PYRROLOBENZODIAZEPINE CONJUGATES
20210380605 · 2021-12-09
Inventors
Cpc classification
A61K47/6835
HUMAN NECESSITIES
C07K5/06034
CHEMISTRY; METALLURGY
A61K47/65
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
C07K5/06026
CHEMISTRY; METALLURGY
A61K47/6855
HUMAN NECESSITIES
C07D519/00
CHEMISTRY; METALLURGY
International classification
C07D519/00
CHEMISTRY; METALLURGY
A61K47/65
HUMAN NECESSITIES
Abstract
A compound of formula (I) and salts and solvates thereof, wherein R.sup.L is a linker for connection to a cell binding agent, which is formula (IIa) wherein Q is a tripeptide residue of formula (A), where x is 1 or 2, —C(═O)-Q.sup.x-NH— is a dipeptide residue; X is: formula (B), where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5; and G.sup.L is a linker for connecting to a Ligand Unit.
##STR00001##
Claims
1. A compound of formula I: ##STR00125## and salts and solvates thereof, wherein: D represents either group D1 or D2: ##STR00126## the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (ib) C.sub.1-5 saturated aliphatic alkyl; (ic) C.sub.3-6 saturated cycloalkyl; (id) ##STR00127## wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5; (ie) ##STR00128## wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if) ##STR00129## where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and C3, R.sup.2 is selected from H, OH, F, diF and ##STR00130## where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; D′ represents either group D′1 or D′2: ##STR00131## wherein the dotted line indicates the optional presence of a double bond between C2′ and C3′; when there is a double bond present between C2′ and C3′, R.sup.22 is selected from the group consisting of: (iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (iib) C.sub.1-5 saturated aliphatic alkyl; (iic) C.sub.3-6 saturated cycloalkyl; (iid) ##STR00132## wherein each of R.sup.21, R.sup.22a and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.22 group is no more than 5; (iie) ##STR00133## wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (iif) ##STR00134## where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2′ and C3′, R.sup.22 is selected from H, OH, F, diF and ##STR00135## where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; 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 ##STR00136## wherein Q is a tripeptide residue of formula: ##STR00137## where x is 1 or 2, and —C(═O)-Q.sup.X-NH— is a dipeptide residue; X is: ##STR00138## 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; either (a) R.sup.30 is H, and R.sup.31 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or (b) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R.sup.30 is H and R.sup.31 is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or (d) R.sup.30 is H and R.sup.31 is H or ═O; or (e) R.sup.31 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.30 is selected from: ##STR00139## where R.sup.Z is selected from: ##STR00140## (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 both Y and Y′ are O, R″ is either C.sub.3-7 alkylene or a group of formula: ##STR00141## where r is 1 or 2.
3. A compound according to claim 1, wherein R.sup.6 is H, R.sup.9 is H and R.sup.7 is a C.sub.1-4 alkyloxy group.
4. A compound according to claim 1, wherein D is D1, there is a double bond between C2 and C3, and R.sup.2 is: (a) phenyl, which bears one to three substituent groups, selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl; (b) methyl; or (c) a group of formula: ##STR00142## wherein the total number of carbon atoms in the R.sup.2 group is no more than 4.
5. A compound according to claim 1, wherein D is D1 there is a single bond between C2 and C3, and R.sup.2 is: (a) H; or (b) ##STR00143## and R.sup.16a and R.sup.16b are both H.
6. A compound according to claim 1, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.22 is: (a) phenyl, which bears one to three substituent groups, selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl; (b) methyl; or (c) a group of formula: ##STR00144## wherein the total number of carbon atoms in the R.sup.22a group is no more than 4.
7. A compound according to claim 1, wherein D′ is D′1, there is a single bond between C2′ and C3′, and R.sup.22 is: (a) H; or (b) ##STR00145## and R.sup.26a and R.sup.26b are both H.
8. 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, Y′ is the same group as Y, R.sup.22 (if present) is the same group as R.sup.2 (if present).
9. A compound according to claim 1, wherein: (a) R.sup.30 is H, and R.sup.31 is OH; (b) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R.sup.30 is H, and R.sup.31 is H.
10. A compound according to claim 1, which is of formula Ia-1, Ia-2 or Ia-3: ##STR00146## where R.sup.2a and R.sup.12a are the same and are selected from: ##STR00147## 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; (a) R.sup.11b is OH and/or (b) C(═O)—Q.sup.x—NH— is a dipeptide residue selected from .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Ala-.sup.NH.
12. (canceled)
13. A compound according to claim 1 wherein: (a) x is 1 or 2, and/or (b) a is 0, c is 1 and d is 2, and b is 0, 4 or 8, or (c) a, b and c are 0 and d is 2 or 5.
14-16. (canceled)
17. A compound according to claims 1, wherein G.sup.L is selected from: ##STR00148## ##STR00149## where Ar represents a C.sub.5-6 arylene group and where Hal represents I, Br or Cl.
18. A compound according to claim 17, wherein G.sup.L is G.sup.LI-1.
19. A conjugate of formula I:
L−(D.sup.L).sub.p (I) wherein L is a Ligand unit, D.sup.L is a Drug Linker unit of formula I′: ##STR00150## wherein D, R.sup.2, R.sup.6, R.sup.7, R.sup.9, R.sup.11b, Y, R″, Y′, D′, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.22, R.sup.30 and R.sup.31, including the presence or absence of double bonds between C2 and C3 and C2′ and C3′ respectively, are as defined in claim 1; R.sup.LL is a linker for connection to a cell binding agent, which is: ##STR00151## where Q and X are as defined in claim 1 and G.sup.LL is a linker connected to the Ligand Unit; wherein p is an integer of from 1 to 20.
20. A conjugate according to claim 19, wherein G.sup.LL is selected from: ##STR00152## ##STR00153## where Ar represents a C.sub.5-6 arylene group.
21. A conjugate according to claim 20, wherein G.sup.LL is G.sup.LL1-1.
22. A conjugate according to claim 19, wherein the Ligand Unit is an antibody or an active fragment thereof for a tumour-associated antigen.
23. (canceled)
24. A pharmaceutical composition comprising the conjugate of claim 19, and a pharmaceutically acceptable diluent, carrier or excipient.
25. (canceled)
26. A method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate according to claim 19.
Description
BRIEF DESCRIPTION OF FIGURES
[0197]
[0198]
GENERAL SYNTHETIC ROUTES
[0199] 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
[0200] Compounds of the present invention of formula I:
##STR00027##
can be synthesised from a compound of Formula 2:
##STR00028##
where R.sup.2, R.sup.6, R.sup.7, R.sup.9, R.sup.6′, R.sup.7, R.sup.9, R.sup.11b, R.sup.22, R.sup.30, R.sup.31, Y, Y′ and R″ are as defined for compounds of formula I, and R.sup.LL is a precursor of R.sup.L. R.sup.LL will typically be a portion of R.sup.L, such as a group of formula IIIa′:
##STR00029##
In such a case, the reaction involves adding the group G.sup.L.
[0201] The compounds of Formula 2 may be made by deprotecting compounds of Formula 3:
##STR00030##
where R.sup.2, R.sup.6, R.sup.7, R.sup.9, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.11b, R.sup.22, R.sup.30, R.sup.31, Y, Y′ and R″ are as defined for compounds of formula I, R″ is a protected version of R″, and the Prot represents an appropriate carboxy/hydroxy protecting group. Depending on the nature of R.sup.30 and R.sup.31, these may need to be in protected form at points during the synthesis.
[0202] Compounds of formula 3 may be made by ring-closure of compounds of Formula 4:
##STR00031##
where the ring closure is carried out by oxidation, e.g. Swern.
[0203] Compounds of formula 4 can be synthesised from compounds of formula 5:
##STR00032##
by step-wise addition of the two amino protecting groups. Step-wise addition can be achieved by simple protection of one amino group (e.g. by Fmoc), followed by installation of a desired protecting group at the other amino group. This can be followed by removal of the simple protecting group, and then installation of the other desired amino protecting group.
[0204] Compounds of Formula 5 can be synthesised by known methods, such as those disclosed in WO 2011/130598.
Synthesis of Drug Conjugates
[0205] 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.
Further Preferences
[0206] 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.
[0207] 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.
[0208] In some embodiments, R.sup.22 is the same as R.sup.2.
N10′-C11′
[0209] In some embodiment, R.sup.30 is H, and R.sup.31 is OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl. In some of these embodiments, R.sup.31 is OH. In others of these embodiments, R.sup.31 is OR.sup.A, where R.sup.A is C.sub.1-4 alkyl. In some of these embodiments, R.sup.A is methyl.
[0210] In some embodiments, R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
[0211] In some embodiments, R.sup.30 is H and R.sup.31 is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation. In some of these embodiments, M is a monovalent pharmaceutically acceptable cation, and may be Na*. Furthermore, in some embodiments z is 3.
[0212] In some embodiments, R.sup.30 is H and R.sup.31 is H.
[0213] In some embodiments, R.sup.30 is H and R.sup.31 is ═O.
[0214] In some embodiments where R.sup.30 is (e-iii), there may be an additional nitro group on the benzene ring, e.g. ortho to R.sup.Z.
[0215] In some embodiments, R.sup.31 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.30 is selected from:
##STR00033## ##STR00034##
where —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.
[0216] In one embodiment, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: [0217] -Phe-Lys-, [0218] -Val-Ala-, [0219] -Val-Lys-, [0220] -Ala-Lys-, [0221] -Val-Cit-, [0222] -Phe-Cit-, [0223] -Leu-Cit-, [0224] -Ile-Cit-, [0225] -Phe-Arg-, [0226] -Trp-Cit-,
where Cit is citrulline.
[0227] Preferably, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: [0228] -Phe-Lys-, [0229] -Val-Ala-, [0230] -Val-Lys-, [0231] -Ala-Lys-, [0232] -Val-Cit-.
[0233] Most preferably, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is -Phe-Lys- or -Val-Ala-.
[0234] 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.
[0235] 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.
[0236] 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′.
[0237] 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′.
[0238] 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.
[0239] 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.
[0240] Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality: [0241] Arg: Z, Mtr, Tos; [0242] Asn: Trt, Xan; [0243] Asp: Bzl, t-Bu; [0244] Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; [0245] Glu: Bzl, t-Bu; [0246] Gln: Trt, Xan; [0247] His: Boc, Dnp, Tos, Trt; [0248] Lys: Boc, Z—Cl, Fmoc, Z, Alloc; [0249] Ser: Bzl, TBDMS, TBDPS; [0250] Thr: Bz; [0251] Trp: Boc; [0252] Tyr: Bzl, Z, Z—Br.
[0253] 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.
[0254] 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, lie, Leu, Met, Phe, Pro, and Val.
[0255] It is particularly preferred in the present invention, that —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH— is the same dipeptide comprised in R.sup.L.
[0256] Other preferred R.sup.30 groups include:
##STR00035##
Dimer Link
[0257] In some embodiments, Y and Y′ are both O.
[0258] 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.
[0259] In other embodiments, R″ is a group of formula:
##STR00036##
where r is 1 or 2.
R.sup.6 to R.sup.9
[0260] In some embodiments, R.sup.9 is H.
[0261] 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.
[0262] 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 C.sub.6 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.
[0263] These embodiments and preferences apply to R.sup.9′, R.sup.6′ and R.sup.7′ respectively.
D and D′
[0264] In some embodiments, D and D′ are D1 and D′1 respectively.
[0265] In some embodiments, D and D′ are D2 and D′2 respectively.
R.SUP.2
[0266] When there is a double bond present between C2 and C3, R.sup.2 is selected from:
(a) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(b) C.sub.1-5 saturated aliphatic alkyl;
(c) C.sub.3-6 saturated cycloalkyl;
(d)
##STR00037##
wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;
(e)
##STR00038##
wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and
(f)
##STR00039##
where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
[0267] When R.sup.2 is a C.sub.5-10 aryl group, it may be a C.sub.5-7 aryl group. A C.sub.5-7 aryl group may be a phenyl group or a C.sub.5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R.sup.2 is preferably phenyl. In other embodiments, R.sup.2 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
[0268] When R.sup.2 is a C.sub.5-10 aryl group, it may be a C.sub.8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
[0269] When R.sup.2 is a C.sub.5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
[0270] Where R.sup.2 is C.sub.5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C.sub.5-7 aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.
[0271] Where R.sup.2 is a C.sub.8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R.sup.2 Substituents, when R.sup.2 is a C.sub.5-10 Aryl Group
[0272] If a substituent on R.sup.2 when R.sup.2 is a C.sub.5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
[0273] If a substituent on R.sup.2 when R.sup.2 is a C.sub.5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C.sub.1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C.sub.5-7 aryloxy group (e.g. phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
[0274] If a substituent on R.sup.2 when R.sup.2 is a C.sub.5-10 aryl group is C.sub.1-7 alkyl, it may preferably be a C.sub.1-4 alkyl group (e.g. methyl, ethyl, propyl, butyl).
[0275] If a substituent on R.sup.2 when R.sup.2 is a C.sub.5-10 aryl group is C.sub.3-7 heterocyclyl, it may in some embodiments be C.sub.6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C.sub.1-4 alkyl groups. If the C.sub.6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
[0276] If a substituent on R.sup.2 when R.sup.2 is a C.sub.5-10 aryl group is bis-oxy-C.sub.1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
[0277] If a substituent on R.sup.2 when R.sup.2 is a C.sub.5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
[0278] Particularly preferred substituents when R.sup.2 is a C.sub.5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methylthiophenyl. Other particularly preferred substituents for R.sup.2 are dimethylaminopropyloxy and carboxy.
[0279] Particularly preferred substituted R.sup.2 groups when R.sup.2 is a C.sub.5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R.sup.2 group is 4-nitrophenyl. R.sup.2 groups of particular interest include 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
[0280] When R.sup.2 is C.sub.1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
[0281] When R.sup.2 is C.sub.3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
[0282] When R.sup.2 is
##STR00040##
each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5. In some embodiments, the total number of carbon atoms in the R.sup.2 group is no more than 4 or no more than 3.
[0283] In some embodiments, one of R.sup.11, R.sup.12 and R.sup.13 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[0284] In other embodiments, two of R.sup.11, R.sup.12 and R.sup.13 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[0285] In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.
[0286] In some embodiments, R.sup.11 is H.
[0287] In some embodiments, R.sup.12 is H.
[0288] In some embodiments, R.sup.13 is H.
[0289] In some embodiments, R.sup.11 and R.sup.12 are H.
[0290] In some embodiments, R.sup.11 and R.sup.13 are H.
[0291] In some embodiments, R.sup.12 and R.sup.13 are H.
[0292] An R.sup.2 group of particular interest is:
##STR00041##
[0293] When R.sup.2 is
##STR00042##
one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
[0294] When R.sup.2 is
##STR00043##
R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
[0295] In some embodiments, R.sup.14 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R.sup.14 is selected from H and methyl.
[0296] When there is a single bond present between C2 and C3,
[0297] R.sup.2 is H, OH, F, diF or
##STR00044##
where R.sup.16 and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C, alkyl ester.
[0298] In some embodiments, R.sup.2 is H.
[0299] In some embodiments, R.sup.2 is OH.
[0300] In some embodiments, R.sup.2 is
##STR00045##
[0301] In some embodiments, it is preferred that R.sup.16a and R.sup.16b are both H.
[0302] In other embodiments, it is preferred that R.sup.16a and R.sup.16b are both methyl.
[0303] In further embodiments, it is preferred that one of R.sup.16a and R.sup.16b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl.
R.SUP.22
[0304] When there is a double bond present between C2′ and C3, R.sup.22 is selected from:
(a) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(b) C.sub.1-5 saturated aliphatic alkyl;
(c) C.sub.3-6 saturated cycloalkyl;
(d)
##STR00046##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.22 group is no more than 5;
(e)
##STR00047##
wherein one of R.sup.25 and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and
(f)
##STR00048##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
[0305] When R.sup.22 is a C.sub.5-10 aryl group, it may be a C.sub.5-7 aryl group. A C.sub.5-7 aryl group may be a phenyl group or a C.sub.5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R.sup.22 is preferably phenyl. In other embodiments, R.sup.22 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
[0306] When R.sup.22 is a C.sub.5-10 aryl group, it may be a C.sub.8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
[0307] When R.sup.22 is a C.sub.5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
[0308] Where R.sup.22 is C.sub.5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C.sub.5-7 aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.
[0309] Where R.sup.22 is a C.sub.8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R.sup.22 Substituents, when R.sup.22 is a C.sub.5-10 Aryl Group
[0310] If a substituent on R.sup.22 when R.sup.22 is a C.sub.5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
[0311] If a substituent on R.sup.22 when R.sup.22 is a C.sub.5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C.sub.1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C.sub.5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
[0312] If a substituent on R.sup.22 when R.sup.22 is a C.sub.5-10 aryl group is C.sub.1-7 alkyl, it may preferably be a C.sub.1-4 alkyl group (e.g. methyl, ethyl, propyl, butyl).
[0313] If a substituent on R.sup.22 when R.sup.22 is a C.sub.5-10 aryl group is C.sub.3-7 heterocyclyl, it may in some embodiments be C.sub.6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C.sub.1-4 alkyl groups. If the C.sub.6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
[0314] If a substituent on R.sup.22 when R.sup.22 is a C.sub.5-10 aryl group is bis-oxy-C.sub.1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
[0315] If a substituent on R.sup.22 when R.sup.22 is a C.sub.5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
[0316] Particularly preferred substituents when R.sup.22 is a C.sub.5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methylthiophenyl. Other particularly preferred substituents for R.sup.22 are dimethylaminopropyloxy and carboxy.
[0317] Particularly preferred substituted R.sup.22 groups when R.sup.22 is a C.sub.5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R.sup.22 group is 4-nitrophenyl. R.sup.22 groups of particular interest include 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
[0318] When R.sup.22 is C.sub.1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
[0319] When R.sup.22 is C.sub.3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
[0320] When R.sup.22 is
##STR00049##
each of R.sup.21, R.sup.22a and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.22 group is no more than 5. In some embodiments, the total number of carbon atoms in the R.sup.22 group is no more than 4 or no more than 3.
[0321] In some embodiments, one of R.sup.21, R.sup.22a and R.sup.23 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[0322] In other embodiments, two of R.sup.21, R.sup.22a and R.sup.23 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[0323] In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.
[0324] In some embodiments, R.sup.21 is H.
[0325] In some embodiments, R.sup.22a is H.
[0326] In some embodiments, R.sup.23 is H.
[0327] In some embodiments, R.sup.21 and R.sup.22a are H.
[0328] In some embodiments, R.sup.21 and R.sup.23 are H.
[0329] In some embodiments, R.sup.22a and R.sup.23 are H.
[0330] An R.sup.22 group of particular interest is:
##STR00050##
[0331] When R.sup.22 is
##STR00051##
one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
[0332] When R.sup.22 is
##STR00052##
R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
[0333] In some embodiments, R.sup.24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R.sup.24 is selected from H and methyl.
[0334] When there is a single bond present between C2′ and C3′,
[0335] R.sup.22 is H, OH, F, diF or
##STR00053##
where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.24 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester.
[0336] In some embodiments, R.sup.22 is H.
[0337] In some embodiments, R.sup.22 is OH.
[0338] In some embodiments, R.sup.22 is
##STR00054##
[0339] In some embodiments, it is preferred that R.sup.26a and R.sup.2b are bath H.
[0340] In other embodiments, it is preferred that R.sup.26a and R.sup.26b are both methyl.
[0341] In further embodiments, it is preferred that one of R.sup.26a and R.sup.26b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl.
R.SUP.11b
[0342] In some embodiments, R.sup.11b is OH.
[0343] 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.
[0344] In some embodiments of the first aspect of the present invention are of formula Ia-1, Ia-2 or Ia-3:
##STR00055##
where R.sup.2a and R.sup.12a are the same and are selected from:
##STR00056##
[0345] R.sup.1a is selected from methyl and benzyl;
[0346] R.sup.L and R.sup.11b are as defined above.
[0347] In some embodiments of the present invention both R.sup.2 and R.sup.22 comprise no more than 3 carbon atoms.
[0348] Thus in these embodiments where there is a double bond present between C2 and C3, R.sup.2 may be selected from:
##STR00057##
[0349] Thus in these embodiments where there is no double bond present between C2 and C3, R.sup.2 may be selected from:
##STR00058##
[0350] Thus in these embodiments where there is a double bond present between C2′ and C3′, R.sup.22 may be selected from:
##STR00059##
[0351] Thus in these embodiments where there is no double bond present between C2′ and C3′, R.sup.22 may be selected from:
##STR00060##
[0352] In some of these embodiments both R.sup.2 and R.sup.22 comprise no more than 2 carbon atoms.
[0353] Thus in these embodiments where there is a double bond present between C2 and C3, R.sup.2 may be selected from:
##STR00061##
[0354] Thus in these embodiments where there is no double bond present between C2 and C3, R.sup.2 may be selected from:
##STR00062##
[0355] Thus in these embodiments where there is a double bond present between C2′ and C3′, R.sup.22 may be selected from:
##STR00063##
[0356] Thus in these embodiments where there is no double bond present between C2′ and C3′, R.sup.22 may be selected from:
##STR00064##
[0357] In further of these embodiments both R.sup.2 and R.sup.22 comprise no more than 1 carbon atom.
[0358] Thus in these embodiments where there is a double bond present between C2 and C3, R.sup.2 may be methyl. Thus in these embodiments where there is no double band present between C2 and C3, R.sup.2 may be selected from:
##STR00065##
[0359] Thus in these embodiments where there is a double band present between C2′ and C3′, R.sup.22 may be methyl. Thus in these embodiments where there is no double band present between C2′ and C3′, R.sup.22 may be selected from:
##STR00066##
[0360] These embodiments and preferences also apply to the second aspect of the invention.
Linker (R.sup.L/R.sup.LL)
G.SUP.L
[0361] G.sup.L may be selected from
##STR00067## ##STR00068##
where Ar represents a C.sub.5-6 arylene group, e.g. phenylene.
[0362] 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.
G.SUP.LL
[0363] G.sup.LL may be selected from:
##STR00069## ##STR00070##
where Ar represents a C.sub.5-6 arylene group, e.g. phenylene.
[0364] 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.
X
[0365] X is:
##STR00071##
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.
[0366] 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.
[0367] In some embodiments of X, a, b and c are 0 and d is 2 or 5.
Q.SUP.X
[0368] Q.sup.X 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.
[0369] In one embodiment, —C(═O)-Q.sup.X-NH— is selected from: [0370] .sup.CO-Phe-Lys-1, [0371] .sup.COVal-Ala-1, [0372] .sup.COVal-Lys-1, [0373] .sup.COAla-Lys-NH, [0374] .sup.COVal-Cit-.sup.NH, [0375] .sup.COPhe-Cit-.sup.NH, [0376] .sup.CO-Leu-Cit-.sup.NH, [0377] .sup.COIle-Cit-.sup.NH, [0378] .sup.COPhe-Arg-.sup.NH, and [0379] .sup.CO-Trp-Cit-.sup.NH;
where Cit is citrulline.
[0380] Preferably, —C(═O)-Q.sup.X-NH— is selected from: [0381] .sup.CO-Phe-Lys-.sup.NH, [0382] .sup.CO-Val-Ala-.sup.NH, [0383] .sup.CO-Val-Lys-.sup.NH, [0384] .sup.CO-Ala-Lys-.sup.NH, [0385] .sup.COVal-Cit-.sup.NH.
[0386] Most preferably, —C(═O)-Q.sup.X-NH— is selected from .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Ala-.sup.NH.
[0387] Other dipeptide combinations of interest include: [0388] .sup.CO-Gly-Gly-NH. [0389] .sup.CO-Pro-Pro-.sup.NH, and [0390] .sup.CO-Val-Glu-.sup.NH.
[0391] 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.
[0392] In Q, x can be 1 or 2.
[0393] In some embodiments, x in Q is 1.
[0394] In some embodiments, x in Q is 2
[0395] Thus, in some embodiments Q is:
##STR00072##
[0396] In Q, the carboxy group may be in the following stereochemical arrangement relative to neighbouring groups:
##STR00073##
such that it is derived from the appropriate natural amino acid.
[0397] In some embodiments of the present invention, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
##STR00074##
[0398] In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
##STR00075##
[0399] Compounds of particular interest include those of the examples.
EXAMPLES
[0400] 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.
[0401] 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 seconds' 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, 2 μ, 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).
[0402] 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.) dimensions: 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 rate was 20.0 mL/minute and detection was at 254 and 280 nm.
Intermediate Synthesis
(S)-5-((9H-fluoren-9-yl)methoxy)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-5-oxopentanoic acid 3
[0403] ##STR00076##
(R)-5-((9H-fluoren-9-yl)methoxy).sub.4-((tert-butoxycarbonyl)amino)-5-oxopentanoic acid 1 (0.271 g, 0.64 mmol, 1 eq.) was dissolved in a solution of trifluoroacetic acid in DCM (4 mL, 20% v/v). Stirred at room temperature for 2 h. The solution was evaporated, azeotroping with toluene (×3) to remove trifluoroacetic acid. The resulting TFA salt 2 was suspended in DCM (10 mL) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (0.196 g, 0.64 mmol, 1 eq.) and DIPEA (0.25 g, 333 μl, 1.9 mmol, 3 eq.) were added. The mixture was stirred at room temperature for 18 h, diluted with DCM (50 mL) and extracted with 1M citric acid solution (2×50 mL), brine (100 mL), dried (MgSO.sub.4) and evaporated under reduced pressure to give the product 3 as a white foam (0.313 g, 95%). Analytical Data: RT 1.49 min; MS (ES.sup.+) m/z (relative intensity) 519 ([M+H].sup.+, 100)
(S)-33-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31-dioxo-7,10,13,16,19,22,25,28-octaoxa-4,32-diazahexatriacontan-36-oic acid 4
[0404] ##STR00077##
[0405] (S)-5-((9H-fluoren-9-yl)methoxy)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoic acid 1 (1.254 g, 2.9 mmol, 1 eq.) was dissolved in a solution of trifluoroacetic acid in DCM (18 mL, 20% v/v). Stirred at room temperature for 4 h. The solution was evaporated, azeotroping with toluene (×3) to remove trifluoroacetic acid giving the product as a white solid. A portion of the resulting glutamic acid TFA salt 2 (0.313 g, 0.61 mmol, 1eq.) was suspended in DCM (30 mL) and 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oate (0.418 g, 0.61 mmol, 1 eq.) and DIPEA (0.234 g, 317 μl, 1.8 mmol, 3 eq.) were added. The mixture was stirred at room temperature for 18 h. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography [CHCl.sub.3/MeOH 0% to 8% in 1% increments] to give the product as a colourless oil (0.22 g) along with fractions mixed with unreacted TFA salt. The mixed fractions were dissolved in DCM, washed with water, brine, dried (MgSO.sub.4) and evaporated under reduced pressure to give further product (0.109 g). Combined yield of 4 (0.329 g, 51%).
(4S)-4-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]-5-(9H-fluoren-9-ylmethoxy)-5-oxopentanoic acid 5
[0406] ##STR00078##
5 was synthesised by an analogous method to 3 and 4 above.
4-((S)-2-((S)-2-amino-3-methylbutenamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c[1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (I10)
[0407] ##STR00079##
(a) ((Propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone) (I2)
[0408] DMF (12 drops) was added to a stirred suspension of the bis-nitrobenzoic acid 11 (10 g, 21.5 mmol) and oxalyl chloride (5.6 mL, 8.2 g, 64.5 mmol) in anhydrous DCM (150 mL). Following initial effervescence the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. The majority of solvent was removed by evaporation in vacuo and the resulting concentrated solution was re-dissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by vacuum filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40° C. The solid acid chloride was added portion-wise to a stirred suspension of (S)-(+)-2-pyrrolidinemethanol (5.0 g, 4.9 mL, 49.5 mmol) and TEA (15.0 mL, 10.9 g, 108 mmol) in DCM (100 mL) at −40° C. (dry ice/CH.sub.3CN). After 1 hour stirring, the reaction was complete as judged by LC/MS with exclusively desired product at retention time 1.33 minutes, ES+m/z 655 [M+Na].sup.+, 633 [M+H].sup.+. The mixture was diluted with DCM (100 mL) and washed with 1N HCl (2×50 mL), saturated NaHCO.sub.3 (3×40 mL), brine (50 mL), dried (MgSO.sub.4), filtered and the solvent evaporated in vacuo to give the pure product 12 as a yellow solid (13.6 g, 100% yield).
(b) ((2S,2′S)-(4,4′-(propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitrobenzoyl))bis(pyrrolidine-1,2-diyl))bis(methylene) diacetate (I3)
[0409] A solution of Ac.sub.2O (4.47 mL, 4.83 g, 47.3 mmol) in dry DCM (25 mL) was added drop-wise to a stirred solution of the bis-alcohol 12 (13.6 g, 21.5 mmol), DMAP (263 mg, 2.15 mmol) and pyridine (4.17 mL, 4.08 g, 51.6 mmol) in dry DCM (125 mL) at 0° C. (ice/acetone) under an argon atmosphere. The reaction mixture was allowed to warm-up and after 1 hour at room temperature analysis by LC/MS revealed completion of reaction and clean conversion to desired product at retention time 1.55 minutes, ES+m/z 740 [M+Na].sup.+, 717 [M+H].sup.+. The mixture was diluted with DCM (20 mL) and washed with 1N HCl (2×100 mL), H.sub.2O (25 mL), brine (50 mL), dried (MgSO.sub.4), filtered and the solvent evaporated in vacuo to give the crude bis-acetate 13 as a yellow solid (14.4 g, 94% yield) which was of satisfactory purity to be carried through to the next step without further purification.
(c) ((2S,2'S)-(4,4′-(propane-1,3-diylbis(oxy))bis(2-amino-5-methoxybenzoyl))bis(pyrrolidine-1,2-diyl))bis(methylene) diacetate (I4)
[0410] A sample of 10% Pd—C (132 mg) was treated carefully with EtOAc (10 mL) to give a slurry which was added to a solution of the nitro compound 13 (1.32 g, 1.84 mmol) in EtOAc (20 mL) and EtOH (30 mL) in a hydrogenation vessel. Using Parr® apparatus, the mixture was treated with hydrogen gas to 10 psi and shaken at room temperature then degassed in vacuo, this process was repeated a further two times. The vessel was filled with hydrogen gas to 45 psi, shaken and the pressure maintained upon consumption of hydrogen. Analysis by LC/MS showed the reaction was incomplete after 3 hours and was left shaking at 45 psi for 3 days (the weekend) after which time satisfactory conversion to product was achieved, retention time=1.32 minutes, ES+m/z 657 [M+H].sup.+. The reaction mixture was degassed in vacuo and then filtered through a Celite® pad. The filtrate was evaporated in vacuo, the resulting residue re-dissolved in DCM (30 mL), dried (MgSO.sub.4), filtered and the solvent evaporated in vacuo to give the crude bis-aniline 14 as a yellowish foam (1.1 g, 91% yield) which contained an 8% impurity but was carried through to the next step without further purification.
(d) ((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-((tert-butoxycarbonyl)amino)-2-methoxyphenoxy)propoxy)-2-amino-5-methoxybenzoyl)pyrrolidin-2-yl)methyl acetate (I5)
[0411] Boc.sub.2O (330 mg, 1.51 mmol) was added to a stirred solution of the bis-aniline 14 (1.1 g, 1.68 mmol) in dry THF (10 mL). The reaction mixture was heated and stirred at 75° C. for 16 hours. Analysis by LC/MS revealed desired mono Boc product 15 at retention time 1.58 minutes, 1%=50, ES+m/z 779 [M+Na].sup.+, 757 [M+H].sup.+ along with unreacted starting material at retention time 1.32 minutes, 1%=30, and bis-Boc material at retention time 1.81 minutes, 1%=21, ES+m/z 879 [M+Na].sup.+, 857 [M+H].sup.+. The reaction mixture was allowed to cool to room temperature and the THF removed by evaporation in vacuo. Purification by Isolera™ (DCM/MeOH, SNAP Ultra 50 g, 100 mL per minute) provided the mono Boc product 15 as an orange foam (519 mg, 46% yield based on Boc.sub.2O, eluting at 97% DCM/MeOH) unreacted bis-aniline 14 (285 mg, eluting at 95% DCM/MeOH) and bis-Boc (248 mg, eluting at 98% DCM/MeOH).
(e) ((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)pyrrolidine-1-carbonyl)-5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-2-((tert-butoxycarbonyl)amino)-5-methoxybenzoyl)pyrrolidin-2-yl)methyl acetate (I7)
[0412] Triphosgene (380 mg, 1.28 mmol) was added to a stirred solution of the mono Boc product I5 (2.69 g, 3.56 mmol) and TEA (1.09 mL, 791 mg, 7.83 mmol) in dry DCM (30 mL) at room temperature. After stirring for 10 minutes under argon, analysis by LC/MS revealed complete conversion to isocyanate (sampled in MeOH to give methyl carbamate, retention time 1.66 minutes, ES+m/z 837 [M+Na].sup.+, 815 [M+H].sup.+). The mixture was treated with additional TEA (740 μL, 539 mg, 5.34 mmol) followed by the addition of linker 16 (1.34 g, 3.56 mmol). After 2 hours stirring under argon, LC/MS revealed satisfactory conversion to carbamate I7 (retention time 1.74 minutes, (ES+) m/z 1182 [M+Na].sup.+, 1160 [M+H].sup.+). The mixture was diluted with DCM (80 mL) and washed with saturated NH.sub.4Cl (2×30 mL), H.sub.2O (30 mL), brine (50 mL), dried (MgSO.sub.4), filtered and evaporated in vacuo to give the crude product. Purification by Isolera™ (Hexane/EtOAc, SNAP Ultra 100 g, 100 mL per minute) provided the pure carbamate I7 (eluting at 65% Hexane/EtOAc) as a yellow foam (2.95 g, 71% yield).
(f) tert-butyl (5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (I8)
[0413] Solid K.sub.2CO.sub.3(1.75 g, 12.7 mmol) was added to a stirred solution of the acetate-protected compound 17 (2.93 g, 2.53 mmol) in MeOH (60 mL) and H.sub.2O (12 mL). After 1 hour stirring at room temperature the reaction was deemed to be complete as judged by LC/MS with desired product at retention time 1.57 minutes, ES+m/z 1098 [M+Na].sup.+, 1076 [M+H].sup.+. The MeOH was removed by evaporation in vacuo and the resulting residue was partitioned between water (75 mL) and DCM (75 mL). The layers were separated and the aqueous phase was extracted with DCM (3×25 mL). The combined organic layers were washed with water (3×50 mL), brine (60 mL), dried (MgSO.sub.4), filtered and evaporated in vacuo to provide the crude product. Purification by Isolera™ (DCM/MeOH, SNAP Ultra 100 g, 100 mL per minute) the bis-alcohol 18 (eluting at 97% DCM/MeOH) as a white foam (2.44 g, 90% yield).
(g) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (I9)
[0414] A solution of anhydrous DMSO (710 μL, 780 mg, 9.99 mmol) in dry DCM (20 mL) was added drop-wise to a stirred solution of oxalyl chloride (2.72 mL of a 2.0M solution in DCM, 5.44 mmol) in dry DCM (20 mL) at −45° C. (dry ice/CH.sub.3CN) under an argon atmosphere. After 15 minutes stirring at −45° C., the reaction mixture was treated drop-wise with a solution of the bis-alcohol 18 (2.44 g, 2.27 mmol) in dry DCM (30 mL). After stirring at −45° C. for a further 1 hour, the reaction mixture was treated drop-wise with a solution of TEA (3.16 mL, 2.29 g, 22.7 mmol) in dry DCM (20 mL). The reaction mixture was allowed to warm to room temperature over a period of 1.5 hours and diluted with DCM (100 mL) then washed with saturated NH.sub.4Cl (2×50 mL), saturated NaHCO.sub.3 (50 mL), water (30 mL), brine (50 mL), dried (MgSO.sub.4), filtered and evaporated in vacuo to give the crude product. Purification by Isolera™ (DCM/MeOH, SNAP Ultra 100 g, 100 mL per minute) gave the cyclised compound I9 (eluting at 95.7% DCM/MeOH) as a yellowish foam (1.61 g, 66% yield): LC/MS I9 at retention time 1.46 minutes, ES+m/z 1072 [M+H].sup.+, 1094 [M+Na].sup.+.
(h) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c[1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo(2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (I10)
[0415] Pd(PPh.sub.3).sub.4 (6.47 mg, 5.6 μmol) was added to a stirred solution of pyrrolidine (29 μL, 25 mg, 0.35 mmol) and the Alloc compound 19 (300 mg, 0.28 mmol) in dry DCM (10 mL). After stirring for 4 hours under argon at room temperature, analysis by LC/MS revealed reaction completion with desired product observed at retention time 1.10 minutes, ES+, m/z 1010 [M+Na].sup.+, 988 [M+H].sup.+. The reaction mixture was diluted with DCM (30 mL) then washed with saturated NH.sub.4Cl (2×20 mL), brine (30 mL), dried (MgSO.sub.4), filtered and evaporated in vacuo to give the crude product. Trituration with diethyl ether followed by evaporation in vacuo gave the crude amine I10 (261 mg, 95% yield) which was carried through to the next step without further purification or analysis.
Example 1
[0416] ##STR00080##
[0417] Compound 6 is Compound 23 of WO2014/057074
a) 4-((S)-2-((S)-2-((S)-5-((9H-fluoren-9-yl)methoxy)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-5-oxopentanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 7
[0418] Acid 6 (0.153 g, 0.295 mmol, 1.1 eq.) and EDCI hydrochloride (0.077 g, 0.4 mmol, 1.5 eq.) were added to a solution of SG3305 (0.247 g, 0.27 mmol, 1 eq.) in CHCl.sub.3 (20 mL). The solution was stirred at room temperature for 3 h when reaction was shown to be complete by LCMS. The reaction mixture was diluted with CHCl.sub.3 (100 mL), washed with water (100 mL), brine (100 mL), dried (MgSO.sub.4) and evaporated under reduced pressure. The product was purified by flash column chromatography [CHCIa/MeOH 0% to 4% in 1% increments] to give the product 7 as a yellow solid (0.275 g, 72%). Analytical Data: RT 1.68 min; MS (ES*) m/z (relative intensity) 1422 ([M+H].sup.+, 2)
b) N2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-N5-((S)-1-(((S)-1-((4-((((11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamine 8
[0419] A solution of N-Methyl pyrrolidine in CHCl.sub.3 (2 mL, 10% v/v, 30 eq.) was added to a solution of Fmoc protected compound 7 (0.130 g, 91 μmol, 1eq) in CHCl.sub.3 (2 mL). The solution was stirred at room temperature for 5 h. The reaction mixture was evaporated under reduced pressure triturating with diethyl ether (×3). Purification by prep HPLC followed by lyophilisation gave the product as a white foam (0.041 g, 36%). Analytical Data: RT 1.48 min; MS (ES.sup.+) m/z (relative intensity) 1245 ([M+H].sup.+)
Example 2
[0420] ##STR00081##
a) 4-((2S,5S,10S)-10-(((9H-fluoren-9-yl)methoxy)carbonyl)-42-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-54isopropyl-2-methyl-4,7,12,40-tetraoxo-15,18,21,24,27,30,33,36-octaoxa-3,6,11,39-tetraazadotetracontanamido)benzyl (11s,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-(5H)-carboxylate 9
[0421] Acid 4 (0.108 g, 0.2 mmol, 1 eq.) and EDCI hydrochloride (0.057 g, 0.3 mmol, 1.5 eq.) were added to a solution of 6 (0.184 g, 0.2 mmol, 1 eq.) in CHCl.sub.3 (10 mL). The solution was stirred at roam temperature for 18 h when reaction was shown to be complete by LCMS. The reaction mixture was diluted with CHCl.sub.3 (100 mL), washed with water (100 mL), brine (100 mL), dried (MgSO.sub.4) and evaporated under reduced pressure. The product was purified by flash column chromatography [CHCl.sub.3/MeOH 0%] to give the product as a colourless oil (0.064 g, 18%).
b) N2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)-N5-((S)-1-(((S)-1-((4-((((11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamine 10
[0422] A solution of N-Methyl pyrrolidine in CHCl.sub.3 (0.9 mL, 10% v/v, 30 eq.) was added to a solution of Fmoc protected compound 9 (0.055 g, 30 μmol, 1eq) in CHCl.sub.3 (2 mL). The solution was stirred at room temperature for 18 h. The reaction mixture was evaporated under reduced pressure triturating with diethyl ether (×3). Purification by prep HPLC followed by lyophilisation gave the product as a white foam (0.0125 g, 35%). Analytical Data: RT 1.37 min; MS (ES.sup.+) m/z (relative intensity) 1626 ([M+H].sup.+)
Example 3
[0423] ##STR00082## ##STR00083##
a) General Method for Amide Coupling (110 to 11)
[0424] EDCI.Math.HCl (0.28 mmol, 1.25eq) was added to a solution of 110 (0.22 mmol, 1.0eq) and 2 (0.27 mmol, 1.2eq) in chloroform (10 mL) and the resulting mixture stirred at room temperature for 2 hrs. The reaction mixture was washed with water (20 mL), dried (biotage) and evaporated to leave a white solid which was purified by column.
i) 4-((S)-2-((S)-2-((S)-5-((9H-fluoren-9-yl)methoxy)-4-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-5-oxopentanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-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-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-e][1,4]diazepine-10(5H)-carboxylate 11a
[0425] (methanol/DCM, 4/96 v/v) to leave 11a as a white solid. Yield=275 mg (86%). LC/MS rt 1.80 min m/z (1448.0) M+H.
ii) 4-((S)-2-((S)-2-((S)-5-((9H-fluoren-9-yl)methoxy)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-5-oxopentanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-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-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 11b
[0426] (methanol/DCM, 4/96 v/v) to leave 11b as a white solid. Yield=256 mg (81%). LC/MS rt 1.85 min m/z (1489.7) M+H.
b) General Method for Boc Deprotection of 11 to 12
[0427] TFA (1.8 mL) and water (0.2 mL) were added to a solution of 3 (0.18 mmol) in water (0.2 mL) and stirred at 0° C. for 30 mins. The solvent was removed under vacuum and saturated sodium hydrogen carbonate was added to neutralise the reaction, along with DCM to aid solubility. The organic phase was removed, dried (biotage) and evaporated. The resulting residue was purified by column.
i) 4-((S)-2-((S)-2-((S)-5-((9H-fluoren-9-yl)methoxy)-4-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-5-oxopentanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 12a
[0428] (gradient: methanol/DCM, 2/98 to 7/93 vN) to leave 12a as a white solid. Yield=256 mg (81%). LC/MS rt 1.85 min m/z (1489.7) M+H.
ii) 4-((S)-2-((S)-2-((S)-5-((9H-fluoren-9-yl)methoxy)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-5-oxopentanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate 13b
[0429] (methanol/chloroform, 5/95 v/v) to leave 13b as a white solid. Yield=181 mg (79%). LC/MS rt 1.79 min m/z (1317.1) M+H.
c) General Method of Acid Deprotection of 12 to 13
[0430] 1-Methylpyrrolidine (250 μL) was added to a solution of 12 (0.13 mmol) in DMF (2.5 mL) and stirred at room temperature for 10 mins. The solvent was removed under vacuum and the residue purified by prep HPLC (30-35% MeCN/water+0.1% formic acid over 10 mins). Fractions containing product were freeze dried to give an off-white solid.
i) N2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-N5-((S)-1-(((S)-1-((4-((((11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamine 13a
[0431] Yield=23 mg (15%). LC/MS rt 1.46 min m/z (1151.3) M+H.
ii) N2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-N5-((S)-1-(((S)-1-((4-((((11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamine 13b
[0432] Yield=60 mg (38%). LC/MS rt 1.50 min m/z (1193.3) M+H.
Example 4—Conjugation
Conjugate Tratuzumab-13a (ConjA)
[0433] A 50 mM solution of
[0434] 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 ConjA at 214 nm and 330 nm (Compound 13a specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 13a, consistent with a drug-per-antibody ratio (DAR) of 1.77 molecules of Compound 13a per antibody.
[0435] 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 ConjA at 280 nm shows a monomer purity of 98%. UHPLC SEC analysis gives a concentration of final ConjA at 1.96 mg/mL in 11.5 mL, obtained mass of ConjA is 22.6 mg (75% yield).
Conjugate Tratuzumab-13a (ConjB)
[0436] A 50 mM solution of
[0437] 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 ConjB at 214 nm and 330 nm (Compound 13a specific) shows a mixture of unconjugated light chains, light chains attached to a single molecule of Compound 13a, unconjugated heavy chains and heavy chains attached to up to three molecules of Compound 13a, consistent with a drug-per-antibody ratio (DAR) of 7.82 molecules of Compound 13a per antibody.
[0438] 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 ConjB at 280 nm shows a monomer purity of 96.5%. UHPLC SEC analysis gives a concentration of final ConjA at 2.40 mg/mL in 11.9 mL, obtained mass of ConjB is 28.6 mg (95% yield).
Herceptin-C239i Antibody
[0439] Herceptin antibodies were engineered to have cysteine inserted between the 239 and 240 positions were produced following the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 5 10.1021/acs.molpharmaceut.6b00995).
Conjugate Herceptin-8 (ConjC)
[0440] Herceptin-C239i antibody (30 mg) was loaded onto solid support and reduced, reoxidised, conjugated to Compound 8, purified, released from the resin and formulated onto 25 mM Histidine, 200 mM Sucrose, Tween-20 0.02%, pH 6.0 according to patent #US 2014/038041 A1.
[0441] 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 ConjC at 214 nm and 330 nm (Compound 8 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 8, consistent with a drug-per-antibody ratio (DAR) of 1.9 molecules of Compound 8 per antibody.
[0442] 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 ConjC at 280 nm shows a monomer purity of 95%. UHPLC SEC analysis gives a concentration of ConjC at 2.2 mg/mL in 5.85 mL, obtained mass of ConjC is 12.9 mg (43% yield).
Example 5—Xenograft Testing
NCI-N87 Xenografted Mice
[0443] Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17/lcr-Prkdcscid, Charles River) were ten weeks old with a body weight (BW) range of 16.5 to 21.1 grams on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm CI), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on irradiated Enricho'cobs™ Laboratory Animal Bedding in static micro-isolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and 40-60% humidity. CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.
Tumour Cell Culture
[0444] Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin sulfate and 25 μg/mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37° C., in an atmosphere of 5% CO.sub.2 and 95% air.
In Vivo Implantation and Tumour Growth
[0445] The NCI-N87 cells used for implantation were harvested during log phase growth and Re-suspended in phosphate buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumour implant, each test mouse was injected subcutaneously in the right flank with 1×10.sup.7 cells (0.1 mL cell suspension), and tumour growth was monitored as the average size approached the target range of 100 to 150 mm.sup.3. Fourteen days later, designated as Day 1 of the study, mice were sorted according to calculated tumour size into groups each consisting of ten animals with individual tumour volumes ranging from 108 to 144 mm.sup.3 and group mean tumour volumes of 115 mm.sup.3.
[0446] Tumours were measured in two dimensions using calipers, and volume was calculated using the formula:
where w=width and l=length, in mm, of the tumour. Tumour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumour volume.
Treatment
[0447] Treatment began on Day 1 in groups of 10 mice (n=10) with established subcutaneous NCI-N87 tumours (108-144 mm.sup.3). ConjA (6 mg/kg) and ConjB (3 mg/kg) were administered intravenously once on Day 1 (qd×1). A vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 83. Each mouse was euthanized when its tumour reached the endpoint volume of 800 mm.sup.3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse.
[0448] The results are illustrated in
Endpoint and Tumor Growth Delay (TGD) Analysis
[0449] 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.
Tumour Growth Inhibition
[0450] 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:
[0451] 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.
MTV and Criteria for Regression Responses
[0452] 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.
Toxicity
[0453] 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.
Statistical and Graphical Analyses
[0454] 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≤5 0.05, very significant (“**”) at 0.001<P≤5 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-00001 Median MTV (n), n TTE T − C % TGD Day 82 Vehicle — 10 45.7 — — — ConjA 6 mg/kg 10 77.8 32.1 70 446 (4) ConjB 3 mg/kg 10 82 36.3 79 486 (7)
TABLE-US-00002 BW PR CR TFS Nadir TR NTRm NTR Vehicle 0 0 0 −0.5% (3) 0 0 0 ConjA 0 0 0 — 0 0 0 ConjB 0 0 0 — 0 0 0
[0455] A further study was carried out with ConjB being dosed at 4 mg/kg, in an analogous manner to the above. The study completed on Day 79, and the results are shown in
TABLE-US-00003 Median MTV (n), n TTE T − C % TGD Day 79 Vehicle — 10 24.8 — — — ConjB 4 mg/kg 10 79 54.2 218 —
TABLE-US-00004 BW PR CR TFS Nadir TR NTRm NTR Vehicle 0 0 0 −2.0% (2) 0 0 0 ConjA 0 0 0 −8.0% (50) 0 0 0
Example 6—ADC Cytotoxicity Method MTS
[0456] The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining, and counted using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2×10.sup.5/ml, dispensed (50 μl per well) into 96-well flat-bottom plates.
[0457] A stock solution (1 ml) of ConjC (20 μg/ml) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 8×10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 μl into 900 μl of cell culture medium. ADC dilution was dispensed (50 μl per well) into 4 replicate wells of the 96-well plate, containing 50 μl cell suspension seeded the day previously. Control wells received 50 μl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37° C. in a CO.sub.2-gassed incubator for the exposure time.
[0458] At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 μl per well) into each well and incubated for 4 hours at 37° C. in the CO.sub.2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC.sub.50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.
[0459] ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87. MDA-MB-468 and NCI-N87 were cultured in RPMI 1640 with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum.
TABLE-US-00005 EC.sub.50 (μM) NCI-N87 MDA-MB-468 ConjC 0.0003081 1.869
[0460] All documents and other references mentioned above are herein incorporated by reference.
EMBODIMENTS OF INVENTION
[0461] 1. A compound of formula I:
##STR00084##
and salts and solvates thereof, wherein:
D represents either group D1 or D2:
##STR00085##
the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of:
(ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(ib) C.sub.1-5 saturated aliphatic alkyl;
(ic) C.sub.3-6 saturated cycloalkyl;
(id)
##STR00086##
wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;
(ie)
##STR00087##
wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(if)
##STR00088##
where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2 and C3,
[0462] R.sup.2 is selected from H, OH, F, diF and
##STR00089##
where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;
D′ represents either group D′1 or D′2:
##STR00090##
wherein the dotted line indicates the optional presence of a double bond between C2′ and C3′;
when there is a double bond present between C2′ and C3′, R.sup.22 is selected from the group consisting of:
(iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
(iib) C.sub.1-5 saturated aliphatic alkyl;
(iic) C.sub.3-6 saturated cycloalkyl;
(iid)
##STR00091##
wherein each of R.sup.21, R.sup.22a and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.22 group is no more than 5;
(iie)
##STR00092##
wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(iif)
##STR00093##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
when there is a single bond present between C2′ and C3′,
R.sup.22 is selected from H, OH, F, diF and
##STR00094##
where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;
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, NRN2 (where R.sup.N 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
##STR00095##
wherein
[0463] Q is a tripeptide residue of formula:
##STR00096##
where x is 1 or 2, —C(═O)-Q.sup.X-NH— is a dipeptide residue;
[0464] X is:
##STR00097##
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;
either [0465] (a) R.sup.3 is H, and R.sup.31 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or [0466] (b) R.sup.3 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or [0467] (c) R.sup.3 is H and R.sup.31 is SOM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or [0468] (d) R.sup.3 is H and R.sup.31 is H or ═O; or [0469] (e) R.sup.31 is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.30 is selected from:
##STR00098##
where R.sup.Z is selected from:
##STR00099## [0470] (z-ii) OC(═O)CH.sub.3; [0471] (z-iii) NO.sub.2; [0472] (z-iv) OMe; [0473] (z-v) glucoronide; [0474] (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 0.
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:
##STR00100##
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 D is D1, there is a double bond between C2 and C3, and R.sup.2 is a C.sub.5-7 aryl group.
10. A compound according to statement 9, wherein R.sup.2 is phenyl.
11. A compound according to any one of statements 1 to 8, wherein D is D1, there is a double bond between C2 and C3, and R.sup.2 is a C.sub.8-10 aryl group.
12. A compound according to any one of statements 9 to 11, wherein R.sup.2 bears one to three substituent groups.
13. A compound according to any one of statements 9 to 12, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.
14. A compound according to any one of statements 1 to 8, wherein D is D1, there is a double bond between C2 and C3, and R.sup.2 is a C.sub.1-5 saturated aliphatic alkyl group.
15. A compound according to statement 14, wherein R.sup.2 is methyl, ethyl or propyl.
16. A compound according to any one of statements 1 to 7, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.3-6 saturated cycloalkyl group.
17. A compound according to statement 16, wherein R.sup.2 is cyclopropyl.
18. A compound according to any one of statements 1 to 8, wherein D is D1, there is a double bond between C2 and C3, and R.sup.2 is a group of formula:
##STR00101##
19. A compound according to statement 18, wherein the total number of carbon atoms in the R.sup.2 group is no more than 4.
20. A compound according to statement 19, wherein the total number of carbon atoms in the R.sup.2 group is no more than 3.
21. A compound according to any one of statements 18 to 20, wherein one of R.sup.11, R.sup.12 and R.sup.13 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
22. A compound according to any one of statements 18 to 20, wherein two of R.sup.11, R.sup.12 and R.sup.13 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
23. A compound according to any one of statements 1 to 8, wherein D is D1, there is a double bond between C2 and C3, and R.sup.2 is a group of formula:
##STR00102##
24. A compound according to statement 23, wherein R.sup.2 is the group:
##STR00103##
25. A compound according to any one of statements 1 to 8, wherein D is D1, there is a double bond between C2 and C3, and R.sup.2 is a group of formula:
##STR00104##
26. A compound according to statement 25, wherein R.sup.14 is selected from H, methyl, ethyl, ethenyl and ethynyl.
27. A compound according to statement 26, wherein R.sup.14 is selected from H and methyl.
28. A compound according to any one of statements 1 to 8, wherein D is D1, there is a single bond between C2 and C3, and R.sup.2 is H.
29. A compound according to any one of statements 1 to 8, wherein D is D1, there is a single bond between C2 and C3, R.sup.2 is
##STR00105##
and R.sup.16a and R.sup.16b are both H.
30. A compound according to any one of statements 1 to 8, wherein D is D1, there is a single bond between C2 and C3, R.sup.2 is
##STR00106##
and R.sup.16a and R.sup.16b are both methyl.
31. A compound according to any one of statements 1 to 8, wherein D is D1, there is a single bond between C2 and C3, R.sup.2 is
##STR00107##
one of R.sup.16a and R.sup.16b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
32. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.22 is a C.sub.5-7 aryl group.
33. A compound according to statement 32, wherein R.sup.22 is phenyl.
34. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.22 is a C.sub.8-10 aryl group.
35. A compound according to any one of statements 32 to 34, wherein R.sup.2 bears one to three substituent groups.
36. A compound according to any one of statements 32 to 35, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.
37. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.1 is a C.sub.1-5 saturated aliphatic alkyl group.
38. A compound according to statement 37, wherein R.sup.1 is methyl, ethyl or propyl.
39. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.22 is a C.sub.3-6 saturated cycloalkyl group.
40. A compound according to statement 39, wherein R.sup.1 is cyclopropyl.
41. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.22 is a group of formula:
##STR00108##
42. A compound according to statement 41, wherein the total number of carbon atoms in the R.sup.1 group is no more than 4.
43. A compound according to statement 42, wherein the total number of carbon atoms in the R.sup.1 group is no more than 3.
44. A compound according to any one of statements 41 to 43, wherein one of R.sup.21, R.sup.22a and R.sup.23 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
45. A compound according to any one of statements 41 to 43, wherein two of R.sup.21, R.sup.22a and R.sup.23 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
46. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.22 is a group of formula:
##STR00109##
47. A compound according to statement 46, wherein R.sup.2 is the group:
##STR00110##
48. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a double bond between C2′ and C3′, and R.sup.1 is a group of formula:
##STR00111##
49. A compound according to statement 48, wherein R.sup.24 is selected from H, methyl, ethyl, ethenyl and ethynyl.
50. A compound according to statement 49, wherein R.sup.24 is selected from H and methyl.
51. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a single bond between C2′ and C3′, and R.sup.22 is H.
52. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a single bond between C2′ and C3′, R.sup.22 is
##STR00112##
and R.sup.26a and R.sup.26b are both H.
53. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a single bond between C2′ and C3′, R.sup.22 is
##STR00113##
and R.sup.26a and R.sup.26b are both methyl.
54. A compound according to any one of statements 1 to 31, wherein D′ is D′1, there is a single bond between C2′ and C3′, R.sup.22 is
##STR00114##
one of R.sup.26a and R.sup.26b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
55. A compound according to any one of statements 1 to 54, wherein R.sup.7 is selected from the same groups as R.sup.6, R.sup.7′ is selected from the same groups as R.sup.7, R.sup.9′ is selected from the same groups as R.sup.9 and Y is selected from the same groups as Y.
56. A compound according to statement 55, wherein R.sup.6′ is the same groups as R.sup.6, Rr is the same groups as R.sup.7, R.sup.9′ is the same groups as R.sup.9 and Y is the same groups as Y.
57. A compound according to any one of statements 1 to 56, wherein R.sup.2 is the same group as R.sup.2.
59. A compound according to any one of statements 1 to 57, wherein R.sup.3 is H, and R.sup.31 is OH.
60. A compound according to any one of statements 1 to 57, wherein R.sup.0 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
61. A compound according to any one of statements 1 to 57, wherein R.sup.0 is H, and R.sup.31 is H.
62. A compound according to any one of statements 1 to 57, wherein R.sup.0 is H, and R.sup.31 is ═O.
63. A compound according to any one of statements 1 to 57, wherein R.sup.31 is OH or OR.sup.A and R.sup.30 is selected from:
##STR00115## ##STR00116##
where —C(═Oy-X.sub.1—NHC(═O)X.sub.2—NH— represent a dipeptide.
64. A compound according to statement 63, 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-.
65. The conjugate according to statement 64, wherein —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, and -Val-Ala-.
66. A compound according to statement 1, which is of formula Ia-1, Ia-2 or Ia-3
##STR00117##
where R.sup.2a and R.sup.12a are the same and are selected from:
##STR00118##
R.sup.1a is selected from methyl and benzyl;
R.sup.L and R.sup.11b are as defined in statement 1.
67. A compound according to any one of statements 1 to 66, wherein R.sup.11b is OH.
68. A compound according to any one of statements 1 to 66, wherein R.sup.11b is OR.sup.A, where R.sup.A is C.sub.1-4 alkyl.
69. A compound according to statement 68, wherein R.sup.A is methyl.
70. A compound according to any one of statements 1 to 69, wherein —C(═O)-Q.sup.x-NH— is a dipeptide residue selected from: [0475] .sup.COPhe-Lys-.sup.NH, [0476] .sup.COVal-Ala-.sup.NH, [0477] .sup.CO-Val-Lys-.sup.NH, [0478] .sup.CO-Ala-Lys-.sup.NH, [0479] .sup.CO-Val-Cit-.sup.NH, [0480] .sup.CO-Phe-Cit-.sup.NH, [0481] .sup.CO-Leu-Cit-.sup.NH, [0482] .sup.CO-Ile-Cit-.sup.NH, [0483] .sup.CO-Phe-Arg-.sup.NH, and [0484] .sup.CO-Trp-Cit-.sup.NH.
71. A compound according to statement 70, wherein —C(═O)-Q.sup.X-NH— is selected from .sup.CO-Phe-Lys-N, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Ala-.sup.NH.
72. A compound according to any one of statements 1 to 71 wherein x is 1.
73. A compound according to any one of statements 1 to 71 wherein x is 2.
74. A compound according to any one of statements 1 to 73, wherein a is 0 to 3.
75. A compound according to statement 74, wherein a is 0.
76. A compound according to any one of statements 1 to 75, wherein b is 0 to 12.
77. A compound according to statement 76, wherein b is 0 to 8.
78. A compound according to any one of statements 1 to 77, wherein d is 0 to 3.
79. A compound according to statement 78, wherein d is 2.
80. A compound according to any one of statements 1 to 73, wherein a is 0, c is 1 and d is 2, and b is from 0, 4 or 8.
81. A compound according to any one of statements 1 to 73, wherein a, b and c are 0 and d is 2 or 5.
82. A compound according to any one of statements 1 to 81, wherein G.sup.L is selected from:
##STR00119## ##STR00120##
where Ar represents a C.sub.5-6 arylene group.
83. A compound according to statement 82, wherein Ar is a phenylene group.
84. A compound according to either statement 82 or statement 83, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.
85. A compound according to statement 84, wherein G.sup.L is G.sup.L1-1.
86. A conjugate of formula I:
L−(D.sup.L).sub.p (1)
wherein L is a Ligand unit, D is a Drug Linker unit of formula I′:
##STR00121##
wherein D, R.sup.2, R.sup.6, R.sup.7, R.sup.9, R.sup.11b, Y, R″, Y′, D′, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.22, R.sup.30 and R.sup.31, including the presence or absence of double bonds between C2 and C3 and C2′ and C3′ respectively, are as defined in any one of statements 1 to 69;
R.sup.LL is a linker for connection to a cell binding agent, which is:
##STR00122##
where Q and X are as defined in any one of statements 1 and 70 to 81 and G.sup.LL is a linker connected to the Ligand Unit;
wherein p is an integer of from 1 to 20.
87. A conjugate according to statement 86, wherein G.sup.LL is selected from:
##STR00123## ##STR00124##
where Ar represents a C.sub.5-6 arylene group.
88. A conjugate according to statement 87, wherein Ar is a phenylene group.
89. A compound according to either statement 87 or statement 88, wherein G.sup.LL is selected from G.sup.LL1-1and G.sup.LL1-2.
90. A compound according to statement 89, wherein G.sup.LL is G.sup.LL1-1.
91. A conjugate according to any one of statements 86 to 90, wherein the Ligand Unit is an antibody or an active fragment thereof.
92. The conjugate according to statement 91, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.
93. The conjugate according to statement 94 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;
[0485] (8) PSCA hlg;
(9) ETBR;
(10) MSG783;
(11) STEAP2;
(12) TrpM4;
(13) CRIPTO;
(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) EGFRvII;
(46) CD33;
(47) CD19;
(48) IL2R.SUP.A.;
(49) AXL;
(50) CD30-TNFRSF8;
(51) BCMA-TNFRSFI7;
(52) CT Ags-CTA;
(53) CD174 (Lewis Y)-FUT3;
(54) CLECI4A;
(55) GRP78-HSPA5;
(56) CD70;
[0486] (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-HAVCRI;
[0487] (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).
[0488] 94. The conjugate of any one of statements 91 to 93 wherein the antibody or antibody fragment is a cysteine-engineered antibody.
95. The conjugate according to any one of statements 86 to 94 wherein p is an integer from 1 to 8.
96. The conjugate according to statement 95, wherein p is 1, 2, 3, or 4.
97. A composition comprising a mixture of conjugates according to any one of statements 86 to 96, wherein the average p in the mixture of conjugate compounds is about 1 to about 8.
98. The conjugate according to any one of statements 86 to 96, for use in therapy.
99. A pharmaceutical composition comprising the conjugate of any one of statements 86 to 96 a pharmaceutically acceptable diluent, carrier or excipient.
100. The conjugate according to any one of statements 86 to 96 or the pharmaceutical composition according to statement 99, for use in the treatment of a proliferative disease in a subject.
101. The conjugate for use according to statement 100, wherein the disease treated is cancer.
102. Use of a conjugate according to any one of statements 86 to 96 or a pharmaceutical according to statement 99 in a method of medical treatment.
103. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 99.
104. The method of statement 103 wherein the method of medical treatment is for treating cancer.
105. The method of statement 104, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.
106. Use of a conjugate according to any one of statements 86 to 96 in a method of manufacture of a medicament for the treatment of a proliferative disease.
107. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to any one of statements 86 to 96 or a pharmaceutical composition according to statement 99.