20 g, 4-90 mmol) in tetrahydrofuran (50 mL) and methanol (50 mL) was charged with iron (III) chloride hexahydrate (0.80 g, 2.90 mmol), activated charcoal (2.60 g, 221 mmol) and hydrazine (2.90 mL, 58.9 mmol). The reaction mixture was then stirred at reflux (85? C.) for 16 h. The mixture was subsequently allowed to cool to room temperature and filtered through a plug of celite. The filter cake was washed with ethyl acetate and methanol and then concentrated in vacuo to give the title compound (1.7 g, 83%) as brown solid.
[1185] .sup.1H NMR (400 MHz, MeOD) 37.48 (s, 1H), 7.46 (s, 1H), 7.41-7.33 (m, 4H), 7.20-7.18 (m, 3H), 6.84 (s, 1H), 6.56 (s, 1H), 5.11 (s, 2H), 4.61 (s, 1H), 4.54-4.40 (m, 1H), 3.77 (s, 3H), 3.62-3.54 (m, 2H), 3.19 (dd, J=16.2, 5.9 Hz, 2H), 2.92-2.80 (m, 2H); .sup.13C NMR (100 MHz, MeOD) 3169.1, 149.8, 141.0, 135.5, 130.7, 129.0, 128.7, 128.6, 128.5, 128.4, 128.2, 127.4, 127.0, 126.7, 110.1, 109.1, 71.0, 68.7, 64.8, 56.4, 50.3, 27.9; MS (ES+): m/z=419 (M+H).sup.+; LCMS (Method B): t.sub.R=3.50 min.
Example 49: Allyl (S)-(5-(benzyloxy)-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamate (46)
[1186] ##STR00156##
[1187] A solution of (S)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(3-(hydroxymethyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (45) (1.500 g, 3.6 mmol) and anhydrous pyridine (696 L, 8.97 mmol) in anhydrous dichloromethane (50 mL) at -10? C. was slowly charged with a solution of allylchloroformate (343 ?L, 3.23 mmol) in anhydrous dichloromethane (30 mL). The reaction mixture was stirred at room temperature for 30 min and then sequentially washed with a saturated aqueous solution of copper (II) sulfate (500 mL), water (50 mL) and a saturated aqueous solution of sodium hydrogen carbonate (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 20%), to give the title compound (1.47 g, 81%) as an off-white solid.
[1188] .sup.1H NMR (400 MHz, MeOD) 38.14 (s, 1H), 7.81 (s, 1H), 7.51 (s, 1H), 7.49 (s, 1H), 7.42-7.32 (m, 4H), 7.23-7.17 (m, 3H), 6.82 (s, 1H), 5.97-5.87 (m, 1H), 5.33 (dq, J=17.2, 1.5 Hz, 1H), 5.22 (dq, J=10.6, 1.3 Hz, 1H), 5.19 (s, 2H), 4.68-4.64 (m, 1H), 4.61 (dd, J=5.5, 1.3 Hz, 2H), 4.44 (br. s, 2H), 3.82 (s, 3H), 3.70-3.64 (m, 1H), 3.21-3.15 (m, 1H), 2.74 (br. s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.4, 152.9, 148.7, 144.1, 140.1, 135.3, 131.4, 130.5, 129.1, 128.1, 127.5, 127.0, 126.7, 125.9, 125.5, 117.9, 116.8, 109.6, 105.7, 69.7, 67.4, 66.0, 64.7, 55.3, 53.8, 26.8; MS (ES+): m/z=503 (M+H).sup.+; LCMS (Method B): t.sub.R=3.95 min.
Example 50: Allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-14-oxo-6,6a7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14M-carboxylate
[1189] (47)
##STR00157##
[1190] A solution of allyl (S)-(5-(benzyloxy)-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydro-isoquinoline-2-carbonyl)-4-methoxyphenyl)carbamate (46) (1.4 g, 2.78 mmol) in dichloromethane (80 mL) was charged with 2,2,6,6-tetramethyl-1-piperidinyloxy (44 mg, 0.28 mmol) and (diacetoxyiodo)benzene (1.0 g, 3.33 mmol). The reaction mixture was stirred at room temperature for 16 h and was then sequentially washed with a saturated aqueous solution of sodium metabisulfite (40 mL), a saturated aqueous solution of sodium hydrogen carbonate (40 mL), water (30 mL) and brine (30 mL). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 20%), to give the title compound (1.2 g, 86%) as an off-white solid.
[1191] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.44-7.31 (m, 6H), 7.28-7.26 (m, 5H), 6.72 (s, 1H), 5.70-5.61 (m, 1H), 5.31 (d, J=9.8 Hz, 1H), 5.20-5.17 (m, 1H), 5.11-5.07 (m, 3H), 4.83 (d, J=15.6 Hz, 1H), 4.58 (d, J=15.6 Hz, 1H), 4.48-4.34 (m, 2H), 3.94 (s, 3H), 3.74-3.69 (m, 1H), 3.17-3.05 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.0, 149.0, 136.2, 134.3, 133.7, 131.8, 126.7, 128.2, 127.9, 127.8, 127.3, 126.7, 118.1, 114.0, 111.2, 84.8, 71.0, 66.7, 56.2, 53.5, 50.8, 44.3, 30.2; MS (ES+): m/z=501 (M+H).sup.+; LCMS (Method B): t.sub.R=3.80 min.
Example 0.1: Allyl (6aS)-3,6-dihydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (48)
[1192] ##STR00158##
[1193] A solution of allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (47) (1.100 g, 2.20 mmol) in anhydrous dichloromethane (20 mL) was charged with a solution of boron trichloride (1 M in hexane, 4.4 mL, 4.4 mmol) at ?78? C. The resulting mixture was stirred for 5 h at ?78? C. and then quenched via dropwise addition of water (5 mL). An aqueous acetic acid solution (50 mL) was added to adjust to pH=3, and the resulting mixture was then extracted with ethyl acetate (2?60 mL). The combined organic extracts were then concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 30%), to give the title compound (860 mg, 95%) as a pale yellow solid.
[1194] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.29-7.26 (m, 6H), 6.76 (s, 1H), 6.02 (s, 1H), 5.84-5.75 (m, 1H), 5.34-5.31 (m, 1H), 5.17-5.13 (m, 2H), 4.83 (d, J=15.6 Hz, 1H), 4.64-4.56 (m, 2H), 4.46-4.43 (m, 1H), 3.95 (s, 3H), 3.75-3.70 (m, 1H), 3.19-3.06 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.0, 159.4, 148.0, 146.0, 134.3, 133.7, 131.8, 127.9, 127.8, 127.3, 126.7, 118.1, 115.3, 110.6, 84.8, 66.8, 56.3, 44.3, 31.0, 30.2; MS (ES+): m/z=411 (M+H).sup.+; LCMS (Method B): t.sub.R=3.15 min.
Example 52: Allyl (6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (49)
[1195] ##STR00159##
[1196] A solution of allyl (6aS)-3,6-dihydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo-[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (48) (300 mg, 0.73 mmol) in N,N-dimethylformamide (3 mL) was charged with methyl 4-bromobutanoate (166 ?L, 1.31 mmol) and potassium carbonate (151 mg, 1.10 mmol) and stirred at room temperature under an inert atmosphere of argon for 20 h. The reaction mixture was diluted with water
[1197] (30 mL) and extracted with ethyl acetate (3?20 mL). The combined organic extracts were then washed with brine (20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound (368 mg, 99%) as a yellow oil, which was carried through to the subsequent step without further purification.
[1198] .sup.1H NMR (400 MHz, CDCl.sub.3) 37.30 (s, 5H), 6.75 (br. s, 1H), 5.86-5.74 (m, 1H), 5.38 (d, J=9.8 Hz, 1H), 5.13 (d, J=11.3 Hz, 2H) 4.83 (d, J=15.6 Hz, 1H), 4.43 (br. s, 1H), 4.08 (q, J=5.9 Hz, 2H), 3.94-3.91 (m, 3H), 3.71 (s, 3H), 3.50 (t, J=6.4 Hz, 2H) 3.07-3.20 (m, 2H), 2.56-2.59 (m, 2H), 2.25-2.17 (m, 4H); .sup.13C NMR (100 MHz, CDCl.sub.3) 3173.3, 172.9, 169.0, 148.7, 134.3, 133.8, 131.9, 127.7, 127.7, 127.1, 126.6, 124.9, 117.7, 113.6, 111.1, 84.7, 67.9, 66.5, 56.0, 55.8, 51.6, 51.6, 44.2, 32.6, 30.3, 27.7, 24.2; MS (ES+): m/z=511 (M+H).sup.+, MS (ES?): m/z=509 (M?1).sup.?; LCMS (Method B): t.sub.R=3.63 min, LCMS (Method A): t.sub.R=6.97 min.
Example 53: Allyl (6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-66a,7,12-tetrahydrobenzo[5,6][1,4]-diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (50)
[1199] ##STR00160##
[1200] A solution of allyl (6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (49)
[1201] (367 mg, 0.72 mmol) in ethyl acetate (2 mL) was charged with p-toluenesulfonic acid monohydrate (3.7 mg, 1% w/w) and 3,4-dihydro-2H-pyran (657 ?L, 7.20 mmol). The resulting mixture was stirred at room temperature for 20 h, then diluted with ethyl acetate
[1202] (15 mL) and subsequently washed with a saturated aqueous solution of sodium hydrogen carbonate (10 mL), water (15 mL) and brine (15 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Column chromatography (silica gel), eluting ethyl acetate/petroleum ether (50%, isocratic) afforded the title compound (390 mg, 91%) as a light-yellow gel.
[1203] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.73-7.70 (m, 1H), 7.56-7.52 (m, 1H), 7.30-7.28 (m, 1H), 7.23 (d, J=8.2 Hz, 1H), 6.86 (s, 1H), 6.60 (s, 1H), 5.81-5.63 (m, 1H), 5.46 (d, J=9.4 Hz, 1H), 5.11-5.03 (m, 2H), 4.79 (d, J=15.6 Hz, 1H), 4.74-4.48 (m, 2H), 4.48-4.31 (m, 1H), 4.28-4.18 (m, 2H), 4.06 (q, J=6.0 Hz, 2H), 3.91 (s, 3H), 3.69 (s, 3H), 3.63-3.51, (m, 2H), 3.25-3.17 (m, 1H), 3.12-3.05 (m, 1H), 2.62-2.47 (m, 2H), 2.26-2.10 (m, 2H), 1.90-1.65 (m, 3H), 1.58 (d, J=10.9 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 173.4, 169.2, 167.7, 149.2, 134.7, 132.4, 130.8, 128.8, 126.5, 117.2, 117.1, 114.5, 113.9, 111.2, 110.8, 99.9, 90.2, 67.7, 68.1, 66.3, 63.6, 56.1, 51.6, 44.2, 31.1, 30.4, 28.9, 25.2, 23.7, 23.0, 20.1, 10.9; MS (ES+): m/z=595 (M+H).sup.+; LCMS (Method B): t.sub.R=4.35 min, LCMS (Method A): t.sub.R=8.27 min.
Example 54: 4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]-diazepino[1,2-b]isoquinolin-3-yl)oxy)butanoic acid (51)
[1204] ##STR00161##
[1205] A solution of allyl (6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (50) (332 mg, 0.55 mmol) in 1,4-dioxane (1 mL) was charged with an aqueous solution of sodium hydroxide (1 M, 1.20 mL, 1.2 mmol) and stirred at room temperature for 15 h. The reaction mixture was then concentrated in vacuo, whereupon water (10 mL) was added and the suspension was acidified to pH=1 with an aqueous solution of citric acid (1 M). The aqueous layer was then extracted with ethyl acetate (3?15 mL) and the combined organic extracts were then washed with brine (15 mL) and concentrated in vacuo to give the title compound (278 mg, 87%) as a white solid.
[1206] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.78-7.69 (m, 1H), 7.60-7.53 (m, 1H), 7.32-7.30 (m, 1H), 7.30 (br. s, 1H), 6.89 (s, 1H), 6.61 (br. s, 1H), 5.82-5.62 (m, 1H), 5.47 (d, J=9.8 Hz, 1H), 5.13-5.03 (m, 2H), 4.82 (d, J=16.0 Hz, 1H), 4.73-4.55 (m, 2H), 4.30-4.20 (m, 2H), 4.18-4.06 (m, 2H), 3.99 (dd, J=10.7, 5.3 Hz, 1H), 3.93 (s, 3H), 3.80-3.68 (m, 1H), 3.60 (br. s, 1H), 3.21 (d, J=3.1 Hz, 1H), 3.15-3.08 (m, 1H), 2.61 (q, J=7.3 Hz, 2H), 2.18 (quin, J=6.6 Hz, 2H), 1.89-1.67 (m, 3H), 1.65-1.53 (m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 177.8, 169.4, 169.4, 167.8, 149.2, 149.0, 134.6, 132.4, 130.9, 128.8, 127.7, 127.5, 126.8, 126.5, 117.2, 68.1, 67.6, 66.3, 63.3, 56.1, 44.2, 38.7, 31.1, 30.3, 28.9, 25.3, 23.7, 23.0, 20.0, 14.0, 10.9; MS (ES+): m/z=581 (M+H).sup.+, MS (ES?): m/z=579 (M?1).sup.?; LCMS (Method B): t.sub.R=3.93 min, LCMS (Method A): t.sub.R=7.53 min.
Example 55: Allyl (6aS)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-1H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (2)
[1207] ##STR00162##
[1208] A solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6, 6a,7,12,14-hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)oxy)butanoic acid (51) (109 mg, 0.188 mmol) in N,N-dimethylacetamide (4 mL) was charged with (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (2) (38.0 mg, 0.188 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (108 mg, 0.56 mmol) and stirred at room temperature under argon for 18 h. The reaction mixture was subsequently quenched with a saturated aqueous solution of sodium hydrogen carbonate, then extracted with ethyl acetate (3?60 mL). The combined organic extracts were then washed with brine (80 mL), dried over magnesium sulfate and concentrated in vacuo. Column chromatography (silica), eluting with ethyl acetate/hexane (from 25% to 100%) afforded the title compound (62 mg, 46%) as a grey oil.
[1209] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.27 (d, J=9.0 Hz, 1H), 7.64 (d, J=8.2, 1H), 7.52-7.48 (m, 1H), 7.38-7.34 (m, 1H), 7.27-7.18 (m, 6H), 6.94 (s, 1H), 5.70-5.63 (m, 1H), 5.04-4.95 (m, 2H), 4.79-4.48 (m, 2H), 4.35-4.19 (m, 4H), 4.03-3.88 (m, 6H), 3.68 (br. s, 1H), 3.59-3.37 (m, 2H), 3.16-2.72 (m, 4H), 2.39-2.28 (m, 2H), 1.70-1.41 (m, 8H), 1.33-1.28 (m, 2H); MS (ES?): m/z=794 (M?1).sup.?; LCMS (Method C): t.sub.R=4.15 min.
Example 56: (S)-3-(4-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-7,12-dihydrobenzo[5,6][1,4]-diazepino[1,2-b]isoquinolin-14(6aH)-one (q3)
[1210] ##STR00163##
[1211] A solution of allyl (6aS)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo-[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (52) (32 mg, 0.041 mmol) in dichloromethane (4 mL) was charged with tetrakis-(triphenylphosphine)palladium(0) (4 mg) and pyrrolidine (10 ?L) and then stirred at room temperature under argon. After approximately 10 min, the resulting mixture was concentrated in vacuo and immediately purified by column chromatography, eluting with methanol/ethyl acetate (from 0% to 5%), to give the title compound (3 mg, 12%) as a yellow oil.
[1212] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.38 (br s, 1H), 8.21 (d, J=8.2 Hz, 1H), 8.14 (s, 1H), 7.80 (d, J=8.6 Hz, 1H), 7.72 (d, J=7.8 Hz, 1H), 7.69 (d, J=7.4 Hz, 1H), 7.61 (d, J=6.6 Hz, 1H), 7.58-7.48 (m, 2H), 7.44 (s, 1H), 7.38-7.28 (m, 2H), 6.84 (s, 1H), 4.89 (d, J=15.2 Hz, 1H), 4.56 (dd, J=15.2, 2.5 Hz, 1H), 4.40-4.31 (m, 2H), 4.23 (dt, J=6.3, 3.3 Hz, 2H), 4.03-3.98 (m, 1H), 3.95-3.89 (m, 1H), 3.87 (s, 3H), 3.75-3.67 (m, 1H), 3.34-3.29 (m, 2H), 2.79-2.69 (m, 1H), 2.26-2.21 (m, 2H), 1.62-1.53 (m, 1H), 1.40-1.35 (m, 1H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 170.0, 167.4, 159.0, 155.6, 151.1, 146.8, 145.6, 134.4, 134.0, 131.9, 130.4, 128.6, 128.5, 127.9, 127.7, 127.0, 126.2, 123.3, 122.7, 122.4, 114.4, 112.1, 110.3, 110.0, 104.5, 67.8, 55.4, 52.9, 49.4, 43.2, 31.6, 30.2, 26.0; MS (ES+): m/z=610 (M+H).sup.+; LCMS (Method C): t.sub.R=3.55 min.
[1213] Reaction Scheme for Preparing Compound (55)
##STR00164##
Example 57: Allyl (6S,6aS)-3-(4-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (54)
[1214] ##STR00165##
[1215] A solution of allyl (6S,6aS)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)-oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (17) (177 mg, 0.237 mmol) in dichloromethane (3 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (141 mg, 0.710 mmol), 4-(dimethylamino)-pyridine (32 mg, 0.26 mmol) and triethylamine (115 ?L, 0.83 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently washed with water (2?10 mL), dried over magnesium sulfate and concentrated in vacuo, to give the title compound
[1216] (95 mg, 46%) as a yellow oil, which was used in the subsequent step without further purification.
[1217] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.48 (s, 1H), 7.99 (d, J=8.6 Hz, 1H), 7.84 (d, J=8.2 Hz, 1H), 7.64 (t, J=7.4 Hz, 1H), 7.56-7.50 (m, 1H), 7.30 (s, 1H), 6.78 (s, 1H), 6.62 (d, J=5.9 Hz, 1H), 6.31 (d, J=9.4 Hz, 1H), 5.95-5.80 (m, 1H), 5.26-5.14 (m, 3H), 4.81-4.56 (m, 3H), 4.49-4.35 (m, 4H), 4.34-4.18 (m, 4H), 4.10-4.05 (m, 2H), 4.01 (s, 3H), 3.99-3.94 (br, 2H), 3.74 (br, 4H), 3.64-3.57 (m, 2H), 2.72-2.59 (m, 4H), 2.50 (s, 3H), 2.45-2.38 (m, 3H), 1.94-1.73 (m, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 170.7, 169.1, 157.8, 151.7, 149.3, 148.3, 141.0, 139.3, 132.0, 129.7, 127.6, 124.9, 124.9, 124.8, 122.6, 122.4, 120.8, 117.2, 114., 11.8, 108.2, 106.5, 95.3, 84.1, 68.0, 66.4, 63.2, 57.7, 56.0, 54.7, 53.0, 46.6, 46.1, 45.7, 42.4, 31.9, 30.7, 29.2, 25.2, 23.8, 22.9, 18.1; MS (ES+): m/z=874 (M+H).sup.+; LCMS (Method C): t.sub.R=3.00 min.
Example 58: (S)-1-(Chloromethyl)-3-(4-(((S)-2-methoxy-12-oxo-6a,7,8,9,10, 12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 4-methylpiperazine-1-carboxylate (55)
[1218] ##STR00166##
[1219] A solution of allyl (6S,6aS)-3-(4-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (54) (95 mg, 0.11 mmol) in dichloromethane (0.5 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (13 mg) and pyrrolidine (11 ?L) and then stirred at room temperature under argon. After 5 min, the resulting mixture was concentrated in vacuo and purified by column chromatography (silica), eluting with ethyl acetate (100%), followed by triethylamine/ethyl acetate (3%), then triethylamine/methanol/ethyl acetate (from 3:5:95 to 3:10:90), to give the title compound
[1220] (48 mg, 65%) as a creamy solid.
[1221] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 8.38 (s, 1H), 7.96 (d, J=5.9 Hz, 1H), 7.93-7.88 (m, 2H), 7.56-7.50 (m, 1H), 7.44-7.39 (m, 1H), 7.33 (s, 1H), 6.81 (s, 1H), 4.44-4.32 (m, 2H), 4.29-4.09 (m, 3H), 4.03 (dd, J=10.9, 3.1 Hz, 1H), 3.86 (s, 3H), 3.82-3.72 (m, 2H), 3.55 (br, 2H), 3.33-3.28 (m, 2H), 3.24-3.20 (m, 1H), 3.18-3.12 (m, 1H), 2.89-2.78 (m, 1H), 2.76-2.66 (m, 1H), 2.52 (br, 2H), 2.45 (br, 2H), 2.40-2.36 (m, 1H), 2.31 (s, 3H), 2.20 (t, J=6.6 Hz, 1H), 2.18-2.09 (m, 1H), 2.00-1.89 (m, 1H), 1.81-1.75 (m, 2H), 1.73-1.55 (m, 2H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 170.6, 166.7, 163.9, 153.0, 150.9, 147.9, 141.6, 1400.4, 130.0, 127.3, 124.6, 124.4, 122.9, 122.5, 120.9, 117.1, 111.8, 110.7, 110.0, 67.9, 55.4, 54.6, 52.8, 49.6, 47.9, 46.9, 45.7, 45.5, 45.3, 39.1, 31.5, 25.2, 24.2, 23.0, 18.2; MS (ES+): m/z=688 (M+H).sup.+; LCMS (Method C): t.sub.R=2.72 min.
[1222] Reaction Scheme for Preparing Compound (57)
##STR00167##
Example 59: Allyl (6S,6aS)-3-((6-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbon yl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (56)
[1223] ##STR00168##
[1224] A solution of allyl (6S,6aS)-3-((6-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (23) (49 mg, 0.063 mmol) in dichloromethane (5 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (38 mg, 0.19 mmol), 4-(dimethyl-amino)pyridine (8.5 mg, 0.069 mmol) and triethylamine (30 ?L, 0.22 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently washed with water (2?10 mL), dried over magnesium sulfate and concentrated in vacuo, to give the title compound (45 mg, 79%) as a brown oil, which was used in the subsequent step without further purification.
[1225] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.35 (s, 1H), 7.85 (d, J=8.2 Hz, 1H), 7.71 (d, J=8.2 Hz, 1H), 7.54-7.49 (m, 1H), 7.41 (t, J=7.8 Hz, 1H), 7.16 (s, 1H), 6.51 (s, 1H), 6.18 (d, J=9.4 Hz, 1H), 6.01 (d, J=10.2 Hz, 1H), 5.83-5.69 (m, 1H), 5.16-5.00 (m, 2H), 4.68-4.54 (m, 1H), 4.34-4.20 (m, 3H), 4.13-3.93 (m, 4H), 3.89 (s, 3H), 3.73 (br, 1H), 3.65 (br, 3H), 3.51-3.45 (m, 2H), 3.11-3.01 (m, 1H), 2.87 (br, 1H), 2.57 (br, 6H), 2.41 (s, 3H), 2.37-2.31 (m, 2H), 1.97-1.87 (m, 2H), 1.84-1.71 (m, 6H), 1.68-1.44 (m, 10H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 172.1, 169.2, 153.3, 149.3, 148.3, 143.9, 139.7, 133.6, 132.0, 127.6, 125.7, 124.8, 122.7, 122.4, 121.0, 120.8, 117.9, 113.7, 110.9, 108.1, 94.0, 93.6, 68.9, 66.4, 63.2, 60.1, 56.1, 54.5, 53.4, 53.1, 46.0, 42.5, 38.8, 35.7, 30.7, 28.9, 25.7, 25.3, 24.2, 23.0, 20.0, 18.2; MS (ES+): m/z=902 (M+H).sup.+; LCMS (Method C): t.sub.R=3.18 min.
Example 60: (S)-1-(Chloromethyl)-3-(6-(((S)-2-methoxy-12-oxo-6a,7,8,9, 10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 4-methylpiperazine-1-carboxylate (57)
[1226] ##STR00169##
[1227] A solution of allyl (6S,6aS)-3-((6-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (56) (45 mg, 0.050 mmol) in dichloromethane (5 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (6 mg) and pyrrolidine (5 ?L) and then stirred at room temperature under argon. After 5 min, the resulting mixture was concentrated in vacuo and purified by column chromatography (silica), eluting with ethyl acetate (10%), followed by triethylamine/ethyl acetate (3%), then triethylamine/methanol/ethyl acetate (from 3:5:95 to 3:10:90), to give the title compound
[1228] (18 mg, 500%) as a creamy solid.
[1229] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 8.38 (s, 1H), 7.97 (d, J=5.5 Hz, 1H), 7.92 (dd, J=8.4, 4.1 Hz, 2H), 7.55 (t, J=7.6 Hz, 1H), 7.45-7.40 (m, 1H), 7.33 (s, 1H), 6.77 (s, 1H), 4.46-4.35 (m, 2H), 4.27 (br, 1H), 4.17-4.10 (m, 2H), 4.08-4.02 (m, 2H), 3.86 (s, 3H), 3.85-3.79 (m, 2H), 3.80-3.74 (m, 2H), 3.56 (br s, 2H), 3.19-3.11 (m, 1H), 2.70-2.62 (m, 1H), 2.54 (br s, 2H), 2.46 (br s, 2H), 2.32 (s, 3H), 2.18-2.10 (m, 1H), 1.93-1.86 (m, 2H), 1.85-1.76 (m, 6H), 1.69-1.59 (m, 4H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 172.6, 168.7, 163.9, 153.0, 151.0, 147.9, 147.0, 142.7, 140.4, 132.5, 127.3, 124.3, 122.9, 122.5, 121.2, 117.3, 116.7, 111.8, 110.0, 109.8, 68.5, 55.4, 52.8, 49.6, 49.2, 47.9, 46.9, 46.5, 45.4, 39.1, 35.1, 31.9, 25.5, 24.2, 24.0, 23.0, 18.2; MS (ES+): m/z=716 (M+H).sup.+; LCMS (Method C): t.sub.R=2.83 min.
[1230] Reaction Scheme for Preparing Compound (59)
##STR00170##
Example 61: Allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxo-ethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-(12H)-carboxylate (58)
[1231] ##STR00171##
[1232] A solution of allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (41) (65 mg, 0.090 mmol) in dichloromethane (5 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (54 mg, 0.27 mmol), 4-(dimethylamino)-pyridine (12 mg, 0.098 mmol) and triethylamine (41 ?L, 0.31 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently washed with water (2?10 mL), dried over magnesium sulfate and concentrated in vacuo, to give the title compound (50 mg, 66%) as a brown oil, which was used in the subsequent step without further purification.
[1233] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.27 (s, 1H), 7.79 (d, J=8.6 Hz, 1H), 7.62 (d, J=8.2 Hz, 1H), 7.43 (t, J=7.6 Hz, 1H), 7.38-7.32 (m, 1H), 7.31-7.26 (m, 3H), 7.23-7.17 (m, 1H), 7.10 (s, 1H), 6.64 (s, 1H), 5.84 (d, J=10.2 Hz, 1H), 5.70-5.53 (m, 1H), 5.04 (br, 4H), 4.27 (d, J=9.4 Hz, 2H), 4.15-4.08 (m, 1H), 3.95 (br s, 1H), 3.80 (s, 3H), 3.69-3.64 (m, 1H), 3.57 (br s, 4H), 3.41-3.30 (m, 2H), 3.02-2.92 (m, 1H), 2.47 (br s, 4H), 2.39-2.35 (m, 3H), 2.32 (s, 3H), 1.98-1.89 (m, 2H), 1.74-1.60 (m, 2H), 1.59-1.53 (m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.2, 168.9, 153.3, 149.6, 149.6, 149.0, 148.2, 140.8, 140.7, 136.8, 134.1, 131.9, 129.6, 129.0, 128.9, 127.9, 127.5, 126.0, 124.9, 124.8, 122.5, 121.2, 117.6, 116.2, 114.6, 110.9, 110.6, 106.3, 101.4, 82.2, 70.8, 66.4, 56.0, 55.5, 54.5, 54.0, 53.1, 48.4, 45.9, 45.6, 38.6, 29.6, 22.9, 18.2; MS (ES+): m/z=852 (M+H).sup.+; LCMS (Method C): t.sub.R=2.97 min.
Example 62: (S)-1-(Chloromethyl)-3-(2-(3-((((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)-methyl)phenyl)acetyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 4-methylpiperazine-1-carboxylate (59)
[1234] ##STR00172##
[1235] A solution of allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (58) (50 mg, 0.059 mmol) in dichloromethane (5 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (7 mg) and pyrrolidine (6 ?L) and then stirred at room temperature under argon. After 5 min, the resulting mixture was concentrated in vacuo and purified by column chromatography (silica), eluting with ethyl acetate (100%), followed by triethylamine/ethyl acetate (3%), then triethylamine/methanol/ethyl acetate (from 3:5:95 to 3:10:90), to give the title compound (12 mg, 27%) as a creamy solid.
[1236] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 8.35 (s, 1H), 7.96-7.90 (m, 3H), 7.59-7.52 (m, 2H), 7.45 (d, J=8.6 Hz, 1H), 7.41 (d, J=3.5 Hz, 1H), 7.37 (br, 2H), 7.34 (d, J=2.0 Hz, 1H), 6.85 (d, J=5.5 Hz, 1H), 5.26-5.25 (m, 2H), 4.51-4.38 (m, 2H), 4.26 (br, 1H), 4.13 (d, J=12.1 Hz, 1H), 4.01 (br, 4H), 3.89-3.82 (m, 5H), 3.80-3.70 (m, 4H), 3.55 (br, 4H), 2.53 (br, 2H), 2.46 (br, 2H), 2.31 (s, 3H), 1.66-1.57 (m, 2H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 168.1, 164.0, 157.4, 153.3, 151.3, 150.5, 148.0, 146.4, 143.4, 140.2, 135.3, 132.8, 129.1, 128.6, 127.4, 126.0, 124.5, 123.6, 123.0, 122.5, 118.4, 116.6, 111.8, 110.7, 110.6, 70.3, 55.4, 54.5, 53.1, 49.5, 47.9, 46.7, 45.6, 45.4, 45.3, 39.1, 25.2, 24.1, 18.2; MS (ES+): m/z=750 (M+H).sup.+; LCMS (Method C): t.sub.R=2.90 min; HRMS calculated for [C42H.sub.45ClN.sub.5O.sub.6].sup.+: 750.3053, found: 750.3033.
[1237] Reaction scheme for preparing compound (64)
##STR00173##
Example 63: Allyl (6aS)-3-(4-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (60)
[1238] ##STR00174##
[1239] A solution of allyl (6aS)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo-[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (52)
[1240] (30 mg, 0.038 mmol) in dichloromethane (4 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (22.5 mg, 0.113 mmol), 4-(dimethylamino)-pyridine (5 mg, 0.042 mmol) and triethylamine (17 ?L, 0.13 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently washed with water (2?10 mL), dried over magnesium sulfate and concentrated in vacuo, to give the title compound
[1241] (27 mg, 77%) as a brown oil, which was used in the subsequent step without further purification.
[1242] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 8.36 (d, J=2.7 Hz, 1H), 7.97-7.92 (m, 2H), 7.93-7.87 (m, 1H), 7.57-7.51 (m, 1H), 7.44-7.39 (m, 1H), 7.34-7.28 (m, 3H), 7.14 (d, J=7.8 Hz, 1H), 6.98 (s, 1H), 5.83-5.67 (m, 1H), 5.64-5.56 (m, 1H), 5.42 (d, J=9.4 Hz, 1H), 5.12-4.96 (m, 2H), 4.76-4.66 (m, 1H), 4.51-4.40 (m, 2H), 4.39-4.25 (m, 3H), 4.17 (br, 2H), 4.08-4.02 (m, 1H), 4.00-3.91 (m, 1H), 3.86 (s, 3H), 3.84-3.77 (m, 2H), 3.68-3.48 (m, 4H), 3.20-3.06 (m, 3H), 2.89-2.79 (m, 1H), 2.57-2.42 (m, 4H), 2.30 (s, 3H), 2.23-2.15 (m, 2H), 2.06-2.01 (m, 2H), 1.80-1.66 (m, 2H), 1.61-1.43 (m, 4H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 171.1, 161.8, 153.0, 149.2, 149.0, 147.2, 144.6, 143.5, 136.0, 135.4, 134.8, 127.9, 127.7, 127.6, 127.3, 126.9, 126.8, 126.4, 123.0, 122.9, 122.5, 120.9, 120.7, 116.4, 114.8, 110.7, 109.6, 107.3, 102.8, 97.2, 67.9, 65.8, 63.2, 59.5, 55.5, 52.8, 49.6, 49.1, 46.9, 45.4, 43.5, 41.9, 35.2, 33.7, 31.5, 30.9, 30.1, 25.2; MS (ES+): m/z=922 (M+H).sup.+; LCMS (Method C): t.sub.R=3.20 min.
Example 64: (S)-1-(Chloromethyl)-3-(4-(((S)-2-methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)oxy)butanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 4-methylpiperazine-1-carboxylate (61)
[1243] ##STR00175##
[1244] A solution of allyl (6aS)-3-(4-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-14-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (600) (27 mg, 0.029 mmol) in dichloromethane (4 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (3.3 mg) and pyrrolidine (3 ?L) and then stirred at room temperature under argon. After 5 min, the resulting mixture was concentrated in vacuo and purified by column chromatography (silica), eluting with ethyl acetate (100%), followed by triethylamine/ethyl acetate (3%), then triethylamine/methanol/ethyl acetate (from 3:5:95 to 3:10:90), to give the title compound
[1245] (9 mg, 41%) as a brown solid.
[1246] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 8.38 (s, 1H), 7.96-7.89 (m, 3H), 7.56 (t, J=7.6 Hz, 1H), 7.49 (t, J=4.5 Hz, 1H), 7.47-7.42 (m, 2H), 7.36-7.27 (m, 3H), 6.84 (s, 1H), 4.88 (d, J=15.2, 1H), 4.56 (d, J=15.2 Hz, 1H), 4.48-4.36 (m, 3H), 4.33-4.19 (m, 3H), 4.15-4.03 (m, 2H), 3.94-3.89 (m, 1H), 3.87 (s, 3H), 3.84-3.76 (m, 4H), 3.55 (br, 2H), 3.32 (t, J=5.3 Hz, 1H), 2.57-2.41 (m, 4H), 2.31 (s, 3H), 2.27-2.19 (m, 2H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 170.6, 165.6, 162.4, 153.0, 149.0, 151.1, 147.9, 145.8, 136.5, 135.3, 134.4, 128.8, 127.9, 127.7, 127.0, 126.2, 124.4, 122.9, 122.5, 112.2, 116.0, 113.9, 112.2, 110.7, 110.3, 67.9, 55.4, 54.5, 52.9, 49.9, 49.4, 45.5, 43.2, 41.9, 31.5, 30.2, 24.1; MS (ES+): m/z=736 (M+H).sup.+; LCMS (Method C): t.sub.R=2.88 min.
[1247] Further Reaction Schemes
##STR00176## ##STR00177##
##STR00178## ##STR00179##
##STR00180##
##STR00181##
##STR00182##
##STR00183##
##STR00184## ##STR00185##
##STR00186## ##STR00187##
##STR00188## ##STR00189##
##STR00190## ##STR00191##
##STR00192## ##STR00193## ##STR00194##
##STR00195##
##STR00196##
##STR00197##
##STR00198##
##STR00199##
##STR00200##
##STR00201##
##STR00202## ##STR00203## ##STR00204##
##STR00205## ##STR00206##
Example 65: 4-hydroxy-5-methoxy-2-nitrobenzaldehde (65)
[1248] ##STR00207##
[1249] A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (32) (100 g, 348 mmol) in glacial acetic acid (800 mL) was charged with an aqueous solution of hydrobromic acid (48% v/v, 88.0 mL, 522 mmol) and heated to 85? C., with stirring for 1 h, after which the reaction was judged to be complete by TLC. After allowing the resulting mixture to cool to room temperature, it was then diluted in water (1.60 L), and the resulting precipitate filtered, and washed with cold water (100 mL?3) to give the title compound (50.0 g, 73%) as a yellow solid, which was used immediately in the subsequent step without further purification.
[1250] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 11.11 (br s, 1H), 10.15 (br s, 1H), 7.50 (s, 1H), 7.35 (s, 1H), 3.94 (s, 3H); MS (ES?): m/z=196 (M?H).sup.?; LCMS (Method B): t.sub.R=2.55 min.
Example 66: 5-Methoxy-2-nitro-4-((triisopropylsilyl)ox)benzaldehyde (66)
[1251] ##STR00208##
[1252] A mixture of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde (65) (50.0 g, 254 mmol), triisopropylsilyl chloride (59.7 mL, 279 mmol) and imidazole (51.8 g, 761 mmol) was heated and stirred at 100? C. for 30 min. The reaction mixture was poured onto ice-water and extracted with ethyl acetate (500 mL?3). The organic extract was dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (5%) to give the title compound (57.5 g, 64%) as a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 10.42 (s, 1H), 7.59 (s, 1H), 7.40 (s, 1H), 3.95 (s, 3H), 1.33-1.24 (m, 3H), 1.07 (s, 18H).
Example 67: 5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (67)
[1253] ##STR00209##
[1254] A solution of sodium chlorite (80%, 46.0 g, 407 mmol) and sodium phosphate monobasic dihydrate (35.5 g, 228 mmol) in water (200 mL) was added to a solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (66) (57.5 g, 163 mmol) in tetrahydrofuran
[1255] (800 mL) at room temperature. Hydrogen peroxide (30% w/w, 235 mL, 2.28 mol) was immediately added to the vigorously stirred biphasic mixture. The starting material dissolved, and the temperature of the reaction mixture rose to 45? C. After 30 min, the reaction was judged to have completed by TLC. The mixture was subsequently acidified to pH=3-4 with citric acid and extracted with ethyl acetate (500 mL?3). The combined organic extracts were washed with water (150 mL) and brine (150 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (10%) then methanol/dichloromethane (10%) to afford the title compound
[1256] (38.0 g, 63%) as a yellow oil.
[1257] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.81 (s, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 3.91 (s, 3H), 1.26 (q, J=7.4 Hz, 3H), 1.09 (d, J=7.4 Hz, 18H); MS (ES?): m/z=368 (M?H).sup.?; LCMS (Method D): t.sub.R=4.75 min.
Example 68: (S)-(2-(Hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4 ((triisopropyvlsilyl)oxy)phenyl)methanone (68)
[1258] ##STR00210##
[1259] A solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (67) (28.0 g, 75.8 mmol), HATU (31.7 g, 83.4 mmol) and dry triethylamine (44 mL) in dry dichloromethane (300 mL) was stirred at room temperature for 30 min. (S)-Piperidin-2-ylmethanol (11.4 g, 98.5 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was partitioned between dichloromethane (500 mL?2) and water (too mL). The combined organic extracts were then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 75%), to give the title compound (20.0 g, 57%) as a yellow solid.
[1260] .sup.1H NMR (400 MHz, CDCl.sub.3) mixture of rotamers, ? 7.68-7.65 (m, 1H), 7.03-6.65 (m, 1H), 5.04-4.69 (m, 1H), 4.12-4.05 (m, 0.4H), 4.01-3.95 (m, 0.5H), 3.92-3.89 (m, 2.6H), 3.83-3.74 (m, 1.5H), 3.64-3.59 (m, 0.4H), 3.45-3.40 (m, 0.3H), 3.21-3.01 (m, 1.4H), 2.87-2.79 (m, 0.4H), 1.97-1.94 (m, 0.6H), 1.88-1.77 (m, 0.6H), 1.73-1.62 (m, 3H), 1.56-1.44 (m, 2H), 1.29-1.24 (m, 3H), 1.09 (d, J=7.3 Hz, 18H); MS (ES+): m/z=467 (M+H).sup.+; LCMS (Method B): t.sub.R=4.75 min.
Example 69: (S)-(2-Amino-3-methoxy-4-((triisopropylsilyl)ox)phenyl)(2-(hydroxymethyl)piperidin-1-yl)methanone (69)
[1261] ##STR00211##
[1262] A solution of (S)-(2-(hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4 ((triisopropylsilyl)oxy)phenyl)methanone (68) (1.00 g, 2.14 mmol) in tetrahydrofuran (5 mL) was charged with palladium on activated charcoal (10 wt. % basis, 100 mg), ammonium formate (1.10 g, 17.1 mmol) and water (1 mL), and stirred at room temperature, under argon, for 2 h. The resulting mixture was filtered through celite, the filter cake was washed with ethyl acetate (50 mL) and water (50 mL) and the filtrate separated. The organic phase was then extracted with brine (50 mL?2), and dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 67%), gave the title compound (892 mg, 95%) as a yellow solid.
[1263] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.67 (s, 1H), 6.30 (s, 1H), 4.00-3.81 (m, 4H), 3.72 (s, 3H), 3.57 (s, 1H), 3.08 (s, 1H), 1.68-1.64 (m, 4H), 1.57-1.43 (m, 2H), 1.28-1.17 (m, 3H), 1.08 (d, J=7.4 Hz, 18H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.8, 147.9, 143.7, 133.2, 112.5, 110.0, 109.5, 68.7, 61.0, 56.4, 30.9, 25.8, 19.9, 17.9, 12.9; MS (ES+): m/z=437 (M+H).sup.+; LCMS (Method C): t.sub.R=4.07 min.
Example 70: Allyl (S)-(2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (70)
[1264] ##STR00212##
[1265] A solution of (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxylmethyl)piperidin-1-yl)methanone (69) (892 mg, 2.04 mmol) in dichloromethane (4 mL) was cooled to ?10? C. and charged with pyridine (380 ?L) and allyl chloroformate (228 ?L, 2.14 mmol), dropwise under argon. After 35 min, the reaction mixture was diluted with dichloromethane (10 mL), then extracted with a saturated aqueous solution of copper sulfate (10 mL?2) and brine (10 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 75%), gave the title compound (907 mg, 85%) as a pale yellow solid.
[1266] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.08 (s, 1H), 7.62 (s, 1H), 6.75 (s, 1H), 5.92 (ddt, J=17.2, 10.7, 5.5 Hz, 1H), 5.32 (dt, J=17.3, 1.7 Hz, 1H), 5.20 (dt, J=10.6, 1.4 Hz, 1H), 4.61 (dt, J=5.5, 1.5 Hz, 2H), 3.88 (t, J=10.7 Hz, 1H), 3.76 (s, 3H), 3.61-3.57 (m, 1H), 3.20-3.02 (m, 2H), 2.03 (s, 1H), 1.65-1.62 (m, 3H), 1.53-1.40 (m, 2H), 1.29-1.24 (m, 4H), 1.11-1.08 (m, 18H); MS (ES+): m/z=522 (M+H).sup.+; LCMS (Method A): t.sub.R=9.62 min.
Example 71: Allyl (6aS)-6-hydroxy-2-methoxy-12-oxo-3-((triisopropyl-silyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (71)
[1267] ##STR00213##
[1268] A solution of allyl (S)-(2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (70) (17.0 g, 32.7 mmol) in dichloromethane (150 mL) was charged with (diacetoxyiodo)benzene (12.6 g, 39.2 mmol) and 2,2,6,6-tetramethylpiperidine 1-oxyl (510 mg, 3.30 mmol), and stirred at room temperature for 16 h. The resulting mixture was then diluted with dichloromethane (350 mL), and sequentially washed with a saturated aqueous solution of sodium metabisulfite (100 mL) and a saturated aqueous solution of sodium hydrogen carbonate (100 mL). The organic extract was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 25% to 500%), to give the title compound
[1269] (13.0 g, 77%) as a pale yellow solid.
[1270] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.13 (s, 1H), 6.65 (s, 1H), 5.90 (d, J=10.3 Hz, 1H), 5.76 (s, 1H), 5.14 (t, J=12.1 Hz, 2H), 4.59 (dd, J=13.1, 5.3 Hz, 1H), 4.44 (dd, J=12.9, 5.1 Hz, 1H), 4.34 (dt, J=13.5, 4.1 Hz, 1H), 3.83 (s, 3H), 3.77 (br, 1H), 3.45 (ddd, J=10.1, 5.9, 4.0 Hz, 1H), 3.10-2.99 (m, 1H), 2.09-1.98 (m, 1H), 1.82-1.67 (m, 2H), 1.67-1.56 (m, 3H), 1.28-1.15 (m, 3H), 1.06 (dd, J=7.4, 2.5 Hz, 18H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.2, 156.2, 150.6, 147.6, 131.9, 127.0, 125.7, 121.2, 118.2, 110.9, 82.3, 66.9, 55.5, 55.3, 38.6, 23.2, 23.0, 18.2, 17.8, 12.8; MS (ES+): m/z=519 (M+H).sup.+; LCMS (Method A): t.sub.R=8.67 min.
Example 72: Allyl (6aS)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (72)
[1271] ##STR00214##
[1272] A solution of allyl (6aS)-6-hydroxy-2-methoxy-12-oxo-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (71) (14.0 g, 27.0 mmol) in tetrahydrofuran (130 mL) was charged with 3,4-dihydro-2H-pyran (24.6 g, 270 mmol) and p-toluenesulfonic acid monohydrate (140 mg, 0.76 mmol), and stirred for 18 h at room temperature. The resulting mixture was then diluted with ethyl acetate
[1273] (360 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate
[1274] (200 mL) and brine (100 mL). The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (17%), gave the title compound (12.5 g, 77%) as a yellow oil.
[1275] .sup.1H NMR (400 MHz, CDCl.sub.3), 1:1 mixture of diastereomers, ? 7.13 (s, 0.4H), 7.10 (s, 0.5H), 6.90 (s, 0.5H), 6.52 (s, 0.4H), 6.15 (d, J=10.0 Hz, 0.4H), 5.98 (d, J=10.0 Hz, 0.5H), 5.80-5.68 (m, 1H), 5.17-4.94 (m, 3H), 4.64-4.21 (m, 3H), 3.91-3.85 (m, 1H), 3.83 (d, J=1.8 Hz, 3H), 3.66-3.39 (m, 2H), 3.14-3.00 (m, 1H), 2.08-1.87 (m, 1H), 1.83-1.33 (m, 12H), 1.26-1.19 (m, 3H), 1.08-1.05 (m, 18H); MS (ES+): m/z=603 (M+H).sup.+; LCMS (Method A): t.sub.R=9.95 min.
Example 73: Allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (73)
[1276] ##STR00215##
[1277] A solution of allyl (6aS)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (72) (10.5 g, 17.4 mmol) in tetrahydrofuran (33 mL) was charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 26.1 mL, 26.1 mmol) at room temperature and stirred, to give an instantaneous orange colour. After 10 min, the reaction mixture was concentrated in vacuo, then immediately purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 100%), to give the title compound (7.13 g, 92%) as a white solid.
[1278] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 7.16 (s, 0.4H), 7.13 (s, 0.6H), 6.91 (br, 0.5H), 6.62 (br, 0.3H), 6.16 (d, J=10.1 Hz, 0.4H), 5.99 (d, J=10.1 Hz, 0.6H), 5.76 (ddd, J=15.8, 10.3, 5.0 Hz, 0.8H), 5.14-5.03 (m, 2H), 4.98 (br, 0.5H), 4.61 (dd, J=13.7, 4.9 Hz, 0.8H), 4.55-4.48 (m, 0.3H), 4.48 (br, 0.3H), 4.40 (dd, J=13.9, 5.2 Hz, 0.5H), 4.32-4.20 (m, 1H), 3.90 (s, 3H), 3.88-3.81 (m, 1H), 3.63 (br, 0.4H), 3.59-3.52 (m, 0.6H), 3.49-3.43 (m, 1H), 3.11-2.98 (m, 1H), 1.97-1.89 (m, 1H), 1.80-1.47 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, 8169.4, 169.2, 155.8, 147.9, 147.7, 146.6, 146.4, 132.2, 132.0, 128.4, 128.2, 125.9, 125.5, 117.0, 116.4, 115.8, 110.2, 109.8, 100.5, 95.3, 88.0, 84.1, 66.5, 66.3, 63.8, 63.2, 60.4, 56.2, 56.1, 55.4, 55.2, 38.8, 38.8, 31.0, 30.6, 25.2, 25.1, 23.22, 23.1, 23.0, 23.0, 20.1, 19.7, 18.4, 18.2; MS (ES+): m/z=447 (M+H).sup.+; LCMS (Method A): t.sub.R=6.87 min.
Example 74: 3-(Methoxycarbonyl)-4-phenylbut-3-enoic acid (74)
[1279] ##STR00216##
[1280] A solution of benzaldehyde (100 g, 942 mmol) and dimethyl succinate (206 g, 1.41 mol) in tert-butanol (500 mL) was added to a refluxing solution of potassium tert-butoxide (158 g, 1.41 mol) in tert-butanol (1.5 L) over 1 h. The mixture was then stirred for a further 30 min before being allowed to cool to room temperature. After concentrating in vacuo, the resulting residue was diluted with water (500 mL) and extracted with ethyl acetate (500 mL). The aqueous phase was then acidified to pH=4-5 with an aqueous solution of hydrochloric acid (6 M), then extracted with ethyl acetate (1 L). The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (300 g, impure) as a yellow oil which was used in the subsequent step without further purification.
[1281] MS (ES+): m/z=221 (M+H).sup.+; LCMS (Method F): t.sub.R=3.23 min.
Example 75: Methyl 4-hydroxy-2-naphthoate (75)
[1282] ##STR00217##
[1283] A solution of 3-(me oxycarbonyl)-4-phenylbut-3-enoic acid (74) (300 g) and trifluoroacetic anhydride (99.3 mL, 714 mmol) in tetrahydrofuran (1.5 L) was stirred at 70? C. for 5 h, after which, consumption of starting material was confirmed by TLC. The reaction mixture was then concentrated in vacuo, adjusted to pH=8-9 with an aqueous solution of sodium hydroxide (1 M) and extracted with ethyl acetate (1 L). The organic phase was then dried over sodium sulfate and concentrated in vacuo. Recrystallisation from ethyl acetate/petroleum spirit, 40-60? C. (10%) gave the title compound (100 g, 53%) as a yellow solid.
[1284] MS (ES+): m/z=202 (M+H).sup.+; LCMS (Method F): t.sub.R=3.55 min.
Example 76: Methyl 4-(benzyloxy)-2-naphthoate (76)
[1285] ##STR00218##
[1286] A solution of methyl 4-hydroxy-2-naphthoate (75) (200 g, 990 mmol), benzyl bromide (203 g, 1.19 mol) and caesium carbonate (386 g, 1.19 mol) in N,N-dimethylformamide (800 mL) was stirred at 90? C. for 16 h, after which TLC confirmed consumption of starting material. The mixture was diluted in ethyl acetate (1.5 L), washed with water (1 ?L?2), then brine (500 mL), dried over sodium sulfate and concentrated in vacuo to give the title compound (250 g, 86%) as a white solid, which was used in the subsequent step without further purification.
Example 77: 4-(Benzyloxy)-2-naphthoic acid (77)
[1287] ##STR00219##
[1288] A solution of methyl 4-(benzyloxy)-2-naphthoate (76) (250 g, 856 mmol) in toluene (500 mL) was charged with an aqueous solution of sodium hydroxide (12 M, 300 mL) and heated to 100? C. for 16 h, after which TLC confirmed the consumption of starting material. The organic phase was separated and concentrated in vacuo. The residue was then taken up into ethyl acetate (1.5 L) and acidified to pH=2 with an aqueous solution of hydrochloric acid (6 M). The organic phase was separated, dried over sodium sulfate and concentrated in vacuo. Recrystallization from ethyl acetate/petroleum spirit, 40-60? C.
[1289] (10%) gave the title compound (90 g, 32%) as a white solid.
[1290] MS (ES+): m/z=279 (M+H).sup.+; LCMS (Method F): t.sub.R=4.09 min.
Example 78: tert-Butyl (4-(benzyloxy)naphthalen-2-yl)carbamate (78)
[1291] ##STR00220##
[1292] A solution of 4-(benzyloxy)-2-naphthoic acid (77) (50.0 g, 180 mmol), diphenyl phosphoryl azide (41.5 mL, 234 mmol) and triethylamine (28.9 mL, 270 mmol) in toluene
[1293] (300 mL) was stirred at room temperature for 1 h, after which TLC showed consumption of starting material. tert-Butanol (200 mL) was added and the resulting mixture was stirred at 90? C. for 17 h. This was then diluted with ethyl acetate (1.5 L) and water (500 mL). The organic phase was separated, dried over sodium sulfate, filtered and concentrated in vacuo. Recrystallization from ethyl acetate/petroleum spirit, 40-60? C. (10%) gave the title compound (35 g, 56%) as a pink solid.
[1294] MS (ES+): m/z=350 (M+H).sup.+; LCMS (Method F): t.sub.R=4.67 min.
Example 79: tert-Butyl (4-(benzyloxy)-1-iodonaphthalen-2-yl)carbamate (79)
[1295] ##STR00221##
[1296] A mixture of tert-butyl (4-(benzyloxy)naphthalen-2-yl)carbamate (78) (55.0 g, 157 mmol), iodic acid (5.50 g, 31.5 mmol) and iodine (16.0 g, 63 mmol) in methanol (400 mL) and water (100 mL) was stirred at 80? C. for 5 h, after which TLC showed consumption of starting material. The mixture was diluted with water (1.0 L) and filtered. The resulting cake was washed with methanol (200 mL) and concentrated in vacuo to give the title compound (72 g, 96%) as a brown solid.
[1297] MS (ES+): m/z=476 (M+H).sup.+; LCMS (Method E): t.sub.R=4.91 min.
Example 80: tert-Butyl (R)-(4-(benzyloxy)-1-iodonaphthalen-2-yl)(oxiran-2-ylmethyl)carbamate (80)
[1298] ##STR00222##
[1299] A solution of tert-butyl (4-(benzyloxy)-1-iodonaphthalen-2-yl)carbamate (79) (52 g, 109 mmol) in N,N-dimethylformamide (500 mL) was charged with sodium hydride (60% dispersion in mineral oil, 17 g, 425 mmol) and stirred at room temperature for 30 min, after which (S)-oxiran-2-ylmethyl 3-nitrobenzenesulfonate (51 g, 197 mmol) was added and the resulting mixture stirred for a further 3 h. TLC confirmed consumption of starting material. The reaction mixture was poured cautiously onto ice-water (500 mL) and extracted with ethyl acetate (1.0 L). The organic phase was separated, and washed with water (500 mL) and brine (300 mL), then dried over sodium sulfate and concentrated in vacuo to give the title compound (55 g, 95%) as a white solid.
[1300] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.33-8.32 (m, 1H), 8.41-8.20 (m, 1H), 7.59-7.48 (m, 4H), 7.45-7.33 (m, 3H), 6.94-6.83 (m, 1H), 5.28 (s, 2H), 4.15-4.09 (m, 1H), 3.50-3.42 (m, 1H), 3.14-3.13 (m, 1H), 2.82-2.60 (m, 1H), 2.41 (ddd, J=12.4, 4.8, 2.8 Hz, 1H), 1.33-1.31 (m, 9H).
Example 81: tert-Butyl (S)-3-(benzyloxy)-1-(hydroxymethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (81)
[1301] ##STR00223##
[1302] Zinc chloride (1 M in tetrahydrofuran, 28 mL) was diluted in anhydrous tetrahydrofuran
[1303] (40 mL) and cooled to 0? C., under an inert atmosphere of argon. A solution of methyl lithium (1.6 M in diethyl ether, 70.6 mL) was then added to the cooled mixture, dropwise, and stirred for 30 min, before cooling further to ?78? C. (Trimethylsilyl)isothiocyanate (4 mL, 28.2 mmol) was added dropwise to the reaction mixture at ?78? C., before warming to 0? C. for 30 min and then again cooling to ?78? C. A solution of tert-butyl (R)-(4-(benzyloxy)-1-iodonaphthalen-2-yl)(oxiran-2-ylmethyl)carbamate (800) (10 g, 18.8 mmol) in tetrahydrofuran (20 mL) was added dropwise to the reaction mixture at ?78? C. for 30 min, then warmed to 0? C. for 1 h, followed by room temperature for 30 min. After quenching with a saturated aqueous solution of ammonium chloride, the mixture was extracted with dichloromethane (500 mL?3) and the combined organics were washed with brine (100 mL), dried over sodium sulfate and concentrated in vacuo to give the title compound (10 g, impure), which was used in the subsequent step without further purification.
[1304] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.29 (d, J=8.4 Hz, 1H), 7.90 (s, 1H), 7.71 (d, J=8.2 Hz, 1H), 7.55 (d, J=6.8 Hz, 2H), 7.51-7.40 (m, 3H), 7.36-7.32 (m, 2H), 5.27 (s, 2H), 4.22 (d, J=11.4 Hz, 1H), 4.13 (t, J=10.0 Hz, 1H), 4.01-3.95 (m, 1H), 3.85 (bs, 1H), 3.81-3.73 (m, 1H), 1.60 (s, 9H); MS (ES+): m/z=406 (M+H).sup.+; LCMS (Method F): t.sub.R=4.69 min.
Example 82: tert-Butyl (S)-5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (82)
[1305] ##STR00224##
[1306] A solution of tert-butyl (S)-5-(benzyloxy)-1-(hydroxymethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (81) (10.0 g, 12.4 mmol), carbon tetrachloride (30 mL) and triphenylphosphine (3.90 g, 14.8 mmol) in dichloromethane (50 mL) was stirred at room temperature for 2 h, after which, TLC showed consumption of starting material. The reaction mixture was then concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (10%), followed by recrystallisation from dichloromethane/petroleum spirit, 40-60? C. (90%) gave the title compound (1.47 g, 28%) as a white solid. [?]D.sup.23=?14.50 (c 0.470, CH.sub.2Cl.sub.2); .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.29 (d, J=8.4 Hz, 1H), 7.86 (s, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.58-7.30 (m, 7H), 5.27 (s, 2H), 4.27-4.24 (m, 1H), 4.13 (t, J=10.6 Hz, 1H), 4.01-3.87 (m, 2H), 3.44 (t, J=10.4 Hz, 1H), 1.61 (s, 9H); MS (ES+): m/z=424 (M+H).sup.+; LCMS (Method D): t.sub.R=4.27 min.
Example 83: (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11)
[1307] ##STR00225##
[1308] A solution of tert-butyl (S)-5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (82) (100 mg, 0.236 mmol) in anhydrous dichloromethane
[1309] (3 mL) was charged with boron trichloride (1 M solution in dichloromethane, 708 ?L, 0.708 mmol), in a dropwise manner via syringe, at room temperature and stirred under an inert atmosphere of argon. The resulting orange solution was stirred for 5 min before being quenched by cautious addition of methanol (5 mL), then concentrated in vacuo. The residue was charged again with methanol (5 mL) and re-concentrated in vacuo. Diethyl ether (5 mL) was then charged and the residue concentrated in vacuo once again. The residue was then subjected to high vacuum for 30 min to give the title compound (55 mg, impure) as a pale green crystalline solid (unstable), which was used immediately in the subsequent step (amide coupling) without further purification.
[1310] MS (ES+): m/z=234 (M+H).sup.+; LCMS (Method C): t.sub.R=2.62 min.
Example 84: tert-Butyl (8bR,9aS)-4-oxo-9,9a-dihydro-1H-benzo[e]cyclopropa[c]indole-2(4H)-carboxylate (83)
[1311] ##STR00226##
[1312] A solution of tert-butyl (S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (to) (20 mg, 0.060 mmol) in anhydrous N,N-dimethylacetamide (1.0 mL) was cooled to 0? C. and charged with potassium carbonate (58.0 mg, 0.419 mmol) and stirred at this temperature for 25 min. The reaction mixture was then quenched (cold) with a saturated aqueous solution of sodium hydrogen carbonate and the resulting slurry extracted twice with ethyl acetate. The combined organic extracts were then dried over magnesium sulfate and concentrated in vacuo before purification was enacted by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C.
[1313] (25%, isocratic) to give the title compound (14 mg, 79%) as a yellow solid.
[1314] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.22 (dd, J=7.9, 1.1 Hz, 1H), 7.49 (dd, J=7.7, 1.4 Hz, 1H), 7.39 (dt, J=7.6, 1.2 Hz, 1H), 6.86 (dd, J=7.8, 0.6 Hz, 1H), 6.82 (br s, 1H), 4.04-3.96 (m, 2H), 2.79-2.73 (m, 1H), 1.62 (dd, J=7.7, 4.4 Hz, 1H), 1.57 (s, 9H), 1.47 (t, J=4.7 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 186.1, 159.7, 151.7, 140.2, 132.7, 131.8, 126.9, 126.5, 120.9, 108.7, 83.5, 52.9, 33.5, 28.2, 23.4, 14.1; MS (ES+): m/z=298 (M+H).sup.+; LCMS (Method C): t.sub.R=3.37 min.
Example 85: tert-Butyl (S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (84)
[1315] ##STR00227##
[1316] A solution of tert-butyl (S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (10) (50 mg, 0.15 mmol) in dichloromethane (5 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (89 mg, 0.45 mmol), 4-(dimethylamino)pyridine (20 mg, 0.17 mmol) and triethylamine (73 ?L, 0.52 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently washed with water (2?10 mL), dried over magnesium sulfate and concentrated in vacuo, to give the title compound (59 mg, 86%) as a yellow solid.
[1317] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.05 (br s, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.68 (d, J=8.3 Hz, 1H), 7.51-7.45 (m, 1H), 7.38-7.33 (m, 1H), 4.28-4.21 (br, 1H), 4.15-4.07 (m, 1H), 4.03-3.96 (m, 1H), 3.94-3.88 (m, 1H), 3.72 (t, J=4.9 Hz, 2H), 3.68-3.60 (m, 2H), 3.45 (t, J=100.8 Hz, 1H), 2.61-2.50 (m, 4H), 2.31 (s, 3H), 1.57 (s, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 153.4, 152.4, 148.4, 148.3, 130.2, 127.6, 124.2, 124.1, 122.6, 122.3, 120.1, 109.3, 81.2, 54.6, 54.2, 48.5, 46.3, 46.1, 45.8, 28.4; MS (ES+): m/z=460 (M+H).sup.+; LCMS (Method C): t.sub.R=3.00 min.
General Procedure A: O-Alkylation of Pyridinobenzodiazepines
[1318] ##STR00228##
[1319] A solution of allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (73) (1 equiv.) in N,N-dimethylformamide (0.1 M) was charged with potassium carbonate (1.5 equiv.) and organohalide (1.2 equiv.) and stirred at room temperature, whilst monitoring by TLC and LCMS. Once the reaction was judged to be complete, it was diluted into ethyl acetate and washed twice with cold brine. The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo, and then purified by flash column chromatography (silica).
[1320] General Procedure B: Hydrolysis of Pyridinobenzodiazepine Methyl Esters
##STR00229##
[1321] A solution of methyl ester (1 equiv.) in tetrahydrofuran (0.1 M) was charged with an aqueous solution of sodium hydroxide (0.5 M, 4 equiv.) and stirred at room temperature, whilst monitoring by TLC and LCMS. Once the reaction was judged to be complete, the mixture was then partially concentrated in vacuo (to remove tetrahydrofuran), then diluted into ethyl acetate and acidified to pH=3-4 with a saturated aqueous solution of citric acid. The organic layer was separated, and the aqueous layer washed with ethyl acetate. The combined organic extracts were then washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting residue was used in the next step without any further purification.
General Procedure C: Amide Coupling of (S)-Seco-CBI to Pyridinobenzodiazepine Carboxylic Acids
[1322] ##STR00230##
[1323] A solution of pyridinobenzodiazepine carboxylic acid (1 equiv.) in N,N-dimethylacetamide
[1324] (0.1 M) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (1.4 equiv.), followed by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (4 equiv.) and stirred at room temperature for 16 h. The resulting mixture was diluted into ethyl acetate and washed with cold brine (twice), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification was enacted by flash column chromatography (silica).
General Procedure D: Deprotection of N-Alloc, O-THP Pyridinobenzodiazepines
[1325] ##STR00231##
A solution of protected pyridinobenzodiazepine (1 equiv.) in dichloromethane (0.1 M) was charged with pyrrolidine (1.2 equiv.), and tetrakis(triphenylphosphine) palladium(0) (0.1 equiv.) and stirred at room temperature whilst monitoring by TLC and LCMS. After the reaction was judged to be complete (approx. 10 min), the mixture was diluted in dichloromethane and filtered through a pad of celite. The filtrate was concentrated in vacuo, then charged with diethyl ether and concentrated again. Diethyl ether was charged once more, and the residue concentrated in vacuo for a third time. Purification was enacted by flash column chromatography (silica).
Example 86: Allyl (6aS)-2-methoxy-3-5-((3-(2-methoxy-2-oxoethyl)benzyl)-oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (85)
[1326] ##STR00232##
[1327] General Procedure A was followed, using allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (73) (1.00 g, 2.24 mmol), 3-(bromomethyl)-benzeneacetic acid methyl ester (653 mg, 2.69 mmol), potassium carbonate (464 mg, 3.36 mmol) and N,N-dimethylformamide (5 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (66%) gave the title compound (980 mg, 74%) as a colourless gel.
[1328] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 7.34-7.31 (m, 2H), 7.24-7.21 (m, 1H), 7.19 (s, 1H), 7.16 (s, 1H), 6.89 (s, 1H), 6.53 (br, 1H), 6.14 (d, J=10.4 Hz, 1H), 5.99 (d, J=10.0 Hz, 1H), 5.74-5.61 (m, 1H), 5.19-4.98 (m, 4H), 4.54 (dd, J=13.5, 4.9 Hz, 1H), 4.40-4.20 (m, 1H), 3.92-3.78 (m, 4H), 3.67 (s, 3H), 3.61 (s, 3H), 3.46 (dt, J=9.8, 4.9 Hz, 1H), 3.11-2.98 (m, 1H), 1.84-1.41 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, ? 171.8, 171.7, 169.3, 169.1, 149.9, 149.6, 149.3, 136.8, 136.7, 134.4, 134.3, 132.2, 132.0, 129.0, 128.8, 128.1, 128.0, 127.7, 126.1, 126.0, 116.9, 115.1, 114.8, 110.8, 110.4, 100.3, 95.2, 88.1, 84.2, 71.2, 70.8, 66.4, 66.2, 63.9, 63.1, 56.1, 56.1, 55.4, 55.2, 52.0, 41.1, 41.0, 38.8, 38.8, 31.0, 30.5, 25.2, 25.2, 23.2, 23.0, 22.9, 20.0, 19.6, 18.4, 18.2; MS (ES+): m/z=609 (M+H).sup.+; LCMS (Method A): t.sub.R=7.83 min.
Example 87: 2-(3-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo-[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (86)
[1329] ##STR00233##
[1330] General Procedure B was followed, using allyl (6aS)-2-methoxy-3-((3-(2-methoxy-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (85) (980 mg, 1.61 mmol), sodium hydroxide (0.5 M, aq., 6.4 mL, 3.22 mmol) and tetrahydrofuran (3.2 mL). The title compound (843 mg, 88%) was isolated as a white solid, which was used in the next step without further purification.
[1331] MS (ES+): m/z=595 (M+H).sup.+; LCMS (Method A): t.sub.R=7.12 min.
Example 88: Allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (87)
[1332] ##STR00234##
[1333] General Procedure C was followed using 2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (29) (843 mg, 1.42 mmol), (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (86) (536 mg, 1.98 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.14 g, 5.95 mmol) and N,N-dimethylaectamide (2.8 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (67%) gave the title compound (926 mg, 81%) as a green oil.
[1334] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 10.01 (br s, 1H), 8.34 (d, J=16.2 Hz, 1H), 8.23 (dd, J=8.2, 3.3 Hz, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.46 (dd, J=17.7, 7.3 Hz, 2H), 7.38-7.29 (m, 4H), 7.17-7.11 (m, 1H), 6.86 (s, 0.5H), 6.56 (s, 0.5H), 6.14 (d, J=9.6 Hz, 1H), 5.97 (d, J=10.0 Hz, 1H), 5.58 (dd, J=16.4, 11.2 Hz, 1H), 5.17-4.90 (m, 5H), 4.53-4.42 (m, 0.5H), 4.39 (br, 0.5H), 4.29 (d, J=10.8 Hz, 2H), 3.96-3.89 (m, 3H), 3.82 (s, 3H), 3.79 (s, 2H), 3.60 (br, 1H) 3.52-3.36 (m, 2H), 3.27 (q, J=10.7 Hz, 1H), 3.12-2.97 (m, 1H), 1.84-1.36 (m, 12H); MS (ES+): m/z=8100 (M+H).sup.+; LCMS (Method A): t.sub.R=8.67 min.
Example 89: (S)-3-((3-(2-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)ox)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (42)
[1335] ##STR00235##
[1336] General Procedure D was followed, using allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (87) (96 mg, 0.12 mmol), tetrakis(triphenylphosphine)palladium(0) (14 mg, 0.012 mmol), pyrrolidine (12 ?L, 0.14 mmol) and dichloromethane (5 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%), followed by trituration from dichloromethane/diethyl ether, gave the title compound (30 mg, 41%) as a white solid.
[1337] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.34 (br s, 1H), 8.21 (d, J=8.6 Hz, 1H), 8.10 (br s, 1H), 7.92 (d, J=5.9 Hz, 1H), 7.80 (d, J=7.8 Hz, 1H), 7.75-7.67 (m, 1H), 7.65-7.59 (m, 1H), 7.57-7.50 (m, 2H), 7.40 (d, J=7.0 Hz, 1H), 7.38-7.34 (m, 2H), 6.84 (br, 1H), 5.26-5.12 (m, 1H), 5.13-5.06 (m, 1H), 4.45-4.39 (m, 2H), 4.37-4.31 (m, 2H), 4.14-4.09 (m, 2H), 4.00-3.93 (m, 2H), 3.82 (s, 3H), 3.73-3.70 (s, 1H), 3.67-3.60 (m, 1H), 2.50 (t, J=7.4 Hz, 1H), 2.32 (dt, J=7.4, 2.0 Hz, 1H), 1.82-1.77 (m, 2H), 1.64-1.55 (br, 2H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 169.1, 166.8, 163.9, 150.5, 148.0, 140.2, 135.4, 131.8, 129.1, 128.7, 128.6, 128.5, 126.0, 123.3, 122.9, 122.4, 121.5, 114.6, 111.8, 110.6, 100.3, 70.2, 55.4, 53.2, 49.6, 46.8, 42.7, 41.7, 39.2, 29.7, 24.1, 22.9, 18.1; MS (ES+): m/z=624 (M+H).sup.+; LCMS (Method C): t.sub.R=4.02 min, LCMS (Method A): t.sub.R=7.60 min; HRMS calculated for [C.sub.36H.sub.35ClN.sub.3O.sub.5].sup.+: 624.2260, found: 624.2254.
Example 90: Allyl (6aS)-2-methoxy-3-((2-(methoxycarbonyl)benzo[b]-thiophen-3-yl)methoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (88)
[1338] ##STR00236##
[1339] General Procedure A was followed using allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1, 2-a][1,4]diazepine-5(12H)-carboxylate (73) (1.00 g, 2.24 mmol), methyl 3-(bromomethyl)-1-benzothiophene-2-carboxylate (766 mg, 2.69 mmol), potassium carbonate (464 mg, 3.36 mmol) and N,N-dimethylformamide (4.5 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (64%) gave the title compound (615 mg, 84%) as a white solid.
[1340] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 8.26 (d, J=7.9 Hz, 0.4H), 8.20 (d, J=7.8 Hz, 0.6H), 7.81 (dd, J=7.6, 5.3 Hz, 1H), 7.50-7.37 (m, 2H), 7.15 (d, J=2.9 Hz, 1H), 7.09 (s, 0.6H), 6.87 (s, 0.4H), 6.15 (d, J=10.1 Hz, 0.4H), 6.01 (d, J=9.9 Hz, 0.6H), 5.93 (d, J=12.8 Hz, 0.4H), 5.85-5.61 (m, 2.4H), 5.16-4.92 (m, 3H), 4.57 (d, J=13.7 Hz, 0.6H), 4.44 (dd, J=14.4, 9.9 Hz, 1H), 4.35-4.20 (m, 1H), 4.00-3.80 (m, 7.6H), 3.68-3.56 (m, 1H), 3.54-3.41 (m, 1H), 3.05 (t, J=11.9 Hz, 1H), 1.81-1.46 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, ? 169.3, 169.1, 163.2, 162.9, 155.8, 149.6, 140.4, 140.3, 139.1, 127.5, 127.3, 126.8, 125.4, 125.3, 125.0, 124.9, 122.4, 117.0, 115.6, 114.9, 110.5, 99.9, 95.2, 87.9, 84.0, 66.4, 66.3, 63.6, 63.4, 63.1, 62.1, 60.3, 56.1, 56.0, 55.4, 55.3, 52.5, 52.4, 38.8, 30.9, 30.6, 25.3, 25.2, 23.3, 23.2, 23.0, 22.9, 19.8, 19.7, 18.4, 18.2; MS (ES+): m/z=651 (M+H).sup.+; LCMS (Method A): t.sub.R=8.80 min and t.sub.R=8.98.
Example 91: 3-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-g-yl)oxy)methyl)benzo[b]thiophene-2-carboxylic acid (89)
[1341] ##STR00237##
[1342] General Procedure B was followed, using allyl (6aS)-2-methoxy-3-((2-(methoxycarbonyl)benzo[b]thiophen-3-yl)methoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,1-hexahydrobenzo[e]pyrido[1,2-a][1, 4]diazepine-5(12H)-carboxylate (88) (559 mg, 0.859 mmol), sodium hydroxide (0.5 M, aq., 3.4 mL, 1.72 mmol) and tetrahydrofuran (1.7 mL). The title compound (559 mg, impure) was isolated as a cream solid, which was used in the next step without further purification.
[1343] MS (ES+): m/z=637 (M+H).sup.+; LCMS (Method A): t.sub.R=7.68 min.
Example 92: Allyl (6aS)-3-((2-((S)-1-(chloromethyl)-5-hydroxy-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)benzo[b]thiophen-3-yl)methoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-(12aH)-carboxylate (90)
[1344] ##STR00238##
[1345] General Procedure C was followed, using 3-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5, 6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)benzo[b]thiophene-2-carboxylic acid (89) (200 mg, 0.314 mmol), (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (96 mg, 0.354 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (203 mg, 1.06 mmol) and N,N-dimethylaectamide (1 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (58%) gave the title compound (150 mg, 56%) as a green solid.
[1346] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.63 (br, 1H), 8.17 (t, J=7.4 Hz, 1H), 8.00 (dd, J=11.7, 5.5 Hz, 1H), 7.83 (dd, J=12.6, 5.7 Hz, 1H), 7.58 (d, J=8.3 Hz, 1H), 7.44 (dt, J=7.4, 5.1 Hz, 3H), 7.33-7.25 (m, 1H), 7.22-6.90 (m, 3H), 6.12 (d, J=9.3 Hz, 1H), 5.94 (d, J=10.0 Hz, 1H), 5.59 (ddd, J=15.8, 10.6, 5.2 Hz, 1H), 5.46 (q, J=12.2 Hz, 2H), 5.01 (d, J=14.9 Hz, 1H), 4.92 (d, J=11.1 Hz, 1H), 4.51-4.16 (m, 4H), 3.91-3.67 (m, 3H), 3.58-3.51 (m, 3H), 3.49-3.31 (m, 3H), 3.12-2.93 (m, 1H), 1.93-1.14 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 169.3, 163.2, 155.8, 155.1, 149.8, 149.6, 149.5, 149.4, 14.6, 139.4, 137.9, 134.4, 132.1, 131.9, 129.9, 127.6, 125.2, 124.0, 123.7, 123.5, 123.2, 122.8, 122.1, 117.0, 116.2, 115.6, 110.8, 110.5, 100.0, 94.9, 88.1, 66.3, 64.4, 63.8, 62.9, 55.8, 55.5, 46.2, 42.1, 38.8, 30.9, 25.2, 25.0, 23.2, 22.9, 19.1; MS (ES+): m/z=852 (M+H).sup.+; LCMS (Method A): t.sub.R=8.93 min.
Example 93: (S)-3-((2-((S)-1-(Chloromethyl)-5-hydroxy-2,3-dihydro-H-benzo[e]indole-3-carbonyl)benzo[b]thiophen-3-yl)methoxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (91)
[1347] ##STR00239##
[1348] General Procedure D was followed, using allyl (6aS)-3-((2-((S)-1-(chloromethyl)-5-hydroxy-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)benzo[b]thiophen-3-yl)methoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (90) (150 mg, 0.176 mmol), tetrakis(triphenylphosphine)palladium(0) (20 mg, 0.018 mmol), pyrrolidine (18 ?L, 0.211 mmol) and dichloromethane (1 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (89%), followed by trituration from dichloromethane/diethyl ether, gave the title compound (43 mg, 37%) as an off-white solid.
[1349] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.37 (br, 1H), 8.28-8.16 (m, 2H), 8.09-8.04 (m, 1H), 7.85 (d, J=8.3 Hz, 2H), 7.60-7.49 (m, 4H), 7.43-7.36 (m, 1H), 7.25 (s, 1H), 6.97 (s, 1H), 5.58 (s, 2H), 4.45-4.27 (m, 2H), 4.17-4.06 (m, 2H), 4.00 (d, J=11.2 Hz, 1H), 3.84-3.73 (m, 1H), 3.63 (s, 3H), 3.51 (s, 1H), 3.10 (t, J=12.8 Hz, 1H), 1.85-1.55 (m, 6H); MS (ES+): m/z=666 (M+H).sup.+; LCMS (Method A): t.sub.R=7.45 min.
Example 94: Allyl (6aS)-2-methoxy-3-((6-(2-methoxy-2-oxoethyl)pyridin-2-yl)methoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12Ha)-carboxylate (q2)
[1350] ##STR00240##
[1351] General Procedure A was followed, using allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (73) (100 mg, 0.224 mmol), methyl 2-[6-(chloromethyl)-2-pyridyl]acetate hydrochloride (46 mg, 0.195 mmol), potassium carbonate (61 mg, 0.44 mmol) and N,N-dimethylformamide (2 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (84%) gave the title compound (100 mg, impure) as a white solid.
[1352] MS (ES+): m/z=610 (M+H).sup.+; LCMS (Method C): t.sub.R=3.93 min
Example 95: 2-(6-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-566a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)pyridin-2-yl)acetic acid (q3)
[1353] ##STR00241##
[1354] General Procedure B was followed, using allyl (6aS)-2-methoxy-3-((6-(2-methoxy-2-oxoethyl)pyridin-2-yl)methoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (92) (100 mg, 0.16 mmol), sodium hydroxide (0.5 M, aq., 0.65 mL, 0.32 mmol) and tetrahydrofuran (3 mL). The title compound (95 mg, impure) was isolated as a white solid, which was used in the next step without further purification.
[1355] MS (ES+): m/z=596 (M+H).sup.+; LCMS (Method C): t.sub.R=3.55 min.
Example 96: Allyl (6aS)-3-((6-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)pyridin-2-yl)methoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (94)
[1356] ##STR00242##
[1357] General Procedure C was followed, using 2-(6-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7, 8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)pyridin-2-yl)acetic acid (93) (95 mg, 0.16 mmol), (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (43 mg, 0.16 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (123 mg, 0.64 mmol) and N,N-dimethylaectamide (2 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (87%) gave the title compound (43 mg, 33%) as a green solid.
[1358] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 8.26 (dd, J=14.3, 8.9 Hz, 1H), 8.00 (s, 1H), 7.68 (t, J=7.8 Hz, 1H), 7.59 (dd, J=14.1, 6.0 Hz, 1H), 7.48 (t, J=9.7 Hz, 1H), 7.42-7.31 (m, 2H), 7.20-7.14 (m, 1H), 7.04 (d, J=7.7 Hz, 1H), 6.85 (br, 1H), 6.17-6.10 (m, 1H), 6.01-5.90 (m, 1H), 5.68-5.52 (m, 1H), 5.27-5.16 (m, 2H), 5.07-4.89 (m, 2H), 4.76-4.69 (m, 1H), 4.48 (d, J=9.6 Hz, 1H), 4.36-4.24 (m, 1H), 4.22-4.02 (m, 2H), 3.96-3.84 (m, 4H), 3.85-3.67 (m, 2H), 3.58-3.33 (m, 2H), 3.28 (td, J=10.7, 4.7 Hz, 1H), 3.12-2.96 (m, 1H), 2.93 (s, 3H), 1.96-1.33 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, ? 169.3, 162.5, 156.8, 156.0, 155.7, 155.2, 149.2, 141.0, 137.9, 137.1, 132.1, 129.9, 127.5, 123.9, 123.4, 122.9, 122.4, 122.2, 122.0, 117.0, 114.9, 110.5, 100.7, 100.1, 88.0, 71.4, 66.5, 66.3, 63.0, 57.8, 56.1, 53.8, 52.2, 50.6, 46.0, 38.9, 36.5, 31.4, 30.9, 25.2, 23.2, 18.4; MS (ES+): m/z=811 (M+H).sup.+; LCMS (Method C): t.sub.R=4.38 min.
Example 97: (S)-3-((6-(2-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)pyridin-2-yl)methoxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (95)
[1359] ##STR00243##
[1360] General Procedure D was followed, using allyl (6aS)-3-((6-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)pyridin-2-yl)methoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9, -hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (94) (43 mg, 0.050 mmol), tetrakis(triphenylphosphine)palladium(0) (6 mg, 0.005 mmol), pyrrolidine (5 ?L, 0.211 mmol) and dichloromethane (1 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 75% to 100%), followed by ethyl acetate/methanol (7%), gave the title compound (12 mg, 36%) as a white solid.
[1361] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.43 (br, 1H), 8.21 (d, J=8.4 Hz, 1H), 8.06 (s, 1H), 7.84 (d, J=3.0 Hz, 1H), 7.83 (s, 1H), 7.81 (br, 1H), 7.55-7.52 (m, 1H), 7.51 (d, J=8.2 Hz, 1H), 7.41 (d, J=7.8 Hz, 1H), 7.37-7.32 (m, 2H), 6.84 (s, 1H), 5.32-5.23 (m, 2H), 4.69 (dd, J=100.8, 1.8 Hz, 1H), 4.50-4.43 (m, 1H), 4.13 (ddd, J=15.7, 12.9, 6.3 Hz, 4H), 3.97 (dd, J=10.9, 3.1 Hz, 1H), 3.87 (s, 3H), 3.79-3.76 (m, 1H), 3.73-3.65 (m, 2H), 1.96-1.53 (m, 6H); MS (ES+): m/z=625 (M+H).sup.+; LCMS (Method A): t.sub.R=6.97 min.
Example 98: Allyl (6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (96)
[1362] ##STR00244##
[1363] General Procedure A was followed, using allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (73) (670 mg, 1.50 mmol), ethyl 6-bromohexanoate (280 ?L, 1.58 mmol), potassium carbonate (311 mg, 2.25 mmol) and N,N-dimethylformamide (2 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (55%) gave the title compound (749 mg, 85%) as a yellow oil.
[1364] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 7.16 (m, 1H), 6.50 (s, 1H), 6.10 (m, 1H), 5.81-5.76 (m, 1H), 5.14-5.03 (m, 2H), 4.69-4.57 (m, 2H), 4.47-4.37 (m, 1H), 4.34-4.26 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 4.01-3.94 (m, 3H), 3.90 (s, 3H), 3.68-3.62 (m, 1H), 3.68-3.46 (m, 2H), 3.12-3.03 (m, 1H), 2.33 (t, J=7.4 Hz, 2H), 1.89-1.66 (m, 11H), 1.57-1.47 (m, 6H), 1.25 (t, J=7.1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, ? 173.5, 173.4, 169.3, 169.1, 155.8, 150.0, 149.3, 149.0, 132.2, 132.0, 127.7, 126.2, 125.7, 116.9, 114.2, 113.7, 110.6, 110.2, 100.0, 98.9, 95.3, 88.0, 84.2, 68.8, 68.6, 66.2, 65.8, 63.9, 63.1, 60.1, 56.1, 56.0, 55.3, 55.2, 38.7, 34.2, 34.1, 31.0, 30.7, 28.6, 28.5, 25.5, 25.2, 25.0, 23.2, 23.1, 23.0, 22.9, 19.9, 19.6, 18.3, 18.1, 14.2; MS (ES+): m/z=589 (M+H).sup.+; LCMS (Method B): t.sub.R=4.32 min.
Example 9q: 6-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-H-pyran-2-yl)ox)-5,6,6a,7,8,9,1,12-octahydrobenzo[e]. pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (97)
[1365] ##STR00245##
[1366] General Procedure B was followed, using allyl (6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (96) (713 mg, 1.21 mmol), sodium hydroxide (0.5 M, aq., 6.06 mL, 3.03 mmol) and tetrahydrofuran (14 mL). The title compound (412 mg, 61%) was isolated as a white solid, which was used in the next step without further purification.
[1367] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.18 (s, 1H), 6.19 (s, 1H), 6.18-5.99 (m, 1H), 5.81-5.71 (m, 1H), 5.12-5.02 (m, 2H), 4.67-4.51 (m, 1H), 4.48-4.36 (m, 1H), 4.31-4.23 (m, 1H), 4.00-3.88 (m, 7H), 3.66-3.46 (m, 2H), 3.12-3.02 (m, 1H), 2.36 (t, J=7.4 Hz, 2H), 1.81-1.79 (m, 2H), 1.75-1.65 (m, 10H), 1.55-1.49 (m, 7H); MS (ES+): m/z=561 (M+H).sup.+; LCMS (Method B): t.sub.R=3.78 min.
Example 100: Allyl (6aS)-3-((6-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepine-5(12H)-carboxylate (98)
[1368] ##STR00246##
[1369] General Procedure C was followed, using 6-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (97) (400 mg, 0.713 mmol), (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (193 mg, 0.713 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (537 mg, 2.80 mmol) and N,N-dimethylaectamide (3 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (70%) gave the title compound (337 mg, 61%) as a grey solid.
[1370] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.34 (d, J=7.2 Hz, 1H), 8.29 (d, J=8.2 Hz, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.54-7.48 (m, 1H), 7.39-7.33 (m, 1H), 7.18 (s, 1H), 6.58 (s, 1H), 6.19 (d, J=10.0 Hz, 1H), 6.01 (d, J=10.0 Hz, 1H), 5.81-5.66 (m, 1H), 5.17-4.99 (m, 3H), 4.68-4.42 (m, 2H), 4.35-4.24 (m, 3H), 4.09-4.01 (m, 3H), 3.88 (s, 3H), 3.85-3.80 (m, 1H), 3.67-3.60 (m, 1H), 3.52-3.46 (m, 1H), 3.42 (t, J=11 Hz, 1H), 3.13-3.02 (m, 1H), 2.74-2.55 (m, 2H), 2.01-1.88 (m, 6H), 1.82-1.61 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.2, 163.7, 156.3, 155.1, 149.3, 146.6, 141.2, 131.6, 130.0, 127.6, 125.4, 123.9, 123.5, 122.7, 122.0, 117.9, 116.0, 114.6, 110.5, 108.0, 106.4, 100.4, 94.7, 69.1, 66.8, 63.0, 60.4, 56.1, 55.9, 52.3, 46.9, 46.3, 42.3, 35.7, 31.9, 29.7, 29.4, 25.5, 25.4, 25.2, 23.1, 22.7; MS (ES?): m/z=774 (M?1).sup.?, MS (ES+): m/z=798 (M+Na).sup.+; LCMS (Method C): t.sub.R=3.93 min.
Example 101: (S)-3-((6-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo-[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (99)
[1371] ##STR00247##
[1372] General Procedure D was followed, using allyl (6aS)-3-((6-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (98) (337 mg, 0.434 mmol), tetrakis(triphenyl-phosphine)palladium(0) (50 mg, 0.043 mmol), pyrrolidine (43 ?L, 0.521 mmol) and dichloromethane (1 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 30% to 100%), gave the title compound (161 mg, 63%) as a white solid.
[1373] .sup.1H NMR (400 MHz, acetone-d.sub.6) 39.31 (br s, 1H), 8.21 (d, J=8.2 Hz, 1H), 8.13 (s, 1H), 7.97 (d, J=5.5 Hz, 1H), 7.80 (d, J=8.6 Hz, 1H), 7.74-7.67 (m, 1H), 7.63-7.49 (m, 1H), 7.33 (s, 1H), 6.78 (s, 1H), 4.39-4.30 (m, 2H), 4.19-4.11 (m, 3H), 4.10-4.05 (m, 1H), 4.01 (dd, J=11.0, 3.5 Hz, 1H), 3.85 (s, 3H), 3.79-3.68 (m, 2H), 3.16 (td, J=11.3, 3.1 Hz, 1H), 2.70-2.56 (m, 2H), 2.18-2.10 (m, 1H), 2.02-1.95 (m, 1H), 1.94-1.76 (m, 6H), 1.70-1.60 (m, 4H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 166.8, 163.9, 154.4, 151.0, 147.9, 140.3, 130.3, 127.2, 123.3, 122.7, 122.3, 121.1, 114.4, 111.7, 109.8, 100.4, 68.5, 55.4, 53.0, 49.6, 46.9, 41.7, 39.1, 35.3, 25.5, 24.1, 24.0, 22.9, 18.1; MS (ES+): m/z=590 (M+H).sup.+; LCMS (Method A): t.sub.R=7.20 min.
Example 102: Allyl (6aS)-2-methoxy-3-((8-methoxy-8-oxooctyl)ox)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (100)
[1374] ##STR00248##
[1375] General Procedure A was followed, using allyl (6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (73) (150 mg, 0.34 mmol), methyl 8-bromooctanoate (70 ?L, 0.36 mmol), potassium carbonate (70 mg, 0.51 mmol) and N,N-dimethylformamide
[1376] (2 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 10% to 60%) gave the title compound (182 mg, 91%) as a yellow oil.
[1377] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, 87.14 (s, 1H), 7.11 (s, 1H), 6.80 (br, 1H), 6.16 (d, J=10.0 Hz, 1H), 5.98 (d, J=10.0 Hz, 1H), 5.74 (ddd, J=16.1, 10.4, 5.0 Hz, 1H), 5.06 (dd, J=23.1, 12.6 Hz, 3H), 4.68-4.52 (m, 3H), 4.48-4.34 (m, 2H), 4.30-4.20 (m, 1H), 3.99-3.92 (m, 4H), 3.49-3.42 (m, 1H), 3.08-2.97 (m, 2H), 2.27 (t, J=7.5 Hz, 2H), 1.86-1.31 (m, 22H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, ? 174.1, 169.4, 169.2, 155.8, 150.1, 149.3, 149.0, 132.2, 132.0, 127.8, 126.1, 116.9, 114.2, 113.7, 110.6, 110.2, 100.1, 88.1, 84.2, 69.1, 68.9, 66.4, 65.8, 63.9, 63.2, 56.1, 56.0, 55.3, 51.4, 38.8, 38.7, 34.0, 33.9, 31.0, 29.0, 28.9, 28.8, 28.7, 25.8, 25.7, 25.2, 24.8, 24.7, 23.2, 23.0, 22.9, 19.9, 18.2; MS (ES+): m/z=603 (M+H).sup.+; LCMS (Method C): t.sub.R=4.55 min.
Example 103: 8-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)octanoic acid (101)
[1378] ##STR00249##
[1379] General Procedure B was followed, using allyl (6aS)-2-methoxy-3-((8-methoxy-8-oxooctyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9, 10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (100) (182 mg, 0.302 mmol), sodium hydroxide (0.5 M, aq., 12 mL, 0.604 mmol) and tetrahydrofuran (3 mL). The title compound (177 mg, quant.) was isolated as a white solid, which was used in the next step without further purification.
[1380] MS (ES+): m/z=589 (M+H).sup.+; LCMS (Method C): t.sub.R=4.03 min.
Example 104: Allyl (6aS)-3-((8-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro 3H-benzo[e]indol-3-yl)-8-oxooctyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)ox)-6.6a,7,89,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (102)
[1381] ##STR00250##
[1382] General Procedure C was followed, using 8-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)octanoic acid (45) (177 mg, 0.301 mmol), (S)-1-(Chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (81 mg, 0.301 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (231 mg, 1.20 mmol) and N,N-dimethylaectamide (2 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 100%) gave the title compound (64 mg, 26%) as a grey solid.
[1383] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, 310.16 (s, 1H), 8.39 (s, 1H), 8.29 (d, J=8.3 Hz, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.55-7.45 (m, 1H), 7.43-7.31 (m, 1H), 7.17-7.12 (m, 1H), 6.84 (s, 1H), 6.50 (s, 1H), 6.18 (d, J=9.7 Hz, 1H), 6.00 (d, J=10.1 Hz, 1H), 5.80-5.68 (m, 1H), 5.17-4.98 (m, 2H), 4.70-4.51 (m, 1H), 4.50-4.13 (m, 4H), 3.95-3.88 (m, 7H), 3.66-3.43 (m, 2H), 3.39 (t, J=10.7 Hz, 1H), 3.11-3.01 (m, 1H), 2.66-2.45 (m, 2H), 1.97-1.39 (m, 22H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, 6173.1, 169.5, 169.3, 155.4, 149.4, 149.1, 141.2, 129.9, 128.9, 127.8, 127.5, 123.9, 123.3, 122.9, 121.9, 116.9, 114.5, 114.3, 100.6, 100.1, 88.1, 84.2, 69.2, 68.9, 66.5, 66.2, 63.9, 63.2, 62.6, 56.1, 53.5, 46.3, 42.2, 38.9, 36.4, 31.0, 29.7, 29.3, 29.2, 29.1, 28.9, 25.8, 25.7, 25.2, 24.9, 23.2, 23.0, 22.9, 19.9, 19.6; MS (ES+): m/z=804 (M+H).sup.+; LCMS (Method C): t.sub.R=4.75 min.
Example 105: (S)-3-((8-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-H-benzo[e]indol-3-yl)-8-oxooctyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (103)
[1384] ##STR00251##
[1385] General Procedure D was followed, using allyl (6aS)-3-((8-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-8-oxooctyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate (102) (64 mg, 0.080 mmol), tetrakis(triphenyl-phosphine)palladium(0) (9 mg, 0.008 mmol), pyrrolidine (8 ?L, 0.095 mmol) and dichloromethane (1 mL). Chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (97%), gave the title compound (8 mg, 16%) as a white solid.
[1386] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.36 (s, 1H), 8.20 (d, J=8.5 Hz, 1H), 8.10 (s, 1H), 7.96 (d, J=5.7 Hz, 1H), 7.78 (d, J=8.3 Hz, 1H), 7.49 (t, J=7.6 Hz, 1H), 7.32 (s, 1H), 7.31-7.27 (m, 2H), 6.75 (s, 1H), 4.34 (d, J=9.7 Hz, 2H), 4.16-4.08 (m, 2H), 4.05-3.95 (m, 2H), 3.86 (s, 3H), 3.79-3.75 (m, 1H), 3.73-3.66 (m, 1H), 3.19-3.11 (m, 1H), 2.66-2.48 (m, 2H), 1.88-1.70 (m, 8H), 1.58-1.42 (m, 8H); MS (ES+): m/z=618 (M+H).sup.+; LCMS (Method A): t.sub.R=7.70 min.
[1387] General Procedure E: Synthesis of Protected N-Methyl Piperazine Carbamates
##STR00252##
[1388] A solution of (S)-seco-CBI phenol (1 equiv.) in dichloromethane (0.1 M) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (1 equiv.), followed by 4-(dimethylamino)pyridine (1.1 equiv.) and triethylamine (3.5 equiv.) and the resulting mixture stirred at room temperature. After the reaction was judged complete by TLC and LCMS (approx. 10 min), the mixture was concentrated in vacuo, then charged with diethyl ether and concentrated once again. The residue was then loaded directly onto silica and purified by flash column chromatography (eluent basified with 5% triethylamine).
[1389] General Procedure F: Deprotection of N-Alloc, O-THP Pyridinobenzodiazepine N-Methyl Piperazine Carbamates
##STR00253##
[1390] A solution of protected N-Alloc, O-THP pyridinobenzodiazepine N-methyl piperazine carbamate (1 equiv.) in dichloromethane (0.1 M) was charged with pyrrolidine (1.2 equiv.), and tetrakis(triphenylphosphine)palladium(0) (0.1 equiv.) and stirred at room temperature whilst monitoring by TLC and LCMS. After the reaction was judged to be complete (approx. 10 min), the mixture was diluted in dichloromethane and filtered through a pad of celite. The filtrate was concentrated in vacuo, then charged with diethyl ether and concentrated again. Diethyl ether was charged once more, and the residue concentrated in vacuo for a third time. The residue was then loaded directly onto silica and purified by flash column chromatography (eluent basified with approx. 5% triethylamine).
Example 106: Allyl (6aS)-3-hydroxy-2,6-dimethoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate
[1391] ##STR00254##
[1392] A solution of allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (47) (100 mg, 0.199 mmol) in dichloromethane (1 mL) was charged with boron trichloride (1 M solution in dichloromethane, 600 ?L, 0.600 mmol) and the resulting suspension was stirred at room temperature for 10 min, then methanol (2 mL) was added to the reaction mixture which was irradiated with microwaves 60 min at 55? C. The reaction mixture was subsequently filtered through a cotton pad that was washed with dichloromethane and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with petroleum spirit 40-60? C./ethyl acetate (1:0 to 0:1) gave the title compound (40 mg, 48%) as a cream powder.
[1393] MS (ES+): m/z=424 (M+H).sup.+; LCMS (Method B): t.sub.R=3.53 min.
Example 107: Allyl (6aS)-6-hydroxy-2-methoxy-3-((3-(2-methoxy-2-oxoethyl)benzyl)oxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]-diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (105)
[1394] ##STR00255##
[1395] A solution of allyl (6aS)-3,6-dihydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (104) (385 mg, 0.94 mmol) in N,N-dimethylformamide (1 mL) was charged with potassium carbonate
[1396] (195 mg, 1.41 mmol) and 3-(bromomethyl)-benzeneacetic acid methyl ester (241 mg, 0.99 mmol) and the resulting mixture stirred for 16 h. The mixture was then diluted in ethyl acetate (50 mL) and washed with cold brine (2?20 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (70%) gave the title compound (202 mg, 32%) as a yellow solid.
[1397] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.36-7.28 (m, 3H), 7.27-7.19 (m, 6H), 6.70 (s, 1H), 5.67 (br, 1H), 5.29 (dd, J=8.7, 5.0 Hz, 1H), 5.12-5.04 (m, 4H), 4.79 (d, J=15.7 Hz, 1H), 4.56 (d, J=15.5 Hz, 1H), 4.50-4.30 (m, 2H), 3.90 (s, 3H), 3.70-3.65 (m, 4H), 3.61 (d, J=7.1 Hz, 2H), 3.08 (qd, J=15.3, 4.8 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 172.0, 168.9, 155.7, 150.1, 148.9, 136.5, 131.9, 129.1, 128.9, 128.2, 127.8, 127.7, 127.2, 126.6, 118.0, 114.3, 111.2, 84.8, 71.0, 66.6, 60.4, 56.2, 55.7, 52.1, 44.2, 40.9, 30.2, 21.0; MS (ES+): m/z=573 (M+H).sup.+; LCMS (Method C): t.sub.R=3.78 min.
Example 108: 2-(3-((((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazepino[1,2-b]-isoquinolin-3-yl)oxy)methyl)phenyl)acetic acid (106)
[1398] ##STR00256##
[1399] A solution of ally methoxy-2-oxoethyl)benzyl)-oxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (105) (200 mg, 0.35 mmol) in tetrahydrofuran (4.5 mL) was charged with an aqueous solution of sodium hydroxide (1 M, 900 ?L, 0.90 mmol) and stirred for 2 h. The mixture was then partially concentrated in vacuo, taken up into ethyl acetate (50 mL), and adjusted to pH=3-4 with an aqueous solution of citric acid (1 M). After separating the organic phase, the aqueous phase was extracted with ethyl acetate (50 mL) and the combined organic extracts dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound (175 mg, 90%) as a white solid, which was used in the subsequent step without further purification.
[1400] MS (ES+): m/z=559 (M+H).sup.+; LCMS (Method C): t.sub.R=3.52 min.
Example 109: Allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-14-oxo-6,6a,7, 12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-isoquinoline-5(14H)-carboxylate (107)
[1401] ##STR00257##
[1402] A solution of 2-(3-((((6aS)-5-((allyloxy)carbonyl)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)oxy)methyl)phenyl)acetic acid (106) (175 mg, 0.313 mmol) in N,N-dimethylacetamide
[1403] (4 mL) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (95 mg, 0.352 mmol), followed by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (268 mg, 1.40 mmol) and the resulting mixture stirred at room temperature for 16 h. The reaction mixture was subsequently diluted into ethyl acetate (50 mL) and washed with cold brine (2?20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 40% to 100%) afforded the title compound
[1404] (114 mg, 42%) as an off-white solid.
[1405] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 9.07 (s, 1H), 8.90 (s, 1H), 8.25-8.19 (m, 2H), 8.14-8.07 (m, 1H), 7.66-7.58 (m, 1H), 7.56-7.46 (m, 1H), 7.45-7.30 (m, 4H), 7.27-7.18 (m, 4H), 7.12 (d, J=6.6 Hz, 1H), 6.75 (s, 1H), 5.63 (ddd, J=22.4, 10.4, 5.2 Hz, 1H), 5.47-5.30 (m, 1H), 5.28-5.20 (m, 1H), 5.08-4.98 (m, 3H), 4.71 (dd, J=15.6, 6.6 Hz, 1H), 4.58 (dd, J=15.3, 5.1 Hz, 1H), 4.53-4.43 (m, 1H), 4.42-4.29 (m, 1H), 4.42-4.29 (m, 2H), 4.26-4.16 (m, 1H), 3.97 (br, 1H), 3.96-3.82 (m, 5H), 3.48 (br, 1H), 3.37 (t, J=10.7 Hz, 1H), 2.96 (d, J=4.7 Hz, 2H); MS (ES+): m/z=774 (M+H).sup.+; LCMS (Method C): t.sub.R=4.18 min.
Example 110: (S)-3-((3-(2-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-1-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-14(6aH)-one (108)
[1406] ##STR00258##
[1407] A solution of allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5(14H)-carboxylate (106) (114 mg, 0.15 mmol) in dichloromethane (0.5 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (17 mg, 0.015 mmol) and pyrrolidine (15 ?L, 0.18 mmol) and stirred at room temperature. After the reaction was judged to be complete by TLC and LCMS, the mixture was concentrated in vacuo, charged with diethyl ether and concentrated in vacuo again. Purification by flash column chromatography (silica), eluting to with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%) gave the title compound
[1408] (39 mg, 39%) as a white solid.
[1409] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.76 (br, 1H), 8.20 (d, J=8.4 Hz, 1H), 8.06 (s, 1H), 7.80-7.74 (m, 1H), 7.52-7.49 (m, 1H), 7.47 (br, 1H), 7.47-7.45 (m, 2H), 7.44 (d, J=1.4 Hz, 1H), 7.42-7.40 (m, 2H), 7.38 (s, 1H), 7.37-7.29 (m, 3H), 7.28 (d, J=6.3 Hz, 1H), 6.87 (s, 1H), 5.21 (q, J=12.4 Hz, 2H), 4.88 (d, J=15.3 Hz, 1H), 4.59-4.49 (m, 1H), 4.41 (d, J=10.5 Hz, 1H), 4.36-4.28 (m, 1H), 4.10 (br, 1H), 4.00-3.95 (m, 1H), 3.93-3.91 (m, 1H), 3.88 (dd, J=11.3, 5.3 Hz, 1H), 3.83 (s, 3H), 3.73 (dd, J=5.3, 3.9 Hz, 1H), 3.66-3.60 (m, 1H), 3.29-3.25 (m, 2H); MS (ES+): m/z=672 (M+H).sup.+; LCMS (Method A): t.sub.R=7.80 min.
Example 11: (S)-(2-(Hydroxymethyl)indolin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (109)
[1410] ##STR00259##
[1411] A solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (67) (1.00 g, 2.71 mmol) in dichloromethane (25 mL) was charged with (S)-(+)-2-indolinemethanol (404 mg, 2.71 mmol), HATU (0.54 g, 4.06 mmol) and N,N-diisopropylethylamine (875 mg, 6.77 mmol). The reaction mixture was stirred at room temperature for 3 h and then diluted with water (100 mL) and extracted with dichloromethane (100 mL?2). The combined organic extracts were then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 10% to 100%) to afford the title compound
[1412] (800 mg, 58%) as a yellow oil.
[1413] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.22-7.14 (m, 1H), 7.08-7.00 (m, 1H), 6.95-6.90 (m, 1H), 6.80-6.70 (m, 1H), 5.69-5.65 (m, 1H), 5.23-5.06 (m, 1H), 4.00-3.82 (m, 3H), 2.80 (s, 5H), 2.04 (s, 1H), 1.34-1.25 (m, 3H), 1.15-1.10 (m, 18H); MS (ES+): m/z=501 (M+H).sup.+; LCMS (Method E): t.sub.R=4.45 min.
Example 112: (S)-(2-Amino-1-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)indolin-1-yl)methanone (110)
[1414] ##STR00260##
[1415] A solution of (S)-(2-(hydroxymethyl)indolin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (109) (800 mg, 1.60 mmol) in methanol (10 mL) was charged with palladium (10 wt. % loading on carbon, 80 mg). The mixture was stirred at room temperature under an atmosphere of hydrogen for 16 h then filtered through a pad of Celite. The resulting cake was then washed with ethyl acetate (50 mL) and concentrated under reduced pressure. The residue was then purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 20% to 50%) to afford the title compound (500 mg, 66%) as a yellow oil.
[1416] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 37.22 (d, J=6.8 Hz, 1H), 7.08 (s, 1H), 7.00-6.93 (m, 2H), 6.75 (s, 1H), 6.37 (d, J=2.8 Hz, 1H), 4.98-4.88 (m, 3H), 4.61-4.57 (m, 1H), 3.58 (s, 3H), 3.47-3.44 (m, 1H), 3.32-3.26 (m, 1H), 3.01-2.97 (m, 1H), 2.69 (s, 1H), 1.27-1.21 (m, 3H), 1.08 (d, J=7.2 Hz, 18H); MS (ES+): m/z=471 (M+H).sup.+; LCMS (Method D): t.sub.R=2.98 min.
Example 113: Allyl (S)-(2-(2-(hydroxymethyl)indoline-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (111)
[1417] ##STR00261##
[1418] A solution of (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)indolin-1-yl)methanone (110) (470 mg, 1.00 mmol) in dichloromethane
[1419] (10 mL) at ?10? C. was charged with anhydrous pyridine (158 mg, 2.00 mmol) and allyl chloroformate (127 mg, 1.05 mmol). After 30 min, the reaction was judged to be complete by TLC and was then diluted with dichloromethane (100 mL), washed with a saturated aqueous solution of copper sulfate (100 mL), water (100 mL) and a saturated aqueous solution of sodium hydrogen carbonate (10 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (5%) to afford the title compound (400 mg, 72%) as a yellow oil.
[1420] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.36 (s, 1H), 7.74 (s, 1H), 7.19 (d, J=7.2 Hz, 1H), 6.97-6.87 (m, 2H), 6.72 (s, 1H), 6.40 (s, 1H), 5.98-5.87 (m, 1H), 5.31 (d, J=16.8 Hz, 1H), 5.22 (d, J=10.4 Hz, 1H), 4.94-4.91 (m, 1H), 4.60 (d, J=5.6 Hz, 2H), 3.76 (d, J=6.0 Hz, 2H), 3.54 (s, 3H), 3.45-3.38 (m, 1H), 2.81-2.76 (m, 1H), 1.35-1.28 (m, 3H), 1.12 (d, J=7.6 Hz, 18H); MS (ES+): m/z=555 (M+H).sup.+; LCMS (Method D): t.sub.R=2.78 min.
Example 114: Allyl (12aS)-12-hydroxy-8-methoxy-6-oxo-9-((triisopropyl-silyl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-(11(12H)-carboxylate (112)
[1421] ##STR00262##
[1422] A solution of allyl (S)-(2-(2-(hydroxymethyl)indoline-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (111) (391 mg, 0.71 mmol) in dichloromethane
[1423] (13 mL) was charged with 2,2,6,6-tetramethylpiperidine 1-oxyl (11 mg, 0.07 mmol) and (diacetoxyiodo)benzene (274 mg, 0.85 mmol). The reaction mixture was stirred at room temperature for 18 h and then diluted in dichloromethane (40 mL), washed with a saturated aqueous solution of sodium metabisulfite (10 mL), then a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and lastly, brine (10 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (10%) to afford the title compound (290 mg, 74%) as a yellow oil.
[1424] .sup.1H NMR (500 MHz, CDCl.sub.3) ? 8.19 (d, J=8.0 Hz, 1H), 7.21 (d, J=7.5 Hz, 1H), 7.08 (t, J=7.5 Hz, 1H), 6.71 (s, 1H), 5.78 (s, 1H), 5.73 (d, J=10.0 Hz, 1H), 5.20-5.14 (m, 2H), 4.62-4.58 (m, 1H), 4.46 (s, 1H), 4.15-4.06 (m, 2H), 3.86-3.84 (m, 3H), 3.49-3.43 (m, 1H), 3.21 (d, J=17.0 Hz, 1H), 2.04 (d, J=2.0 Hz, 1H), 1.28-1.21 (m, 3H), 1.09-1.08 (m, 18H); MS (ES+): m/z=553 (M+H).sup.+; LCMS (Method D): t.sub.R=2.68 min.
Example 115: Allyl (12aS)-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-5-((triisopropylsilyl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]-diazepino[1,2-a]indole-11(12H)-carboxylate (113)
[1425] ##STR00263##
[1426] A solution of allyl (12aS)-12-hydroxy-8-methoxy-6-oxo-9-((triisopropylsilyl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate (112) (280 mg, 0.510 mmol) in tetrahydrofuran (5 mL) was charged with 3,4-dihydro-2H-pyran (429 mg, 5.10 mmol) and p-toluenesulfonic acid monohydrate (3 mg, 1% w/w). The reaction mixture was stirred at room temperature for 18 h and then diluted with ethyl acetate (30 mL), washed with a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and brine (10 mL), then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (20%) to afford the title compound (300 mg, 92%) as a colourless oil.
[1427] .sup.1H NMR (500 MHz, CDCl.sub.3) ? 8.20-8.13 (m, 1H), 7.25-7.21 (m, 2H), 7.08-6.61 (m, 2H), 5.94 (m, 1H), 5.78-5.66 (m, 1H), 5.13-5.04 (m, 2H), 4.96-4.94 (m, 2H), 4.89 (d, J=6.0 Hz, 1H), 3.86 (d, J=2.0 Hz, 3H), 3.65-3.61 (m, 1H), 3.47-3.42 (m, 1H), 2.07-2.01 (m, 1H), 1.98-1.95 (m, 1H), 1.79-1.73 (m, 6H), 1.33-1.26 (m, 3H), 1.11-1.08 (m, 18H); MS (ES+): m/z=637 (M+H).sup.+; LCMS (Method D): t.sub.R=4.43 min.
Example 116: Allyl (12aS)-9-hydroxy-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate (114)
[1428] ##STR00264##
[1429] A solution of allyl (12aS)-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-9-((triisopropylsilyl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate (113) (290 mg, 0.46 mmol) in tetrahydrofuran (5 mL) under an inert atmosphere of nitrogen was charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 0.65 mL, 0.65 mmol). The mixture was stirred at room temperature for 1 h and then charged with water (10 mL), extracted with ethyl acetate (30 mL?2) and the combined organic phases washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (20%) to afford the title compound (100 mg, 45%) as a white solid.
[1430] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 38.16 (dd, J=21.0, 8.0 Hz, 1H), 7.28 (d, J=5.0 Hz, 1H), 7.25-7.20 (m, 1H), 7.10-7.05 (m, 1H), 6.77 (m, 1H), 6.02 (s, 1H), 5.81-5.72 (m, 1H), 5.20-5.14 (m, 1H), 5.13-4.84 (m, 1H), 4.66-4.48 (m, 2H), 4.15-4.07 (m, 1H), 3.95 (s, 3H), 3.60-3.42 (m, 2H), 3.30-3.18 (m, 1H), 1.88-1.54 (m, 7H), 1.29-1.24 (m, 1H); MS (ES+): m/z=481 (M+H).sup.+; LCMS (Method E): t.sub.R=2.78 min.
Example 117: Allyl (12aS)-8-methoxy-5-((3-(2-methoxy-2-oxoethyl)benzyl)-oxy)-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-12a,13-dihydro-6H-benzo-[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate (115)
[1431] ##STR00265##
[1432] A solution of allyl (12aS)-9-hydroxy-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate
[1433] (114) (250 mg, 0.52 mmol) in N,N-dimethylformamide (2 mL) was charged with potassium carbonate (108 mg, 0.78 mmol) and 3-(bromomethyl)-benzeneacetic acid methyl ester (134 mg, 0.55 mmol) and stirred at room temperature, whilst monitoring by TLC and LCMS. Once the reaction was judged to be complete, it was diluted into ethyl acetate and washed twice with cold brine. The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo, and then purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 50%) to give the title compound (223 mg, 67%) as a white solid.
[1434] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, 88.10 (dt, J=17.3, 8.5 Hz, 1H), 7.33-7.28 (m, 3H), 7.05-6.98 (m, 1H), 6.92 (s, 1H), 6.58 (br, 1H), 5.95 (d, J=9.2 Hz, 1H), 5.84 (d, J=9.6 Hz, 1H), 5.72-5.57 (m, 1H), 5.17-4.98 (m, 4H), 4.56-4.44 (m, 1H), 4.41 (br, 1H), 4.31 (d, J=9.6 Hz, 1H), 4.10-3.97 (m, 1H), 3.84-3.77 (m, 1H), 3.88 (s, 3H), 3.64 (s, 3H), 3.59 (s, 2H), 3.57-3.51 (m, 1H), 3.41 (dt, J=16.7, 10.9 Hz, 2H), 3.22 (d, J=17.3 Hz, 1H), 3.02 (d, J=16.2 Hz, 1H), 1.80-1.27 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, 8171.7, 165.9, 165.8, 155.5, 150.5, 150.3, 149.5, 149.3, 136.6, 131.9, 129.9, 129.0, 128.9, 127.6, 125.0, 117.1, 115.3, 115.1, 110.9, 100.4, 96.5, 91.4, 88.3, 71.2, 70.8, 66.5, 66.3, 63.9, 61.0, 56.2, 56.1, 52.0, 41.0, 40.9, 32.5, 32.0, 31.1, 30.9, 25.2, 20.2; MS (ES+): m/z=643 (M+H).sup.+; LCMS (Method A): t.sub.R=8.67 min.
Example 118: 2-(3-((((12aS)-11-((Allyloxy)carbonyl)-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-11,12,12a,13-tetrahydro-6H-benzo[5,6]-[1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)acetic acid (116)
[1435] ##STR00266##
[1436] A solution of allyl (12aS)-8-methoxy-9-((3-(2-methoxy-2-oxoethyl)benzyl)oxy)-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate (115) (223 mg, 0.36 mmol) in tetrahydrofuran (4 mL) was charged with an aqueous solution of sodium hydroxide (1 M, 720 ?L, 0.72 mmol) and stirred at room temperature, whilst monitoring by TLC and LCMS. Once the reaction was judged to be complete, the mixture was then partially concentrated in vacuo (to remove tetrahydrofuran), then diluted into ethyl acetate and acidified to pH=3-4 with a saturated aqueous solution of citric acid. The organic layer was separated, and the aqueous layer washed with ethyl acetate. The combined organic extracts were then washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting white solid was used in the next step without any further purification.
[1437] MS (ES+): m/z=629 (M+H).sup.+; LCMS (Method A): t.sub.R=7.97 min.
Example 119: Allyl (12aS)-9-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-aH-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-8-methoxy-6-oxo-12-((tetrahydro-1H-pyran-2-yl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]-diazepino[1,2-a]indole-11(12H)-carboxylate (117)
[1438] ##STR00267##
[1439] A solution of 2-(3-((((12aS)-11-((allyloxy)carbonyl)-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)acetic acid (116) (85 mg, 0.14 mmol) in N,N-dimethylacetamide (1 mL) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (38 mg, 0.14 mmol), followed by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (107 mg, 0.56 mmol) and stirred at room temperature for 16 h. The resulting mixture was diluted into ethyl acetate and washed with cold brine (twice), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification was enacted by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (56%), to give the title compound (68 mg, 600%) as a grey solid.
[1440] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, ? 10.03 (br, 0.5H), 9.75 (br, 0.5H), 8.40 (d, J=17.5 Hz, 1H), 8.24 (d, J=3.8 Hz, 1H), 8.15 (d, J=7.8 Hz, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.50 (dd, J=12.9, 5.7 Hz, 2H), 7.36 (dd, J=9.6, 5.3 Hz, 3H), 7.27-7.22 (m, 2H), 7.19 (t, J=6.9 Hz, 1H), 7.07 (t, J=7.4 Hz, 1H), 6.95 (s, 0.5H), 6.65 (s, 0.5H), 6.04-5.94 (m, 1H), 5.86 (d, J=9.4 Hz, 1H), 5.66-5.51 (m, 1H), 5.21-5.09 (m, 3H), 5.00 (d, J=13.4 Hz, 2H), 4.52-4.39 (m, 1H), 4.32 (t, J=10.0 Hz, 1H), 4.17-3.99 (m, 3H), 3.98-3.87 (m, 4H), 3.87-3.81 (m, 5H), 3.58-3.51 (m, 1H), 3.51-3.36 (m, 1H), 3.35-3.18 (m, 1H), 1.77-1.30 (m, 6H); MS (ES+): m/z=845 (M+H).sup.+; LCMS (Method A): t.sub.R=9.00 min.
Example 120: (S)-9-((3-(2-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-8-methoxy-12?,?-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one (118)
[1441] ##STR00268##
A solution of allyl (12aS)-9-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-8-methoxy-6-oxo-12-((tetrahydro-2H-pyran-2-yl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate (117) (68 mg, 0.081 mmol) in dichloromethane (1 mL) was charged with pyrrolidine (11 ?L, 0.18 mmol), and tetrakis(triphenylphosphine)palladium(0) (9 mg, 0.008 mmol) and stirred at room temperature whilst monitoring by TLC and LCMS. After the reaction was judged to be complete (15 min), the mixture was diluted in dichloromethane and filtered through a pad of celite. The filtrate was concentrated in vacuo, then charged with diethyl ether and concentrated again. Diethyl ether was charged once more, and the residue concentrated in vacuo for a third time. Purification was enacted by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%), to give the title compound (30 mg, 58%) as a pale yellow solid.
[1442] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 10.31 (s, 1H), 8.07 (d, J=8.0 Hz, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.95 (d, J=4.5 Hz, 1H), 7.91 (s, 1H), 7.72 (d, J=8.2 Hz, 1H), 7.43 (t, J=7.6 Hz, 1H), 7.38-7.32 (m, 4H), 7.30-7.24 (m, 2H), 7.23-7.11 (m, 2H), 7.09-7.01 (m, 1H), 6.92 (s, 1H), 5.20 (d, J=11.9 Hz, 1H), 5.12 (d, J=11.8 Hz, 1H), 5.00 (br, 1H), 4.48 (dt, J=10.1, 5.1 Hz, 1H), 4.32-4.20 (m, 2H), 4.05 (br, 2H), 3.89 (s, 3H), 3.78 (s, 2H), 3.73-3.63 (m, 1H), 3.55-3.50 (m, 1H); MS (ES+): m/z=658 (M+H).sup.+; LCMS (Method A): t.sub.R=7.82 min.
Example 121: Methyl (2S,4R)-4-hydroxy-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidine-2-carboxylate (119)
[1443] ##STR00269##
[1444] A solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (67) (136 g, 0.37 mol) in dichloromethane (1 L) was charged with methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate hydrochloride (67.2 g, 0.37 mol), HATU (271 g, 0.74 mol) and N,N-diisopropylethylamine (166.4 g, 1.29 mol) and the resulting mixture stirred for 1 h. Water (2 L) was then added, and dichloromethane (1.5 L). The phases were separated, and the aqueous layer extracted with dichloromethane (1.5 L). The combined organic phases were then dried over sodium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (500%), gave the title compound (114 g, 62%) as a yellow solid.
[1445] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.68 (s, 1H), 6.82-6.76 (m, 1H), 4.86 (t, J=8.0 Hz, 1H), 4.60-4.43 (m, 1H), 3.92-3.87 (m, 3H), 3.81-3.79 (m, 3H), 3.56-3.48 (m, 2H), 3.19-3.15 (m, 1H), 2.46-2.34 (m, 1H), 2.20-2.14 (m, 1H), 1.32-1.22 (m, 3H), 1.10-1.07 (m, 18H); MS (ES+): m/z=497 (M+H).sup.+; LCMS (Method D): t.sub.R=2.17 min.
Example 122: ((2S,4R)-4-Hydroxy-2-(hydroxymethyl)pyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (120)
[1446] ##STR00270##
[1447] A solution of methyl (2S,4R)-4-hydroxy-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidine-2-carboxylate (119) (114 g, 0.23 mol) in tetrahydrofuran (1.1 L) was charged slowly with lithium borohydride (2 M in tetrahydrofuran, 460 mL, 0.92 mol) at 0? C. under nitrogen and stirred for 2 h. The reaction was then quenched by cautious addition of water (2 L) and extracted with ethyl acetate (2?1.5 L). The combined organic phases were then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (67%), gave the title compound (89.2 g, 83%) as a yellow oil.
[1448] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.68 (s, 1H), 6.82 (s, 1H), 4.60-4.51 (m, 1H), 4.36 (s, 1H), 4.02-3.99 (m, 1H), 3.92 (s, 3H), 3.83-3.72 (m, 1H), 3.35-3.29 (m, 1H), 3.16-3.13 (m, 1H), 2.22-2.17 (m, 1H), 2.00-1.88 (m, 1H), 1.29-1.24 (m, 3H), 1.12-1.05 (m, 18H); MS (ES+): m/z=469 (M+H).sup.+; LCMS (Method D): t.sub.R=1.75 min.
Example 123: ((2S,4R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)-oxy)phenyl)methanone (121)
[1449] ##STR00271##
[1450] A solution of ((2S,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (120) (75.0 g, 0.16 mol) in dichloromethane
[1451] (750 mL) was charged with triethylamine (194.5 g, 1.92 mol) and tert-butyldimethylsilyl chloride (169.0 g, 1.12 mol). The reaction mixture was stirred at room temperature for 2 h, then diluted with water (2 L) and extracted with dichloromethane (2?1 L). The combined to organic phases were dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (50%), to give the title compound (57.7 g, 62%) as a yellow solid.
[1452] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.70-7.66 (m, 1H), 6.79-6.73 (m, 1H), 4.54 (s, 1H), 4.41 (s, 1H), 3.89-3.87 (m, 5H), 3.79-3.78 (m, 1H), 3.37-3.34 (m, 1H), 3.09-3.07 (m, 1H), 2.35-2.30 (m, 1H), 2.12-2.08 (m, 1H), 1.29-1.27 (m, 3H), 1.10-1.08 (m, 18H), 0.91 (s, 9H), 0.10 (d, J=4.0 Hz, 6H); MS (ES+): m/z=605 (M+Na).sup.+; LCMS (Method D): t.sub.R=3.90 min.
Example 124: (S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidin-3-one (122)
[1453] ##STR00272##
[1454] A solution of ((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (121) (50.0 g, 85.8 mmol) in dichloromethane (500 mL) was charged with 2,2,6,6-tetramethyl-1-piperidinyloxy (1.34 g, 8.60 mmol) and (diacetoxyiodo)benzene (33.2 g, 103.0 mmol) and the resulting mixture stirred at room temperature for 12 h. The mixture was then diluted into water (2 L) and extracted with dichloromethane (1?2 L). The combined organic phases were then dried over sodium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (25%), gave the title compound (48.8 g, 98%) as a yellow oil.
[1455] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.72-7.68 (m, 1H), 6.79-6.71 (m, 1H), 4.97 (d, J=7.6 Hz, 1H), 4.28 (d, J=8.0 Hz, 1H), 3.88 (s, 3H), 3.71 (d, J=8.4 Hz, 1H), 3.65-3.61 (m, 1H), 3.44 (d, J=14.0 Hz, 1H), 2.80-2.74 (m, 1H), 2.56-2.52 (m, 1H), 1.26-1.25 (m, 3H), 1.10-1.09 (m, 18H), 0.85 (s, 9H), 0.08 (d, J=10.0 Hz, 6H); MS (ES+): m/z=603 (M+Na).sup.+; LCMS (Method D): t.sub.R=4.17 min.
Example 125: (S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (123)
[1456] ##STR00273##
[1457] A solution of (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidin-3-one (122) (30.0 g, 51.7 mmol) in dichloromethane (300 mL) was cooled to ?50? C. and charged with 2,6-lutidine (33.2 g, 310.1 mmol) under nitrogen. Triflic anhydride (43.8 g, 155.1 mmol) was then added and the resulting mixture stirred at the same temperature for 1.5 h, after which water (1 L) was added. The mixture was then extracted with dichloromethane (2?500 mL), and the combined organic phases dried over sodium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (10%), gave the title compound (30.0 g, 81%) as a brown oil.
[1458] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.71 (s, 1H), 6.75 (s, 1H), 6.05 (s, 1H), 4.78 (s, 1H), 3.93-3.88 (m, 5H), 3.21-3.11 (m, 1H), 3.01-2.96 (m, 1H), 1.29-1.27 (m, 3H), 1.11 (s, 9H), 1.10 (s, 9H), 0.91 (s, 9H), 0.11 (d, J=1.6 Hz, 6H); MS (ES+): m/z=735 (M+Na).sup.+; LCMS (Method D): t.sub.R=2.45 min.
Example 126: (S)-(2-(((tert-Butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)-phenyl)methanone (124)
[1459] ##STR00274##
[1460] A solution of (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate
[1461] (123) (30.0 g, 42.1 mmol) in 1,4-dioxane (300 mL) was charged with methylboronic acid (8.82 g, 147.0 mmol), silver (I) oxide (39.0 g, 168.0 mmol), potassium phosphate (54.0 g, 252.0 mmol), triphenylarsine (5.16 g, 16.8 mmol) and bis(benzonitrile)palladium(II) chloride (1.62 g, 4.21 mmol). The reaction mixture was stirred at 110? C. for 10 min under nitrogen. It was then filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C.
[1462] (17%), to afford the title compound (15.8 g, 65%) as a brown oil.
[1463] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.70 (s, 1H), 6.77 (s, 1H), 5.51-5.50 (m, 1H), 4.67-4.65 (m, 1H), 3.92-3.86 (m, 5H), 2.82-2.72 (m, 1H), 2.57-2.52 (m, 1H), 1.62 (d, J=1.2 Hz, 3H), 1.30-1.26 (m, 3H), 1.11 (s, 9H), 1.09 (s, 9H), 0.90 (s, 9H), 0.10 (d, J=2.4 Hz, 6H); MS (ES+): m/z=601 (M+Na).sup.+; LCMS (Method D): t.sub.R=2.44 min.
Example 127: (S)-(2-Amino-5-methoxy-4-((triisopropylsilyl)ox)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-H-pyrrol-1-yl)methanone (125)
[1464] ##STR00275##
[1465] A solution of (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (124) (15.0 g, 25.9 mmol) in ethanol (160 mL) and water (40 mL) was charged with iron (7.24 g, 129.7 mmol) and ammonium chloride (6.93 g, 129.7 mmol). The reaction mixture was stirred at 80? C. for 2 h under nitrogen and then filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (10%), to afford the title compound (10.1 g, 76%) as a yellow oil.
[1466] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.72 (s, 1H), 6.26 (s, 1H), 6.15 (s, 1H), 4.63-4.61 (m, 1H), 3.93-3.87 (m, 1H), 3.78-3.77 (m, 1H), 3.70 (s, 3H), 2.76-2.66 (m, 1H), 2.55-2.50 (m, 1H), 1.67 (s, 3H), 1.25-1.23 (m, 3H), 1.09-1.07 (m, 18H), 0.88 (s, 9H), 0.06-0.04 (m, 6H); MS (ES+): m/z=549 (M+H).sup.+; LCMS (Method D): t.sub.R=2.56 min.
Example 128: Allyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (126)
[1467] ##STR00276##
[1468] A solution of (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone (125)
[1469] (10.0 g, 18.2 mmol) in dichloromethane (100 mL) was charged with pyridine (2.88 g, 36.5 mmol). Allyl chloroformate (2.30 g, 19.1 mmol) was then added at ?5? C. The reaction mixture was stirred at ?5? C. for 30 min and then diluted with water (500 mL) and extracted with dichloromethane (2?300 mL). The combined organic phases were then dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (10%), to afford the title compound (10.7 g, 93%) as a yellow oil.
[1470] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.96 (s, 1H), 7.20 (s, 1H), 6.81 (s, 1H), 6.02 (s, 1H), 5.95-5.86 (m, 1H), 5.31-5.27 (m, 1H), 5.21-5.17 (m, 1H), 4.57-4.42 (m, 3H), 3.83-3.67 (m, 5H), 2.75-2.64 (m, 1H), 2.43-2.35 (m, 1H), 1.62 (s, 3H), 1.25-1.21 (m, 3H), 1.06-1.03 (m, 18H), 0.86 (s, 9H), 0.05-0.03 (m, 6H); MS (ES+): m/z=633 (M+H).sup.+; LCMS (Method D): t.sub.R=3.57 min.
Example 129: Allyl (S)-(2-(2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate
[1471] (127)
##STR00277##
[1472] A solution of allyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (126) (9.70 g, 15.3 mmol) in acetic acid/methanol/tetrahydrofuran/water (7:1:1:2, 110 mL) was stirred at room temperature for 2 h. The reaction mixture was diluted with a saturated aqueous solution of sodium hydrogen carbonate (400 mL) and extracted with ethyl acetate (2?300 mL). The combined organic phases were then dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (25%), to afford the title compound
[1473] (7.60 g, 95%) as a colourless oil.
[1474] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.93 (s, 1H), 7.18 (s, 1H), 6.87 (s, 1H), 5.95-5.86 (m, 2H), 5.34-5.26 (m, 1H), 5.21-5.17 (m, 1H), 4.84-4.81 (m, 1H), 4.55-4.39 (m, 3H), 3.74 (s, 3H), 3.69-3.60 (m, 1H), 3.56-3.48 (m, 1H), 2.73-2.62 (m, 1H), 2.45-2.37 (m, 1H), 1.62 (s, 3H), 1.25-1.22 (m, 3H), 1.06-1.04 (m, 18H); MS (ES+): m/z=519 (M+H)*; LCMS (Method D): t.sub.R=2.67 min.
Example 130: Allyl (11aS)-11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (128)
[1475] ##STR00278##
[1476] A solution of allyl (S)-(2-(2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (127) (7.60 g, 14.7 mmol) in dichloromethane (80 mL) was charged with 2,2,6,6-tetramethyl-1-piperidinyloxy (0.23 g, 1.47 mmol) and (diacetoxyiodo)benzene (5.19 g, 16.1 mmol). The reaction mixture was stirred at room temperature for 12 h and then diluted with water (300 mL) and extracted with dichloromethane (2?300 mL). The combined organic phases were then dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C.
[1477] (25%) to afford the title compound (3.90 g, 51%) as a light-green oil.
[1478] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.19 (s, 1H), 6.72-6.67 (m, 2H), 5.81-5.71 (m, 2H), 5.23-5.08 (m, 2H), 4.61-4.56 (m, 1H), 4.49-4.42 (m, 1H), 3.84 (s, 3H), 3.66-3.55 (m, 1H), 3.02-2.90 (m, 1H), 2.63-2.54 (m, 1H), 2.29-2.21 (m, 1H), 1.79-1.75 (m, 3H), 1.22-1.20 (m, 3H), 1.08-1.06 (m, 18H); MS (ES+): m/z=517 (M+H).sup.+; LCMS (Method D): t.sub.R=2.09 min.
Example 131: Allyl (11aS)-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4a]diazepine-10(5H)-carboxylate (12)
[1479] ##STR00279##
[1480] A solution of allyl (11aS)-11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (128) (3.90 g, 7.55 mmol) in tetrahydrofuran (50 mL) was charged with 3,4-dihydro-2H-pyran (6.35 g, 75.5 mmol) and p-toluenesulfonic acid (0.13 g, 0.75 mmol). The reaction mixture was stirred at room temperature for 12 h and then diluted into water (300 mL) and extracted to with ethyl acetate (2?300 mL). The combined organic phases were then dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C.
[1481] (20%) to afford the title compound (4.30 g, 95%) as a colourless oil.
[1482] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.19-7.16 (m, 1H), 6.93-6.57 (m, 2H), 6.01-5.86 (m, 1H), 5.75-5.71 (m, 1H), 5.20-4.97 (m, 3H), 4.62-4.52 (m, 1H), 4.47-4.29 (m, 1H), 3.96-3.88 (m, 1H), 3.84 (d, J=2.0 Hz, 3H), 3.80-3.73 (m, 1H), 3.65-3.50 (m, 1H), 2.99-2.89 (m, 1H), 2.65-2.41 (m, 1H), 1.80-1.71 (m, 5H), 1.58-1.46 (m, 4H), 1.25-1.17 (m, 3H), 1.10-1.05 (m, 18H); MS (ES+): m/z=601 (M+H).sup.+; LCMS (Method D): t.sub.R=3.90 min.
Example 132: Allyl (11aS)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)ox)-11,11a-dihydro-H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (130)
[1483] ##STR00280##
[1484] A solution of allyl (11aS)-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (129) (4.30 g, 7.16 mmol) in tetrahydrofuran (35 mL) was charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 9.3 mL, 9.31 mmol). The reaction mixture was stirred at room temperature for 1 h under nitrogen and then diluted into water
[1485] (200 mL) and extracted with ethyl acetate (2?200 mL). The combined organic phases were dried over sodium sulfate, filtered and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (50%) to afford the title compound (2.50 g, 78%) as a white solid.
[1486] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.23-7.20 (m, 1H), 6.99-6.66 (m, 2H), 6.07-5.86 (m, 2H), 5.83-5.68 (m, 1H), 5.19-4.98 (m, 3H), 4.68-4.54 (m, 1H), 4.50-4.35 (m, 1H), 3.97-3.89 (m, 4H), 3.85-3.75 (m, 1H), 3.65-3.53 (m, 1H), 2.99-2.90 (m, 1H), 2.70-2.40 (m, 1H), 1.80-1.72 (m, 5H), 1.58-1.49 (m, 4H); MS (ES+): m/z=445 (M+H).sup.+; LCMS (Method D): t.sub.R=1.00 min.
Example 133: Allyl (1aS)-7-methoxy-8-((3-(2-methoxy-2-oxoethyl)benzyl)-oxy)-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (131)
[1487] ##STR00281##
[1488] A solution of allyl (11aS)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate
[1489] (130) (444 mg, 0.999 mmol) in N,N-dimethylformamide (2 mL) was charged with potassium carbonate (207 mg, 1.50 mmol) and 3-(bromomethyl)-benzeneacetic acid methyl ester (466 mg, 1.92 mmol) and stirred at room temperature overnight. The reaction mixture was then diluted into ethyl acetate (200 mL) and washed with cold brine (2?100 mL). The organic phase was then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 100%) gave the title compound (452 mg, 75%) as a white solid.
[1490] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of diastereomers, 87.32-7.30 (m, 2H), 7.30 (d, J=1.9 Hz, 1H), 7.24 (d, J=2.5 Hz, 1H), 7.23-7.19 (m, 1H), 6.91 (s, 1H), 6.66 (s, 1H), 5.98 (d, J=9.5 Hz, 1H), 5.86 (d, J=9.3 Hz, 1H), 5.75-5.57 (m, 1H), 5.15-4.95 (m, 4H), 4.52 (d, J=13.5 Hz, 1H), 4.33 (d, J=14.1 Hz, 1H), 3.89 (s, 3H), 3.88-3.72 (m, 2H), 3.66 (s, 3H), 3.61 (s, 2H), 3.48-3.40 (m, 1H), 2.99-2.86 (m, 1H), 2.61 (d, J=15.8 Hz, 0.5H), 2.43 (d, J=16.9 Hz, 0.5H), 1.74 (s, 3H), 1.73-1.68 (m, 1H), 1.56-1.46 (m, 4H), 1.39-1.30 (m, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of diastereomers, 6171.7, 163.2, 163.1, 149.4, 149.2, 136.7, 136.6, 134.4, 134.3, 132.2, 132.1, 131.9, 129.0, 128.9, 128.8, 128.1, 126.2, 126.1, 123.5, 123.3, 121.1, 116.9, 115.4, 115.2, 110.6, 100.2, 92.2, 70.8, 66.3, 63.8, 63.6, 59.5, 56.2, 56.1, 52.0, 41.0, 39.3, 38.8, 31.0, 25.2, 20.0, 19.9, 13.7, 13.6; MS (ES+): m/z=607 (M+H).sup.+; LCMS (Method A): t.sub.R=8.18 min.
Example 134: 2-(3-((((11aS)-10-((Allyloxy)carbonyl)-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-5,10,11,11a-tetrahydro-1H-benzo-[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)phenyl)acetic acid (132)
[1491] ##STR00282##
[1492] A solution of allyl (11aS)-7-methoxy-8-((3-(2-methoxy-2-oxoethyl)benzyl)oxy)-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (131) (450 mg, 0.74 mmol) in tetrahydrofuran (18 mL) was charged with an aqueous solution of sodium hydroxide (1 M, 4 mL) and stirred at room temperature for 4 h. A saturated aqueous solution of citric acid was then added until pH=4 and the resulting mixture was extracted with ethyl acetate (2?100 mL). The combined organic phases were then dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting residue was then used in the subsequent step without further purification.
[1493] MS (ES+): m/z=593 (M+H).sup.+; LCMS (Method A): t.sub.R=7.42 min.
Example 135: Allyl (11aS)-8-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H-carboxylate (133)
[1494] ##STR00283##
[1495] A solution of 2-(3-((((11aS)-10-((allyloxy)carbonyl)-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)phenyl)acetic acid (132) (439 mg, 0.74 mmol) in N,N-dimethylacetamide (3 mL) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (200 mg, 0.74 mmol), followed immediately by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (567 mg, 2.96 mmol) and the resulting mixture stirred at room temperature for 16 h. The reaction mixture was subsequently diluted into ethyl acetate (100 mL) and washed with cold brine (2?20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%), gave the title compound (369 mg, 62%) as a grey solid.
[1496] .sup.1H NMR (400 MHz, DMSO-d.sub.6), mixture of diastereomers, ? 10.31 (s, 1H), 8.03 (d, J=8.3 Hz, 1H), 7.91 (s, 1H), 7.72 (d, J=8.3 Hz, 1H), 7.43 (t, J=7.6 Hz, 1H), 7.39-7.23 (m, 5H), 7.04 (s, 2H), 7.04 (s, 2H), 6.95 (s, 0.5H), 6.87 (s, 0.5H), 6.58 (s, 1H), 5.74 (dd, J=190.2, 13.1 Hz, 2H), 5.13-4.87 (m, 4H), 4.43 (br, 1H), 4.27 (dt, J=190.7, 12.8 Hz, 2H), 4.07 (br, 1H), 3.88 (d, J=3.6 Hz, 2H), 3.77-3.73 (m, 4H), 3.69 (dt, J=10.9, 6.9 Hz, 2H), 3.42 (dd, J=190.8, 13.3 Hz, 1H), 2.95-2.80 (m, 1H), 2.50 (d, J=15.5 Hz, 0.5H), 2.40 (d, J=19.8 Hz, 0.5H), 1.69 (s, 3H), 1.64-1.23 (m, 6H); MS (ES+): m/z=809 (M+H).sup.+; LCMS (Method A): t.sub.R=8.77 min.
Example 136: (S)-8-((3-(2-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-7-methoxy-2-methyl-11,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one (134)
[1497] ##STR00284##
[1498] A solution of allyl (11aS)-8-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-7-methoxy-2-methyl-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (133) (100 mg, 0.12 mmol) in dichloromethane (2 mL) was charged with pyrrolidine (22 ?L, 0.264 mmol) and tetrakis(triphenylphosphine)palladium(0) (14 mg, 0.012 mmol) and the resulting mixture stirred for 10 min, after which it was diluted into dichloromethane (10 mL), filtered through a plug of celite. The filter cake was then washed with dichloromethane and the filtrate concentrated in vacuo. Diethyl ether (500 mL) was then charged and the mixture concentrated again. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%), gave the title compound (45 mg, 58%) as a yellow solid.
[1499] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 10.31 (s, 1H), 8.03 (d, J=8.4 Hz, 1H), 7.90 (s, 1H), 7.83 (d, J=4.0 Hz, 1H), 7.72 (d, J=8.2 Hz, 1H), 7.61-7.53 (m, 1H), 7.53-7.47 (m, 1H), 7.44 (t, J=7.5 Hz, 1H), 7.38-7.22 (m, 5H), 6.87 (s, 1H), 6.66 (s, 1H), 5.18-5.06 (m, 1H), 4.97 (d, J=5.8 Hz, 1H), 4.34-4.16 (m, 2H), 4.08 (br, 1H), 3.88 (s, 3H), 3.77-3.65 (m, 2H), 3.64-3.61 (m, 1H), 3.59-3.57 (m, 1H), 3.33 (q, J=7.0 Hz, 1H), 2.95 (br, 1H), 1.69 (s, 3H); MS (ES+): m/z=623 (M+H).sup.+; LCMS (Method A): t.sub.R=7.37 min.
Example 137: Methyl (S)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)-benzoyl)piperidine-2-carboxylate (135)
[1500] ##STR00285##
[1501] A solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (67) (2.00 g, 5.41 mmol) in dichloromethane (12 mL) was charged with triethylamine (3.2 mL, 22.9 mmol) and HATU (2.16 g, 5.68 mmol) and stirred for 5 min at room temperature before methyl
[1502] (2S)-piperidinecarboxylate hydrochloride (972 mg, 5.41 mmol) was added. The resulting mixture was stirred for 16 h before diluting into ethyl acetate (200 mL) and washing with a saturated aqueous solution of sodium hydrogen carbonate (100 mL?2), followed by an aqueous solution of acetic acid (1% v/v, 100 mL) and brine (100 mL). After drying over magnesium sulfate, filtering and concentrating in vacuo, the residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 10% to 50%) to give the title compound (1.27 g, 95%) as a yellow solid.
[1503] .sup.1H NMR (400 MHz, CDCl.sub.3), mixture of rotamers, ? 7.66 (s, 0.7H), 7.64 (s, 0.3H), 6.77 (s, 0.3H), 6.76 (s, 0.5H), 5.60 (d, J=4.7 Hz, 0.5H), 4.76 (d, J=12.8 Hz, 0.3H), 3.89 (s, 3H), 3.81 (s, 1.3H), 3.78 (s, 1.7H), 3.71-3.69 (m, 1H), 3.28-3.13 (m, 1.5H), 2.84 (td, J=13.1, 3.5 Hz, 0.3H), 2.30 (d, J=12.6 Hz, 0.7H), 1.93-1.82 (m, 0.7H), 1.80-1.63 (m, 2.4H), 1.34-1.17 (m, 4.8H), 1.07 (dd, J=6.5, 5.0 Hz, 18H); .sup.13C NMR (100 MHz, CDCl.sub.3), mixture of rotamers, 8171.6, 171.1, 167.6, 167.0, 156.3, 156.0, 145.7, 137.5, 137.3, 127.2, 126.9, 116.0, 115.9, 109.6, 109.5, 60.3, 58.2, 56.1, 56.0, 52.4, 52.3, 51.9, 45.4, 39.6, 30.9, 27.1, 26.1, 25.0, 24.1, 21.4, 21.2, 17.8, 17.7; MS (ES+): m/z=495 (M+H).sup.+; LCMS (Method A): t.sub.R=9.32 min.
Example 138: (S)-3-Hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione (136)
[1504] ##STR00286##
[1505] A solution of methyl (S)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-piperidine-2-carboxylate (135) (1.20 g, 2.43 mmol) in tetrahydrofuran (10 mL) was charged with ammonium formate (1.25 g, 19.9 mmol), followed by palladium on activated charcoal (10 wt. % basis, 125 mg) and water (2 mL). The resulting mixture was heated to 65? C., under argon, for 16 h. When the reaction was judged to be complete by TLC and LCMS, it was diluted into ethyl acetate (200 mL) and filtered over celite. The filter cake was washed with ethyl acetate (100 mL) and water (100 mL) and the filtrate phases separated. The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo. The aqueous phase was frozen and lyophilised to dryness, then charged with ethyl acetate
[1506] (100 mL), sonicated for 10 min, filtered and the filtrate concentrated in vacuo. The combined red residues were purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 10% to 100%), to give the title compound
[1507] (268 mg, 40%) as a cream solid.
[1508] .sup.1H NMR (400 MHz, MeOD) ? 7.26 (s, 1H), 6.51 (s, 1H), 4.41 (dt, J=13.2, 3.6 Hz, 1H), 4.20 (dd, J=6.3, 3.5 Hz, 1H), 3.87 (s, 3H), 2.93 (ddd, J=13.5, 12.1, 3.9 Hz, 1H), 2.23-2.12 (m, 1H), 1.99-1.86 (m, 1H), 1.86-1.76 (m, 1H), 1.75-1.63 (m, 2H), 1.62-1.55 (m, 1H); .sup.13C NMR (too MHz, MeOD) ? 171.6, 169.2, 150.7, 145.2, 131.6, 117.7, 111.9, 106.8, 55.2, 51.5, 39.9, 22.9, 22.1, 18.7; MS (ES+): m/z=277 (M+H).sup.+; LCMS (Method A): t.sub.R=4.87 min.
Example 139: Methyl (S)-2-(3-(((2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)-acetate (137)
[1509] ##STR00287##
[1510] A solution of (S)-3-hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione (136) (167 mg, 0.604 mmol) in N,N-dimethylformamide (1.2 mL) was charged with potassium carbonate (100 mg, 0.725 mmol) and 3-(bromomethyl)-benzeneacetic acid methyl ester (147 mg, 0.604 mmol). The resulting mixture was stirred for 4 h, before it was diluted into ethyl acetate (100 mL) and washed with cold brine (2?50 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 100%), gave the title compound (197 mg, 47%) as a white solid.
[1511] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.80 (s, 1H), 7.33 (s, 1H), 7.31-7.25 (m, 3H), 7.19-7.14 (m, 1H), 6.50 (s, 1H), 5.02 (S, 2H), 4.45 (dt, J=13.6, 3.8 Hz, 1H), 4.11-4.06 (m, 1H), 3.85 (s, 3H), 3.65 (s, 3H), 3.59 (s, 2H), 2.92 (td, J=13.5, 3.9 Hz, 1H), 2.17 (dt, J=140.9, 7.1 Hz, 1H), 1.97-1.82 (m, 1H), 1.82-1.71 (m, 1H), 1.71-1.60 (m, 2H), 1.61-1.47 (m, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 172.1, 171.5, 168.2, 151.1, 146.8, 136.2, 134.3, 130.3, 129.2, 129.0, 128.7, 126.3, 119.7, 112.6, 105.6, 70.8, 56.2, 52.2, 51.3, 40.8, 40.2, 23.1, 22.7, 19.1; MS (ES+): m/z=439 (M+H).sup.+; LCMS (Method A): t.sub.R=6.30 min.
Example 140: (S)-2-(3-(((2-Methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid
[1512] (138)
##STR00288##
[1513] A solution of methyl (S)-2-(3-(((2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetate (137) (115 mg, 0.262 mmol) in tetrahydrofuran (1 mL) was charged with an aqueous solution of sodium hydroxide (0.5 M, 1 mL, 0.525 mmol) and the resulting mixture stirred at room temperature for 1.5 h. After the reaction was judged to be complete by TLC and LCMS, it was adjusted to pH=4 with a saturated aqueous solution of citric acid and extracted with ethyl acetate (2?100 mL). The combined organic extracts were then dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting white solid was employed in the subsequent step without further purification.
[1514] MS (ES+): m/z=425 (M+H).sup.+; LCMS (Method A): t.sub.R=5.80 min.
Example 141: (S)-3-((3-(2-((S)-1-(Chloromethyl)-3-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione (139)
[1515] ##STR00289##
[1516] A solution of (S)-2-(3-(((2-Methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (138) (111 mg, 0.262 mmol) in N,N-dimethylacetamide (1 mL) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (127 mg, 0.472 mmol), followed by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (271 mg, 1.42 mmol) and the resulting mixture stirred at room temperature for 16 h. The reaction mixture was subsequently diluted into ethyl acetate (100 mL) and washed with cold brine (2?20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (90%), followed by trituration in dichloromethane/diethyl ether afforded the title compound (71 mg, 43%) as a cream solid.
[1517] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.97 (s, 1H), 8.87 (s, 1H), 8.41 (s, 1H), 8.26 (d, J=8.2 Hz, 1H), 7.53 (d, J=8.3 Hz, 1H), 7.47-7.39 (m, 2H), 7.39-7.31 (m, 3H), 7.19 (dd, J=9.7, 5.4 Hz, 1H), 6.91 (d, J=7.6 Hz, 1H), 6.65 (s, 1H), 5.45 (d, J=13.1 Hz, 1H), 5.07 (d, J=13.1 Hz, 1H), 4.45 (dd, J=9.8, 3.7 Hz, 1H), 4.22 (d, J=10.5 Hz, 1H), 4.18-4.12 (m, 1H), 4.11-3.90 (m, 4H), 3.87 (s, 3H), 3.83 (d, J=2.8 Hz, 1H), 3.43 (d, J=16.8 Hz, 1H), 2.92 (td, J=13.5, 3.8 Hz, 1H), 2.25 (d, J=13.0 Hz, 1H), 1.91-1.48 (m, 5H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.3, 169.1, 168.0, 155.1, 150.0, 147.4, 141.2, 135.5, 133.4, 132.7, 129.9, 129.6, 129.5, 129.3, 126.5, 123.7, 123.4, 122.4, 122.0, 119.9, 114.4, 112.2, 108.4, 100.5, 71.5, 64.3, 56.2, 53.3, 51.2, 46.5, 42.2., 40.3, 23.2, 22.8, 19.2; MS (ES+): m/z=640 (M+H).sup.+; LCMS (Method A): t.sub.R=7.25 min.
Example 142: (S)-1-(Chloromethyl)-3-(2-(3-((((S)-2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 4-methylpiperazine-1-carboxylate (140)
[1518] ##STR00290##
[1519] A solution of (S)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo-[e]pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione (139) (40 mg, 0.062 mmol) in dichloromethane (1 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (12 mg, 0.062 mmol), 4-(dimethylamino)pyridine (8.4 mg, 0.069 mmol) and then triethylamine
[1520] (30 ?L, 0.22 mmol). The resulting mixture was stirred at room temperature for 1 h, after which it was concentrated in vacuo, then charged with diethyl ether and concentrated again, then charged with diethyl ether once more and concentrated once again, to give a white solid residue, which was purified by flash column chromatography (silica), eluting with ethyl acetate (100%), then triethylamine/ethyl acetate (5%), and finally with triethylamine/methanol/ethyl acetate (5:5:90) to give the title compound (20 mg, 42%) as a cream solid.
[1521] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.35 (s, 1H), 8.25 (br, 1H), 7.84 (d, J=8.4 Hz, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.47 (t, J=7.5 Hz, 1H), 7.40 (d, J=8.0 Hz, 1H), 7.36 (t, J=6.0 Hz, 1H), 7.34-7.27 (m, 3H), 7.25-7.21 (m, 1H), 6.41 (s, 1H), 5.08 (s, 2H), 4.45 (d, J=13.3 Hz, 1H), 4.31 (d, J=10.7 Hz, 1H), 4.19-4.12 (m, 1H), 4.05 (t, J=6.6 Hz, 2H), 3.87 (s, 3H), 3.85-3.78 (m, 4H), 3.61 (br, 2H), 3.37 (t, J=10.6 Hz, 1H), 3.02-2.84 (m, 2H), 2.59-2.43 (m, 4H), 2.37 (s, 3H), 2.20-2.12 (m, 1H), 1.91-1.83 (m, 1H), 1.81-1.73 (m, 1H), 1.71-1.50 (m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.0, 169.2, 168.2, 153.5, 151.0, 148.3, 146.7, 1400.8, 136.5, 134.3, 134.2, 130.3, 129.7, 129.1, 126.1, 125.1, 122.6, 122.5, 121.2, 119.7, 118.4, 122.6, 111.0, 105.6, 70.8, 57.7, 56.2, 53.2, 51.2, 46.1, 46.0, 45.7, 44.6, 42.4, 40.1, 23.2, 22.6, 19.1; MS (ES+): m/z=766 (M+H).sup.+; LCMS (Method A): t.sub.R=5.97 min.
Example 143: tert-Butyl (S)-3-hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (141)
[1522] ##STR00291##
[1523] A solution of tert-Butyl (S)-5-(benzyloxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (82) (100 mg, 0.236 mmol) in tetrahydrofuran (4 mL) was charged with ammonium formate (119 mg, 1.89 mmol) and palladium on activated charcoal (10 wt. % basis, 100 mg), followed by water (1 mL). The resulting mixture was heated to 65? C., under argon, for 3 h, after which TLC and LCMS showed completion of the reaction. After allowing the mixture to cool to ambient conditions, it was filtered through a pad of celite. The resulting filter cake was then washed with ethyl acetate (100 mL), water
[1524] (100 mL) and methanol (10 mL). The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (100 mL). The combined organic phases were then dried over magnesium sulfate, filtered and concentrated in vacuo to give a green solid residue, which was recrystallised from ethyl acetate/petroleum spirit, 40-60? C. to give the title compound (68 mg, 96%) as a white solid. This was then used in the subsequent step without any further purification.
[1525] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.23-8.15 (m, 1H), 7.80 (br, 1H), 7.68 (d, J=8.4 Hz, 1H), 7.52-7.42 (m, 2H), 7.37-7.28 (m, 1H), 4.03-3.88 (m, 1H), 3.83-3.65 (m, 1H), 3.42 (t, J=11.0 Hz, 1H), 1.58 (s, 9H), 1.38 (d, J=6.8 Hz, 3H); MS (ES?): m/z=298 (M?1).sup.?; LCMS (Method A): t.sub.R=7.80 min.
Example 144: (S)-1-Methyl-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride
[1526] (142)
##STR00292##
[1527] A solution of tert-butyl (S)-5-hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indole-3-carboxylate (141) (68 mg, 0.227 mmol) in 1,4-dioxane (1 mL) was charged with hydrochloric acid (4 M in 1,4-dioxane, 1 mL) and stirred at room temperature for 4 h, after which LCMS confirmed completion of reaction. The resulting mixture was concentrated in vacuo, then charged with diethyl ether and concentrated again, then subjected to strong vacuum for 1 h. The resulting navy blue residue (unstable) was used in the next step without further purification.
[1528] MS (ES+): m/z=200 (M+H).sup.+; LCMS (Method A): t.sub.R=4.70 min.
Example 145: (S)-3-((3-(2-((S)-5-Hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-6,2(5H,6aH)-dione (14)
[1529] ##STR00293##
A solution of (S)-2-(3-(((2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (138) (74 mg, 0.174 mmol) in N,N-dimethylacetamide (1 mL) was charged to (S)-1-methyl-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (142) (54 mg, 0.227 mmol), followed immediately by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (100 mg, 0.523 mmol), and the resulting blue-green solution was stirred at room temperature for 16 h, whereupon it was diluted into ethyl acetate (100 mL) and washed with cold brine (2?50 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (98%) gave the title compound (35 mg, 33%) as a white solid.
[1530] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.05 (s, 1H), 8.65 (s, 1H), 8.46 (s, 1H), 8.27 (d, J=8.3 Hz, 1H), 7.64 (d, J=8.2 Hz, 1H), 7.48 (s, 1H), 7.45-7.39 (m, 1H), 7.35 (td, J=8.2, 1.2 Hz, 2H), 7.30 (s, 1H), 7.22 (t, J=7.6 Hz, 1H), 6.97 (d, J=7.4 Hz, 1H), 6.68 (s, 1H), 5.50 (d, J=13.1 Hz, 1H), 5.10 (d, J=13.1 Hz, 1H), 4.46 (dd, J=13.6, 3.7 Hz, 1H), 4.18 (dt, J=11.0, 9.5 Hz, 2H), 3.93-3.89 (m, 4H), 3.85-3.76 (m, 3H), 3.50 (d, J=16.9 Hz, 1H), 2.93 (td, J=13.4, 3.8 Hz, 1H), 2.27 (d, J=12.8 Hz, 1H), 1.89-1.51 (m, 4H), 1.40 (d, J=6.7 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.3, 168.8, 168.0, 153.8, 149.9, 147.4, 139.6, 135.4, 133.7, 133.0, 129.9, 129.7, 129.3, 126.5, 123.4, 123.1, 122.7, 120.6, 119.9, 112.2, 108.8, 100.5, 71.6, 57.4, 56.2, 51.2, 41.9, 40.3, 33.7, 23.2, 22.9, 21.8, 19.3; MS (ES+): m/z=606 (M+H).sup.+; LCMS (Method A): t.sub.R=7.27 min.
Example 146: Allyl (6aS)-3-(3-(2-((S)-5-hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,89,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (144)
[1531] ##STR00294##
[1532] A solution of 2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10, 12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (86) (167 mg, 0.28 mmol) in N,N-dimethylacetamide (2 mL) was charged to (S)-1-methyl-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (142)
[1533] (65 mg, 0.28 mmol), followed immediately by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (215 mg, 1.12 mmol), and the resulting mixture was to stirred at room temperature for 5 h. TLC and LCMS showed completion of reaction, whereupon the mixture was diluted into ethyl acetate (100 mL), and washed with cold brine (2?50 mL). The organic phase was then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification was enacted by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (80%) to give the title compound (116 mg, 53%) as a grey solid.
[1534] MS (ES+): m/z=776 (M+H).sup.+; LCMS (Method A): t.sub.R=8.18 min.
Example 147: (S)-3-((3-(2-((S)-5-Hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (145)
[1535] ##STR00295##
[1536] A solution of allyl (6aS)-3-((3-(2-((S)-5-hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (144) (116 mg, 0.15 mmol) in dichloromethane (1 mL) was charged with pyrrolidine (10 ?L) and tetrakis(triphenylphosphine)palladium(0) (3 mg) and the resulting mixture stirred for 25 min, after which it was diluted into dichloromethane (10 mL), filtered through a plug of celite, wash with dichloromethane and concentrated in vacuo. Diethyl ether (50 mL) was then charged and the mixture concentrated again. Purification by flash column chromatography (silica), eluting with methanol/ethyl acetate (3%) gave the title compound
[1537] (54 mg, 61%) as a white solid.
[1538] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.91 (br, 1H), 8.35 (s, 1H), 8.22 (d, J=8.3 Hz, 1H), 7.76 (d, J=5.7 Hz, 1H), 7.64 (d, J=8.3 Hz, 1H), 7.47-7.40 (m, 3H), 7.37-7.29 (m, 4H), 6.75 (s, 1H), 5.15 (q, J=12.7 Hz, 2H), 4.24-4.10 (m, 2H), 3.90 (d, J=3.0 Hz, 2H), 3.85 (s, 3H), 3.76 (dd, J=14.0, 3.7 Hz, 1H), 3.73-3.61 (m, 2H), 3.25-3.14 (m, 1H), 1.97-1.59 (m, 6H), 1.28 (d, J=6.8 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 170.0, 167.5, 163.1, 154.0, 150.2, 148.2, 139.7, 136.9, 134.3, 129.8, 129.3, 128.7, 127.9, 126.7, 126.1, 123.7, 123.1, 123.0, 122.6, 121.4, 120.9, 111.7, 110.9, 100.8, 70.5, 57.7, 56.1, 49.5, 43.7, 39.7, 33.6, 24.4, 22.9, 21.4, 18.3; MS (ES+): m/z=590 (M+H).sup.+; LCMS (Method A): t.sub.R=7.13 min.
Example 148: Methyl (S)-1-(5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoyl)piperidine-2-carboxylate (146)
[1539] ##STR00296##
[1540] A slurry of 5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (3) (520 mg, 1.66 mmol) in dichloromethane (3.3 mL) was charged with triethylamine (972 ?L, 6.97 mmol) and stirred. The resulting yellow solution was then charged with HATU (663 mg, 1.74 mmol) and stirred at room temperature for 5 min before adding methyl (2S)-piperidinecarboxylate hydrochloride (298 mg, 1.66 mmol) and stirring at room temperature for 4 h, after which the mixture was diluted into dichloromethane (100 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate (50 mL) followed by an aqueous solution of acetic acid (1% v/v, 10000 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo, to give the title compound (653 mg, 90%) as a yellow solid, which was used in the subsequent step without further purification.
[1541] MS (ES+): m/z=439 (M+H).sup.+; LCMS (Method A): t.sub.R=7.25 min.
Example 140: Methyl (S)-4-((2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoate (147)
[1542] ##STR00297##
[1543] A solution of methyl (S)-1-(5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoyl)piperidine-2-carboxylate (146) (621 mg, 1.42 mmol) in tetrahydrofuran (3 mL) was charged with ammonium formate (715 mg, 11.3 mmol) and palladium on activated charcoal (10 wt. % basis, 62 mg), followed by water (1 mL) and the resulting mixture stirred under argon at 65? C. for 16 h. The mixture was then filtered through a pad of celite, and the filter cake washed with ethyl acetate (100 mL) and water (100 mL). The organic phase was then separated, washed twice with brine (50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%), followed by methanol/ethyl acetate (from 0% to 65%) gave the title compound (52 mg, 10%) as an amber oil.
[1544] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.77 (s, 1H), 7.30 (s, 1H), 6.49 (s, 1H), 4.45 (d, J=13.6 Hz, 1H), 4.10-4.06 (m, 1H), 3.99 (t, J=6.3 Hz, 2H), 3.82 (s, 3H), 3.71 (d, J=7.3 Hz, 1H), 3.63 (s, 3H), 2.98-2.86 (m, 1H), 2.48 (t, J=7.1 Hz, 2H), 2.23-2.14 (m, 1H), 1.95-1.82 (m, 1H), 1.82-1.46 (m, 5H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 173.4, 171.4, 168.3, 151.3, 146.6, 130.4, 119.5, 112.5, 104.7, 67.8, 56.1, 51.6, 51.3, 400.1, 38.6, 30.2, 24.1, 23.1, 22.7; MS (ES+): m/z=377 (M+H).sup.+; LCMS (Method C): t.sub.R=2.93 min.
Example 150: (S)-4-((2-Methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (148)
[1545] ##STR00298##
[1546] A solution of methyl (S)-4-((2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoate (147) (55 mg, 0.146 mmol) in tetrahydrofuran (1 mL) was charged with an aqueous solution of sodium hydroxide (0.5 M, 0.58 mL, 0.29 mmol) and stirred at room temperature for 2 h, upon which TLC and LCMS showed completion of reaction. The reaction mixture was then concentrated in vacuo, and taken up into water (500 mL) and ethyl acetate (50 mL), then acidified to pH=1 with an aqueous solution of hydrochloric acid (1 M). The phases were separated, and the aqueous extract was washed with ethyl acetate (100 mL). The combined organic extracts were then dried over magnesium sulfate, filtered, and concentrated in vacuo, the residue of which was then used in the subsequent step without further purification.
[1547] MS (ES+): m/z=363 (M+H).sup.+; LCMS (Method C): t.sub.R=2.55 min.
Example 151: (S)-3-(4-((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione (149)
[1548] ##STR00299##
[1549] A solution of (S)-4-((2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (148) (53 mg, 0.146 mmol) in N,N-dimethylacetamide (1 mL) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (64 mg, 0.236 mmol), followed immediately by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (84 mg, 0.439 mmol) and the resulting mixture stirred at room temperature for 16 h. The mixture was then taken up into ethyl acetate (100 mL) and washed with cold brine (2?50 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 15% to 100%) gave the title compound (5.6 mg, 7%) as a green solid.
[1550] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.27 (br, 2H), 8.11 (s, 1H), 7.61 (d, J=8.6 Hz, 1H), 7.53-7.47 (m, 1H), 7.39-7.32 (m, 1H), 7.27 (s, 1H), 7.26 (br, 1H), 6.39 (s, 1H), 4.45 (d, J=14.1 Hz, 1H), 4.18-4.04 (m, 2H), 3.98 (br, 1H), 3.89 (d, J=13.2 Hz, 2H), 3.73 (s, 3H), 3.35 (t, J=11.5 Hz, 1H), 2.96-2.85 (m, 2H), 2.69 (br, 1H), 2.40-2.20 (m, 1H), 2.16-2.08 (m, 1H), 1.96-1.73 (m, 4H), 1.70-1.48 (m, 4H); MS (ES+): m/z=578 (M+H).sup.+; LCMS (Method C): t.sub.R=3.42 min.
Example 152: (S)-1-(Chloromethyl)-3-(4-(((S)-2-methoxy-6,12-dioxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanol)-2,3-dihydro-1H-benzo[e]indol-5-yl 4-methylpiperazine-1-carboxylate (150)
[1551] ##STR00300##
[1552] A solution of (S)-3-(4-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione (149) (2.7 mg, 0.0047 mmol) in dichloromethane (100 ?L) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (2.8 mg, 0.014 mmol), 4-(dimethylamino)pyridine (1.2 mg, 0.0098 mmol) and triethylamine (2.3 ?L, 0.016 mmol) and stirred at room temperature for 1.5 h. The reaction mixture was subsequently diluted into dichloromethane (10 mL) washed with a saturated aqueous solution of sodium hydrogen carbonate (2?5 mL), dried over magnesium sulfate and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with ethyl acetate (10%), followed by triethylamine/methanol/ethyl acetate (from 5:0:1 to 5:1:0), to give the title compound (1.6 mg, 48%) as a white solid.
[1553] .sup.1H NMR (400 MHz, MeOD) ?8.23 (s, 1H), 8.06 (d, J=6.3 Hz, 1H), 7.86 (dd, J=17.9, 8.4 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 7.46 (t, J=8.0 Hz, 1H), 7.26 (s, 1H), 6.77 (d, J=6.6 Hz, 1H), 6.66 (s, 1H), 4.42-4.36 (m, 3H), 4.21-4.16 (m, 3H), 3.89 (br, 2H), 3.78 (s, 3H), 3.65-3.59 (m, 2H), 2.96-2.80 (m, 2H), 2.75-2.70 (m, 1H), 2.65-2.50 (m, 4H), 2.38 (s, 3H), 2.25 (t, J=6.4 Hz, 2H), 2.19-2.11 (m, 1H), 1.85-1.55 (m, 8H); MS (ES+): m/z=704 (M+H).sup.+; LCMS (Method C): t.sub.R=2.83 min.
Example 153: Allyl (6aS)-3-(4-((S)-5-hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (151)
[1554] ##STR00301##
[1555] A solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (100 mg, 0.188 mmol) in N,N-dimethylacetamide (0.5 mL) was charged to (S)-1-methyl-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (142) (54 mg, 0.227 mmol), followed immediately by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (87 mg, 0.454 mmol) and the resulting mixture stirred at room temperature overnight. The reaction mixture was then diluted into ethyl acetate (100 mL) and washed with cold brine (2?50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 10% to 100%) gave the title compound (36 mg, 22%) as a green solid.
[1556] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.53 (br, 1H), 8.28-8.23 (m, 2H), 7.66 (d, J=8.4 Hz, 1H), 7.45 (t, J=7.6 Hz, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.18 (s, 1H), 7.03 (s, 1H), 6.15 (d, J=11.0 Hz, 1H), 5.97 (d, J=9.7 Hz, 1H), 5.70 (br, 1H), 5.26-4.93 (m, 3H), 4.60-4.45 (m, 1H), 4.24 (d, J=13.1 Hz, 3H), 4.15 (d, J=10.3 Hz, 1H), 3.87 (s, 3H), 3.80 (d, J=10.5 Hz, 3H), 3.65 (br, 1H), 3.47 (dd, J=10.1, 6.1 Hz, 1H), 3.08 (d, J=13.2 Hz, 1H), 2.79-2.57 (m, 2H), 2.30 (dd, J=13.3, 7.0 Hz, 2H), 1.82-1.36 (m, 12H), 1.35 (d, J=6.6 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.3, 169.3, 156.2, 153.7, 149.2, 143.1, 139.7, 132.0, 129.9, 126.8, 123.0, 122.6, 120.7, 117.2, 114.3, 110.6, 110.3, 100.5, 94.6, 83.9, 68.1, 62.8, 60.4, 57.4, 56.1, 55.7, 38.8, 33.6, 30.9, 30.4, 25.2, 23.3, 21.7, 21.0, 18.2; MS (ES+): m/z=714 (M+H).sup.+; LCMS (Method C): t.sub.R=3.92 min.
Example 1.54: (S)-3-(4-((S)-5-Hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1.4]diazepin-12(6aH)-one (152)
[1557] ##STR00302##
[1558] A solution of allyl (6aS)-3-(4-((S)-5-hydroxy-1-methyl-1,2-dihydro-3H-benzo[e]indol-3-yl)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (151) (36 mg, 0.050 mmol) in dichloromethane (1 mL) was charged with pyrrolidine (5 ?L, 0.061 mmol), and tetrakis(triphenylphosphine)palladium(0) (5.8 mg, 0.005 mmol) and stirred at room temperature for 40 min, whereupon it was concentrated in vacuo. Purification by flash column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 100%) gave the title compound (16 mg, 59%) as a cream solid.
[1559] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.20 (s, 1H), 8.19 (d, J=8.4 Hz, 1H), 8.13 (s, 1H), 7.97 (d, J=5.7 Hz, 1H), 7.75 (d, J=8.3 Hz, 1H), 7.46 (t, J=7.5 Hz, 1H), 7.34 (s, 1H), 7.33-7.28 (m, 1H), 6.82 (s, 1H), 4.36 (t, J=9.5 Hz, 1H), 4.26-4.05 (m, 3H), 3.93 (d, J=10.2 Hz, 1H), 3.86 (s, 3H), 3.82 (br, 1H), 3.79-3.71 (m, 2H), 3.21-3.11 (m, 1H), 2.73-2.62 (m, 1H), 2.22 (t, J=6.6 Hz, 2H), 2.00-1.55 (m, 6H), 1.34 (d, J=6.8 Hz, 3H); MS (ES+): m/z=528 (M+H).sup.+; LCMS (Method A): t.sub.R=7.02 min.
Example 155: ((Allyloxy)carbonyl)-L-valine (153)
[1560] ##STR00303##
[1561] A solution of L-valine (33.0 g, 282 mmol) and potassium carbonate (58.4 g, 423 mmol) in tetrahydrofuran (500 mL) and water (500 mL) was charged with allyl chloroformate (40.75 g, 338 mmol) dropwise, and stirred at room temperature for 18h. The resulting mixture was partially concentrated in vacuo, then extracted with diethyl ether (300 mL). The aqueous phase was acidified to pH=2 with concentrated hydrochloric acid, then extracted with dichloromethane (3?300 mL). The combined organic phases were then washed with brine, dried over magnesium sulfate, filtered and then concentrated in vacuo to give the title compound (53.0 g, 94%) as a colourless oil.
[1562] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 12.55 (s, 1H), 7.40 (d, J=8.6 Hz, 1H), 5.91 (ddt, J=16.2, 10.6, 5.2 Hz, 1H), 5.30 (dd, J=17.2, 1.8 Hz, 1H), 5.18 (dd, J=1.6, 1.8 Hz, 1H), 4.52-4.44 (m, 2H), 3.85 (dd, J=8.6, 6.0 Hz, 1H), 2.09-1.99 (m, 1H), 0.90-0.86 (m, 6H); MS (ES+): m/z=202 (M+H).sup.+; LCMS (Method F): t.sub.R=3.10 min.
Example 156: 2,5-Dioxopyrrolidin-1-yl ((allyloxy)carbonyl)-L-valinate (154)
[1563] ##STR00304##
[1564] A solution of ((allyloxy)carbonyl)-L-valine (153) (53.0 g, 263 mmol) and N-hydroxysuccinimide (30.3 g, 263 mmol) in anhydrous tetrahydrofuran (1 L) was charged with N,N-dicyclohexylcarbodiimide (54.4 g, 263 mmol) and the resulting mixture stirred at room temperature for 18 h. The mixture was then filtered, and the residue washed with tetrahydrofuran (300 mL). The combined filtrate was then concentrated in vacuo. Dichloromethane was then added to the residue and the resulting slurry was left to stand at 0? C. The suspension was filtered and washed with cold dichloromethane. The filtrate was then concentrated in vacuo to afford the title compound (60.0 g, 76%) as a viscous, colourless oil.
[1565] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.99 (d, J=8.2 Hz, 1H), 5.93 (ddt, J=16.2, 10.6, 5.2 Hz, 1H), 5.38-5.26 (m, 1H), 5.19 (d, J=10.6 Hz, 1H), 4.53 (d, J=5.4 Hz, 2H), 4.33 (dd, J=8.2, 6.2 Hz, 1H), 2.81 (s, 4H), 2.19 (q, J=6.8 Hz, 1H), 1.01 (d, J=6.8 Hz, 6H); MS (ES+): m/z=299 (M+H).sup.+; LCMS (Method F): t.sub.R=3.57 min.
Example 157: ((Allyloxy)carbonyl)-L-valyl-L-alanine (155)
[1566] ##STR00305##
[1567] A solution of L-alanine (18.8 g, 211 mmol) and sodium hydrogen carbonate (18.6 g, 221 mmol) in tetrahydrofuran (100 mL) and water (200 mL) was charged with a solution of 2,5-dioxopyrrolidin-1-yl ((allyloxy)carbonyl)-L-valinate (154) (60.0 g, 201 mmol) in tetrahydrofuran (100 mL). The resulting mixture was stirred for 72 h and then partially concentrated in vacuo. A saturated aqueous solution of citric acid was used to acidify to pH=3-4, and the mixture was then extracted with ethyl acetate (6?150 mL). The combined organic extracts were washed with water (200 mL), brine (200 mL) and dried over magnesium sulfate, then filtered and concentrated in vacuo to afford a white solid, which was triturated with diethyl ether, giving the title compound (38.0 g, 69%) as a white powder.
[1568] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 12.47 (s, 1H), 8.17 (d, J=7.0 Hz, 1H), 7.15 (d, J=9.0 Hz, 1H), 5.93-5.85 (m, 1H), 5.29 (d, J=17.2 Hz, 1H), 5.17 (d, J=10.4 Hz, 1H), 4.46 (d, J=5.2 Hz, 2H), 4.22-4.15 (m, 1H), 3.87 (t, J=8.0 Hz, 1H), 1.95 (dd, J=140.6, 7.6 Hz, 1H), 1.26 (d, J=7.2 Hz, 3H), 0.88 (d, J=6.8 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H); MS (ES+): m/z=273 (M+H).sup.+; LCMS (Method F): t.sub.R=2.67 min.
Example 158: Allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (156)
[1569] ##STR00306##
[1570] A solution of ((allyloxy)carbonyl)-L-valyl-L-alanine (155) (38.0 g, 140 mmol) in anhydrous tetrahydrofuran (800 mL) was charged with (4-aminophenyl)methanol (18.1 g, 147 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (36.2 g, 147 mmol). The resulting mixture was stirred at room temperature for 72 h. The solvent was then evaporated in vacuo to give a pale brown solid. This residue was then triturated with diethyl ether and filtered, washed with an excess of diethyl ether to afford the title compound (40.0 g, 76%) as a white solid.
[1571] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 9.89 (s, 1H), 8.13 (d, J=7.2 Hz, 1H), 7.53 (d, J=8.0 Hz, 2H), 7.24 (dd, J=8.8, 2.6 Hz, 3H), 5.91 (td, J=100.8, 5.2 Hz, 1H), 5.30 (d, J=17.0 Hz, 1H), 5.17 (d, J=10.4 Hz, 1H), 5.08 (d, J=5.6 Hz, 1H), 4.52-4.45 (m, 2H), 4.43 (d, J=3.6 Hz, 3H), 3.94-3.82 (m, 1H), 1.98 (d, J=6.8 Hz, 1H), 1.30 (d, J=7.0 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.8 Hz, 3H); MS (ES+): m/z=378 (M+H).sup.+; LCMS (Method F): t.sub.R=2.98 min.
Example 159: Allyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (157)
[1572] ##STR00307##
A solution of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (156) (40.0 g, 106 mmol) in anhydrous tetrahydrofuran (1 L) was charged with bis(4-nitrophenyl) carbonate (64.5 g, 212 mmol) and triethylamine (21.5 g, 212 mmol). The resulting mixture was stirred at room temperature for 1.5 h and then concentrated in vacuo. The mixture was triturated with ethyl acetate (2?100 mL) and methanol/dichloromethane (10%, 3?50 mL) to afford the title compound (20.0 g, 35%) as a white solid.
[1573] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 100.007 (s, 1H), 8.31 (d, J=8.6 Hz, 2H), 8.17 (d, J=6.8 Hz, 1H), 7.64 (d, J=8.2 Hz, 2H), 7.56 (d, J=8.6 Hz, 2H), 7.41 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.8 Hz, 1H), 5.94-5.86 (m, 1H), 5.30 (d, J=17.0 Hz, 1H), 5.24 (s, 2H), 5.17 (d, J=10.4 Hz, 1H), 4.48 (d, J=5.2 Hz, 2H), 4.45-4.32 (m, 1H), 3.90 (t, J=7.8 Hz, 1H), 1.98 (dd, J=8.6, 5.2 Hz, 1H), 1.32 (d, J=7.0 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.8 Hz, 3H); MS (ES+): m/z=543 (M+H).sup.+; LCMS (Method F): t.sub.R=3.83 min.
Example 160: 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3-methylbutan-amido)propanamido)benzyl (2-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (158)
[1574] ##STR00308##
[1575] A solution of (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)piperidin-1-yl)methanone (69) (5.0 g, 11.5 mmol) in N,N-dimethylformamide (23 mL) was charged with allyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (157) (6.2 g, 11.5 mmol) and 1-hydroxybenzotriazole hydrate (1.5 g, 11.5 mmol), and the resulting mixture was heated to 60? C. under argon, for 16 h. The mixture was allowed to cool to room temperature, then diluted into ethyl acetate (500 mL) and washed with cold brine (3?100 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 10% to 90%) gave the title compound (7.49 g, 78%) as a beige solid.
[1576] .sup.1H NMR (400 MHz, CDCl.sub.3) 39.12 (br, 1H), 8.13 (br, 1H), 7.96 (s, 2H), 7.47 (d, J=8.3 Hz, 2H), 7.22 (d, J=8.0 Hz, 2H), 6.73 (s, 1H), 5.80 (d, J=8.2 Hz, 1H), 5.24 (d, J=17.3 Hz, 1H), 5.14 (d, J=10.2 Hz, 1H), 5.04 (q, J=12.4 Hz, 2H), 4.67-4.58 (m, 1H), 4.55-4.44 (m, 2H), 4.09 (dd, J=12.3, 6.6 Hz, 1H), 3.80 (t, J=100.6 Hz, 1H), 3.71 (s, 3H), 3.53 (br, 1H), 2.14-1.98 (m, 1H), 1.65-1.50 (m, 4H), 1.34 (d, J=6.9 Hz, 3H), 1.28-1.15 (m, 4H), 1.05 (d, J=7.4 Hz, 22H), 0.87 (dd, J=11.4, 6.8 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.9, 170.6, 170.5, 162.6, 156.5, 153.9, 137.9, 137.1, 132.5, 132.1, 128.7, 127.5, 120.1, 119.8, 117.7, 111.0, 66.2, 65.9, 60.3, 56.3, 55.9, 49.5, 36.5, 31.4, 31.2, 25.6, 19.8, 19.6, 19.2, 17.9, 17.8, 12.8; MS (ES+): m/z=840 (M+H).sup.+; LCMS (Method A): t.sub.R=9.53 min.
Example 161: 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (5-hydroxy-2-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxyphenyl)carbamate (159)
[1577] ##STR00309##
[1578] A solution of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-propanamido)benzyl (2-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (158) (1.89 g, 2.25 mmol) in tetrahydrofuran (6 mL) was charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 3.38 mL, 3.38 mmol) and stirred at room temperature for 10 min. The reaction mixture was subsequently concentrated in vacuo to give an orange solid, which was purified by flash column chromatography (silica), eluting with methanol/ethyl acetate (from 0% to 10%) to give the title compound (1.41 g, 92%) as a cream solid.
[1579] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.18 (s, 1H), 8.25 (s, 1H), 7.66 (d, J=6.9 Hz, 1H), 7.46 (s, 1H), 7.36 (d, J=8.0 Hz, 2H), 7.14 (d, J=8.2 Hz, 2H), 6.73 (s, 1H), 5.85 (ddd, J=16.2, 11.2, 6.6 Hz, 2H), 5.25 (d, J=17.3 Hz, 1H), 5.15 (d, J=10.6 Hz, 1H), 5.08-4.94 (m, 2H), 4.73-4.61 (m, 1H), 4.59-4.43 (m, 2H), 4.11 (t, J=7.2 Hz, 1H), 3.97 (br, 1H), 3.88-3.76 (m, 1H), 3.74 (s, 3H), 3.51 (ddd, J=17.5, 10.7, 9.8 Hz, 1H), 2.93 (br, 1H), 2.48 (br, 1H), 2.03 (dd, J=12.7, 7.3 Hz, 2H), 1.59 (br, 4H), 1.35 (d, J=6.8 Hz, 5H), 0.87 (apparent t, J=6.7 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 172.1, 170.9, 170.5, 156.6, 154.3, 147.6, 143.5, 137.7, 132.5, 132.2, 129.5, 128.6, 120.1, 117.8, 110.2, 66.3, 65.9, 60.3, 56.2, 50.6, 49.6, 31.3, 25.6, 19.5, 19.1, 17.9, 17.8; MS (ES+): m/z=684 (M+H).sup.+; LCMS (Method A): t.sub.R=6.13 min.
Example 162: Methyl 2-(3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)methyl)phenyl)acetate (160)
[1580] ##STR00310##
[1581] A solution of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (5-hydroxy-2-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxyphenyl)carbamate (159) (2.18 g, 3.19 mmol) in N,N-dimethylformamide (7 mL) was charged with potassium carbonate (661 mg, 4.78 mmol) and 3-(bromomethyl)-benzeneacetic acid methyl ester (813 mg, 3.35 mmol) and the resulting mixture stirred at room temperature for 16 h. The mixture was then diluted into ethyl acetate (500 mL) and washed with cold brine (2?100 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with methanol/ethyl acetate (from 0% to 10%) gave the title compound (2.03 g, 75%) as a pale yellow solid.
[1582] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.01 (s, 1H), 8.27 (s, 1H), 7.67 (s, 1H), 7.48 (d, J=8.2 Hz, 2H), 7.44-7.18 (m, 7H), 6.80 (s, 1H), 5.93-5.80 (m, 1H), 5.74 (d, J=8.1 Hz, 1H), 5.26 (d, J=14.5 Hz, 1H), 5.17 (d, J=10.2 Hz, 1H), 5.06 (q, J=12.4 Hz, 3H), 4.69-4.60 (m, 1H), 4.60-4.46 (m, 2H), 3.83 (br, 1H), 3.79 (s, 3H), 3.66 (s, 3H), 3.61 (s, 2H), 3.51 (br, 1H), 2.93 (br, 1H), 2.09-2.04 (m, 1H), 1.66-1.54 (m, 4H), 1.40 (br, 1H), 1.36 (d, J=6.9 Hz, 3H), 0.89 (dd, J=10.0, 6.8 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.9, 171.2, 170.5, 156.5, 154.1, 149.5, 145.3, 137.9, 136.7, 134.2, 132.5, 132.1, 129.0, 128.8, 128.7, 128.6, 126.6, 119.9, 117.9, 70.6, 66.4, 66.0, 60.4, 57.7, 56.4, 52.1, 49.6, 41.1, 31.2, 25.7, 21.0, 19.6, 19.2, 17.9, 17.8; MS (ES+): m/z=846 (M+H).sup.+; LCMS (Method A): t.sub.R=7.10 min.
Example 163: 2-(3-(5-((((4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)methyl)-phenyl)acetic acid (161)
[1583] ##STR00311##
[1584] A solution of methyl 2-(3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)methyl)phenyl)acetate (160)
[1585] (2.03 g, 2.40 mmol) in tetrahydrofuran (34 mL) was charged with an aqueous solution of sodium hydroxide (0.5 M, 9.6 mL, 4.80 mmol) dropwise, and stirred at room temperature. The reaction progress was closely monitored by LCMS and after 2 h, quenched by cautious addition of a saturated aqueous solution of citric acid (adjusted to pH=3-4). The resulting mixture was partially concentrated in vacuo, then extracted with ethyl acetate (2?250 mL). The combined organic phases were washed with brine (100 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with methanol/ethyl acetate (from 2% to 10%) gave the title compound (1.19 g, 59%) as a pale yellow solid.
[1586] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.96 (s, 1H), 8.07 (s, 1H), 7.43 (d, J=7.8 Hz, 2H), 7.34 (d, J=7.5 Hz, 1H), 7.29 (d, J=7.4 Hz, 1H), 7.27-7.20 (m, 4H), 7.17 (d, J=7.8 Hz, 1H), 6.74 (s, 1H), 5.92-5.81 (m, 1H), 5.67-5.57 (m, 1H), 5.27 (d, J=17.1 Hz, 1H), 5.18 (d, J=10.8 Hz, 1H), 5.11-4.94 (m, 4H), 4.71-4.57 (m, 1H), 4.53 (t, J=5.1 Hz, 2H), 4.05 (t, J=6.6 Hz, 1H), 3.89-3.81 (m, 1H), 3.76 (s, 3H), 3.65-3.53 (m, 3H), 2.94 (br, 1H), 2.75-2.15 (br, 6H), 2.03 (dd, J=16.9, 11.5 Hz, 2H), 1.59-1.42 (br, 3H), 1.35 (d, J=7.0 Hz, 3H), 0.88 (apparent t, J=7.4 Hz, 6H); MS (ES+): m/z=832 (M+H).sup.+; LCMS (Method A): t.sub.R=6.75 min.
Example 164: 2-(3-((((6aS)-5-(((4-((S)-2-((S)-2-(((Allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (162)
[1587] ##STR00312##
[1588] A solution of 2-(3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)methyl)phenyl)acetic acid
[1589] (161) (670 mg, 0.805 mmol) in anhydrous dichloromethane (5 mL) was cooled to ?5? C. and charged with Dess-Martin periodinane (678 mg, 1.60 mmol). After 15 min, the reaction mixture was allowed to warm to room temperature and stirred for a further 1 h, whilst monitoring the reaction progress by LCMS. The reaction was quenched by addition of a saturated aqueous solution of sodium metabisulfite, then extracted with dichloromethane (2?100 mL). The combined organic phases were dried over magnesium sulfate and concentrated in vacuo. The resulting residue was then purified by flash column chromatography (silica), eluting with methanol/ethyl acetate (from 0% to 10%) to give the title compound (431 mg, 65%) as an off-white solid.
[1590] MS (ES?): m/z=828 (M?1).sup.?, MS (ES+): m/z=830 (M+H).sup.+; LCMS (Method A): t.sub.R=6.63 min.
Example 165: 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (163)
[1591] ##STR00313##
[1592] A solution of 2-(3-((((6aS)-5-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetic acid (162) (253 mg, 0.305 mmol) in N,N-dimethylacetamide
[1593] (1 mL) was charged to (S)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol hydrochloride (11) (115 mg, 0.427 mmol), followed immediately by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (263 mg, 1.37 mmol) and the resulting mixture was sonicated, then stirred at room temperature for 3 h. The mixture was then diluted into ethyl acetate (100 mL) and washed with cold brine (2?20 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 50% to 100%) gave the title compound (195 mg, 61%) as a pale green solid.
[1594] MS (ES+): m/z=1045 (M+H).sup.+; LCMS (Method A): t.sub.R=7.78 min.
Example 166: 4-((S)-2-((S)-2-(((Allyloxy)carbonyl)amino)-3-methylbutan-amido)propanamido)benzyl (6aS)-3-(3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (164)
[1595] ##STR00314##
[1596] A solution of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-propanamido)benzyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (163) (153 mg, 0.146 mmol) in dichloromethane (2 mL) was charged with 4-methyl-1-piperazinecarbonyl chloride hydrochloride (29 mg, 0.146 mmol), 4-(dimethylamino)-pyridine (20 mg, 0.16 mmol) and triethylamine (71 ?L, 0.51 mmol) and stirred at room temperature for 18 h. The reaction mixture was subsequently concentrated in vacuo, then charged with diethyl ether
[1597] (10 mL) and concentrated again. Purification by flash column chromatography (silica), eluting with ethyl acetate (100%), then triethylamine/ethyl acetate (5%), then methanol/triethylamine/ethyl acetate (from 0:5:95 to 10:5:90), gave the title compound
[1598] (133 mg, 78%) as a white solid.
[1599] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.42 (s, 1H), 8.40 (s, 1H), 7.92 (apparent t, J=7.3 Hz, 2H), 7.76 (s, 1H), 7.62 (d, J=8.3 Hz, 2H), 7.55 (t, J=7.6 Hz, 1H), 7.55 (t, J=7.6 Hz, 1H), 7.46-7.41 (m, 1H), 7.36-7.30 (m, 4H), 7.22 (d, J=7.3 Hz, 2H), 7.08 (s, 1H), 6.73 (s, 1H), 6.52 (d, J=7.4 Hz, 1H), 5.97 (d, J=10.2 Hz, 1H), 5.89 (ddd, J=15.9, 10.5, 5.3 Hz, 1H), 5.27 (d, J=17.2 Hz, 1H), 5.12 (d, J=10.6 Hz, 1H), 4.95-4.83 (m, 1H), 4.75 (d, J=11.5 Hz, 1H), 4.54-4.44 (m, 5H), 4.30-4.21 (m, 2H), 4.07-3.97 (m, 4H), 3.82 (br, 2H), 3.80 (s, 3H), 3.55 (br, 2H), 3.40-3.32 (m, 2H), 3.30 (s, 2H), 2.54-2.37 (m, 5H), 2.28 (s, 3H), 1.79-1.48 (m, 6H), 1.33 (d, J=6.7 Hz, 3H), 0.93 (dd, J=13.7, 7.0 Hz, 6H); MS (ES+): m/z=1171 (M+H).sup.+; LCMS (Method A): t.sub.R=6.33 min.
Example 167: 4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)-benzyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (165)
[1600] ##STR00315##
[1601] A solution of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (164) (100 mg, 0.085 mmol) in dichloromethane (5 mL) was charged with pyrrolidine (8 ?L, 0.094 mmol) and tetrakis(triphenylphosphine)palladium(0) (10 mg, 0.009 mmol) and the resulting mixture stirred for 40 min, whilst monitoring the reaction progress by LCMS. The mixture was then diluted into dichloromethane (15 mL) and filtered through a plug of celite. The filter cake was washed with dichloromethane (20 mL) and ethyl acetate (20 mL) and the combined filtrates concentrated in vacuo. The resulting residue was then charged with diethyl ether (20 mL) and concentrated again, then subjected to strong vacuum for 1 h, giving the title compound as a white solid, which was employed in the subsequent step immediately, without further purification.
[1602] .sup.1H NMR (400 MHz, MeOD) ?8.28 (s, 1H), 7.85 (d, J=9.2 Hz, 2H), 7.55 (d, J=7.8 Hz, 1H), 7.47 (s, 3H), 7.36-7.22 (m, 4H), 7.18-7.06 (m, 2H), 6.68 (s, 1H), 5.97-5.91 (m, 1H), 5.14 (br, 1H), 4.90 (br, 1H), 4.52-4.30 (m, 2H), 4.26-4.15 (m, 2H), 3.93 (d, J=9.1 Hz, 2H), 3.84 (dd, J=140.7, 7.4 Hz, 3H), 3.79 (s, 3H), 3.68-3.52 (m, 3H), 3.39-3.32 (m, 1H), 3.19-3.11 (m, 1H), 2.60-2.46 (m, 4H), 2.36 (s, 3H), 1.99 (dd, J=11.3, 3.8 Hz, 4H), 1.90 (d, J=6.0 Hz, 3H), 1.79-1.45 (m, 5H), 1.40-1.34 (m, 3H), 1.28 (br, 1H), 0.97-0.80 (m, 7H); MS (ES+): m/z=1087 (M+H).sup.+; LCMS (Method A): t.sub.R=5.45 min.
Example 168: 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (166)
[1603] ##STR00316##
[1604] A solution of 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-methylpiperazine-1-carbonyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (165) (93 mg, 0.085 mmol) in dichloromethane (10 mL) was charged with 6-maleimidohexanoic acid (18 mg, 0.085 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (16 mg, 0.085 mmol) and stirred at room temperature for 4 h. The resulting mixture was then concentrated partially in vacuo, and then loaded directly onto silica. Purification by flash column chromatography (silica), eluting with ethyl acetate (100%), then triethylamine/ethyl acetate (5%), then methanol/triethylamine/ethyl acetate (from 0:5:95 to 10:5:90), gave the title compound (66 mg, 61% over two steps) as a white solid.
[1605] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.38 (s, 1H), 8.37 (s, 1H), 7.91 (apparent t, J=7.5 Hz, 2H), 7-77 (s, 1H), 7.66 (d, J=8.4 Hz, 2H), 7.54 (t, J=7.7 Hz, 1H), 7.45-7.37 (m, 3H), 7.30 (br, 4H), 7.22 (d, J=7.7 Hz, 2H), 7.07 (s, 1H), 6.80 (s, 2H), 6.73 (s, 1H), 5.96 (d, J=10.2 Hz, 1H), 5.24 (d, J=11.5 Hz, 1H), 4.89 (d, J=12.9 Hz, 1H), 4.74 (d, J=12.1 Hz, 1H), 4.47 (br, 3H), 4.30-4.21 (m, 3H), 4.06-3.93 (m, 3H), 3.81 (br, 2H), 3.79 (s, 3H), 3.53 (br, 2H), 3.40 (t, J=7.1 Hz, 2H), 3.36 (br, 1H), 2.54-2.36 (m, 4H), 2.26 (s, 3H), 2.08 (s, 4H), 1.77-1.46 (m, 9H), 1.32 (d, J=6.4 Hz, 3H), 1.29-1.22 (m, 2H), 1.21-1.17 (m, 1H), 1.06-1.01 (m, 1H), 0.91 (dd, J=9.7, 7.0 Hz, 6H); .sup.13C NMR (100 MHz, acetone-d.sub.6) ? 173.5, 171.3, 170.9, 170.8, 168.5, 153.2, 151.2, 149.0, 148.2, 141.4, 139.0, 136.9, 135.1, 134.2, 131.7, 129.9, 129.2, 128.9, 128.6, 127.4, 126.1, 124.6, 123.0, 122.5, 119.2, 115.0, 81.9, 70.5, 66.7, 58.9, 56.0, 55.4, 54.7, 54.4, 53.2, 49.6, 46.9, 45.4, 41.8, 38.3, 37.2, 35.4, 30.4, 29.7, 26.2, 25.1, 23.1, 23.0, 18.8, 17.7; MS (ES+): m/z=1280 (M+H).sup.+; LCMS (Method A): t.sub.R=6.28 min.
Example 169: Allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-nitrobenzyl)-oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-al 1,4]diazepine-5(12H)-carboxylate (167)
[1606] ##STR00317##
[1607] A solution of allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate
[1608] (87) (468 mg, 0.578 mmol) in N,N-dimethylformamide (2 mL) was charged with 4-nitrobenzyl bromide (151 mg, 0.700 mmol) and potassium carbonate (160 mg, 1.16 mmol) and the resulting mixture stirred at room temperature for 3 h. The mixture was then diluted into ethyl acetate (100 mL) and washed with cold brine (2?20 mL). After drying over magnesium sulfate, filtering and concentrating in vacuo, the residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 30% to 100%) to give the title compound (323 mg, 59%) as a yellow solid.
[1609] MS (ES+): m/z=945 (M+H).sup.+; LCMS (Method A): t.sub.R=9.75 min.
Example 170: Allyl (6aS)-3-((3-(2-((S)-5-((4-aminobenzyl)oxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2a-][1,4]diazepine-5(12aH)-carboxylate (168)
[1610] ##STR00318##
[1611] A solution of allyl (6aS)-3-((3-(2-((S)-1-(chloromethyl)-5-((4-nitrobenzyl)oxy)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (167) (61 mg, 0.065 mmol) in tetrahydrofuran (3 mL) and acetone (2 mL) was charged with water (1 mL), ammonium chloride (207 mg, 3.87 mmol) and zinc powder (127 mg, 1.94 mmol) and the resulting mixture stirred rapidly, at room temperature, under argon, for 1 h. The mixture was then filtered through a pad of celite and the filter cake washed with dichloromethane (100 mL) and water (100 mL). The resulting filtrate was separated, and the organic phase washed with brine (2?50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo, to give a white solid, which was used in the subsequent step without further purification.
[1612] MS (ES+): m/z=915 (M+H).sup.+; LCMS (Method A): t.sub.R=8.78 min.
Example 171: Allyl (6aS)-3-((3-(2-((S)-5-((4-((S)-2-((S)-2-(((allyloxy)-carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)-oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (169)
[1613] ##STR00319##
[1614] A solution of allyl (6aS)-3-((3-(2-((S)-5-((4-aminobenzyl)oxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (168) (59 mg, 0.065 mmol) in dichloromethane (1 mL) was charged with ((allyloxy)carbonyl)-L-valyl-L-alanine (155) (18 mg, 0.065 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (12 mg, 0.065 mmol) and the resulting mixture stirred at room temperature for 16 h. The reaction mixture was subsequently concentrated in vacuo and then loaded directly onto silica, and purified by flash column chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 100%) to give the title compound (38 mg, 51% over two steps) as a cream solid.
[1615] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.89 (d, J=140.6 Hz, 1H), 8.24 (d, J=8.1 Hz, 1H), 8.14 (s, 1H), 7.60 (apparent dd, J=16.5, 8.0 Hz, 3H), 7.51-7.44 (m, 1H), 7.44-7.38 (m, 3H), 7.37-7.28 (m, 5H), 7.15 (d, J=10.4 Hz, 2H), 6.90 (s, 1H), 6.14 (d, J=9.5 Hz, 1H), 5.98 (d, J=10.0 Hz, 1H), 5.94-5.78 (m, 1H), 5.71-5.55 (m, 1H), 5.33-4.90 (m, 8H), 4.76-4.61 (m, 1H), 4.59-4.47 (m, 4H), 4.33 (t, J=9.9 Hz, 2H), 4.19 (t, J=10.3 Hz, 1H), 4.05 (t, J=6.7 Hz, 1H), 3.98 (br, 1H), 3.91-3.86 (m, 3H), 3.84 (s, 3H), 3.83-3.79 (m, 1H), 3.64-3.42 (m, 2H), 3.36 (t, J=10.7 Hz, 1H), 3.04 (t, J=12.2 Hz, 1H), 2.17-2.06 (m, 1H), 1.99-1.89 (m, 2H), 1.82-1.56 (m, 7H), 1.53-1.34 (m, 6H), 1.00-0.89 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) ? 171.8, 171.1, 169.4, 169.1, 155.8, 149.6, 149.3, 141.7, 137.7, 137.0, 136.9, 134.5, 132.4, 129.7, 129.1, 128.4, 128.3, 127.7, 126.3, 123.8, 123.6, 123.3, 122.0, 120.4, 120.1, 120.0, 118.0, 117.0, 115.5, 115.1, 100.3, 98.0, 88.1, 84.2, 70.0, 66.0, 64.3, 60.4, 56.1, 53.4, 49.5, 46.1, 43.4, 42.3, 38.8, 31.0, 30.6, 25.2, 23.2, 23.0, 21.0, 19.2, 19.1; MS (ES+): m/z=1169 (M+H).sup.+; LCMS (Method A): t.sub.R=9.20 min.
Example 172: (S)-2-Amino-N((S)-1-((4-((((S)-1-(chloromethyl)-3-(2-(3-((((S)-2-methoxy-12-oxo-6a,7, 8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (170)
[1616] ##STR00320##
[1617] A solution of ally (6aS)-3-((3-(2-((S)-5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indol-3-yl)-2-oxoethyl)benzyl)oxy)-2-methoxy-2-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1, 2-a][1,4]diazepine-5(12H)-carboxylate
[1618] (169) (40 mg, 0.034 mmol) in dichloromethane (1 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (4 mg), and pyrrolidine (6.3 ?L, 0.075 mmol) and stirred at room temperature for 10 min. The resulting mixture was then diluted into dichloromethane (10 mL) and filtered through a pad of celite. The filter cake was then washed with dichloromethane (10 mL) and the filtrate concentrated in vacuo. Diethyl ether (10 mL) was charged to the residue and the resulting mixture concentrated in vacuo again. Strong vacuum was then applied to the residue for 20 min, before it was employed in the subsequent step without further purification.
[1619] MS (ES+): m/z=899 (M+H).sup.+; LCMS (Method A): t.sub.R=6.43 min.
Example 173: N((S)-1-(((S)-1-((4-((((S)-1-(Chloromethyl)-3-(2-(3-((((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-al 1,4]diazepin-3-yl)oxy)methyl)phenyl)acetyl)-2,3-dihydro-H-benzo[e]indol-5-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (171)
[1620] ##STR00321##
[1621] A solution of (S)-2-amino-N((S)-1-((4-((((S)-1-(chloromethyl)-3-(2-(3-((((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1, 2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (170) (31 mg, 0.034 mmol) in dichloromethane (1 mL) was charged with 6-maleimidohexanoic acid (7.1 mg, 0.034 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
[1622] (6.6 mg, 0.034 mmol) and stirred at room temperature for 1.5 h. More 6-maleimidohexanoic acid (7.1 mg, 0.034 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (6.6 mg, 0.034 mmol) were added, and the mixture stirred for a further 2.5 h. The resulting mixture was then concentrated in vacuo, and then loaded directly onto silica. Purification by flash column chromatography, eluting with ethyl acetate/petroleum spirit, 40-60? C. (from 0% to 100%) then with methanol/ethyl acetate (from 0% to 20%) gave the title compound (16 mg, 43% over two steps) as a yellow solid.
[1623] .sup.1H NMR (400 MHz, acetone-d.sub.6) ? 9.30 (d, J=11.6 Hz, 1H), 8.23 (d, J=4.5 Hz, 2H), 7.90 (d, J=3.6 Hz, 2H), 7.80 (t, J=8.6 Hz, 2H), 7.70 (d, J=6.9 Hz, 1H), 7.55-7.45 (m, 5H), 7.43-7.29 (m, 6H), 6.84 (d, J=3.8 Hz, 1H), 6.78 (s, 1H), 6.72 (s, 1H), 5.25-5.17 (m, 4H), 4.59-4.51 (m, 1H), 4.47-4.33 (m, 2H), 4.22 (t, J=6.6 Hz, 1H), 4.18-4.09 (m, 2H), 4.01-3.91 (m, 4H), 3.83 (s, 3H), 3.75-3.65 (m, 3H), 3.12 (dd, J=17.4, 7.8 Hz, 1H), 2.32-2.21 (m, 3H), 1.84-1.74 (m, 4H), 1.67-1.54 (m, 7H), 1.33-1.26 (m, 3H), 0.97 (dd, J=6.9, 3.4 Hz, 6H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) ? 171.7, 171.4, 171.3, 169.6, 166.7, 165.1, 164.2, 155.0, 150.4, 147.6, 142.4, 140.1, 136.9, 135.8, 134.8, 134.7, 130.1, 129.5, 128.8, 128.6, 124.0, 123.1, 122.6, 121.3, 119.7, 119.5, 116.0, 111.8, 110.4, 98.2, 70.4, 69.8, 56.0, 53.3, 49.7, 48.0, 47.9, 42.4, 41.2, 37.4, 37.3, 30.8, 28.2, 28.1, 26.2, 25.3, 24.1, 23.0, 18.1; MS (ES+): m/z=1092 (M+H).sup.+; LCMS (Method A): t.sub.R=7.90 min.
Example 174: Biological and Biophysical Characterisation
[1624] Cytotoxicity in Cell Lines
[1625] The cytotoxicity of compounds 13, 24, 42, 53, 55, 57, 59 and 61 were evaluated in the FaDu (head and neck cancer) and PC3 (prostate) cell lines using the standard MTIT assay for a 72 hour incubation period (Table 1). Some of the CBI-PDD dimers were extremely cytotoxic reaching IC.sub.50 values of 10 picomolar in some cell lines (e.g., Compound 42 in FaDu). As anticipated, the prodrug forms of the molecules were significantly less active (e.g., Compound 59=4.5 micromolar in FaDu).
TABLE-US-00013 TABLE 1 Cytotoxicity of CBI-PDD analogues in the FaDu and PC3 cell lines. Cytotoxicity Compound FaDu PC3 Number (nM, 72 hour) (nM, 72 hour) 13 1.17 1.43 24 1.83 0.12 42 0.01 0.03 53 0.65 0.02 55 1.5 0.02 57 >10 3 59 4500 900 61 >10 >10
[1626] The cytotoxicity of a further series of compounds was evaluated in the SW48, LIM1215 and SW620 cell-lines (gastric) using the standard MTT assay for a 72 hour incubation period (Table 2). Some of the compounds were extremely cytotoxic reaching IC.sub.50 values of 4 picomolar in some cell lines (e.g., compound 108 in SW48).
TABLE-US-00014 TABLE 2 Cytotoxicity of CBI-PDD analogues in the SW48, LIM1215 and SW620 cell lines. Cytotoxicity Compound SW48 LIM1215 SW620 Number (72 hrs) (72 hours) (72 hours) 10 2916 1271 2002 83 4392 2890 3510 91 2317 2213 2612 99 0.035 0.039 0.045 13 0.28 0.68 0.42 57 0.042 0.41 1.1 108 0.004 0.78 0.045 139 0.95 12 13.5 140 Not active 18.1 Not active 143 Not active Not active Not active 145 32.1 136.9 350
[1627] Biophysical Characterisation
[1628] The ability of 13 to cross-link DNA was determined using an assay involving a linear double-stranded TyrT fragment (
[1629] Following denaturation conditions (treatment with formamide and heating at 65? C. for 5 min) the DNA strands were completely separated (see controls C2 and C4,
[1630] Each compound was tested at 10 different concentrations, and the assay was repeated twice. The cross-linking ability of 13 is shown in
[1631] The ability of a further series of compounds 13, 42, 59, 99 and the PBD dimer Talirine to cross-link DNA was determined using an assay involving a linear double-stranded HexARev fragment (
[1632] The cleavage pattern of three molecules is evident in
[1633] Cleavage Assay
[1634] The ability of 13 and of a series of compounds to cleave double-stranded DNA was investigated using a modification of the previously established DNA footprinting assay (34). Following an overnight incubation of the ligand-DNA complexes, the mixture was mixed with strand separation buffer containing 10 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue, 80% formamide and incubated at 100? C. for 3 min. The mixture was then immediately cooled on ice and run on an 8% denaturing gel. Examination of the obtained gel (
[1635] Similarly, examination of the obtained gel (
[1636] FRET DNA Melting
[1637] FRET DNA melting studies were undertaken on 13 using two fluorescently labelled sequences. The sequences (
[1638] Further FRET DNA melting studies were undertaken on a number of molecules using three fluorescently labelled sequences. The sequences (
[1639] The short duplexes used in this FRET study are relatively unstable in the duplex form with a melting temperature below 30? C. so that, in the absence of ligand, a large part of the melting occurs below the starting temperature of the experiment. However, the inter- and intrastrand cross-links formed by 13 stabilizes the duplex form, producing very large increases in melting temperature with T.sub.m values of ?65? C. for 5-AIIAGAITIT-3 (
[1640] Compound 42 can also form intra-strand cross-links. This is evident in the bottom sequence in
[1641] The melting temperature of each duplex increases significantly in proportion to the concentration of 42 present, providing strong supporting evidence that the compound can produce interstrand (
[1642] A number of other compounds were assessed to understand the contribution of each alkylating unit to the observed melting temperatures for the bis-alkylating molecules. Compound 59 (
[1643] Compound 152 (
[1644] Finally, compounds 83 and 150 were also evaluated for ability to stabilise DNA. Compound 150 possesses a carbamate and inactive G-alkylating moiety, and was therefore expected to cause a very limited degree of DNA stabilisation (see
[1645] Summary of Cross-Linking Data
[1646] Taken together, the cross-linking data presented above provide strong evidence that 13, 42 and 99 produce both intrastrand and interstrand cross-links which appear to form with a high degree of sequence-specificity (e.g.,
[1647] Conjugation
[1648] Stochastic Conjugation
[1649] Conjugation of 165 and 171 to IgG1 Antibody (Forming ADC1)
[1650] Compounds 165 and 171 were conjugated to an IgG1 antibody targeted to Antigen X in a stochastic manner.
[1651] Antibody QC
[1652] The antibody was of good quality with 98.9% monomer content (
[1653] Conjugation of 165 and 171 to IgG1 Antibody
[1654] The conjugation process caused no significant aggregation compared to the starting antibody and contained 96.7% monomer in the case of 165. No free toxin linker could be detected in the ADC sample (see
[1655] Biophysical Characterisation Methodology
[1656] 1. Material
[1657] 1.1. DNA Fragment
[1658] The preparation of the TyrT DNA fragment (
[1659] The radiolabelled DNA fragment was separated from the remainder of the plasmid DNA on a 6% non-denaturing polyacrylamide gel. The gel (20 cm long, 0.3 mm thick) was run at 400 V in 1? TBE running buffer for about 1-2h, until the bromophenol blue had run most of the way down the gel. The glass plates were separated and the position of the labelled DNA fragment was established by short (1 min) exposure to an X-ray film. The relevant band was then cut from the gel and the radiolabelled DNA eluted by adding 300 ?L 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and gently agitating overnight at room temperature. The eluted DNA was finally precipitated with ethanol and re-suspended in a suitable volume of 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA buffer so as to give at least to counts per second/?L on a hand-held Geiger counter. With fresh plasmid and ?-.sup.32P-dATP this process typically generated about 150 ?L of radiolabelled fragment DNA. The absolute concentration of the DNA is not important, and it is typically lower than to nM.
[1660] 1.2. Compounds
[1661] Compounds such as 13 were synthesised as described above and the PBD dimer Talirine was obtained from Aurum Pharmatech LLC. Stock solution was prepared by dissolving the ligands in DMSO to give a concentration of 10 mM. From this stock solution, working solutions of the desired concentration were prepared by diluting with 10 mM Tris-HCl, pH 7.5 containing 10 mM NaCl.
[1662] 2. Cleavage Assay
[1663] 2.1. Preparation of Ligand-DNA Complexes
[1664] Radiolabelled DNA (1.5 ?L) was mixed with 1.5 ?L ligand solution of various concentrations (10 ?M-10 nM) and incubated overnight at 37? C.
[1665] 2.2. Preparation of GA Marker
[1666] Labelled DNA (1.5 ?L) was mixed with 20 ?L sterile water and 5 ?L of denaturing loading solution (80% formamide containing 10 mM EDTA, 10 mM NaOH, 0.01% bromophenol blue). The sample was then incubated at 100? C. for 20 min with the micro-centrifuge tube cap open to allow evaporation.
[1667] 2.3. Cleavage Assay
[1668] Loading solution (4.5 ?L) was added to samples from Section 2.1. The digestion products were boiled for 3 min at 100? C. and quickly cooled on ice prior to electrophoresis. Separation was performed on an 8% denaturing polyacrylamide gel (40 cm long, 0.3 mm thick) at 1500 V for about 2 h until the dye reached the bottom of the gel. The gel plates were then separated, the gels fixed by immersing in 10% (v/v) acetic acid, followed by transfer to Whatmann 3 MM paper and drying under vacuum at 80? C. The dried gel was then exposed to a phosphorimager screen overnight before being scanned using a Typhon FLA 7000 instrument.
[1669] 3. Cross-Linking Assay
[1670] 3.1. Preparation of Ligand-DNA Complexes
[1671] Radiolabelled DNA (1.5 ?L) was mixed with 1.5 ?L ligand solution of various concentrations (10 ?M-10 nM) and incubated overnight at 37? C.
[1672] 3.2 Cross-Linking Assay
[1673] After overnight incubation, the samples were mixed with 7 ?L loading solution (80% formamide containing 10 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue) and incubated at 65? C. for 5 min. Control 1 (C1) for native double-stranded DNA consisted of 1.5 ?L labelled DNA, 1.5 ?L 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 ?L 1? loading dye. Control 2 (C2) for denatured native single-stranded DNA was composed of 1.5 ?L labelled DNA, 1.5 ?L 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA which was incubated at 65? C. for 5 min. Control 3 (C3) for native double-stranded DNA consisted of 1.5 ?L labelled DNA, 1.5 ?L 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 ?L SSB. Control 4 (C4) for denatured native single-stranded DNA was composed of 1.5 ?L labelled DNA, 1.5 ?L 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 ?L SSB which was incubated at 65? C. for 5 min. Separation was performed on a 7.5% denaturing polyacrylamide gel (20 cm long, 0.3 mm thick) at 500V for about 4 h until the dye reached the bottom of the gel. The gel plates were then separated, the gels fixed by immersing in 10% (v/v) acetic acid, followed by transfer to Whatmann 3 MM paper and drying under vacuum at 80? C. The dried gel was then exposed to a phosphorimager screen overnight before scanning using a Typhon FLA 7000 instrument.
[1674] FRET Studies Methodology
[1675] 1. General
[1676] 1.1. Oligonucleotides
[1677] Oligonucleotides were obtained from ATDbio (Southampton, UK) in lyophilised form. They were labelled with a fluorophore molecule (F=fluorescein) at the 5-end and a quencher molecule (Q=dabcyl) at the 3-end of the complementary strand. Each oligonucleotide was dissolved in distilled H.sub.2O to form stock solutions of 100 pM. Working solutions of 5 ?M were prepared by diluting the stock solution with distilled H.sub.2O.
[1678] 1.2. Buffers
[1679] The following buffers were used: 250 mM phosphate buffer pH 7.4 (consisting of sodium dihydrogen phosphate and sodium phosphate diluted in distilled H.sub.2O) and 5 M sodium chloride buffer. All buffers and distilled H.sub.2O were filtered through a 0.2 ?M filter prior to use.
[1680] 1.3. Compound
[1681] For the FRET experiments a stock solutions were prepared by dissolving compound (such as compound 13) in DMSO to give a concentration of 10 mM. From this stock solution, working solutions of the desired concentration were prepared by diluting the stock solution with distilled H.sub.2O.
[1682] 1.4. Preparation of ligand-DNA complexes
[1683] The reaction mixture was comprised of 4 ?L of 250 mM phosphate buffer (final concentration of 50 mM), 4 ?L flourophor and 4 ?L quencher molecule of the appropriate oligonucleotide for a final concentration of 0.2 ?M, 4 ?L 5 M sodium chloride (final concentration of 1 M NaCl), and 4 ?L of distilled H.sub.2O. This mixture was heated in an Eppendorf tube at 90? C. for 1 min and slowly cooled down to room temperature. This process was carried out to anneal the single strands to double-stranded DNA. Following this, 4 ?L of the ligand was added in the desired concentration and the mixture incubated overnight either at room temperature or 4? C. A control sample of DNA only was prepared by mixing 4 ?L 250 mM phosphate buffer (final concentration of 50 mM) with 4 ?L fluorophore-labelled and 4 ?L quencher-labelled oligonucleotides (of the appropriate sequence) to give a final concentration of 0.2 PIM, 4 ?L 5 M sodium chloride (final concentration of 1 M NaCl) and 4 ?L distilled H.sub.2O. This mixture was analysed without prior annealing.
[1684] 1.5. Fluorescence melting
[1685] Fluorescence melting profiles were measured using a Roche LightCycler using a total reaction volume of 20 ?L. Initially, the samples were denatured by heating to 95? C. at a rate of 1? C. min.sup.?1. The samples were then maintained at 95? C. for 5 min before annealing by cooling to 25? C. at 1? C. min.sup.?1. The samples were then held at 25? C. for a further 5 min and finally melted by heating to 95? C. at 1? C. min.sup.?1. Annealing steps and melting steps were all recorded and changes in fluorescence were measured at 520 nm.
[1686] 1.6. Data Analysis
[1687] T.sub.m values were obtained from the first derivates of the melting profiles using the Roche LightCycler software.
[1688] MT Cytotoxicity Methodology
[1689] Tumor cell lines were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and 1 mM sodium pyruvate. 1800 cells per well were seeded in a volume of 180 ?l in a 96-well flat bottom polystyrene plate. The cells were allowed to adhere overnight at 37? C. in a CO.sub.2 incubator. Ligands were initially formulated in DMSO, and stocks stored at ?80? C. They were then further formulated at lox concentration in RPMI1640 medium. 20 ul of diluted samples were added into each treatment well. On each plate, blank wells with no cells, and untreated wells containing cells, were included. Plates were then cultured at 37? C. in a CO.sub.2 incubator for 72 hrs. Cytotoxicity was evaluated using a tetrazolium salt-based assay, the MTT assay. After 72 hours, the supernatant was removed from each well and 200 ?l of a sterile filtered 500 ?g/ml MTT solution in water added to each well. The plates were then incubated at 37? C. in a CO.sub.2 incubator for 4 hrs. The supernatant was then removed and the formazan crystals formed solubilized by adding 150 ?l of DMSO to each well. The plate was then read on a plate reader at 540 nm, and percentage cell survival calculated as follows: ((mean absorbance treated wells at concentration x?mean absorbance blank wells)?(mean absorbance untreated wells at concentration x?mean absorbance blank wells))?100. Data were plotted as concentration in nM vs. % cell survival in Microsoft Excel, and IC.sub.50 values (concentration where cell survival is reduced by a half) were determined from the graph.
[1690] Conjugation Methodology
[1691] All ADC conjugations were completed using a similar methodology, an example of which is provided below. 21.5 mg IgG1 antibody (8.0 mg/ml in PBS) were charged with EDTA to a final concentration of 2 mM. Reduction was attained by adding 1.27 molar equivalents TCEP (10 mM in water) and incubating for 2 hours at 20? C. After 1.5 hours, a reduction in-process test conjugation with Mal-vcMMAE was performed, and analyzed by HIC to test for the reduction level. As the target reduction level had not been reached, another 0.1 molar equivalents TCEP were added and the reduction time extended by 1 hour. After 0.5 hours, a second in-process test was run. After confirmation of the desired reduction level, 20% (v/v) Propylene glycol was added to the reduced antibody followed by 6.4 molar equivalents 165/171 (10 mM stock in DMSO). The solution was incubated for 1 hour at rt. The reaction was quenched by adding 6.4 molar equivalents N-Acetylcysteine (10 mM in water). The ADC was buffer exchanged via G25 into PBS and washed by dead-end filtration (Vivaspin-20, 30 kDa MWCO, 0.0006 m.sup.2) for 10 DVs. Samples were taken for analysis by HIC, SEC, PLRP, free toxin linker, Endosafe, and the concentration was determined using a SEC calibration curve. Aliquotting was carried out under laminar flow, and the product was stored at ?80? C. Only disposable, sterile and pyrogen/DNA/RNA-free plasticware was used.
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