PYRROLOBENZODIAZEPINE CONJUGATES
20210316006 · 2021-10-14
Inventors
Cpc classification
A61K47/6803
HUMAN NECESSITIES
C07K5/06026
CHEMISTRY; METALLURGY
A61K47/6855
HUMAN NECESSITIES
C07K5/06034
CHEMISTRY; METALLURGY
C07D519/00
CHEMISTRY; METALLURGY
International classification
A61K47/68
HUMAN NECESSITIES
Abstract
A compound of formula (I) wherein R.sup.L is a linker for connection to a cell binding agent.
##STR00001##
Claims
1. A compound of formula I: ##STR00114## and salts and solvates thereof, wherein: R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; where R and R′ are independently selected from C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups; R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, selected from O, S, and NR.sup.N2 where R.sup.N2 is H or C.sub.1-4 alkyl, and/or an aromatic ring selected from benzene or pyridine; Y and Y′ are selected from O, S, or NH; R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively; R.sup.11b is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; and R.sup.L is a linker for connection to a cell binding agent, which is selected from: (iiia): ##STR00115## wherein Q is: ##STR00116## where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; X is: ##STR00117## where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5; G.sup.L is a linker for connecting to an antibody or an active fragment thereof; and (iiib): ##STR00118## where R.sup.L1 and R.sup.L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1; either: (a) R.sup.20 is H and R.sup.21 is H; (b) R.sup.20 is H and R.sup.21 is ═O; or (c) R.sup.21 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.20 is selected from: ##STR00119## where R.sup.Z is selected from: ##STR00120## (z-ii) OC(═O)CH.sub.3; (z-iii) NO.sub.2; (z-iv) OMe; (z-v) glucoronide; (z-vi) NH—C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—C(═O)—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, CH.sub.2CH.sub.2OMe, and (CH.sub.2CH.sub.2O).sub.2Me.
2. A compound according to claim 1, wherein: a) both Y and Y′ are O; and/or b) R″ is C.sub.3-7 alkylene or a group of formula: ##STR00121## where r is 1 or 2; c) R.sup.9 is H, R.sup.6 is H and R.sup.7 is a C.sub.1-4 alkyloxy group.
3.-5. (canceled)
6. A compound according to claim 1, wherein R.sup.6′ is the same group as R.sup.6, R.sup.7′ is the same group as R.sup.7, R.sup.9′ is the same group as R.sup.9 and Y′ is the same group as Y.
7. The compound according to claim 1, wherein R.sup.21 is OH or OR.sup.A and R.sup.20 is ##STR00122## and —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.
8. The compound according to claim 7, wherein —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, and -Val-Ala-.
9. The compound according to either claim 7 or 8 wherein R.sup.ZC is (CH.sub.2CH.sub.2O).sub.2Me.
10. A compound according to claim 1, which is of formula Ia, Ib or Ic: ##STR00123## where R.sup.1a is selected from methyl and benzyl; R.sup.L and R.sup.11b are as defined in claim 1.
11. A compound according to claim 1, wherein R.sup.L is of formula IIIa, and Q is a dipeptide residue selected from: .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Ala-.sup.NH, .sup.CO-Val-Lys-.sup.NH, .sup.CO-Ala-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH, .sup.CO-Phe-Cit-.sup.NH, .sup.CO-Leu-Cit-.sup.NH, .sup.CO-Phe-Arg-.sup.NH, and .sup.CO-Trp-Cit-.sup.NH.
12. A compound according to of claim 1, wherein R.sup.L is of formula IIIa and, a is 0, c is 1 and d is 2, and b is from 0 to 8.
13. A compound according to claim 12, wherein b is 0, 4 or 8.
14. A compound according to any claim 1, wherein R.sup.L is of formula IIIa and G.sup.L is selected from: TABLE-US-00007
15. A compound according to claim 14, wherein G.sup.L is G.sup.L1-1.
16. A compound according to claim 1, wherein the compound is of formula Id: ##STR00137## where Q is selected from: (a) —CH.sub.2—; (b) —C.sub.3H.sub.6—; and ##STR00138##
17. A conjugate of formula II:
L-(D.sup.L).sub.p (I) wherein L is an antibody or an active fragment thereof, D.sup.L is a Drug Linker unit of formula I′: ##STR00139## wherein R.sup.6, R.sup.7, R.sup.9, R.sup.11b, Y, R″, Y′, R.sup.6′, R.sup.7, R.sup.9′, R.sup.20 and R.sup.21, are as defined in claim 1; R.sup.LL is a linker for connection to a cell binding agent, which is selected from: (iiia): ##STR00140## where Q and X are as defined in claim 1 and G.sup.LL is a linker connected to an antibody or an active fragment thereof; and ##STR00141## where R.sup.L1 and R.sup.L2 are as defined in claim 1; wherein p is an integer of from 1 to 20.
18. A conjugate according to claim 17, wherein C.sup.LL is selected from: TABLE-US-00008
19. A conjugate according to claim 17, wherein D.sup.L is of formula (Id′): ##STR00155## where Q is selected from: (a) —CH.sub.2—; (b) —C.sub.3H.sub.6—; and ##STR00156##
20. A compound of Formula IV: ##STR00157## wherein R.sup.6, R.sup.7, R.sup.9, Y, R″, Y′, R.sup.6′, R.sup.7 and R.sup.9′, are as defined in claim 1; either: (a) R.sup.30 is H and R.sup.31 is H; (b) R.sup.30 is H and R.sup.31 is ═O; or (c) R.sup.30 and R.sup.31 form a double bond between the N and C atoms to which they are attached.
21. A composition comprising a mixture of conjugates according to claim 17, wherein the average p in the mixture of conjugate compounds is about 1 to about 8.
22. (canceled)
23. A pharmaceutical composition comprising the conjugate of claim 17, and a pharmaceutically acceptable diluent, carrier or excipient.
24.-25. (canceled)
26. A method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate according to claim 17.
Description
BRIEF DESCRIPTION OF FIGURE
[0211]
GENERAL SYNTHETIC ROUTES
[0212] The synthesis of PBD compounds is extensively discussed in the following references, which discussions are incorporated herein by reference:
a) WO 00/12508 (pages 14 to 30);
b) WO 2005/023814 (pages 3 to 10);
c) WO 2004/043963 (pages 28 to 29); and
d) WO 2005/085251 (pages 30 to 39).
Synthesis Route
[0213] Compounds of the present invention of formula I where R.sup.21 is not H or ═O can be synthesised from a compound of Formula 2:
##STR00022##
where R.sup.6, R.sup.7, R.sup.9, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.11b, Y, Y′ and R″ are as defined for compounds of formula I, and R.sup.LL is a protected version of R.sup.L—this method is particularly applicable to compounds of formula I where R.sup.L is of formula IIIa. R.sup.20P is either R.sup.20 or a precursor thereof. For these compounds, R.sup.LL will typically be a portion of R.sup.L, such as a group of formula IIIa′:
##STR00023##
In such as case, the reaction involves adding the ground G.sub.L. The precursor of R.sup.20 may be of a similar structure, when R.sup.20 is:
##STR00024##
and R.sup.Z is NH—C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—R.sup.ZC.
[0214] The compounds of Formula 2 may be made by deprotecting the R.sup.LL group of compounds of Formula 3:
##STR00025##
where R.sup.6, R.sup.7, R.sup.9, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.11b, Y, Y′ and R″ are as defined for compounds of formula I, R.sup.LL-Prot is a protected version of R.sup.LL, and the Port.sup.N represents a simple nitrogen protecting group (e.g. Fmoc, Boc) that is orthogonal to the R.sup.LL protecting group. R.sup.20P may be the same as the R.sup.20P in Formula 2, or a protected version thereof, as appropriate.
[0215] Compounds of formula 3 may be made by ring-closure of compounds of Formula 4:
##STR00026##
where the ring closure is carried out by oxidation, e.g. Swern.
[0216] Compounds of formula 4 can be synthesised from compounds of formula 5:
##STR00027##
by a step-wise addition of two protecting groups. This can be achieved by simple protection of the amino group which will result in the imino bond in the final compound (e.g. by Fmoc, Boc), followed by installation of a desired protecting group at the other amino group.
[0217] Compounds of formula I where R.sup.L is of formula IIIb, may be synthesised in a similar manner, although the complete R.sup.L group may be installed starting from a compound of Formula 5, rather than with the use of a protected precursor.
[0218] Compounds of Formula 5 can be synthesised by known methods, such as those disclosed in WO 2011/130598.
[0219] Alternatively, compounds of Formula 4 can be synthesised by a monomeric route.
[0220] Compounds of the invention where R.sup.21 is H or ═O can be synthesised by a monomeric route, where the monomer containing these groups is full constructed before linking to the remainder of the compound. Reference is made to the routes shown in WO2014/096368.
[0221] Synthesis of Drug Conjugates
[0222] Conjugates can be prepared as previously described. Antibodies can be conjugated to the Drug Linker compound as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of remaining NAC-quenched drug-linker.
[0223] Further Preferences
[0224] The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
[0225] In some embodiments, R.sup.6′, R.sup.7′, R.sup.9′, and Y′ are selected from the same groups as R.sup.6, R.sup.7, R.sup.9, and Y respectively. In some of these embodiments, R.sup.6′, R.sup.7′, R.sup.9′, and Y′ are the same as R.sup.6, R.sup.7, R.sup.9, and Y respectively.
N10′-C11′
[0226] In some embodiments, R.sup.29 is H and R.sup.21 is H.
[0227] In some embodiments, R.sup.29 is H and R.sup.21 is ═O.
[0228] In some embodiments, R.sup.21 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.20 is selected from:
##STR00028##
—C(═O)—X.sub.1—NHC(═O)X.sub.2—NH— represent a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids. The dipeptide may be the site of action for cathepsin-mediated cleavage.
[0229] In one embodiment, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: [0230] -Phe-Lys-, [0231] -Val-Ala-, [0232] -Val-Lys-, [0233] -Ala-Lys-, [0234] -Val-Cit-, [0235] -Phe-Cit-, [0236] -Leu-Cit-, [0237] -Ile-Cit-, [0238] -Phe-Arg-, [0239] -Trp-Cit-
where Cit is citrulline.
[0240] Preferably, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: [0241] -Phe-Lys-, [0242] -Val-Ala-, [0243] -Val-Lys-, [0244] -Ala-Lys-, [0245] -Val-Cit-.
[0246] Most preferably, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is -Phe-Lys- or -Val-Ala-.
[0247] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
[0248] In one embodiment, the amino acid side chain is derivatised, where appropriate. For example, an amino group or carboxy group of an amino acid side chain may be derivatised.
[0249] In one embodiment, an amino group NH.sub.2 of a side chain amino acid, such as lysine, is a derivatised form selected from the group consisting of NHR and NRR′.
[0250] In one embodiment, a carboxy group COOH of a side chain amino acid, such as aspartic acid, is a derivatised form selected from the group consisting of COOR, CONH.sub.2, CONHR and CONRR′.
[0251] In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. The present inventors have established that protected amino acid sequences are cleavable by enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
[0252] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective Groups in Organic Synthesis, Greene and Wuts.
[0253] Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality: [0254] Arg: Z, Mtr, Tos; [0255] Asn: Trt, Xan; [0256] Asp: Bzl, t-Bu; [0257] Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; [0258] Glu: Bzl, t-Bu; [0259] Gln: Trt, Xan; [0260] His: Boc, Dnp, Tos, Trt; [0261] Lys: Boc, Z—Cl, Fmoc, Z, Alloc; [0262] Ser: Bzl, TBDMS, TBDPS; [0263] Thr: Bz; [0264] Trp: Boc; [0265] Tyr: Bzl, Z, Z—Br.
[0266] In one embodiment, the side chain protection is selected to be orthogonal to a group provided as, or as part of, a capping group, where present. Thus, the removal of the side chain protecting group does not remove the capping group, or any protecting group functionality that is part of the capping group.
[0267] In other embodiments of the invention, the amino acids selected are those having no reactive side chain functionality. For example, the amino acids may be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro, and Val.
[0268] It is particularly preferred in the present invention, that if L.sup.1 comprises a dipeptide, then —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH— is the same dipeptide. An example of a preferred group is:
##STR00029##
[0269] Other preferred R.sup.20 groups include:
##STR00030##
[0270] Dimer Link
[0271] In some embodiments, Y and Y′ are both O.
[0272] In some embodiments, R″ is a C.sub.3-7 alkylene group with no substituents. In some of these embodiments, R″ is a C.sub.3, C.sub.5 or C.sub.7 alkylene. In particular, R″ may be a C.sub.3 or C.sub.5 alkylene.
[0273] In other embodiments, R″ is a group of formula:
##STR00031##
where r is 1 or 2.
[0274] The phenylene group may be replaced by a pyridylene group.
[0275] R.sup.6 to R.sup.9
[0276] In some embodiments, R.sup.9 is H.
[0277] In some embodiments, R.sup.6 is selected from H, OH, OR, SH, NH.sub.2, nitro and halo, and may be selected from H or halo. In some of these embodiments R.sup.6 is H.
[0278] In some embodiments, R.sup.7 is selected from H, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, and halo. In some of these embodiments R.sup.7 is selected from H, OH and OR, where R is selected from optionally substituted C.sub.1-7 alkyl, C.sub.3-10 heterocyclyl and C.sub.5-10 aryl groups. R may be more preferably a C.sub.1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C.sub.5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH.sub.2Ph. Other substituents of particular interest are dimethylamino (i.e. —NMe.sub.2); —(OC.sub.2H.sub.4).sub.qOMe, where q is from 0 to 2; nitrogen-containing 06 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.
[0279] These embodiments and preferences apply to R.sup.9′, R.sup.6′ and R.sup.7′ respectively.
[0280] R.sup.11b
[0281] In some embodiments, R.sup.11b is OH.
[0282] In some embodiments, R.sup.11b is OR.sup.A, where R.sup.A is C.sub.1-4 alkyl. In some of these embodiments, R.sup.A is methyl.
[0283] In some embodiments of the first aspect of the present invention are of formula Ia, Ib or Ic:
##STR00032##
[0284] where R.sup.1a is selected from methyl and benzyl;
[0285] R.sup.20, R.sup.21, R.sup.L and R.sup.11b are as defined above.
[0286] These embodiments and preferences also apply to the second and fifth aspects of the invention.
[0287] Linker (R.sup.L)
[0288] In some embodiments, R.sup.L is of formula IIIa.
[0289] In some embodiments, R.sup.LL is of formula IIIa′.
[0290] G.sup.L
[0291] G.sup.L may be selected from
TABLE-US-00001
[0292] where Ar represents a C.sub.5-6 arylene group, e.g. phenylene.
[0293] In some embodiments, G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2. In some of these embodiments, G.sup.L is G.sup.L1-1.
[0294] G.sup.LL
[0295] G.sup.LL may be selected from:
TABLE-US-00002
where Ar represents a C.sub.5-6 arylene group, e.g. phenylene.
[0296] In some embodiments, G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2. In some of these embodiments, G.sup.LL is G.sup.LL1-1.
[0297] X
[0298] X is:
##STR00059##
where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5.
a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0.
b may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is 0 to 12. In some of these embodiments, b is 0 to 8, and may be 0, 2, 4 or 8.
c may be 0 or 1.
d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2.
[0299] In some embodiments of X, a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8.
[0300] Q
[0301] In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a non-natural amino acid.
[0302] In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.
[0303] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
[0304] In one embodiment, Q is selected from: [0305] .sup.CO-Phe-Lys-.sup.NH, [0306] .sup.CO-Val-Ala-.sup.NH, [0307] .sup.CO-Val-Lys-.sup.NH, [0308] .sup.CO-Ala-Lys-.sup.NH, [0309] .sup.CO-Val-Cit-.sup.m, [0310] .sup.CO-Phe-Cit-.sup.NH, [0311] .sup.CO-Leu-Cit-.sup.NH, [0312] .sup.CO-Ile-Cit-.sup.NH, [0313] .sup.CO-Phe-Arg-.sup.NH, and [0314] .sup.CO-Trp-Cit-.sup.NH;
where Cit is citrulline.
[0315] Preferably, Q is selected from: [0316] .sup.CO-Phe-Lys-.sup.NH, [0317] .sup.CO-Val-Ala-.sup.NH, [0318] .sup.CO-Val-Lys-.sup.NH, [0319] .sup.CO-Ala-Lys-.sup.NH, [0320] .sup.coVal-Cit-.sup.NH.
[0321] Most preferably, Q is selected from .sup.co-Phe-Lys-.sup.NH, .sup.co-Val-Cit-.sup.NH and .sup.co-Val-Ala-.sup.NH.
[0322] Other dipeptide combinations of interest include: [0323] .sup.CO-Gly-Gly-.sup.NH, [0324] .sup.CO-Pro-Pro-.sup.NH, and [0325] .sup.CO-Val-Glu-.sup.NH.
[0326] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
[0327] In some embodiments, Q.sup.X is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin.
[0328] In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
[0329] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.
[0330] In some embodiments, R.sup.L is of formula IIIb.
[0331] In some embodiments, R.sup.LL is of formula IIIb′.
[0332] R.sup.L1 and R.sup.L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group.
[0333] In some embodiments, both R.sup.L1 and R.sup.L2 are H.
[0334] In some embodiments, R.sup.L1 is H and R.sup.L2 is methyl.
[0335] In some embodiments, both R.sup.L1 and R.sup.L2 are methyl.
[0336] In some embodiments, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.
[0337] In some embodiments, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.
[0338] In the group IIIb, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group may be in any available position of the ring. In some of these embodiments, it is in the ortho position. In others of these embodiments, it is in the para position.
[0339] In one particular embodiment, the first aspect of the invention comprises a compound of formula Id:
##STR00060##
where Q is selected from:
(a) —CH.SUB.2.—;
[0340] (b) —C.sub.3H.sub.6—; and
##STR00061##
[0341] In one particular embodiment, the second aspect of the invention, the Drug linker (D.sup.L) is of formula (Id′):
##STR00062##
where Q is selected from:
(a) —CH.SUB.2.—;
[0342] (b) —C.sub.3H.sub.6—; and
##STR00063##
[0343] In some embodiments of the present invention, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
##STR00064##
[0344] In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
##STR00065##
[0345] In some embodiments of the present invention, the C2 OH substituent may be in the following stereochemical arrangement relative to neighbouring groups:
##STR00066##
EXAMPLES
General Information
[0346] Flash chromatography was performed using a Biotage Isolera 1™ using gradient elution starting from either 88% hexane/EtOAc or 99.9% DCM/MeOH until all UV active components (detection at 214 and 254 nm) eluted from the column. The gradient was manually held whenever substantial elution of UV active material was observed. Fractions were checked for purity using thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR U.K. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise stated. Pegylated reagents were obtained from Quanta biodesign US via Stratech UK.
[0347] The analytical LC/MS conditions (for reaction monitoring and purity determination) were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (H.sub.2O with 0.1% formic acid) and solvent B (CH.sub.3CN with 0.1% formic acid). Gradient for routine 3-minute run: Initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 second period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. Gradient for 15-minute run: Initial composition 5% B held over 1 minute, then increased from 5% B to 100% B over a 9 minute period. The composition was held for 2 minutes at 100% B, then returned to 5% B in 10 seconds and held there for 2 minutes 50 seconds. The total duration of the gradient run was 15.0 minutes. Flow rate was 0.8 mL/minute (for 3-minute run) and 0.6 mL/minute (for 15-minute run). Detection was at 254 nm. Columns: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm (routine 3-minute run); and ACE Excel 2 C18-AR, 2μ, 3.0×100 mm fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm (15-minute run).
[0348] The preparative HPLC conditions were as follows: Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was carried out on a Shimazdzu Prominence® machine using a Phenomenex® Gemini NX 5μ C18 column (at 50° C.) 150×21.2 mm. Eluents used were solvent A (H.sub.2O with 0.1% formic acid) and solvent B (CH.sub.3CN with 0.1% formic acid). All UFLC experiments were performed with gradient conditions: Initial composition 13% B increased to 60% B over a 15 minute period then increased to 100% B over 2 minutes. The composition was held for 1 minute at 100% B, then returned to 13% B in 0.1 minute and held there for 1.9 minutes. The total duration of the gradient run was 20.0 minutes. Flow was 20.0 mL/minute and detection was at 254 and 280 nm.
Example 1
[0349] ##STR00067##
a) (3S,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy) benzoyl)pyrrolidin-3-yl benzoate 2
[0350] Diethylazodicarboxylate (17.34 g, 0.085 mol, 5.0 eq) was added to a solution of triphenylphosphine (22.49 g, 0.085 mol, 5.0 eq) in THF (300 mL) and stirred at room temperature for 30 min. 1 (10 g, 0.017 mol, 1.0 eq) was added and stirring continued for a further 30 min, until a white ppt had formed. Benzoic acid (2.1 g, 0.017 mol, 1.0 eq) was added, the ppt turned from white to orange and then back to white. After 30 min, the ppt was removed by filtration. The filtrate was evaporated to dryness and purified by flash chromatography (10% ethyl acetate/heptane to remove the excess mitsunobu reagents followed by 20% ethyl acetate/heptane to elute the product as a white solid). Yield=9.5 g (81%). LC/MS rt 2.28 min m/z (687.4) M+H.
b) (3S,5S)-1-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-3-yl benzoate 3
[0351] Zinc dust (18.0 g, 0.27 mol, 20 eq) was added to a solution of 2 in methanol (75 mL) and stirred at room temperature. Formic acid (15 mL) was added which resulted in an exotherm of 35° C. After 10 mins, the zinc was removed by filtering through a short bed of celite, which was then washed with ethyl acetate (250 mL). The combined organic fractions were washed with saturated NaHCO.sub.3 (100 mL) then brine (50 mL). The resulting organic phase was dried (MgSO.sub.4) and evaporated under reduced pressure to leave a yellow residue which was purified by flash chromatography (gradient ethyl acetate/heptane, 15/85 to 20/80 v/v) to yield 3 as a colourless oil, 8.3 g (91%). LC/MS rt 2.24 min m/z (657.3) M+H.
c) (3S,5S)-1-(2-(((allyloxy)carbonyl)amino)-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-3-yl benzoate 4
[0352] Allyl chloroformate (1.65 g, 13.7 mmol, 1.1 eq) was added dropwise to a solution of 3 (8.2 g, 12.4 mmol, 1.0 eq) and pyridine (1.48 g, 18.7 mmol, 5.0 eq) in dichloromethane (75 mL) at 5° C. The reaction mixture was allowed to warm to room temperature and stirred for a further 60 mins. The organic phase was washed successively with 0.1M HCl (20 mL), saturated sodium hydrogen carbonate (20 mL) and brine (10 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure to leave a white solid which was used in the next step without further purification, 8.5 g (92%).
d) (3S,5S)-1-(2-(((allyloxy)carbonyl)amino)-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(hydroxymethyl)pyrrolidin-3-yl benzoate 5
[0353] 4 (8.5 g, 11.5 mmol) was dissolved in a mixture of acetic acid (35 mL), methanol (5 mL), THF (5 mL) and water (10 mL). The resulting solution was stirred at room temperature overnight. The reaction mixture was then poured into ethyl acetate (100 mL) and washed successively with water (2×100 mL), saturated sodium hydrogen carbonate (50 mL) and brine (50 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and the residue purified by flash chromatography (gradient ethyl acetate/heptane, 40/60 to 50/50 v/v) to yield 5 as a white solid, 5.24 g (73%). LC/MS rt 1.98 min m/z (627.5) M+H.
e) Allyl (2S,11S,11aS)-2-(benzoyloxy)-11-hydroxy-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 6
[0354] Oxalyl chloride (2M in DCM, 4.6 mL, 9.20 mmol, 1.1 eq) was added dropwise to a solution of DMSO (1.63 g, 20.9 mmol, 2.5 eq) in dry dichloromethane (75 mL) at −78° C. under an Argon atmosphere. After 15 mins, a solution of 5 (5.24 g, 8.36 mmol, 1.0 eq) in dichloromethane (20 mL) was added dropwise and the reaction mixture stirred for a further 30 mins. Triethyl amine (4.2 g, 41.8 mmol, 5.0 eq) was added and the resulting solution allowed to warm to room temperature and then stirred for a further 60 mins. The organic phase was then washed successively with 0.1M HCl (25 mL), saturated sodium hydrogen carbonate (25 mL) and brine (10 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure to leave a white solid which was used in the next step without further purification, 4.52 g (87%). LC/MS rt 1.90 min m/z (625.3) M+H.
f) Allyl (2S,11S,11aS)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 7
[0355] tert-Butyldimethylsilyl trifluoromethanesulfonate (5.7 g, 21.6 mmol, 3.0 eq) was added dropwise to a solution of 6 (4.5 g, 7.2 mmol, 1.0 eq) and 2,6-lutidine (3.1 g, 28.8 mmol, 4.0 eq) in dichloromethane (100 mL) at 5° C. under an atmosphere of Argon. The reaction mixture was allowed to warm to room temperature and stirred for a further 3 hrs. The organic layer was then washed successively with water (25 mL), saturated sodium hydrogen carbonate (25 mL) and brine (15 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and the residue purified by flash chromatography (ethyl acetate/heptane, 50/50 v/v) to yield 7 as a white solid, 4.0 g (76%). LC/MS rt 2.29 min m/z (739.3) M+H.
g) Allyl (2S,11S,11aS)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 8
[0356] Lithium acetate dihydrate (0.55 g, 5.4 mmol, 1.0 eq) was added to a solution of 7 (4.0 g, 5.4 mmol, 1.0 eq) in DMF/water (98/2, 5 mL) and stirred at room temperature for 5 hrs. The reaction mixture was diluted with ethyl acetate (100 mL) and the organic phase washed successively with 1M citric acid (50 mL) and brine (50 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and the residue purified by flash chromatography (gradient ethyl acetate/heptane, 75/25 to 100/0 v/v) to leave 8 as a white solid, 3.0 g (95%). LC/MS rt 1.80 min m/z (583.4) M+H.
h) General Method for the Dimerisation of 8 to 9
[0357] Potassium carbonate (2.5 eq) was added to a solution of 8 (1.0 g, 1.72 mmol, 2.1 eq) and either 1,3-dibromopropane; 1,5-diiodopentane; or 1,3-bis(bromomethyl)benzene (1.0 eq) in DMF (5 mL). The resulting mixture was stirred at 75° C. for 3 days. After diluting with dichloromethane (25 mL), the inorganics were removed by filtration and the filtrate evaporated to dryness under reduced pressure. The residue was purified by flash chromatography to leave the products as white solids.
i) diallyl 8,8′-(propane-1,3-diylbis(oxy))(2S,2′S,11S,11aS,11′S,11a′S)-bis(2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) 9a
[0358] (gradient: ethyl acetate/heptane, 50/50 to 100/0 v/v). Yield 0.88 g (90%). LC/MS rt 2.17 min m/z (1227.4) M+Na.
ii) diallyl 8,8′-(pentane-1,5-diylbis(oxy))(2S,2′S,11S,11aS,11′S,11a′S)-bis(2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) 9b
[0359] (ethyl acetate). Yield 0.82 g (82%). LC/MS rt 2.20 min m/z (1255.3) M+Na. diallyl 8,8′-((1,3-phenylenebis(methylene))bis(oxy))(2S,2′S,11S,11aS,11′S,11a′S)-bis(2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H—
iii) benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) 9c
[0360] (gradient: ethyl acetate/heptane, 75/25 to 100/0 v/v). Yield 0.9 g (85%). LC/MS rt 2.20 min m/z (1267.8) M+H.
i) General Method for Ester Hydrolysis of 9 to 10
[0361] 1M lithium hydroxide (1 mL) was added to a solution of 9 in methanol (10 mL) and stirred at room temperature for 2 hrs. The methanol was then removed under reduced pressure and the remaining aqueous layer acidified (pH 6) with 1M citric acid. The product was extracted into ethyl acetate (30 mL), dried (MgSO.sub.4) and evaporated under reduced pressure to leave a white solid which was purified by flash chromatography.
i) diallyl 8,8′-(propane-1,3-diylbis(oxy))(2S,2′S,11S,11aS,11′S,11a′S)-bis(11-((tert-butyldimethylsilyl)oxy)-2-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) 10a
[0362] (gradient: methanol/dichloromethane, 2/98 to 4/96 v/v). Yield 0.64 g (89%). LC/MS rt 1.86 min m/z (997.4) M+H.
ii) diallyl 8,8′-(pentane-1,5-diylbis(oxy))(2S,2′S,11S,11aS,11′S,11a′S)-bis(11-((tert-butyldimethylsilyl)oxy)-2-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) 10b
[0363] (methanol/dichloromethane, 5/95 v/v). Yield 0.68 g (93%). LC/MS rt 1.91 min m/z (1025.7) M+H.
iii) diallyl 8,8′-((1,3-phenylenebis(methylene))bis(oxy))(2S,2′S,11S,11aS,11′S,11a′S)-bis(11-((tert-butyldimethylsilyl)oxy)-2-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) 10c
[0364] (gradient: methanol/dichloromethane, 2/98 to 4/96 v/v). Yield 0.54 g (74%). LC/MS rt 1.92 min m/z (1060.5) M+H.
j) General Method Alloc/TBS Deprotection of 10 to 11
[0365] Tetrakis triphenylphosphine palladium(0) (2 mol %) was added to a solution of 10 (1.0 eq) and pyrrolidine (2.5 eq) in dichloromethane and stirred at room temperature for 30 min. The reaction mixture was diluted with dichloromethane and washed with saturated ammonium chloride. The organic phase was dried (MgSO.sub.4) and the solvent removed under reduced pressure. The residue was purified by reverse phase HPLC to leave the product as a white solid.
i) (2S,2′S,11aS,11a′S)-8,8′-(propane-1,3-diylbis(oxy))bis(2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one) 11a
[0366] LC/MS rt 3.84 min m/z (565.3) M+H.
ii) (2S,2′S,11aS,11a′S)-8,8′-(pentane-1,5-diylbis(oxy))bis(2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one) 11b
[0367] LC/MS rt 1.12 min m/z (593.3) M+H.
iii) (2S,2′S,11aS,11a′S)-8,8′-((1,3-phenylenebis(methylene))bis(oxy))bis(2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one) 11c
[0368] LC/MS rt 4.51 min m/z (626.7) M+H.
Example 2
[0369] ##STR00068##
a) (3S,5S)-1-(2-((((4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido) propanamido)benzyl)oxy)carbonyl)amino)-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-3-yl benzoate 12
[0370] Triethylamine (1.35 g, 13.4 mmol, 2.2 eq) was added to a solution of 3 (4.0 g, 6.0 mmol, 1.0 eq) and triphosgene (0.65 g, 2.2 mmol, 0.36 eq) in THF (40 mL) and stirred a room temperature, under an atmosphere of N.sub.2, for 5 min. A suspension of Alloc-Val-Ala-p-aminobenzyl alcohol (2.75 g, 7.3 mmol, 1.2 eq) and triethylamine (0.92 g, 9.1 mmol, 1.5 eq) in THF (25 mL) was added and the resulting mixture heated at 40° C. for 2 hr. The reaction mixture was filtered, the filtrate evaporated to dryness and purified by flash chromatography (methanol/dichloromethane, 2/98 v/v) to yield 12 as a pale yellow solid, 5.0 g (78%). LC/MS rt 2.24 min m/z (1082.4) M+Na.
b) (3S,5S)-1-(2-((((4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido) propanamido)benzyl)oxy)carbonyl)amino)-5-methoxy-4-((triisopropylsilyl)oxy)benzoyl)-5-(hydroxymethyl)pyrrolidin-3-yl benzoate 13
[0371] 12 (8.0 g, 7.5 mmol) was dissolved in a mixture of acetic acid (35 mL), methanol (5 mL), THF (5 mL) and water (10 mL). The resulting solution was stirred at room temperature overnight. The reaction mixture was then poured into ethyl acetate (100 mL) and washed successively with water (2×100 mL), saturated sodium hydrogen carbonate (50 mL) and brine (50 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and the residue purified by flash chromatography (methanol/dichloromethane, 3/97 v/v) to yield 13 as a white solid, 5.6 g (79%). LC/MS rt 1.95 min m/z (946.3) M+H.
c) 4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2S,11S)-2-(benzoyloxy)-11-hydroxy-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 14
[0372] Oxalyl chloride (2M in DCM, 0.83 mL, 1.6 mmol, 1.1 eq) was added dropwise to a solution of DMSO (0.27 mL, 3.7 mmol, 2.5 eq) in dry dichloromethane (20 mL) at −78° C. under an Argon atmosphere. After 15 mins, a solution of 13 (1.43 g, 1.5 mmol, 1.0 eq) in dichloromethane (5 mL) was added dropwise and the reaction mixture stirred for a further 30 mins. Triethyl amine (1.05 mL, 7.5 mmol, 5.0 eq) was added and the resulting solution allowed to warm to room temperature and then stirred for a further 60 mins. The organic phase was then washed successively with 0.1M HCl (15 mL), saturated sodium hydrogen carbonate (15 mL) and brine (10 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and purified by flash chromatography (methanol/dichloromethane 2/98 v/v) to yield 14 as a white solid, 1.1 g (77%). LC/MS rt 1.86 min m/z (944.3) M+H.
d) 4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2S,11S)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 15
[0373] tert-Butyldimethylsilyl trifluoromethanesulfonate (0.92 g, 3.5 mmol, 3.0 eq) was added dropwise to a solution of 14 (1.1 g, 1.16 mmol, 1.0 eq) and 2,6-lutidine (0.5 g, 4.7 mmol, 4.0 eq) in dichloromethane (15 mL) at 5° C. under an atmosphere of Argon. The reaction mixture was allowed to warm to room temperature and stirred for a further 3 hrs. The organic layer was then washed successively with water (25 mL), saturated sodium hydrogen carbonate (25 mL) and brine (15 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and the residue used in the next step without further purification, 1.2 g (97%). LC/MS rt 2.20 min m/z (1058.4) M+H.
e) 4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2S,11S)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 16
[0374] Lithium acetate dihydrate (0.11 g, 1.04 mmol, 1.0 eq) was added to a solution of 15 (1.1 g, 1.04 mmol, 1.0 eq) in DMF/water (98/2, 3 mL) and stirred at room temperature for 5 hrs. The reaction mixture was diluted with ethyl acetate (25 mL) and the organic phase washed successively with 1M citric acid (20 mL) and brine (20 mL). After drying (MgSO.sub.4), the solvent was removed under reduced pressure and the residue purified by flash chromatography (gradient methanol/dichloromethane, 1/99 to 3/97 v/v) to leave 16 as a white solid, 0.82 g (85%). LC/MS rt 1.77 min m/z (902.3) M+H.
f) 4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2S,11S,11aS)-8-(3-(((2S,11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 17
[0375] Potassium carbonate (0.18 g, 1.3 mmol, 2.5 eq) was added to a solution of 8 (1.0 g, 1.1 mmol, 2.1 eq) and 1,3-dibromopropane (0.1 g, 0.05 mmol, 1.0 eq) in DMF (5 mL). The resulting mixture was stirred at 75° C. for 3 days. After diluting with dichloromethane (25 mL), the inorganics were removed by filtration and the filtrate evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (gradient methanol/dichloromethane, 2/98 to 4/96 v/v) to leave 17 as a white solid, 0.77 g (79%). LC/MS rt 2.04 min m/z (1844.3) M+H.
g) 4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl (2S,11S,11aS)-8-(3-(((2S,11S,11aS)-10-(((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido) benzyl)oxy)carbonyl)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-(benzoyloxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 18
[0376] Pd(Ph.sub.3P).sub.4 (21 mg, 5 mol %) was added to a solution of 17 (0.65 g, 0.35 mmol, 1.0 eq) and pyrrolidine (0.15 g, 2.1 mmol, 6.0 eq) in dichloromethane (10 mL) and stirred at room temperature for 60 mins. The reaction mixture was evaporated to dryness and purified by flash chromatography (gradient methanol/dichloromethane, 5/95 to 20/80 v/v) to leave 18 as a white solid, 0.55 g (93%). LC/MS rt 1.39 min m/z (1676.5) M+H.
h) 4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl (2S,11S,11aS)-8-(3-(((2S,11S,11aS)-10-(((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy) carbonyl)-11-((tert-butyldimethylsilyl)oxy)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-((tert-butyldimethyl silyl)oxy)-2-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a] [1,4]diazepine-10(5H)-carboxylate 19
[0377] 1M lithium hydroxide (0.5 mL) was added to a solution of 18 (250 mg, 0.15 mmol) in methanol (3 mL) and stirred at room temperature for 4 hrs. The methanol was removed under reduced pressure and the aqueous phase acidified (pH 4) with 1M citric acid. The resulting solution was purified by reverse phase isolera (acetonitrile/water, 35/65 v/v+0.1% formic acid) to leave 19 as a white solid, 156 mg (71%). LC/MS rt 1.24 min m/z (1468.2) M+H.
i) 4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl (2S,11S,11aS)-8-(3-(((2S,11S,11aS)-10-(((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy) carbonyl)-2,11-dihydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2,11-dihydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 20
[0378] Triethylamine trihydrofluoride (96 mg, 0.59 mmol, 5.0 eq) was added to a solution of 19 (175 mg, 0.12 mmol, 1.0 eq) in THF (10 mL) and stirred at room temperature for 5 days. The solvent was removed under vacuum and the residue purified by prep HPLC to leave 20 as a white solid, 91 mg (62%). LC/MS rt 0.95 min m/z (1239.9) M+H.
##STR00069##
j) 4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl (2S,11S,11aS)-8-(3-(((2S,11S,11aS)-2,11-dihydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-8-yl)oxy)propoxy)-2,11-dihydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 22
[0379] Sodium hydrogen carbonate (6 mg, 0.07 mmol, 1.05 eq) was dissolved in water (0.5 mL) and added to a solution of 20 (91 mg, 0.07 mmol, 1.0 eq) in THF (0.5 mL). PEG.sub.2-COOH NHS ester (18 mg, 0.07 mmol, 1.0 eq) was added and the resulting mixture stirred at room temperature for 20 mins. The reaction mixture was then purified by prep HPLC to give 2 fractions; 21, white solid, 18 mg (16%) and 22, white solid, 41 mg (41%). 21 LC/MS rt 1.28 min m/z (1499.8) M+H. 22 LC/MS rt 1.07 min m/z (1367.1) M−H.
k) 4-((2R,5R)-38-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,34,36-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,35-triazaoctatriacontanamido)benzyl (2S,11S,11aS)-8-(3-(((2S,11S,11aS)-2,11-dihydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2, 5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2, 3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepin-8-yl)oxy)propoxy)-2,11-dihydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 23
[0380] Sodium hydrogen carbonate (3 mg, 0.036 mmol, 1.2 eq) was dissolved in water (0.5 mL) and added to a solution of 22 (41 mg, 0.03 mmol, 1.0 eq) in THF (0.5 mL). Mal-PEG.sub.8-COOH NHS ester (23 mg, 0.033 mmol, 1.1 eq) was added and the resulting mixture stirred at room temperature for 30 mins. The reaction mixture was then purified by prep HPLC to leave 23 as a white solid, 16 mg (28%). LC/MS rt 1.31 min m/z (1944.45) M+H.
Example 3—Conjugation
[0381] Conj-HER-23
[0382] Site-specific tratuzumab (30 mg) was loaded onto solid support and reduced, reoxidised, conjugated to compound 23, purified, released from the resin and formulated onto 25 mM Histidine, 200 mM Sucrose, Tween-20 0.02%, pH 6.0 according to patent US2014/038041A1.
[0383] UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm×2.1 mm mm column eluting with a gradient of water and acetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm (compound 23 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of compound 23, consistent with a drug-per-antibody ratio (DAR) of 1.9 molecules of compound 23 per antibody.
[0384] UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomer purity of greater than 97%. UHPLC SEC analysis gives a concentration of final ADC at 1.7 mg/mL in 10.5 mL, obtained mass of ADC is 17.9 mg (60% yield).
[0385] Conj-HER-23*
[0386] A 10 mM solution of (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (2.1 molar equivalent/antibody, 210 nanomoles, 21 μL) to a 7.5 mL solution of tratuzumab (15 mg, 100 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL. The reduction mixture was allowed to react at +37° C. for 2 hours in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody solution was allowed to cool to room temperature and compound 23 was added as a DMSO solution (10 molar equivalent/antibody, 1.0 micromoles, in 0.75 mL DMSO) to 7.5 mL of this reduced antibody solution (15 mg, 100 nanomoles) for a 10% (v/v) final DMSO concentration and a final antibody concentration of ˜2 mg/mL. The solution was mixed for 1 hour at room temperature. UHPLC analysis showed drug-per-antibody ratio (DAR) was too low therefore conjugation mixture was purified via spin filter centrifugation into PBS+1 mM EDTA using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, and more TCEP (0.8 molar equivalent/antibody, 80 nanomoles, 8 μL) was added to 5 mL of this buffer exchanged conjugation mixture (15 mg antibody, 100 nanomoles) at ˜3 mg/mL antibody concentration. The new reduction mixture was allowed to react at +37° C. for 1.75 hours in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody solution was allowed to cool to room temperature and compound 23 was added as a DMSO solution (3 molar equivalent/antibody, 0.3 micromoles, in 0.5 mL DMSO) to 5 mL of this reduced antibody solution (15 mg, 100 nanomoles) for a 10% (v/v) final DMSO concentration and a final antibody concentration of ˜3 mg/mL. The solution was mixed for 16 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (1.5 micromoles, 15 μL at 100 mM), then purified via spin filter centrifugation into 25 mM Histidine 205 mM Sucrose pH 6.0 buffer using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, sterile-filtered and analysed. Conj-Her-23* was then buffer exchanged into PBS via spin filter centrifugation, sterile filtered and analysed.
[0387] UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of Conj-Her-23* at 214 nm shows a mixture of unconjugated light chains, light chains attached to a single molecule of compound 23, unconjugated heavy chains and heavy chains attached to up to three molecules of compound 23 consistent with a drug-per-antibody ratio (DAR) of 3.87 molecules of compound 23 per antibody.
[0388] UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of Conj-Her-23* at 280 nm shows a monomer purity of 99%. Reduced SDS-PAGE analysis gives a concentration of final Conj-Her-23* at 1.16 mg/mL in 4.4 mL, obtained mass of Conj-Her-23* is 5.1 mg (34% yield).
[0389] Conj-Her-23**
[0390] A 10 mM solution of (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (10 molar equivalent/antibody, 1 micromole, 100 μL) to a 7.5 mL solution of tratuzumab (15 mg, 100 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.0 mg/mL. The reduction mixture was allowed to react at +37° C. for 3 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody solution was allowed to cool to room temperature and diluted with 6 mL more PBS and 1 mM EDTA. Compound 23 was added as a DMSO solution (15 molar equivalent/antibody, 1.5 micromoles, in 1.5 mL DMSO) to 13.5 mL of this reduced antibody solution (15 mg, 100 nanomoles) for a 10% (v/v) final DMSO concentration and a final antibody concentration of 1.0 mg/mL. The solution was mixed for 16 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (7.5 micromoles, 75 μL at 100 mM), then purified via spin filter centrifugation into 25 mM Histidine 205 mM Sucrose pH 6.0 buffer using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, sterile-filtered and analysed. Conj-Her-23** was then buffer exchanged into PBS via spin filter centrifugation, sterile filtered and analysed.
[0391] UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of Conj-Her-23** at 214 nm shows a mixture of unconjugated light chains, light chains attached to a single molecule of compound 23, unconjugated heavy chains and heavy chains attached to up to three molecules of compound 23, consistent with a drug-per-antibody ratio (DAR) of 7.60 molecules of compound 23 per antibody.
[0392] UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of Conj-Her-23** at 280 nm shows a monomer purity of 95%. Reduced SDS-PAGE analysis gives a concentration of final Conj-Her-23** at 0.2 mg/mL in 6 mL, obtained mass of Conj-Her-23** is 1.2 mg (8% yield).
Example 4—In Vivo Assay
[0393] Conjugate Tested: Conj-Her-23
[0394] Female CB.17 SCID mice, aged ten weeks, were injected with 0.1 ml of 1×10.sup.7 NCl—N87 cells in 50% Matrigel subcutaneously in the right flank. When tumours reached an average size of 100-150 mm.sup.3, treatment began. Mice were weighed twice a week. Tumour size was measured twice a week. Animals were monitored individually. The endpoint of the experiment was a tumour volume of 800 mm.sup.3 or 83 days, whichever came first.
[0395] Groups of 10 xenografted mice were injected i.v. with 0.2 ml per 20 g of body weight of antibody drug conjugate (ADC) in phosphate buffered saline (vehicle) or with 0.2 ml per 20 g of body weight of vehicle alone. The concentration of ADC was adjusted to give 0.6 or 6 mg ADC/kg body weight in a single dose.
[0396] The change in normalised tumour volume over time is shown in
[0397] Endpoint and Tumor Growth Delay (TGD) Analysis
[0398] Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 800 mm.sup.3 or at the end of the study (Day 82), whichever came first. Animals that exited the study for tumor volume endpoint were documented as euthanized for tumor progression (TP), with the date of euthanasia. The time to endpoint (TTE) for analysis was calculated for each mouse by the following equation:
where TTE is expressed in days, endpoint volume is expressed in mm.sup.3, b is the intercept, and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. The data set consisted of the first observation that exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded the attainment of this endpoint volume. The calculated TTE is usually less than the TP date, the day on which the animal was euthanized for tumor size. Animals with tumors that did not reach the endpoint volume were assigned a TTE value equal to the last day of the study (Day 82). In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate the TTE. Any animal classified as having died from NTR (non-treatment-related) causes due to accident (NTRa) or due to unknown etiology (NTRu) were excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment-related) deaths or NTRm (non-treatment-related death due to metastasis) were assigned a TTE value equal to the day of death. Treatment outcome was evaluated from tumor growth delay (TGD), which is defined as the increase in the median time to endpoint (TTE) in a treatment group compared to the control group:
TGD=T−C,
expressed in days, or as a percentage of the median TTE of the control group:
where:
T=median TTE for a treatment group, and
C=median TTE for the designated control group.
[0399] Tumour Growth Inhibition
[0400] Tumor growth inhibition (TGI) analysis evaluates the difference in median tumor volumes (MTVs) of treated and control mice. For this study, the endpoint for determining TGI was Day 33, which was the last day that all evaluable control mice remained in the study. The MTV (n), the median tumor volume for the number of animals, n, on the day of TGI analysis, was determined for each group. Percent tumor growth inhibition (% TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:
[0401] The data set for TGI analysis included all animals in a group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.
[0402] MTV and Criteria for Regression Responses
[0403] Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day. The MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume. Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm.sup.3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm.sup.3 for three consecutive measurements during the course of the study. Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.
[0404] Toxicity
[0405] Animals were weighed daily on Days 1-5, then twice per week until the completion of the study. The mice were observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded when observed. Individual body weight was monitored as per protocol, and any animal with weight loss exceeding 30% for one measurement or exceeding 25% for three consecutive measurements was euthanized as a TR death. Group mean body weight loss was also monitored according to CR Discovery Services protocol. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy. A TR classification was also assigned to deaths by unknown causes during the dosing period or within 14 days of the last dose. A death was classified as non-treatment-related (NTR) if there was no evidence that death was related to treatment side effects. NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRm is assigned to deaths thought to result from tumor dissemination by invasion and/or metastasis based on necropsy results; NTRu describes deaths of unknown causes that lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu.
[0406] Statistical and Graphical Analyses
[0407] GraphPad Prism 8.0 for Windows was used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis. The logrank test was employed to assess the significance of the difference between the overall survival experiences of two groups. The logrank test analyzes the individual TTEs for all animals in a group, except those lost to the study due to NTR death. Statistical analyses of the differences between Day 33 median tumor volumes (MTVs) of control and treated groups were accomplished using the Mann-Whitney U-test. For statistical analyses, two-tailed tests were conducted at significance level P=0.05. Prism summarizes test results as not significant (ns) at P>0.05, significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01, and extremely significant (“***”) at P≤0.001. Because tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all levels of significance were described as either significant or not significant within the text of this report. A scatter plot was constructed to show TTE values for individual mice, by group. Tumor growth curves show group median, mean and individual tumor volumes as a function of time, with error bars (when present) indicating one standard error of the mean (SEM). When an animal exited the study due to tumor size, the final tumor volume recorded for the animal was included with the data used to calculate the mean volume at subsequent time points. Tumor growth curves were truncated when tumors in more than 50% of the assessable animals in the group grew to the endpoint volume and excluded the data for animals whose deaths were assessed as NTR. Kaplan-Meier plots show the percentage of animals in each group remaining in the study versus time. The Kaplan-Meier plot and logrank test share the same TTE data sets. Box and whisker plots were constructed to show the Day 33 tumor volume data by group, with the “box” representing the 25th and 75.sup.th percentile of observations, the “line” representing the median of observations, and the “whiskers” representing the extreme observations. Group body weight changes during the study were plotted as percent mean change from Day 1. Body weight plots were truncated after 50% of the assessable animals in a group had exited the study and excluded the data for animals whose deaths were assessed as NTR.
TABLE-US-00003 Median % MTV (n), n TTE T—C TGD Day 82 Vehicle — 10 45.7 — — — Conj-Her-23 0.6 mg/kg 10 74.0 28.2 62 507 (2) Conj-Her-23 6 mg/kg 10 82.0 36.2 79 63 (9)
TABLE-US-00004 BW PR CR TFS Nadir TR NTRm NTR Vehicle 0 0 0 −0.5% (3) 0 0 0 0.6 mg/kg 0 0 0 −0.8* (71) 0 0 0 6 mg/kg 5 1 1 — 0 0 0
[0408] All documents and other references mentioned above are herein incorporated by reference.
EMBODIMENTS OF INVENTION
[0409] 1. A compound of formula I:
##STR00070##
and salts and solvates thereof, wherein:
R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo;
where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups;
R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo;
R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from O, S, or NH;
R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively;
R.sup.11b is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; and
R.sup.L is a linker for connection to a cell binding agent, which is selected from: (iiia):
##STR00071##
wherein
Q is:
[0410] ##STR00072##
where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue;
X is:
[0411] ##STR00073##
where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5;
G.sup.L is a linker for connecting to a Ligand Unit; and
(iiib):
##STR00074##
where R.sup.L1 and R.sup.L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group;
and e is 0 or 1;
either: [0412] (a) R.sup.20 is H and R.sup.21 is H; [0413] (b) R.sup.20 is H and R.sup.21 is ═O; or [0414] (c) R.sup.21 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.20 is selected from:
##STR00075##
where R.sup.Z is selected from:
##STR00076## [0415] (z-ii) OC(═O)CH.sub.3; [0416] (z-iii) NO.sub.2; [0417] (z-iv) OMe; [0418] (z-v) glucoronide; [0419] (z-vi) NH—C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—C(═O)—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, CH.sub.2CH.sub.2OMe, and (CH.sub.2CH.sub.2O).sub.2Me.
2. A compound according to statement 1, wherein both Y and Y′ are O.
3. A compound according to either statement 1 or statement 2, wherein R″ is C.sub.3-7 alkylene.
4. A compound according to either statement 1 or statement 2, wherein R″ is a group of formula:
##STR00077##
where r is 1 or 2.
5. A compound according to any one of statements 1 to 4, wherein R.sup.9 is H.
6. A compound according to any one of statements 1 to 5, wherein R.sup.6 is H.
7. A compound according to any one of statements 1 to 6, wherein R.sup.7 is selected from H, OH and OR.
8. A compound according to statement 7, wherein R.sup.7 is a C.sub.1-4 alkyloxy group.
9. A compound according to any one of statements 1 to 8, wherein R.sup.6′ is the same group as R.sup.6, R.sup.7′ is the same group as R.sup.7, R.sup.9′ is the same group as R.sup.9 and Y′ is the same group as Y.
10. The compound according to any one of statements 1 to 9, wherein R.sup.21 is OH or OR.sup.A and R.sup.29 is selected from:
##STR00078##
11. The compound according to any one of statements 1 to 10, wherein —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.
12. The compound according to statement 11, wherein —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, and -Val-Ala-.
13. The compound according to any one of statements 10 to 12 wherein R.sup.ZC is selected from CH.sub.2CH.sub.2OMe, and (CH.sub.2CH.sub.2O).sub.2Me.
14. The compound according to statement 13 wherein R.sup.ZC is (CH.sub.2CH.sub.2O).sub.2Me.
15. A compound according to statement 1, which is of formula Ia, Ib or Ic:
##STR00079##
where R.sup.1a is selected from methyl and benzyl;
R.sup.L and R.sup.11b are as defined in statement 1.
16. A compound according to any one of statements 1 to 15, wherein R.sup.11b is OH.
17. A compound according to any one of statements 1 to 15, wherein R.sup.11b is OR.sup.A, where R.sup.A is C.sub.1-4 alkyl.
18. A compound according to statement 17, wherein R.sup.A is methyl.
19. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIa, and Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.
20. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIa, and Q is a dipeptide residue selected from: [0420] .sup.CO-Phe-Lys-.sup.NH, [0421] .sup.CO-Val-Ala-.sup.NH, [0422] .sup.CO-Val-Lys-.sup.NH, [0423] .sup.CO-Ala-Lys-.sup.NH, [0424] .sup.CO-Val-Cit-.sup.NH, [0425] .sup.CO-Phe-Cit-.sup.NH, [0426] .sup.CO-Leu-Cit-.sup.NH, [0427] .sup.CO-Ile-Cit-.sup.NH, [0428] .sup.CO-Phe-Arg-.sup.NH, and [0429] .sup.CO-Trp-Cit-.sup.NH.
21. A compound according to statement 20, wherein Q is selected from .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Ala-.sup.NH.
22. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIa, and Q is a tripeptide residue.
23. A compound according to any one of statements 1 to 22, wherein R.sup.L is of formula IIIa and a is 0 to 3.
24. A compound according to statement 23, wherein a is 0.
25. A compound according to any one of statements 1 to 24, wherein R.sup.L is of formula IIIa and b is 0 to 12.
26. A compound according to statement 25, wherein b is 0 to 8.
27. A compound according to any one of statements 1 to 26, wherein R.sup.L is of formula IIIa and d is 0 to 3.
28. A compound according to statement 27, wherein d is 2.
29. A compound according to any one of statements 1 to 22, wherein R.sup.L is of formula IIIa and, a is 0, c is 1 and d is 2, and b is from 0 to 8.
30. A compound according to statement 29, wherein b is 0, 4 or 8.
31. A compound according to any one of statements 1 to 30, wherein R.sup.L is of formula IIIa and G.sup.L is selected from:
TABLE-US-00005
where Ar represents a C.sub.5-6 arylene group.
32. A compound according to statement 31, wherein Ar is a phenylene group.
33. A compound according to either statement 31 or statement 32, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.
34. A compound according to statement 33, wherein G.sup.L is G.sup.L1-1.
35. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIb, and both R.sup.L1 and R.sup.L2 are H.
36. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIb, R.sup.L1 is H and R.sup.L2 is methyl.
37. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIb, and both R.sup.L1 and R.sup.L2 are methyl.
38. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIb, and, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.
39. A compound according to any one of statements 1 to 18, wherein R.sup.L is of formula IIIb, and, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.
40. A compound according to any one of statements 1 to 18 and 35 to 39, wherein R.sup.L is of formula IIIb, and e is 0.
41. A compound according to any one of statements 1 to 18 and 35 to 39, wherein R.sup.L is of formula IIIb, and e is 1.
42. A compound according to statement 41, wherein the nitro group is in the para position.
43. A compound according to statement 1, wherein the compound is of formula Id:
##STR00093##
where Q is selected from:
(a) —CH.SUB.2.—;
[0430] (b) —C.sub.3H.sub.6—; and
##STR00094##
44. A conjugate of formula II:
L-(D.sup.L).sub.p (I)
wherein L is a Ligand unit, D.sup.L is a Drug Linker unit of formula I′:
##STR00095##
wherein R.sup.6, R.sup.7, R.sup.9, R.sup.11b, Y, R″, Y′, R.sup.6′, R.sup.7, R.sup.9′, R.sup.20 and R.sup.21, are as defined in any one of statements 1 to 18;
R.sup.LL is a linker for connection to a cell binding agent, which is selected from:
(iiia):
##STR00096##
where Q and X are as defined in any one of statements 1 and 19 to 21 and G.sup.LL is a linker connected to a Ligand Unit; and
(iiib):
##STR00097##
where R.sup.L1 and R.sup.L2 are as defined in any one of statements 1 and 35 to 39;
wherein p is an integer of from 1 to 20.
45. A conjugate according to statement 44, wherein G.sup.LL is selected from:
TABLE-US-00006
where Ar represents a C.sub.5-6 arylene group.
46. A conjugate according to statement 45, wherein Ar is a phenylene group.
47. A conjugate according to either statement 45 or statement 46, wherein G.sup.LL is selected from G.sup.LL1-1 and GL.sup.L1-2.
48. A conjugate according to statement 47, wherein G.sup.LL is GL.sup.L1-1.
49. A conjugate according to statement 44, wherein D.sup.L is of formula (Id′):
##STR00111##
where Q is selected from:
(a) —CH.SUB.2.—;
[0431] (b) —C.sub.3H.sub.6—; and
##STR00112##
50. A conjugate according to any one of statements 44 to 49, wherein the Ligand Unit is an antibody or an active fragment thereof.
51. The conjugate according to statement 50, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.
52. The conjugate according to statement 51 wherein the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from (1)-(89):
(1) BMPR1B;
(2) E16;
(3) STEAP1;
(4) 0772P;
(5) MPF;
(6) Napi3b;
(7) Sema 5b;
[0432] (8) PSCA hlg;
(9) ETBR;
(10) MSG783;
(11) STEAP2;
(12) TrpM4;
(13) CRI PTO;
(14) CD21;
(15) CD79b;
(16) FcRH2;
(17) HER2;
(18) NCA;
(19) MDP;
(20) IL20R-alpha;
(21) Brevican;
(22) EphB2R;
(23) ASLG659;
(24) PSCA;
(25) GEDA;
(26) BAFF-R;
(27) CD22;
(28) CD79a;
(29) CXCR5;
(30) HLA-DOB;
(31) P2X5;
(32) CD72;
(33) LY64;
(34) FcRH1;
(35) IRTA2;
(36) TENB2;
(37) PSMA-FOLH1;
(38) SST;
(38.1) SSTR2;
(38.2) SSTR5;
(38.3) SSTR1;
(38.4) SSTR3;
(38.5) SSTR4;
(39) ITGAV;
(40) ITGB6;
(41) CEACAM5;
(42) MET;
(43) MUC1;
(44) CA9;
(45) EGFRvIII;
(46) CD33;
(47) CD19;
(48) IL2RA;
(49) AXL;
(50) CD30-TNFRSF8;
(51) BCMA-TNFRSF17;
(52) CT Ags-CTA;
(53) CD174 (Lewis Y)-FUT3;
(54) CLEC14A;
(55) GRP78-HSPA5;
(56) CD70;
[0433] (57) Stem Cell specific antigens;
(58) ASG-5;
(59) ENPP3;
(60) PRR4;
(61) GCC-GUCY2C;
(62) Liv-1-SLC39A6;
(63) 5T4;
(64) CD56-NCMA1;
(65) CanAg;
(66) FOLR1;
(67) GPNMB;
(68) TIM-1-HAVCR1;
[0434] (69) RG-1/Prostate tumor target Mindin-Mindin/RG-1;
(70) B7-H4-VTCN1;
(71) PTK7;
(72) CD37;
(73) CD138-SDC1;
(74) CD74;
(75) Claudins-CLs;
(76) EGFR;
(77) Her3;
(78) RON-MST1R;
(79) EPHA2;
(80) CD20-MS4A1;
(81) Tenascin C-TNC;
(82) FAP;
(83) DKK-1;
(84) CD52;
(85) CS1-SLAMF7;
(86) Endoglin-ENG;
(87) Annexin A1-ANXA1;
(88) V-CAM (CD106)-VCAM1;
(89) ASCT2 (SLC1A5).
[0435] 53. The conjugate of any one of statements 50 to 52 wherein the antibody or antibody fragment is a cysteine-engineered antibody.
54. The conjugate according to any one of statements 44 to 53 wherein p is an integer from 1 to 8.
55. The conjugate according to statement 54, wherein p is 1, 2, 3, or 4.
56. A composition comprising a mixture of conjugates according to any one of statements 44 to 55, wherein the average p in the mixture of conjugate compounds is about 1 to about 8.
57. The conjugate according to any one of statements 44 to 55, for use in therapy.
58. A pharmaceutical composition comprising the conjugate of any one of statements 44 to 55, and a pharmaceutically acceptable diluent, carrier or excipient.
59. The conjugate according to any one of statements 44 to 55 or the pharmaceutical composition according to statement 58, for use in the treatment of a proliferative disease in a subject.
60. The conjugate for use according to statement 61, wherein the disease treated is cancer.
61. Use of a conjugate according to any one of statements 44 to 55 or a pharmaceutical composition according to statement 58 in a method of medical treatment.
62. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 58.
63. The method of statement 62 wherein the method of medical treatment is for treating cancer.
64. The method of statement 63, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.
65. Use of a conjugate according to any one of statements 44 to 55 in a method of manufacture of a medicament for the treatment of a proliferative disease.
66. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to any one of statements 44 to 55 or a pharmaceutical composition according to statement 58.
67. A compound of Formula IV:
##STR00113##
wherein R.sup.6, R.sup.7, R.sup.9, Y, R″, Y′, R.sup.6′, R.sup.7 and R.sup.9′, are as defined in any one of statements 1 to 18;
either: [0436] (a) R.sup.39 is H and R.sup.31 is H; [0437] (b) R.sup.39 is H and R.sup.31 is ═O; or [0438] (c) R.sup.39 and R.sup.31 form a double bond between the N and C atoms to which they are attached.