HK1116777B

HK1116777B - Improved cytotoxic agents comprising new maytansinoids

Info

Publication number: HK1116777B
Application number: HK08107275.3A
Authority: HK
Inventors: W．C．威迪逊; R．V．J．沙利
Original assignee: 伊缪诺金公司
Priority date: 2003-05-20
Filing date: 2008-07-02
Publication date: 2015-07-31

Description

Improved cytotoxic agents containing novel maytansinoids

This application claims the benefit of provisional application No. 60/471,739 filed on 20/5/2003, the disclosure of which is incorporated herein by reference.

Technical Field

The present invention relates to a process for the preparation of improved cytotoxic conjugates (conjugates) containing maytansinoids and cell-binding agents. These conjugates have therapeutic effects when delivered to a specific cell population in a targeted manner. The present invention also relates to a process for preparing maytansinoids having a thiol moiety, which may be used to prepare cytotoxic conjugates. The invention further relates to novel maytansinoids, and to novel intermediates in the synthesis of novel maytansinoids.

Background

There are many reports that have been attempted to specifically target tumor cells with monoclonal Antibody-drug conjugates (Sela et al, Immunoconjugates189-216(C.Vogel, ed.1987); Ghose et al, Targeted Drugs1-22(E.Goldberg, ed.1983); diene et al, Antibody medical Delivery Systems1-23(J.Rodwell, ed.1988); Pietesz et al, Antibody medical Delivery Systems25-53(J.Rodwell, ed.1988); Bumol et al, Antibody medical Delivery Systems55-79(J.Rodwell, ed.1988); cytotoxic Drugs such as methotrexate, daunorubicin, vincristine, vinblastine, vincristine, C, and C.23, and intermediate molecules linked to a murine molecule via monoclonal Antibody-protein kinase (C.46. C.7: C.23-4; such as described by monoclonal Antibody-drug conjugates (C.7: C.23; see et al, 23. 7. intermediate molecules) were linked to a cytotoxic drug such as methotrexate, daunorubicin, doxorubicin, vincristine, a-6, a protein, C.23, 23, 2. medium molecules (C.23; and 5. C.23; see (C.23, C.1988) and C.23; see (C.23; see, et al.23 1080(1980), dextran (Hurwitz et al. appl. biochem.2: 25-35 (1980); manabi et al biochem pharmacol.34: 289 (1985); dialman et al cancer res.46: 4886 (1986) and 4891; shoval et al proc. natl. acad. sci.85: 8276-8280(1988)), or polyglutamic acid (Tsukada et al.j. natl. cane. inst.73: 721-729 (1984); kato et al.j.med.chem.27: 1602-; tsukada et al.Br.J. Cancer 52: 111-116(1985)).

Various linker (linker) techniques have been used to prepare such immunoconjugates, and cleavable linkers and non-cleavable linkers have been investigated. However, in most cases, the full cytotoxic potential of the drug is only observed when the drug molecule can be released from the conjugate in an unmodified manner at the target site.

One of the cleavable linkers that has been used to prepare antibody-drug conjugates is an acid-labile linker based on aconitic acid, which utilizes the acidic environment of different intracellular compartments, such as endosomes and lysosomes encountered during receptor-mediated endocytosis. Shen and Ryser introduced this method to prepare conjugates of daunorubicin and macromolecular carriers (biochem. Biophys. Res. Commun.102: 1048-1054 (1981)). Yang and Reisfeld used the same technique to link daunorubicin to anti-melanoma antibodies (J.Natl.Canc.Inst.80: 1154-1159 (1988)). More recently, Dillman et al also used acid-labile linkers in a similar manner to prepare conjugates of daunorubicin and anti-T cell antibodies (Cancer Res.48: 6097-6102 (1988)).

An alternative method developed by Trouet et al involves linking daunorubicin to antibodies via a peptide spacer arm (peptide spacer arm) (Proc. Natl. Acad. Sci.79: 626-629 (1982)). This is done on the premise that the free drug can be released from such conjugates by the action of lysosomal peptidases.

However, in vitro cytotoxicity experiments revealed that antibody-drug conjugates rarely achieved the same cytotoxic capacity as the free, non-linked drug. This suggests that the mechanism by which drug molecules are released from antibodies is very inefficient. In the field of immunotoxins, it has been shown that conjugates formed by disulfide bonds between monoclonal antibodies and catalytically active protein toxins are more cytotoxic than conjugates containing other linkers. See, Lambert et al.j.biol.chem.260: 12035-12041 (1985); lambert et al.Immunotoxins175-209(A.Frankel, ed.1988); ghetie et al. cancer res.48: 2610-2617(1988). This is attributed to the high glutathione concentration in the cell, which can effectively cleave the disulfide bond between the antibody molecule and the toxin. However, only a few examples have been reported of the use of disulfide bridges in the preparation of conjugates between drugs and macromolecules. Shen et al describe the conversion of methotrexate to mercaptoacetamide derivatives after disulfide linkage to poly-D-lysine (J.biol.chem.260: 10905-10908 (1985)). In addition, some reports describe the preparation of conjugates of the toxic trisulfide-containing drug calicheamicin (calicheamicin) with antibodies (Hinman et al, 53Cancer Res.3336-3342(1993), Hamann et al, Bioconjugate chem., 13, 40-46(2002), Hamann et al, Bioconjugate chem., 13, 47-58 (2002)).

One reason for the lack of disulfide-linked antibody-drug conjugates is that cytotoxic drugs having sulfur atom containing moieties are not always available, which are required to conveniently disulfide-link the drug to the antibody. Furthermore, it is difficult to chemically modify existing drugs without reducing their cytotoxic potential.

Maytansinoids are highly cytotoxic drugs. Maytansine (maytansine) was first isolated from the east African shrub Maytenus serrata by Kupchan et al, and is 100 to 1000 times more cytotoxic than traditional cancer chemotherapeutics such as methotrexate, daunorubicin, and vincristine (U.S. Pat. No. 3,896,111). Subsequently, it was found that some microorganisms also produce maytansinoids, such as maytansinol (maytansinol) and C-3 esters of maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 maytansinol esters and analogues of maytansinol have also been reported (Kupchan et al J. Med. chem.21: 31-37 (1978); Higashide et al Nature 270: 721-. The C-3 ester is prepared from maytansinol, and examples of such maytansinol analogues include maytansinol which has modifications in the aromatic ring (e.g., dechlorinated) or modified maytansinol at C-9, C-14 (e.g., hydroxylated methyl), maytansinol with modifications at C-15, C-18, C-20 and C-4, 5.

Naturally occurring maytansinol C-3 esters and synthetic maytansinol C-3 esters can be divided into two groups:

(a) c-3 esters with simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348 and 4,331,598) and

(b) c-3 esters of derivatives with N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; 5,208,020 and chem. pharm. Bull. 12: 3441 (1984)).

The esters of group (b) were found to be more cytotoxic than the esters of group (a).

Maytansine is a mitotic inhibitor. It has been reported that treatment of L1210 cells with maytansine in vivo results in 67% of the cells accumulating in mitosis. Untreated control cells are reported to exhibit mitotic indices ranging between 3.2% and 5.8% (Sieber et al 43comparative Leukemia Research1975, bibl Haemat.495-500 (1976)). Experiments with sea urchin eggs and clam eggs suggest that maytansine interferes with microtubule formation by inhibiting tubulin (microtubule), the polymerization of tubulin, and thereby inhibits mitosis (reminlard et al, science 189: 1002-1005 (1975)).

In vitro, P388, L1210 and LY5178 murine leukemia cell suspensions have been found to be maytansineThe dose of maytansine is 10^-3To 10^-1μ g/. mu.l, of which the P388 cell line is the most sensitive. Maytansine has also been shown to be an active inhibitor of the in vitro growth of human nasopharyngeal carcinoma cells, and human acute lymphoblastic leukemia line CEM has been reported to be as low as 10^-7The concentration of. mu.g/ml is inhibited (Wolpert-DeFillippes et al. biochem. Pharmacol.24: 1735-1738 (1975)).

In vivo, maytansine has also been shown to be active. Tumor growth in the P388 lymphocytic leukemia system was inhibited in the 50-fold to 100-fold dose range, indicating a high therapeutic index; significant inhibitory activity was also shown for the L1210 murine leukemia system, the human Lewis lung carcinoma system and the human B-16 melanoma system (Kupchan, ped. Proc. 33: 2288-2295 (1974)). In U.S. Pat. nos. 5,208,020 and 5,416,064 and Chari et al, Cancer res, 52: 127-: the maytansinoids used to bind cell-binding agents are described in 8618-8623 (1996). In these conjugates, the cell binding agent is linked to the maytansinoid DM1[ N ] by a disulfide bond^2’-deacetylation-N-^2’(3-mercapto-1-oxopropyl) -maytansine, 1, CAS No.: 139504-50-0, FIG. 1]。

In the above patents, maytansinoids with acylated N-methyl-L-alanine side chains have the formula 2a, b:

in formula 2a, 1 represents an integer of from 1 to 10. Thus, the maytansinoids of formula 2a have a sulfur atom (-CH) attached to an unsubstituted methylene group₂-S-). The sulfhydryl groups in such maytansinoids or disulfide groups in cell-binding agent-maytansinoid conjugates with such maytansinoids linked by disulfide bonds are said to be "non-hindered" in that there are no α -carbons adjacent to the sulfhydryl or disulfide groupsBulky substituents that cause steric hindrance. In formula 2b, m represents 0, 1, 2 or 3. Thus, maytansinoids of formula 2b also have a sulfur atom attached to an unsubstituted methylene group, m =0 and R₂=CH₃Or CH₂CH₃Except for the case (1). If m =0, the maytansinoid has a substituent on the carbon bearing a thiol (thiol) or disulfide (disulfide) functionality after attachment to the cell-binding agent by a disulfide bond. However, since in this case the sulphur atom is in the beta position relative to the carbonyl group, these maytansinoids and conjugates of these maytansinoids with cell-binding agents via disulphide bonds were found to be unstable due to their tendency to undergo beta-elimination.

Summary of The Invention

The present invention is based on the following unexpected findings: the attachment of maytansinoids bearing a sterically hindered thiol group (with one or two substituents on the α -carbon bearing the thiol functional group) to cell-binding agents results in conjugates having greatly improved in vivo anti-tumor activity, as compared to conjugates prepared with the previously described maytansinoids which do not have substituents on the α -carbon bearing the disulfide bond. It was further unexpectedly found that to obtain improved biological activity, steric hindrance optimally occurs on the maytansinoid side of the disulfide bond in the conjugate. In addition, the acyl group in the side chain of the acylated amino acid of the sulfhydryl-bearing maytansinoid must have a linear chain length of at least 3 carbon atoms between the carbonyl group and the sulfur atom of the amide.

These findings indicate that disulfide-linked (disulphide-linked) cell-binding agent-maytansinoid conjugates can be constructed such that substitutions at the two α -carbon atoms bearing the disulfide bond can result in different degrees of steric hindrance on either side of the disulfide bond.

Thus, the present invention describes the synthesis of novel sterically hindered thiol and disulfide containing maytansinoids with one or two alkyl substituents on the alpha-carbon bearing the sulfur atom. In addition, the acyl group of the side chain of the acylated amino acid, between the carbonyl and sulfur atoms of the amide, has a linear chain length of at least 3 carbon atoms.

The preparation and biological evaluation of these novel maytansinoid cell-binding agent conjugates is also described.

In one embodiment of the invention, novel thiol and disulfide containing maytansinoids are described having mono-or di-alkyl substitution at the carbon atom bearing the sulfur atom.

In a second embodiment, the invention discloses a process for the synthesis of these novel maytansinoids.

In a third embodiment, a method for linking these novel maytansinoids to a cell-binding agent is described. These conjugates are useful as therapeutic agents, are specifically delivered to target cells, and are cytotoxic. These conjugates show greatly improved therapeutic effects in animal tumor models compared to previously described formulations.

More specifically, the present invention provides:

maytansinoids with an acylated amino acid side chain bearing an acyl group with a sterically hindered thiol group at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl (desmethyl) group, wherein the acyl carbon atom bearing a thiol function has one or two substituents which are CH₃、C₂H₅Linear or branched alkyl or alkenyl groups having from 1 to 10 carbon atoms, cycloalkyl or cycloalkenyl groups having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aryl or heterocyclic groups, further one of the substituents may be H, and wherein the acyl group is between a carbonyl functional group (carbonyl functionality) and a sulfur atom, having a linear chain length of at least 3 carbon atoms;

a compound represented by formula 4':

wherein:

y' represents

(CR₇CR₈)_l(CR₉=CR₁₀)_p(C≡C)_qA_r(CR₅CR₆)_mD_u(CR₁₁=CR₁₂)_r(C≡C)_sB_t (CR₃R₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or linear alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic groups, and furthermore R₂May be H;

A. b, D is a cycloalkyl or cycloalkenyl group having 3-10 carbon atoms, a simple aryl or substituted aryl or heterocyclic group;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having I to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl or heterocyclic group;

each of l, m, n, o, p, q, r, s, and t is independently 0 or an integer from 1 to 5, provided that at least 2 of l, m, n, o, p, q, r, s, and t are not simultaneously 0.

Z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple aryl or substituted aryl or heterocyclic group.

A compound represented by formula 4', wherein R₁Is methyl, R₂Is H and Z is H.

A compound represented by formula 4', wherein R₁And R₂Is methyl and Z is H.

A compound represented by formula 4', wherein R₁Is methyl, R₂Is H, Z is-SCH₃。

A compound represented by formula 4', wherein R₁And R₂Is methyl, Z is-SCH₃。

A compound represented by formula (I-L), (I-D) or (I-D, L):

wherein:

y represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ, wherein:

R₁and R₂Each is independently CH₃、C₂H₅A linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group, or a heterocyclic aryl or heterocyclic group, and further, R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group, or a heterocyclic aryl or heterocyclic group;

each of l, m, and n is independently an integer from 1 to 5, and further n may be 0:

z is H, SR or-COR, wherein R is a linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, or a simple aryl or substituted aryl or heterocyclic group; and

may represents a maytansinoid bearing a side chain at a C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation group;

the above compound wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, Z is H;

the above compound wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Are each H, l and m are 1, n is 0, Z is H;

the above compound wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, Z is-SCH₃；

The above compound wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Are each H, l and m are 1, n is 0, Z is-SCH₃；

A compound represented by formula 4:

wherein:

y represents (CR)₇R₈)_l(CR₅R₆)_m(CR₃R₄)_nCR₁R₂SZ, wherein:

R₁and R₂Each is independently CH₃、C₂H₅A linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocyclic group, and furthermore, R₂May be H;

each of l, m, and n is independently an integer from 1 to 5, and further n may be 0; and

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple aryl or substituted aryl or heterocyclic group;

a compound of formula 4 wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Each of (a) is H; each of l and m is 1; n is 0; z is H;

a compound of formula 4 wherein R₁And R₂Is methyl; r₅、R₆、R₇、R₈Each of (a) is H; l and m are1; n is 0; z is H;

a compound of formula 4 wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, Z is-SCH₃；

A compound of formula 4 wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Are each H, l and m are 1, n is 0, Z is-SCH₃；

A maytansinoid-cell-binding agent conjugate comprising at least one maytansinoid linked to a cell-binding agent, wherein the maytansinoid is any one of the compounds described above;

any of the maytansinoid-cell-binding agent conjugates described above, wherein the cell-binding agent comprises at least one antibody binding site, which is preferably humanized (humanized) MY9 or resurfaced (resurfaced) MY9, humanized anti-B4 or resurfaced anti-B4, or humanized C242 or resurfaced C242;

a pharmaceutical composition comprising an effective amount of any of the maytansinoid-cell-binding agent conjugates described above, a pharmaceutically acceptable salt or solvate thereof (solvate), and a pharmaceutically acceptable carrier, diluent or excipient;

method for esterifying maytansinoids at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl to give maytansinoids with an acylated amino acid side chain, where the acyl group has a protected thiol functionality, wherein the acyl carbon atom bearing the protected thiol functionality has one or two substituents which are CH₃、C₂H₅A linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocyclic group and furthermore one of the substituents mentioned may be H and wherein the acyl group is at the carbonyl function anda linear chain length of at least 3 carbon atoms between the sulfur atoms, the method comprising reacting maytansinoids at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl with an acylated amino acid in which the acyl group bears a protected thiol group;

a method of esterifying a maytansinoid to produce a maytansinoid ester (maytansinoid ester), which may be represented by formula (IV-L), (IV-D) or (IV-D, L):

wherein:

Y₂represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ₂Wherein:

R₃、R₄、R₅、R₆、R₇and R₈Is independently H, CH₃、C₂H₅A linear cyclic alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl or heterocyclic group;

each of l, m, and n is independently an integer from 1 to 5, and further n may be 0;

Z₂is SR or-COR, wherein R is a linear alkyl group having 1 to 10 carbon atomsOr an alkenyl group, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, or a simple aryl or substituted aryl or heterocyclic group; and

may is a maytansinoid; the method comprises reacting the May at a C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethyl group with a compound of formula (III-L), (III-D) or (III-D, L);

wherein:

R₃、R₄、R₅、R₆、R₇and R₈Is independently H, CH₃、C₂H₅Linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cycloalkyl or cycloalkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic groups;

each of l, m, and n is independently an integer of 1 to 5, and further n may be 0; and

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or simplyAn aryl or substituted aryl or heterocyclic aromatic or heterocyclic group of (a);

the above process wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Each of (a) is H; each of l and m is 1; n is 0;

the above process, wherein the compound of formula (III) is represented by (III-L);

the above process wherein the compound of formula (III-L) is compound 15a (S, S), 15b (S, R) or a mixture of 15a (S, S) and 15b (S, R);

the above process wherein the compound of formula (III-D) is compound 15(R, S), 15(R, R) or a mixture of 15(R, S) and 15(R, R);

the above process wherein the compound of formula (III-D, L) is racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, wherein the carbon center bearing the sulfur atom is either racemic or R or S chiral, to give a compound of structure 15;

the above process, wherein the mixture of 15a (S, S) and 15b (S, R) is prepared by a process comprising:

(1) reacting 4-mercaptopentanoic acid (12) with methyl methanethiol sulfonate to give compound 13;

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with N-methyl-L-alanine to provide a mixture of said compounds 15a (S, S) and 15b (S, R);

the above process wherein compound 15a (S, S) is prepared by a process comprising:

(1) converting (R) -1, 3-butanediol to (S) -4- (methyldithio) pentanoic acid 19;

(2) converting compound 19 to its N-hydroxysuccinimide ester (20); and

(3) reacting compound 20 with N-methyl-L-alanine to give said compound 15a (S, S).

The above process wherein compound 15b (S, R) is prepared by a process comprising:

(1) converting (S) -1, 3-butanediol to (R) -4- (methyldithio) pentanoic acid;

(2) converting compound 24 to its N-hydroxysuccinimide ester (25); and

(3) reacting compound 25 with N-methyl-L-alanine to give said compound 15b (S, R).

The above process, wherein the mixture of 15(R, S) and 15(R, R) is prepared by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with N-methyl-D-alanine to give a mixture of said compounds 15(R, S) and 15(R, R).

The above process wherein racemic N-methylalanine is acylated with a carboxyl group bearing a protected thiol functionality, wherein the carbon center bearing the sulfur atom is either racemic or R or S chiral, to provide a compound of structure 15, which is prepared by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with racemic N-methylalanine to give racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or of R or S chirality, to give compounds of structure 15.

The above process wherein R₁And R₂Is methyl; r₅、R₆、R₇And R₈Each of (a) is H; each of l and m is 1; n is 0;

the above process, wherein the compound of formula (III-L) is compound 10(S) containing N-methyl-L-alanine;

the above process, wherein the compound of formula (III-D) is compound 10(R) containing N-methyl-D-alanine;

the above process, wherein the compound of formula (III-D, L) is compound 10(S, R) containing racemic N-methylalanine;

the above method, wherein the compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine is produced by the process comprising:

(1) reacting isobutylene sulfide (5) with acetonitrile anion to give compound 6;

(2) hydrolyzing the compound 6 to obtain 4-mercapto-4-methylvaleric acid (7);

(3) converting compound 7 to disulfide 8 by reaction with methyl methanethiol sulfonate;

(4) converting compound 8 to its N-hydroxysuccinimide ester 9; and

(5) reacting compound 9 with N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine to give said compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine;

a process for the manufacture of maytansinoids by any of the above processes, which may further comprise separating the diastereomers, if present, and purifying by HPLC on cyano-bonded silica;

a process for making a maytansinoid-cell-binding agent conjugate, comprising making a purified maytansinoid by any of the processes described above, and reacting the purified maytansinoid with a cell-binding agent containing an active disulfide group or a thiol group;

the above method of making a maytansinoid-cell-binding agent conjugate, wherein the active disulfide group is a dithiopyridine group or a substituted dithiopyridine group;

a method of making a maytansinoid-cell-binding agent conjugate, comprising making a purified maytansinoid by any of the methods described above, and reacting the purified maytansinoid with a cell-binding agent containing a maleimide group (maleimido group) or a haloacetyl group (haloacetyl group);

esterifying maytansinol to give formula 4₂' the method of maytansinoid of [ 1 ]:

wherein:

Y₂' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C≡C)_qA_r(CR₅CR₆)_mD_u(CR₁₁=CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear branched or alkyl or alkenyl having 1 to 10 carbon atoms, cycloalkyl having 3 to 10 carbon atomsOr cycloalkenyl, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic radical, in addition, R₂May be H;

A. each of B and D is independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple aryl or substituted aryl group, or a heterocyclic aryl or heterocyclic group;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl group or a heterocyclic group;

each of l, m, n, o, p, q, r, s, and t is independently 0 or an integer from 1 to 5, provided that at least 2 of l, m, n, o, p, q, r, s, and t are not simultaneously 0; and

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple aryl or substituted aryl or heterocyclic group, said method comprising reacting a maytansinol of structure 11 at C-3:

with a compound of formula (III ' -L), (III ' -D), or (III ' -D, L):

wherein:

Y_2，represents

(CR₇CR₈)_l(CR₉=CR₁₀)_p(C≡C)_qA_o(CR₅CR₆)_mD_u(CR₁₁=CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl, and furthermore R₂May be H;

A. each of B and D is independently cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple aryl or substituted aryl, or heterocyclic aryl or heterocycloalkyl;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁and R₁₂Is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl or heterocyclic aryl group or a heterocyclic alkyl group;

each of l, m, n, o, p, q, r, s, t, and u is independently 0 or an integer from 1 to 5, provided that at least 2 of l, m, n, o, p, q, r, s, t, and u are not simultaneously 0; and

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple aryl or substituted aryl or heterocyclic group.

Esterifying maytansinol to give formula 4₂'the method of maytansinoid of' wherein the compound of formula (I) is represented by formula (I-L).

Esterifying maytansinol to give formula 4₂' the process of maytansinoids, wherein R₁Is methyl, R₂Is H.

Esterifying maytansinol to give formula 4₂The maytansinoid of (1):

wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, cycloalkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aryl or heterocyclic radical, and further R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl group or a heterocyclic group;

each of l, m and n is independently an integer from 1 to 5, and further n may be 0;

Z₂is SR or COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic group having 3 to 10 carbon atomsAlkyl or alkenyl groups as such, or simple aryl groups or substituted aryl or heterocyclic groups, wherein the method comprises reacting a maytansinol of formula 11:

with a compound of formula (III-L), (III-D) or (III-D, L) at the C-3 position:

wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or linear alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic radical, and furthermore R₂May be H;

Z₂is SR or COR, wherein R is a group having 1 to 10A linear alkyl or alkenyl group of carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group;

the above method for esterifying maytansinol to obtain maytansinoids of formula 4a, wherein the compound of formula (III) is represented by formula (III-L);

the above process for esterifying maytansinol to give maytansinoids of formula 4a, wherein the compound of formula (III-L) is 15a (S, S), 15b (S, R) or a mixture of 15a (S, S) and 15b (S, R);

the above process for esterifying maytansinol to give maytansinoids of formula 4a, wherein the compound of formula (III-D) is 15(R, S), 15(R, R) or a mixture of 15(R, S) and 15(R, R);

the above process for esterifying maytansinol to give maytansinoids of formula 4a, wherein the compound of formula (III-D, L) is racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or R or S chiral, to give compounds of structure 15;

the above process for esterifying maytansinol to give maytansinoids of formula 4a, wherein a mixture of 15a (S, S) and 15b (S, R) is produced by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

the above process for esterifying maytansinol, wherein the compound 15a (S, S) is produced by a process comprising:

(2) converting compound 19 to its N-hydroxysuccinimide ester (20); and

(3) compound 20 is reacted with N-methyl-L-alanine to give compound 15a (S, S).

The above process for esterifying maytansinol, wherein the compound 15b (S, R) is produced by a process comprising:

(1) converting (S) -1, 3-butanediol to (R) -4- (methyldithio) pentanoic acid 24;

(4) converting compound 24 to its N-hydroxysuccinimide ester (25); and

(5) reacting compound 25 with N-methyl-L-alanine to provide said compound 15b (S, R);

the above process for esterifying maytansinol to give maytansinoids of formula 4a, wherein a mixture of compounds 15(R, S) and 15(R, R) is produced by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

The above process for esterifying maytansinol to obtain maytansinoids of formula 4a, wherein racemic N-methylalanine is acylated with a carboxyl group bearing a protected thiol functionality, wherein the carbon center bearing the sulfur atom is either racemic or R or S chiral, to give a compound of structure 15, which is produced by the process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with racemic N-methylalanine to give racemic N-methylalanine acylated with a carboxyl group having a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or of R or S chirality, to give compounds of structure 15.

The above process for esterifying maytansinol to give maytansinoids of formula 4b, wherein R₁And R₂Is methyl; r₅、R₆、R₇、R₈Each of (a) is H; l and m are 1; and n is 0;

the above process for esterifying maytansinol to obtain maytansinoids of formula 4b, wherein the compound of formula (III-L) is N-methyl-L-alanine-containing compound 10;

the above process for esterifying maytansinol to obtain maytansinoids of formula 4b, wherein the compound of formula (III-D) is N-methyl-D-alanine-containing compound 10;

the above process for esterifying maytansinol to obtain maytansinoids of formula 4b, wherein the compound of formula (III-D, L) is compound 10 containing racemic N-methylalanine;

the above process for esterifying maytansinol to give maytansinoids of formula 4b, wherein compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine is produced by the process comprising:

(2) hydrolyzing the compound 6 to obtain 4-mercapto-4-methylvaleric acid (7);

(4) converting compound 8 to its N-hydroxysuccinimide ester 9; and

(5) reacting compound 9 with N-methyl-L-alanine, N-methyl-D-alanine, or racemic N-methylalanine to give compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine, or racemic N-methylalanine;

the above method of esterifying maytansinol with 10, followed by separation of diastereomers if present, and purification of maytansinoids by HPLC on cyano-bonded silica, further comprising reducing the disulfide bond to give maytansinoids of formula 4 b;

a process for the manufacture of a maytansinoid-cell-binding agent conjugate, comprising manufacturing a purified maytansinoid by any of the above processes for esterifying maytansinol to give a maytansinoid of formula 4b, and reacting the maytansinoid with a cell-binding agent containing a thiol group or an active dithio group, preferably a dithiopyridyl group or a substituted dithiopyridyl group;

a process for the manufacture of a maytansinoid-cell-binding agent conjugate, comprising producing a purified maytansinoid by any of the processes described above for esterifying maytansinol to give a maytansinoid of formula 4b, and reacting the maytansinoid with a cell-binding agent containing a maleimido group or a haloacetyl group.

Methods of treatment using the above conjugates.

A compound of formula (III):

wherein:

R₁and R₂Each is independently CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group, or a heterocyclic aryl or heterocyclic group, and further R₂May be H;

each of l, m and n is independently an integer from 1 to 5, and further n may be 0; and

Compound 10(S), 10(R) or racemic 10;

a method of making compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine, or racemic N-methylalanine, comprising:

(1) reacting isobutylene sulfide (5) with an anion of acetonitrile to obtain compound 6;

(2) hydrolyzing the compound 6 to obtain 4-mercapto-4-methylvaleric acid (7);

(4) converting compound 8 to its N-hydroxysuccinimide ester 9; and

(5) reacting compound 9 with N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine to obtain said compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine.

A mixture of compounds 15a (S, S) and 15b (S, R);

a method of making a mixture of compounds 15a (S, S) and 15b (S, R), comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14; and

mixtures of compounds 15(R, S) and 15(R, R).

A method of making a mixture of compounds 15(R, S) and 15(R, R), comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with N-methyl-D-alanine to provide a mixture of said compounds 15(R, S) and 15(R, R);

racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or of R or S chirality to give compounds of structure 15.

A process for the manufacture of racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or of R or S chirality, to give compounds of structure 15, which process comprises:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with racemic N-methylalanine to give racemic N-methylalanine acylated with a carboxyl group having a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or of R or S chirality to give compounds of structure 15.

Compound 15a (S, S);

compound 15b (S, R);

a method of making compound 15a (S, S), comprising:

(2) converting compound 19 to its N-hydroxysuccinimide ester (20); and

(3) reacting compound 20 with N-methyl-L-alanine to give said compound 15a (S, S);

a method of making compound 15b (S, R), comprising:

(2) converting compound 24 to its N-hydroxysuccinimide ester (25); and

A pharmaceutical composition comprising an effective amount of any of the maytansinoids, pharmaceutically acceptable salts or solvates thereof, and a pharmaceutically acceptable carrier, diluent or excipient;

the pharmaceutical composition containing the maytansinoid compound further comprises an antibody.

A method of inducing cell death in a selected cell population comprising contacting target cells or a tissue containing target cells with an effective amount of any of the maytansinoid-cell-binding agents described above, a salt or solvate thereof.

Brief Description of Drawings

FIG. 1 shows the structure of the previously described maytansinoids.

FIG. 2 shows the structures of some maytansinoids of the invention.

FIGS. 3a-3d show the scheme for the synthesis of representative maytansinoids of the invention.

FIGS. 4a, 4b are graphs showing the in vitro potency (potency) of the novel maytansinoids of the invention.

FIGS. 4c, 4d are graphs comparing the in vitro potency of the novel maytansinoids of the invention with those previously described.

FIGS. 5a-5d show a procedure for preparing a conjugate of a cell binding agent and a maytansinoid of the invention.

Fig. 6 is a graph showing the in vitro effect of the cell-binding agent-maytansinoid conjugates of the invention.

FIG. 7 is a plot comparing the in vivo anti-tumor effects of huC 242-maytansinoid conjugates of the present invention with previously described maytansinoids on anti-HT-29 human colon tumor xenografts.

FIG. 8 is a plot comparing the in vivo anti-tumor effects of huC 242-maytansinoid conjugates of the present invention with previously described maytansinoids against COLO205 human colon tumor xenografts.

FIG. 9 is a plot comparing the in vivo anti-tumor effect of MY 9-6-maytansinoids of the invention with previously described MY9-6 conjugates of maytansinoids against HL60 promyelocytic myeloid leukemia xenografts.

FIG. 10 shows the results of the assessment of the in vitro cytotoxicity of the conjugate humY9-6-DM4 on target HL-60 and non-target Namalwa cells.

FIG. 11 shows the evaluation of the in vivo effect of the conjugate humY9-6-DM4 against human HL-60 xenograft tumors in SCID mice, and compares it with the effect of the previously described maytansinoid humY9-6 conjugate (humY9-6-DM 1).

FIG. 12 shows the results of in vitro cytotoxicity evaluation of the conjugate huB4-DM4 on Ramos target cells and Colo205 non-target cells.

FIG. 13a shows the evaluation of the in vivo effect of the conjugate huB4-DM4 against human Ramos xenograft tumors in SCID mice, and FIG. 13b shows the change in animal body weight during this experiment.

Detailed Description

The present invention discloses novel maytansinoids containing sterically hindered thiols (thiols) and disulfides (disulfides) wherein the alpha-carbon bearing the sulfur atom bears one or two alkyl substituents. Methods for synthesizing these novel maytansinoids are also disclosed. Further disclosed are novel compounds useful as intermediates in the synthesis of these novel maytansinoids. In addition, the present invention discloses the preparation of these novel maytansinoid and cell-binding agents conjugates.

The art reveals that it is extremely difficult to modify existing drugs without reducing their cytotoxic potential. The disclosed invention overcomes this difficulty by teaching methods for synthesizing novel maytansinoid molecules that contain sterically hindered thiol or disulfide moieties (moieities). The disclosed invention retains, and in some cases even enhances, the cytotoxic potential of the previously described maytansinoids.

Maytansinoid-cell-binding agent conjugates allow maytansinoids to be applied in a targeted manner to exert a sufficient cytotoxic effect only on unwanted cells, thus avoiding side effects due to damage to non-targeted healthy cells. Thus, the present invention provides useful agents and novel methods for preparing these agents for eliminating diseased or abnormal cells to be killed or lysed, such as tumor cells (particularly solid tumor cells), virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells (autoantibody-producing cells), activated cells (those cells involved in graft rejection or graft versus host disease), or any other type of diseased or abnormal cells, while showing minimal side effects.

Thus, the present invention teaches methods for producing improved cytotoxic conjugates comprising novel maytansinoids and cell-binding agents with greatly improved biological activity compared to previously described maytansinoids and cell-binding agents. The present invention further teaches a process for the synthesis of maytansinoid derivatives having a sterically hindered thiol or disulfide moiety which allows chemical ligation to a cell-binding agent, which derivatives exhibit high cytotoxicity in the bound form or in the released form or both. The cytotoxic conjugates of the invention comprise one or more maytansinoids linked to a cell-binding agent. In order to link the maytansinoids to cell-binding agents, the maytansinoids must first be modified.

Maytansinoids that can be used in the present invention to produce maytansinoids that can be linked to a cell-binding agent are known in the art and can be isolated from natural sources according to known methods or synthetically prepared according to known methods.

Examples of suitable maytansinoids include maytansinol and maytansinol analogs (analogues). Examples of suitable maytansinol analogues include those having modified aromatic rings and those having modifications at other positions.

Specific examples of suitable maytansinoids having a modified aromatic ring include:

(1) c-19-dechlorinated (U.S. Pat. No. 4,256,746) (prepared by LAH reduction of ansamitocin P2);

(2) c-20-hydroxy (or C-20-demethyl) +/-C-19-dechlorinated (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation with Streptomyces (Streptomyces) or Actinomycetes (Actinomyces), or by dechlorination with LAH); and

(3) c-20-demethoxy group, C-20-acyloxy group (-OCOR), +/-dechlorination (U.S. Pat. No. 4,294,757) (prepared by acylation with acid chloride).

Specific examples of suitable maytansinol analogues with modifications at other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (by reaction of maytansinol with H₂S or P₂S₅Prepared by reaction);

(2) c-14-alkoxymethyl (Demethoxy/CH)₂OR) (U.S. patent No. 4,331,598);

(3) c-14-hydroxymethyl or acyloxymethyl (CH)₂OH or CH₂OAc) (U.S. patent No. 4,450,254) (prepared from Nocardia (Nocardia);

(4) c-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by transformation of Streptomyces with maytansinol);

(5) c-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora);

(6) C-18-N-demethylation (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by Streptomyces maytansinol demethylation); and

(7)4, 5-deoxy (U.S. Pat. No. 4,371,533) (prepared by trichloro-peptide/LAH reduction of maytansinol).

To link the maytansinoid to the cell-binding agent, the maytansinoid comprises a linking moiety. The linking moiety contains a chemical bond that allows the release of the fully active maytansinoid at a specific site. Suitable chemical bonds are known in the art and include disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds. Disulfide bonds are preferred.

The disclosure of U.S. Pat. No. 5,208,020, which is incorporated herein by reference, teaches the generation of maytansinoids bearing these bonds.

According to the invention, the linking moiety comprises a sterically hindered thiol or disulfide moiety.

Particularly preferred maytansinoids comprising a linking moiety comprising an active chemical group are the C-3 esters of maytansinol and analogues thereof, wherein the linking moiety comprises a sterically hindered thiol or disulfide bond.

Many positions on the maytansinoid can be used as the position to chemically link the linking moiety. For example, the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group are all expected to be useful. However, the C-3 position is preferred, and the C-3 position of maytansinol is particularly preferred.

Further, although the synthesis of maytansinol esters with linking moieties is described below based on disulfide bond-containing linking moieties at the C-3 position, those skilled in the art will appreciate that linking moieties with other chemical bonds, as described above, may also be used in the present invention, and likewise, other maytansinoids and other linking sites, as described above, may also be used in the present invention.

FIG. 2 shows the structures of various maytansinoids of the present invention. Synthesis of maytansinoids with sterically hindered thiol or disulfide moieties is described with reference to fig. 3. Many of the following representative methods employ thiol-containing maytansinoids N^2'-deacetyl-N^2'- (4-mercapto-1-oxopentyl) -maytansine (known as DM3) and N^2'-deacetyl-N^2'- (4-methyl-4-mercapto-1-oxo)Pentyl) -maytansine (known as DM 4). DM3(4a) and DM4(4b) are represented by the following structural formulae:

the in vitro cytotoxicity of the novel maytansinoids of the invention containing sterically hindered thiols and disulfides can be assessed by their ability to inhibit proliferation of various unwanted cell lines in vitro (FIG. 4). For example, cell lines such as the human breast cancer cell line SK-Br-3, or the human epidermoid cancer cell line KB can be used to assess the cytotoxicity of these novel maytansinoids. Cells to be evaluated may be exposed to the compound for 72 hours and the viable fraction of the cells determined by direct assay using known methods. Then, from the analysis result, IC can be calculated₅₀The value is obtained.

Production of maytansinoids with sterically hindered thiol or disulfide moieties

The novel maytansinoids of the invention are those having an acylated amino acid side chain bearing an acyl group having a hindered thiol group at the C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl group, wherein the acyl carbon atom bearing the thiol functionality has one or two substituents which are CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl group or a heterocyclic group, further, one of the substituents may be H, and wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl function and the sulfur atom.

Preferably, the maytansinoid compound is represented by formula 4':

wherein:

y' represents

(CR₇CR₈)_l(CR₉=CR₁₀)_p(C≡C)_qA_r(CR₅CR₆)_mD_u(CR₁₁=CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂SZ，

Wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic radical, and furthermore R₂May be H; A. b, D is a cycloalkyl or cycloalkenyl group having 3-10 carbon atoms, a simple or substituted aryl or heterocyclic group;

each of l, m, n, o, p, q, r, s, and t is independently 0 or an integer from 1 to 5, provided that at least 2 of l, m, n, o, p, q, r, s, and t are not simultaneously 0;

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group.

In a preferred embodiment of the compounds represented by 4', R₁Is methyl, R₂Is H and Z is H; r₁And R₂Is methyl and Z is H; r₁Is methyl, R₂Is H and Z is-SCH₃(ii) a Or R₁And R₂Is methyl and Z is-SCH₃。

More preferably, the maytansinoid is a compound represented by formula (I-L), (I-D) or (I-D, L):

wherein:

y represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂SZ, wherein:

R₁and R₂Each is independently CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group, or a heterocyclic aryl or heterocyclic group, and further R₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Is independently H, CH₃、C₂H₅A linear alkyl or alkenyl group having from 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having from 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl or heterocyclic group;

each of l, m, and n is independently an integer from 1 to 5, and further, n may be 0;

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group; and

may represents maytansinoids having a side chain at the C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation.

More preferred are C-3 esters, which are compounds represented by formula 4:

wherein the substituents are as defined above.

Particularly preferred are any of the above compounds, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, and Z is H; those compounds described above wherein R₁And R₂Is methyl, R₅、R₆、R₇And R₈Is H, l and m are 1, n is 0, and Z is H; those compounds in which R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, and Z is-SCH₃(ii) a Those compounds, R₁And R₂Is methyl, R₅、R₆、R₇、R₈Are each H, l and m are 1, n is 0, and Z is-SCH₃. Further, the L-alanyl stereoisomer is preferred because it is most useful for the conjugate of the invention.

Preferred embodiments of formula 4 include DM3 and DM4, i.e., the maytansinoids of formula 4 wherein Z is H, R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, l and m are 1, and n is 0(DM3, compound 4 a); maytansinoids of formula 4, wherein Z is H, R₁And R₂Are all methyl, R₅、R₆、R₇And R₈Is H, l and m are 1, and n is 0(DM4, compound 4 b); maytansinoids of formula 4, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, and Z is-SCH₃(ii) a And maytansinoids of formula 4, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Are each H, l and m are 1, n is 0, and Z is-SCH₃。

Examples of linear alkyl or alkenyl groups having 1 to 10 carbon atoms include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, propenyl, butenyl, and hexenyl.

Examples of branched alkyl or alkenyl groups having 3 to 10 carbon atoms include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethylpropyl, isobutenyl, and isopentenyl.

Examples of the cyclic alkyl or alkenyl group having 3 to 10 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl and cyclohexenyl.

Simple aryl groups include aryl groups having 6 to 10 carbon atoms, and substituted aryl groups include aryl groups having 6 to 10 carbon atoms with at least one alkyl substituent having 1 to 4 carbon atoms, or an alkoxy substituent such as methoxy, ethoxy, or a halogen substituent or a nitro substituent.

Examples of simple aryl groups containing 6 to 10 carbon atoms include phenyl and naphthyl.

Examples of substituted aryl groups include nitrophenyl, dinitrophenyl.

Heterocyclic aryl groups include groups having 3 to 10 membered rings containing one or two heteroatoms selected from N, O or S.

Heterocycloalkyl includes cyclic compounds comprising a 3 to 10 membered ring system containing one or two heteroatoms selected from N, O or S.

Examples of heterocyclic aryl groups include pyridyl, nitropyridyl, pyrolyl, oxazolyl, thienyl, thiazolyl, and furyl.

Examples of heterocycloalkyl groups include dihydrofuran, tetrahydrofuran, tetrahydropyrolyl, piperidinyl, piperazinyl and morpholinyl (morpholino).

Novel maytansinoids with sterically hindered thiol or disulfide moieties may be prepared by the following novel disclosed methods:

synthesis of maytansinoids

FIG. 3a shows the steps in the synthesis of maytansinoids DM4(4 b). The isobutenylsulfide (5) was reacted with an anion of acetonitrile to obtain a mercapto compound 6. Hydrolyzing 6 with alkali to obtain 4-mercapto-4-methyl pentanoic acid (7). The conversion of 7 to disulfide 8 was accomplished by reaction with methyl methanethiol sulfonate (MeSSO2 Me). Conversion of 8 to N-hydroxysuccinimide ester 9 followed by reaction with N-methyl-L-alanine gave carboxylic acid 10, which was purified by silica gel column chromatography. 10 and maytansinol (11) in the presence of N, N' -Dicyclohexylcarbodiimide (DCC) and zinc chloride to give a mixture of N-acyl-N-methyl-L-alanylmaytansinoid L-DM4Sme, (4e) and N-acyl-N-methyl-D-alanylmaytansinoid D-DM4Sme (4 f). The mixture of diastereomers was separated by HPLC using a cyano-bonded column. The desired L-amino acid containing isomer 4e was collected and reduced with dithiothreitol to give thiol-containing L-aminoacyl-maytansinoids DM4(4b), which was purified by HPLC using a cyanogen-bonded column again.

FIG. 3b shows the steps in the synthesis of maytansinoid DM3(4 a).

4-mercaptopentanoic acid (12) was converted to methyl disulfide by reaction with methyl methanethiol sulfonate to give 13. Compound 13 is converted to N-hydroxysuccinimide ester 14, which is then reacted with N-methyl-L-alanine to give carboxylic acid 15, which is purified by silica gel column chromatography. Reacting 15 with maytansinol (11) in the presence of N, N' -Dicyclohexylcarbodiimide (DCC) and zinc chloride to give a mixture of N-acyl-N-methyl-L-alanylmaytansinoid L-DM3SSMe, (4c) and N-acyl-N-methyl-D-alanylmaytansinoid D-DM3SSMe (4D). The mixture of diastereomers was separated by HPLC using a cyano-bonded column. The desired L-amino acid-containing isomer was collected and reduced with dithiothreitol to give maytansinoids DM3(4a) containing a mercapto-L-amino acid, which was purified by HPLC using a cyanogen-bonded column again.

FIGS. 3c and 3d show the synthesis of DM3 with either a (S) -4-methyldithio-1-oxopentyl moiety or a (R) -4-methyldithio-1-oxopentyl moiety. Conversion of (R) -1, 3-butanediol (16) to its ditosylate ester 17 followed by successive reactions with sodium cyanide and potassium ethyl xanthogenate gave nitrile 18 (FIG. 3 c). Basic hydrolysis followed by disulfide exchange gave (S) -4-methyldithio-pentanoic acid 19. 19 into succinimidyl ester 20, followed by reaction with N-methyl-L-alanine to give N-methyl-N- [4- (S) -methyldithio-1-oxo-pentyl ] -S-alanine (15 a). Reaction with maytansinol, as described above for compound 15, gave two diastereomers of L-DM3SMe, 4g and 4 h. Similarly, (S) -1, 3-butanediol (21) was converted to (R) -4-methyldithio-pentanoic acid 24 and then to 15 b. Reaction with maytansinol, as described above, gave two diastereomers of DM3SMe, 4k and 4 l.

Accordingly, the present invention provides a process for esterifying maytansinoids with an acylated amino acid side chain at a C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation wherein the acyl group has a protected thiol functionality wherein the acyl carbon atom bearing the protected thiol functionality has one or two substituents which are CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic group, furthermore one of the substituents may be H, wherein the acyl group is at the carbonyl function and sulfurA linear chain length of at least 3 carbon atoms between atoms, the method comprising reacting a maytansinoid at a C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation group with an acylated amino acid in which the acyl group bears a protected thiol group.

In a preferred embodiment, the present invention provides esterifying maytansinol to give formula 4₂' the method of maytansinoid of [ 1 ]:

wherein:

Y₂' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C≡C)_qA_r(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear branched or alkyl or alkenyl having 1 to 10 carbon atoms, cycloalkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic radical, and furthermore R₂May be H;

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Each independently of the otherIs H, CH₃、C₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl group or a heterocyclic group;

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group, said method comprising reacting a maytansinol of structure 11 at C-3 position

With a compound of formula (III ' -L), (III ' -D) or (III ' -D, L):

wherein:

Y_2，represents

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C≡C)_qA_o(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂SZ₂，

Wherein:

R₁and R₂Each of (1)Independently is CH₃、C₂H₅Linear alkyl or linear alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl, and furthermore R₂May be H;

Z₂is SR or-COR, wherein R is a linear alkyl or linear alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group.

Preferably, the compound of formula (I) is represented by formula (I-L), and, also preferably, R₁Is methyl and R₂Is H.

In a more preferred embodiment, the present invention provides esterifying maytansinol to give formula 4₂The maytansinoid of (1):

wherein:

each of l, m and n is independently an integer of 1 to 5, and further n may be 0;

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group, said method comprising reacting a maytansinol of formula 11 at C-3 position:

with a compound of formula (III-L), (III-D) or (III-D, L):

wherein:

Diastereoisomers can be separated by HPLC on cyano-bonded silica.

In a more preferred embodiment, the present invention provides a process for esterifying maytansinoids to produce a maytansinoid ester represented by formula (IV-L), (IV-D) or (IV-D, L):

wherein:

Z₂is SR or COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group; and

may is a maytansinoid, which process comprises reacting said May at C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethylation with a compound of formula (III-L), (III-D) or (III-D, L):

wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic radical, and furthermore R₂May be H;

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group.

In an even more preferred embodiment, the present invention provides esterifying maytansinol to give formula 4₂The maytansinoid of (1):

wherein:

Z₂is SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple or substituted aryl or heterocyclic group, said method comprising reacting maytansinol at the C-3 position with a compound of formula (III-L), (III-D) or (III-D, L):

wherein:

Preferably, the compound represented by formula (I) is the L stereoisomer.

For the above process, it is preferred that R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, 1 and m are each 1, and n is 0; or R₁And R₂Is methyl, R₅、R₆、R₇And R₈Is H, 1 and m are 1, and n is 0.

In the preparation of DM3, the compound of formula (III-L) is 15a (S, S), 15b (S, R) or a mixture of 15a (S, S) and 15b (S, R); the compound of formula (III-D) is N-methyl-D-alanine acylated with a racemic acyl group or an acyl group having R or S chirality to give compound 15; the compounds of formula (III-D, L) are racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or R or S chiral, to give compounds of structure 15.

The mixture of 15a (S, S) and 15b (S, R) may be produced by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with N-methyl-L-alanine to give a mixture of said compounds 15a (S, S) and 15b (S, R).

Similarly, a mixture of 15(R, S) and 15(R, R) may be produced by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

Racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or R or S chiral, to give compounds of structure 15, which can be produced by a process comprising:

(2) converting compound 13 to its N-hydroxysuccinimide ester 14;

(3) reacting compound 14 with racemic N-methylalanine to give said racemic N-methylalanine acylated with a carboxyl group bearing a protected thiol functionality, in which the carbon center bearing the sulfur atom is either racemic or of R or S chirality, to give compounds of structure 15.

Compound 15a (S, S) can be prepared by a process comprising:

(2) converting compound 19 to its N-hydroxysuccinimide ester (20); and

Compound 15b (S, R) can be prepared by a process comprising:

(2) converting compound 24 to its N-hydroxysuccinimide ester (25); and

(3) reacting compound 25 with N-methyl-L-alanine to provide said compound 15b (S, R);

in the production of DM4, the compound of formula (III-L) is N-methyl-L-alanine-containing compound 10; the compound of formula (III-D) is N-methyl-D-alanine containing compound 10, and the compound of formula (III-D, L) is racemic N-methylalanine containing compound 10.

Compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine is made by the process comprising:

(2) hydrolyzing the compound 6 to obtain 4-mercapto-4-methylvaleric acid (7);

(4) converting compound 8 to its N-hydroxysuccinimide ester 9; and

(5) compound 9 is reacted with N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine to give compound 10 containing N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine.

According to the invention, the compounds of formula III are also novel:

wherein:

Z₂is SR or-COR, where R is a linear, branched or cyclic alkyl radical having from 1 to 10 carbon atoms, orSimple or substituted aryl or heterocyclic groups.

Compounds of formula III can be readily prepared by those skilled in the art using methods similar to those disclosed herein for the preparation of compounds 10 and 15.

In vitro cytotoxicity of maytansinoids

FIG. 4 shows the in vitro cytotoxicity of maytansinoids of the invention. The novel maytansinoids (4c, 4e) with hindered disulfide bonds were highly potent on the cell lines tested. Thus, 4c killed A-375 cells and SK-Br-3 cells, IC₅₀The values are 1.5x10 respectively^-11M and 7.0x10^-12And M. Similarly, maytansinoid 4e is also highly potent, its IC on A-375 cells and SK-Br-3 cells₅₀The values are 3.2x10 respectively^-11M and 9.0x10^-12And M. Comparing the in vitro potency of the sterically hindered thiol-containing maytansinoid 4a of the present invention with the previously described maytansinoid 1 (FIGS. 4c, d), the results show that the novel maytansinoids are 20 to 50 times more potent than the previously described maytansinoids.

Preparation of cell binding agents

The effectiveness of the compounds of the invention as therapeutic agents depends on the careful selection of an appropriate cell-binding agent. The cell binding agent may be any of a variety currently known, or becoming known, including peptidic and non-peptidic substances. Generally, they may be antibodies (particularly monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance.

More specific examples of cell-binding agents that may be used include:

a polyclonal antibody;

a monoclonal antibody;

antibody fragments such as Fab, Fab 'and F (ab')₂、Fv(Parham，J.Immunol.131：2895-2902(1983)；Spring et al.J.Immunol.113：470-478(1974)；Nisonoff et al.Arch.Biochem.Biophys.89：230-244(1960))；

Interferons (e.g., α, β, γ);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgen and estrogen;

growth factors and colony stimulating factors such as EGF, TGF-. alpha.FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984));

transferrin (O' Keefe et al.J.biol.chem.260: 932-937 (1985)); and

vitamins, such as folic acid.

Monoclonal antibody technology allows for the production of extremely specific cell binding agents in the form of specific monoclonal antibodies. Techniques for making monoclonal antibodies, such as whole target cells, antigens isolated from target cells, whole viruses, attenuated whole viruses, and viral proteins such as viral capsid proteins, are well known in the art, and are produced by immunizing a mouse, rat, hamster, or any other mammal with an antigen of interest. Sensitized human cells may also be used. Another method of making monoclonal antibodies is the use of scFv (single chain variable region) phage libraries, particularly human scFv phage libraries (see Griffiths et al, U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al, WO 92/01047; Liming et al, WO 99/06587). Furthermore, resurfaced antibodies disclosed in U.S. Pat. No. 5,639,641 may be used, as may humanized antibodies.

The choice of a suitable cell-binding agent depends on the particular cell population to be targeted, but generally, if a suitable human monoclonal antibody is available, a human monoclonal antibody is preferred.

For example, monoclonal antibody MY9 is murine IgG₁An antibody which specifically binds to the CD33 antigen { j.d. griffin et al8 leukamia res., 521(1984) }, which antibody can be used if the target cell expresses CD33, for example in Acute Myeloid Leukemia (AML). Similarly, monoclonal antibody anti-B4 is a murine IgG₁Which binds to the CD19 antigen { Nadler et al, 131J. Immunol.244-250(1983) } on B cells, which antibodies can be used if the target cell is a B cell expressing this antigen or a diseased cell, for example in non-Hodgkin's lymphoma or chronic lymphoblastic leukemia. Similarly, monoclonal antibody C242, which binds to the CanAg antigen (U.S. patent No. 5,552,293), can be used to treat CanAg-expressing tumors, such as rectal, pancreatic and gastric cancers.

In addition, GM-CSF, which binds to myeloid cells, can be used as a cell-binding agent against diseased cells from acute myeloid leukemia. IL-2, which binds to activated T cells, can be used for the prevention of transplant rejection, for the treatment and prevention of graft versus host disease, and for the treatment of acute T cell leukemia. MSH, which binds to melanocytes, can be used to treat melanoma. Folate can be used to target the upper folate receptor expressed in ovarian and other tumors. Epidermal growth factor can be used to target squamous cancers such as lung squamous cancer and head squamous cancer and cervical squamous cancer. Somatostatin can be used to target neuroblastoma and other tumor types.

Estrogens (or estrogen analogs) or androgens (or androgen analogs) can be used as cell-binding agents, respectively, to successfully target breast and testicular cancer.

Generation of cytotoxic conjugates

The invention also provides maytansinoid-cell-binding agent conjugates comprising at least one maytansinoid linked to a cell-binding agent, wherein the cell-binding agent is linked to the maytansinoid with a thiol or disulfide functional group present on the acyl group of the side chain of an acylated amino acid, the acylation is carried outIn which the acyl group of the acylated amino acid side chain has its thiol or disulfide function located on a carbon atom bearing one or two substituents, which are CH, present at the C-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 demethyl group of maytansinoids₃、C₂H₅Linear or alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic group, furthermore, one of the substituents may be H, and wherein the acyl group has a linear chain length of at least 3 carbon atoms between the carbonyl function and the sulphur atom.

Preferred cell-binding agent conjugates include at least one maytansinoid linked to a cell-binding agent, wherein the maytansinoid is represented by formula 4₁' means:

wherein:

Y₁' is representative of

(CR₇CR₈)_l(CR₉=CR₁₀)_p(C≡C)_qA_r(CR₅CR₆)_mD_u(CR₁₁=CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂S-，

Wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic radical, and furthermore R₂May be H:

R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₁and R₁₂Is independently H, CH₃、c₂H₅A linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl group or a heterocyclic group; and

each of l, m, n, o, p, q, r, s, t, and u is independently 0 or an integer from 1 to 5, provided that at least 2 of l, m, n, o, p, q, r, s, t, and u are not simultaneously 0.

Preferably, R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

A more preferred cell-binding agent conjugate comprises at least one maytansinoid linked to a cell-binding agent, wherein the maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)₁(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

R₁and R₂Each is independently CH₃、C₂H₅Linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenylHeterocyclic aryl or heterocyclic radical, in addition, R₂May be H;

each of l, m and n is independently an integer from 1 to 5, further n may be 0; and

may represents maytansinol with a side chain at the C-3, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation;

more preferred are maytansinoid-cell-binding agent conjugates in which the maytansinoid is represented by formula 4₁Represents:

wherein the substituents are as defined for formula (II) above.

Particularly preferred are any of the above compounds, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; and those of any of the above, wherein R₁And R₂Is methyl, R₅、R₆、R₇And R₈Is H, l and m are 1, and n is 0.

Further, the L-aminoacyl stereoisomer is preferable.

Representative cytotoxic conjugates of the invention are antibodies/maytansinoids, antibody fragments/maytansinoids, Epidermal Growth Factor (EGF)/maytansinoids, Melanocyte Stimulating Hormone (MSH)/maytansinoids, Thyrotropin (TSH)/maytansinoids, growth hormone release inhibiting factor/maytansinoids, folic acid/maytansinoids, estrogens/maytansinoids, estrogen analogs/maytansinoids, androgens/maytansinoids, and androgenic analogs/maytansinoids.

Thiol-containing maytansinoids are reacted with a suitably modified cell-binding agent to produce cytotoxic conjugates. These conjugates can be purified by gel filtration, ion exchange chromatography or by HPLC.

A scheme for preparing conjugates from thiol-containing maytansinoids is shown in figure 5. More specifically (FIG. 5a, b), a solution of the antibody in an aqueous buffer can be incubated with an excess molar amount of an antibody modifying agent such as N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP, 3a) to introduce dithiopyridyl (FIG. 5a), or with N-succinimidyl-4- (2-pyridyldithio) -butyrate (SPDB, 3b) to introduce dithiopyridyl (FIG. 5 b). The modified antibody is then reacted with a thiol-containing maytansinoid (e.g., 4a or 4b) to form a disulfide-linked antibody-maytansinoid conjugate. The maytansinoid-antibody conjugate can then be purified by gel filtration.

Alternatively, the antibody can be incubated with an excess molar amount of an antibody modifying agent, such as 2-iminothiolane, to introduce sulfhydryl groups. The modified antibody is then reacted with an appropriate disulfide-containing maytansinoid to produce a disulfide-linked antibody-maytansinoid conjugate. The maytansinoid-antibody conjugate can then be purified by gel filtration.

The number of maytansinoid molecules bound per antibody molecule (denoted by w in FIGS. 5a to 5d) can be determined by spectrophotometric determination of the ratio of the absorbance at 252nm and 280 nm. By this method, an average of 1-10 maytansinoid molecules per antibody molecule can be linked. The preferred average number of maytansinoids attached per antibody is 2-5, most preferably 3-4.5.

Alternatively, a solution of the antibody in an aqueous buffer can be incubated with an excess molar amount of an antibody modifying agent such as N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC, 26) to introduce the maleimido group (FIG. 5c), or with N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB, 27) to introduce the iodoacetyl group (FIG. 5 d). The modified antibody is then reacted with a thiol-containing maytansinoid (e.g., 4a or 4b) to produce a thioether-linked antibody-maytansinoid conjugate. The maytansinoid-antibody conjugate can then be purified by gel filtration.

The number of maytansinoid molecules bound per antibody molecule can be determined by spectrophotometric analysis as described above.

Accordingly, the present invention provides a process for the manufacture of a maytansinoid-cell-binding agent conjugate, comprising manufacturing a purified maytansinoid by one of the processes described above, and reacting the purified maytansinoid with a cell-binding agent which contains an active disulphide group or a thiol group. Preferably, the active dithio group is a dithiopyridyl group or a substituted dithiopyridyl group. Particularly preferably, the active disulfide group comprises a nitropyridine disulfide group or a dinitropyridine disulfide group.

In another method, purified maytansinoids are reacted with a cell-binding agent containing a maleimido group or a haloacetyl group.

Conjugates of the cell binding agents of the invention with maytansinoids can be evaluated for their ability to inhibit proliferation of various unwanted cell lines in vitro (figure 6). For example, cell lines such as human colon cancer cell line COLO205, human melanoma cell line A-375, and human myeloid leukemia cell line HL60 can be used to evaluate the cytotoxicity of these conjugates. The cells to be evaluated can be exposed to the compound for 24 hours and the viable fraction of the cells determined by known methods using direct assays. Then, from the analysis result, IC can be calculated₅₀The value is obtained.

FIGS. 6, 10 and 12 show the in vitro potency and target specificity of the antibody-maytansinoid conjugates of the invention. Thus, FIG. 6 shows that both huC242-DM3 and huC242-DM4 are highly effective at killing antigen-positive COLO205 cells, with IC₅₀The values are 1.3x10 respectively^-11M and 1.1x10^-11And M. In contrast, antigen-negative A-375 cells were less than about 500-fold sensitive, indicating that the maytansinoid conjugates of the invention are highly potent and highly specific. Similarly, FIGS. 10 and 12 illustrate the high potency and target specificity of the conjugates of maytansinoids of the present invention with antibodies MY9-6 and anti-B4, respectively.

The anti-tumor effect of the hindered thiol-containing maytansinoid-antibody conjugates of the present invention in vivo was compared to the anti-tumor effect of previously described maytansinoid conjugates in vivo in several different human tumor mouse models. In the first model (FIG. 7), SCID mice bearing subcutaneous xenografts of established human colon tumor HT-29 were treated with either the previously described antibody conjugate of maytansinoid DM1 (huC242-DM1) or with two new maytansinoid conjugates (huC242-DM3, huC242-DM 4). Treatment with huC242-DM1 resulted in an 18-day delay in tumor growth. In contrast, the new agent was significantly more effective, with a 28 day delay in tumor growth for huC242-DM3 and a 36 day delay in tumor growth for huC242-DM 4.

In a second model (FIG. 8), mice bearing an established subcutaneous xenograft of human colon tumor COLO205 were treated with the previously described antibody conjugate of maytansinoid DM1 (huC242-DM1) or with two new maytansinoid conjugates (huC242-DM3, huC242-DM 4). Treatment with huC242-DM1 did not result in tumor regression, delaying tumor growth by 20 days. In contrast, the new agents are significantly more effective. In the huC242-DM 3-treated group, tumors completely regressed for 45 days. huC242-DM4 was more effective, resulting in recovery of all treated mice.

In a third model (FIG. 9), mice bearing subcutaneous xenografts of established human myeloid leukemia HL60 were treated with either the previously described antibody conjugate of the maytansinoid DM1 (MY-9-6-DM1) or with two new maytansinoid conjugates (MY-9-6-DM3, MY-9-6-DM 4). Treatment with MY-9-6-DM1 did not result in tumor regression, delaying tumor growth by 5 days. In contrast, the new agents are significantly more effective. Resulting in tumor regression. MY-9-6-DM3 and MY-9-6-DM4 both delayed tumor growth by more than 20 days.

In the fourth model (FIG. 11), the effects of the maytansinoids of the present invention (humY9-6-DM4) and the previously described conjugates of the maytansinoids (humY9-6-DM1) were directly compared in a subcutaneous xenograft model established with HL-60 cells. Treatment with the conjugate of the invention, MY9-6-DM4, at equivalent doses, resulted in complete tumor regression for 85 days. In contrast, the previously described maytansinoid conjugates were much less active, with a tumor growth delay of only about 48 days.

In a fifth model (fig. 13a), the maytansinoid conjugates of the invention with huB4 antibody showed high anti-tumor activity in a dose-dependent manner in a subcutaneous Ramos tumor model. At non-toxic doses, complete tumor regression and recovery were obtained (fig. 13a, b).

The results of the above five effect experiments demonstrate that cell-binding agent conjugates obtained with the sterically hindered thiol-containing maytansinoids of the present invention have greatly improved antitumor activity compared to the previously described maytansinoid-cell-binding agent conjugates.

Compositions and methods of use

The present invention provides a pharmaceutical composition comprising an effective amount of any of the maytansinoid-cell-binding agents of the invention, a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

The invention also provides a method of treatment comprising administering to a subject in need thereof an effective amount of any of the conjugates described above.

Similarly, the invention provides a method for inducing cell death in a selected cell population, the method comprising contacting a target cell or tissue containing a target cell with an effective amount of a cytotoxic agent comprising any of the maytansinoid-cell-binding agents of the invention, a salt or solvate thereof. The target cell is a cell to which the cell-binding agent can bind.

If desired, other active agents, such as other antineoplastic agents, may be administered with the conjugate.

Suitable pharmaceutically acceptable carriers, diluents and excipients are known and their clinical availability can be determined by one of ordinary skill in the art.

Examples of suitable carriers, diluents and/or excipients include: (1) dulbecco's phosphate buffered saline, pH about 7.4, with or without about 1mg/ml to 25mg/ml human serum albumin, (2) 0.9% physiological saline (0.9% w/v NaCl), and (3) 5% (w/v) glucose; and may also include antioxidants such as tryptamine and stabilizers such as tween 20.

Methods for inducing cell death in a selected cell population can be performed in vitro (in vitro), in vivo (in vivo), or ex vivo (ex vivo).

Examples of in vitro applications include, for killed diseased or malignant cells, treatment of autologous bone marrow prior to transplantation of the autologous bone marrow to the same patient; to kill competent T cells and prevent graft versus host disease (GVDH), bone marrow is treated prior to transplantation; cell cultures are treated in order to kill all cells except those that do not express the desired variant of the target antigen, or in order to kill those that express the undesired antigen.

The conditions for non-clinical in vitro applications can be readily determined by one of ordinary skill in the art.

Examples of clinical ex vivo applications are the removal of tumor cells or lymphocytes from bone marrow prior to autologous transplantation in the treatment of cancer or autoimmune diseases, or the removal of T cells and other lymphocytes from autologous or allogeneic bone marrow or tissue prior to transplantation for the prevention of GXDH. The treatment may be performed as follows. Bone marrow is harvested from a patient or other individual and then incubated in serum-containing medium to which the cytotoxic agent of the present invention is added at a concentration ranging from about 10. mu.M to 1pM for about 30 minutes to about 48 hours at about 37 ℃. The exact concentration conditions and incubation time, i.e. dosage, can be readily determined by one of ordinary skill in the art. Following incubation, the bone marrow cells are washed with serum-containing medium and returned intravenously to the patient according to known procedures. Between the time of bone marrow harvest and reinfusion of the treated cells, if the patient receives other treatments such as ablative chemotherapy (ablative chemotherapy) or whole body radiotherapy, standard medical equipment cryopreserves the treated bone marrow cells in liquid nitrogen.

For clinical in vivo use, the cytotoxic agents of the invention will be provided as solutions or lyophilized powders which are tested for sterility and endotoxin levels. Examples of suitable protocols for administering the conjugate are as follows. Conjugates were administered as intravenous pellets weekly for 4 weeks. The bolus dose is administered in 50 to 1000ml of normal physiological saline, to which 5 to 10ml of human serum albumin may be added. The dose per administration is 10. mu.g to 2000mg, given intravenously (daily dose ranging from 100ng to 20 mg/kg). After 4 weeks of treatment, the patient may continue to receive treatment in weekly units. The skilled artisan can determine the specific clinical protocol clinically allowable with respect to route of administration, excipients, diluents, dosage, time, etc.

Depending on the in vivo or ex vivo method of inducing cell death in a selected cell population, examples of medical conditions that may be treated include any type of malignancy, including, for example, lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphoid organ cancers; autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis; transplant rejection, such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; graft versus host disease (graft versus host disease); viral infections, such as CMV infection, HIV infection, AIDS, and the like; and parasitic infections such as giardiasis, amebiasis, schistosomiasis, and other diseases as determined by one of ordinary skill in the art.

Examples

The invention will now be illustrated with reference to non-limiting examples. All percentages, ratios, parts, etc., are by weight unless otherwise indicated. The embodiments described below are for R₁Is CH₃，R₂Is H, R₅、R₆、R₇、R₈Is H, each of l and m is 1 and n is 0. Similar syntheses may be made for other compounds of the invention in which R is₁And R₂Is independently H, CH₃、C₂H₅Or a higher alkyl, alkenyl, or phenyl, substituted phenyl or heterocyclic aryl or heterocyclic group having 1 to 10 carbon atoms; wherein each of l, m and n is an integer from 1 to 5, and further n may also be 0.

All reagents were purchased from Aldrich Chemical co, new jersey, or from other commercial sources. Maytansinol (11) was prepared as previously described (U.S. Pat. No. 6,333,410). Nuclear magnetic resonance (¹H NMR) spectra were obtained on a Bruker400MHz apparatus and mass spectra were obtained using electrospray ionization on a Bruker daltons acquire 3000 apparatus.

Example 1

Synthesis of maytansinoids 4b

4-mercapto-4-methylvaleric acid (7): a stir bar and a 150ml addition funnel were fitted to a 500ml flask. The system was placed under nitrogen atmosphere. 150ml of anhydrous Tetrahydrofuran (THF) and 75ml of 2.5M n-BuLi in hexane (18.7mmol) were added via a catheter and the solution was cooled in a-78 ℃ dry ice/acetone bath.Acetonitrile (7.3g, 9.4ml, 18mmol) was added dropwise over a period of about 5 minutes via syringe. When a white lithium acetonitrile precipitate formed, the reaction was stirred for 30 minutes. Isobutylene sulfide (15g, 17mmol) was dissolved in 100ml of anhydrous THF and added dropwise over about 30 minutes via addition funnel. The cooling bath was removed and the reaction solution was stirred for 3 hours. 38ml of 0.5M HCl was added dropwise while cooling the flask in an ice/water bath. The THF layer was retained and the aqueous layer was washed twice with 75ml ethyl acetate. The THF layer and the ethyl acetate layer were combined, dried over about 20g of anhydrous sodium sulfate, and transferred to a 250ml flask. The solvent was removed by rotary evaporation under vacuum to give crude product 6. Ethanol (30ml) and stir bar were added. The contents were stirred while slowly adding 30ml of a deionized water solution containing 8.0g of NaOH. A reflux condenser was fitted to the flask and placed under an argon atmosphere. The reaction was refluxed overnight and then cooled to room temperature. Deionized water (60mL) was added and the mixture was extracted twice with 25mL of a 2: 1 mixture of ethyl acetate and hexane. The aqueous layer was acidified with concentrated HCl to pH2, then extracted three times with 75ml ethyl acetate. With anhydrous Na₂SO₄The organic layer was dried and the solvent removed by rotary evaporation under vacuum to give 10g of product 7 (39% yield). The resulting material was used without further purification.¹H NMR(CDCl₃)：δ1.38(6H，s)，1.87-1.93(2H，m)，2.08(1H，s)，2.51-2.57(2H，m)。

4-methyl-4- (methyldithio) pentanoic acid (8): in a 250ml flask, a solution of mercaptopentanoic acid 7 (6.0ml, 40mmol) was dissolved in 50ml deionized water. Sodium carbonate (6.4g, 60mmol) was added to the acid at a rate that did not cause excessive foaming and was magnetically stirred. A100 ml addition funnel was fitted to the flask, and the funnel was charged with a solution of methyl methanethiol sulfonate (7.5g, 60mmol) dissolved in 30ml of glass distilled (glass-distilled) 100% ethanol. The flask was cooled in an ice/water bath and the system was maintained under an argon atmosphere. The methyl methanethiol sulfonate solution was added dropwise to the flask as quickly as possible without causing excessive foaming. The cooling bath was removed and the reaction mixture was stirred for an additional 3 hours. The solvent was removed by rotary evaporation under vacuum until approximately 20ml remained. Subsequently, 10m were addedl of saturated sodium bicarbonate and 30ml of deionized water. In a separatory funnel, the mixture was washed three times with 25ml of ethyl acetate. The aqueous layer was adjusted to pH about 2 with 5M HCl and extracted twice with 120ml of ethyl acetate. The organic layers were combined and washed twice with 20ml of a solution consisting of saturated NaCl and 1M HCl in a ratio of 4: 1. Then, the organic layer was dried over 14g of anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under vacuum to obtain 5.4g of product 8 (yield 70%). This material can be carried to the next reaction without further purification.¹H NMR(CDCl₃)：δ1.54(6H，s)，2.5-2.21(2H，m)，2.64(3H，s)，2.69-2.72(2H，m)。MS(M+Na⁺) Calculated values: 217.0, found: 217.1.

n-hydroxysuccinimidyl 4-methyl-4- (methyldithio) pentanoic acid (9):

methyldithiopentanoic acid 8(3g, 15mmol) was dissolved in 20ml of dichloromethane and magnetically stirred with addition of N-hydroxysuccinimide (2.65g, 23mmol), followed by addition of 1- [3- (dimethylamino) propyl hydrochloride]3-Ethylcarbo-diamine (EDC, 4.4g, 23 mmol). The mixture was stirred under argon for 2 hours. The reaction mixture was poured into a 125ml separatory funnel, 40ml of ethyl acetate was added, and the solution was washed twice with 20ml of 50mM potassium phosphate buffer solution, pH6.0, and once with 12ml of saturated sodium chloride. With 14g of anhydrous Na₂SO₄The organic layer was dried and the solvent was removed by rotary evaporation under vacuum to give 4.0g of product 9 (90% yield) which was used without further purification.¹H NMR(CDCl₃)：δ1.30(6H，s)，2.00-2.05(2H，m)，2.39(3H，s)，2.68-2.72(2H，m)，2.73-2.83(4H，m)。MS(M+Na⁺) Calculated values: 314.0, found: 314.1.

N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -L-alanine (10):

in a 125ml flask equipped with a magnetic stir bar, N-methyl-L-alanine (2.85g, 18.0mmol) was dissolved in 50ml of a 1: 1 solution of dimethoxyethane and deionized water. Triethylamine (6.9g, 36mmol) was added,and 9(5.44g, 18mmol) dissolved in 40ml of the same solvent mixture was added dropwise while the solution was stirred vigorously for about 5 minutes. After 2 hours, the reaction mixture was concentrated to about 40ml by rotary evaporation under vacuum, and then 10ml of deionized water and 1M HCl were added to give a pH of about 2. The mixture was poured into a separatory funnel and extracted twice with 50ml of ethyl acetate. The organic layers were combined and subsequently washed with 7ml of saturated sodium chloride solution. With 8.0g of anhydrous Na₂SO₄The organic layer was dried and the solvent was removed by rotary evaporation under vacuum. The residue was kept in a minimum volume of ethyl acetate and purified by chromatography on silica (silica: 40 micron size (flash grade), silica bed: 24X3.0cm, mobile phase: hexane: ethyl acetate: acetic acid 50: 48: 2). The fractions containing the desired product were combined and the solvent was removed in vacuo. The residual acetic acid was removed by dissolving the residue in a minimum volume of ethyl acetate and precipitating the product by quickly dropping hexane while stirring. Hexane was added until no product was detected in the supernatant by TLC analysis. The precipitate was dried in vacuo for 4 hours to give 2.2g of product 10 (yield 51%).¹H NMR(CDCl₃)：δ1.32(6H，s)，1.42(3H，d，J＝7Hz)，1.90-97(2H，m)，2.40(3H，s)，2.42-2.49(2H，m)，2.9(3H，s)，5.15(1H，q，J＝7Hz)。MS(M+Na⁺) Calculated values: 302.1, found: 302.0.

N^2’-deacetyl-N^2’- (4-methyl-4-methyldithio-1-oxopentyl) -maytansine (L-DM4-SMe, 4 e). A solution of maytansinol (11, 25mg, 0.44mmol) and N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -L-alanine (10, 42.0mg, 0.177mmol) in 3mL of dichloromethane was magnetically stirred under argon while adding a solution of dicyclohexylcarbodiimide (DCC, 57.1g, 0.277mmol) in 0.67mL of dichloromethane. After 1 minute, 1M ZnCl was added₂Was added to the solution of (1) (diethyl ether) (0.03ml, 0.03 mmol). The mixture was stirred at room temperature for 2 hours, then 5ml of ethyl acetate were added and the mixture was vacuum filtered through a course filter paper. The filtrate was washed with 2ml of saturated sodium bicarbonate solution and subsequently with 1ml of saturated sodium chloride solutionAnd (6) washing. The organic layer was dried over 2g of anhydrous sodium sulfate. The solvent was removed in vacuo and the residue was purified by silica chromatography using a mixture of dichloromethane and ethanol to remove unreacted maytansinol. The fractions containing the desired product were combined and the solvent was removed in vacuo to give a mixture of diastereomers 4e and 4 f. The residue was taken up in a minimum volume of ethyl acetate and a mixture of hexane, 2-propanol and ethyl acetate in a ratio of 68: 8: 24 was used as the mobile phase at 50cm by 250cm, 10 micron Diazem^TMAnd (5) purifying on a CN column. The flow rate was 118 mL/min. Under these conditions, the desired product 4e eluted at a retention time of 11 minutes, and the undesired enantiomer 4f was retained for 19 minutes. The fractions containing the desired product were combined and the solvent removed in vacuo to yield 12.0mg of product 4e (36% yield).¹H NMR(CDCl₃): δ 0.80(3H, s), 1.28-1.36(13H, m), 1.42-1.46(2H, m), 1.53-1.63(2H, m), 1.64(3H, s), 1.75-1.85(1H, m), 1.90-2.10(1H, m), 2.18(1H, dd, J ═ 3Hz and 14Hz), 2.31(3H, s), 2.40-2.49(1H, m), 2.50-2.65(1H, m), 2.85(3H, s), 3.04(1H, d, J ═ 9Hz), 3.11(1H, d, J ═ 11Hz), 3.23(3H, s), 3.35(3H, s), 3.49(1H, d, J ═ 9), 3.63(1H, d, J ═ 11Hz), 3.23 (3.35 (3H, s), 3.49(1H, d, J ═ 9), 3.63(1H, J ═ 9, 3.9H, J ═ 9, 7, 1H, 5, J ═ 1H, 5, 1H, 5, J ═ 9Hz, 6.21(1H, s), 6.42(1H, dd, J ═ 11Hz and 15Hz), 6.65(1H, d, J ═ 1.5Hz), 6.73(1H, d, J ═ 11Hz), 6.81(1H, d, J ═ 1.5 Hz). High resolution MS (M + H)⁺) Calculated values: 826.3174, found: 826.3150.

N^2’-deacetyl-N^2’- (4-mercapto-4-methyl-1-oxopentyl) -maytansine (L-DM4, 4 b): the disulfide 4e (12mg, 0.015mmol) obtained above was dissolved in 1.0ml of 1: 1 ethyl acetate: methanol. Then, dithiothreitol (18mg, 0.117mmol) dissolved in 0.50mL of 50mM phosphate buffer, pH7.5, was added. The solution was magnetically stirred under argon for 3 hours, then 1mL of 200mM phosphate buffer, pH6.0, was added and the mixture was extracted three times with 2mL of ethyl acetate. The organic layers were combined and dissolved in 1mL of saturated sodium chlorideThe solution was washed, followed by drying over 1g of anhydrous sodium sulfate. The solvent was removed under vacuum, the residue was taken up in a minimum volume of ethyl acetate and a mixture of hexane, 2-propanol and ethyl acetate as mobile phase in a ratio of 70: 8: 22 at 50cm x250cm, 10 micron Diazem^TMAnd (5) purifying on a CN column. The flow rate was 22 mL/min. The desired product 4b eluted at a retention time of 10 minutes. Fractions containing purified 4b were combined and the solvent removed in vacuo to give 11mg of 4b (97% yield).¹H NMR(CDCl₃): δ 0.80(3H, s), 1.19-1.23(1H, m), 1.28-1.36(12H, m), 1.42-1.46(2H, m), 1.53-1.63(2H, m), 1.64(3H, s), 1.75-1.85(1H, m), 1.90-2.10(1H, m), 2.18(1H, dd, J ═ 3Hz and 14Hz), 2.40-2.49(1H, m), 2.50-2.65(2H, m), 2.88(3H, s), 3.04(1H, d, J ═ 9Hz), 3.11(1H, d, J ═ 11Hz), 3.23(3H, s), 3.35(3H, s), 3.49(1H, d, J ═ 9, 3.63(1H, d, J ═ 11Hz), 3.23(3H, s), 3.35(3H, s), 3.49(1H, d, J ═ 9, 3.63 ═ 9, 3.9, 3H, J ═ 4H, 3.9, J ═ 1H, 5(1H, d, J ═ 11Hz), 3.7, 3.9, J ═ 1H, 5, J ═ 1H, J ═ 7, 1H, 5, dd J ═ 9Hz and 15Hz), 6.21(1H, s), 6.42(1H, dd, J ═ 11Hz and 15Hz), 6.65(1H, d, J ═ 1.5Hz), 6.73(1H, d, J ═ 11Hz), 6.81(1H, d, J ═ 1.5 Hz). High resolution MS (M + Na)⁺) Calculated values: 802.3101, found: 802.3116.

example 2

Synthesis of maytansinoids 4a

4-methyldithio-pentanoic acid (13): in a 500mL flask, a solution of 4-mercaptopentanoic acid (12, 16.6mg, 124mmol) was dissolved in 350mL deionized water. The solution was magnetically stirred while sodium carbonate (19.7g, 186mmol) was added to the acid at a rate that did not cause excessive foaming. A250 ml addition funnel was fitted to the flask, which was charged with a solution of methylmercaptan sulfonate (23.4g, 186mmol) dissolved in 220ml of glass distilled 100% ethanol. The flask was cooled in an ice/water bath and the system was maintained under an argon atmosphere. The methyl methanethiol sulfonate solution was added dropwise to the flask as quickly as possible, but not at a rateSo as to cause excessive foaming. The cooling bath was removed and the reaction mixture was stirred for an additional 2 hours. The solvent was removed by rotary evaporation under vacuum until approximately 250ml remained. Subsequently, 30ml of saturated sodium bicarbonate solution and 50ml of deionized water were added. In a separatory funnel, the mixture was washed three times with 200ml of ethyl acetate. The aqueous layer was adjusted to pH about 2 with 5M HCl and extracted twice with 400ml of ethyl acetate. The organic layers were combined and washed with 60ml of a 4: 1 mixture of saturated NaCl solution and 1M HCl, then dried over 50g of anhydrous sodium sulfate and finally the solvent was removed by rotary evaporation under vacuum to give 10.2g of product 13 (45% yield). The material was used in the next reaction without further purification.¹H NMR(CDCl₃)：δ1.36(3H，d，J＝7Hz)，1.84-1.95(H，m)，1.85-2.56(1H，m)，2.42(3H，s)，2.53(2H，t，J＝7Hz)，2.85-2.95(1H，m)，MS(M+Na⁺) Calculated values: 203.3, found: 203.2.

n-hydroxysuccinimidyl 4-methyldithio-pentanoate (14): methyldithio-pentanoic acid (13, 0.75g, 4.16mmol) was dissolved in 7.0ml dichloromethane and magnetically stirred while adding N-hydroxysuccinimide (0.526g, 4.57mmol) followed by 1- [3- (dimethylamino) propyl hydrochloride]-3-ethylcarbodiimide hydrochloride (0.877g, 4.57 mmol). The mixture was stirred under argon for 2.5 hours and then poured into a 60mL separatory funnel containing 20mL of ethyl acetate. The resulting solution was washed twice with 15ml of 50mM potassium phosphate buffer pH6.0 and once with 5ml of saturated sodium chloride. With 8g of anhydrous Na₂SO₄The organic layer was dried and the solvent removed by rotary evaporation under vacuum to give 1.15g of product 14 (87% yield), which was used in the next reaction without further purification.¹HNMRδ1.48(3H，d，J＝7)，2.06(1H，m)，2.17(1H，m)，2.55(3H，s)，2.93(2H，t，J＝7)，2.98(4H，s)，3.15(1H，m)。MS(M+Na⁺) Calculated values: 304.1, found: 304.0.

N-methyl-N- (4-methyldithio-1-oxopentyl) -L-alanine (15): in a 125ml flask equipped with a magnetic stir bar, the N-methyl group was placedL-alanine (0.64g, 6.2mmol) was dissolved in 8ml of a 1: 1 mixture of dimethoxyethane and deionized water. Triethylamine (0.841g, 8.3mmol) was added and the flask stirred vigorously while a solution of 14(1.0g, 3.6mmol) in 8ml of the same solvent mixture was added dropwise over a period of about 5 minutes. After 2 hours, the reaction mixture was concentrated to about 3ml by rotary evaporation under vacuum, and then 15ml of deionized water and 1M HCl were added to give a pH of about 2. The mixture was poured into a 60mL separatory funnel and extracted twice with 15mL of ethyl acetate. The organic layers were combined, washed with 3ml of saturated sodium chloride solution, followed by 8.0g of anhydrous Na₂SO₄Drying and finally, rotary evaporation under vacuum to remove the solvent. The residue was taken up in a minimum volume of ethyl acetate and purified by chromatography on silica (silica: 40 micron flash grade, silica bed: 24X3.0cm, mobile phase hexane: ethyl acetate: acetic acid 50: 48: 2). The fractions containing the desired product 15 were combined and the solvent was removed in vacuo. The residual acetic acid was removed by dissolving the residue in a minimum volume of ethyl acetate and precipitating the product by quickly dropping hexane while stirring. Hexane was added until no product could be detected in the supernatant by TLC analysis. The precipitate was dried in vacuo to give 0.60g of product 15 (62% yield).¹H NMR(CDCl₃)：δ1.35(3H，d，J＝7)，1.41(3H，d，J＝7)，1.94-2.03(2H，m)，2.43(3H，s)，2.50-2.55(2H，m)，2.83-2.93(1H，m)，2.98(3H，s)，5.14(1H，q，J＝7)。MS(M+Na⁺) Calculated values: 288.1, found: 288.1.

N^2’-deacetyl-N^2’- (4-methyldithio-1-oxopentyl) -maytansine (L-DM3-SMe, 4 c): a solution of maytansinol (25mg, 0.44mmol) and 15(42.0mg, 0.177mmol) in 3mL of dichloromethane was magnetically stirred under argon while a solution of dicyclohexylcarbodiimide (DCC, 57.1g, 0.277mmol) in 0.67mL of dichloromethane was added. After 1 minute, 1M ZnCl was added₂Was added to the solution of (1) (diethyl ether) (0.03ml, 0.03 mmol). The mixture was stirred at room temperature for 2 hours, then 5ml of ethyl acetate were added and the mixture was vacuum filtered through a course filter paper. Using 2ml of saturated carbonThe filtrate was washed with sodium hydrogen acid solution and subsequently with 1ml of saturated sodium chloride solution. The organic layer was dried over 2g anhydrous sodium sulfate and then the solvent was removed in vacuo. The residue was purified by silica chromatography using a mixture of dichloromethane and methanol to remove unreacted maytansinol. The fractions containing the desired product were combined and the solvent was removed in vacuo to give a mixture of diastereomers 4c and 4 d. The residue was taken up in a minimum volume of ethyl acetate and mixed in the ratio 68: 8: 24 Hexane, 2-propanol and ethyl acetate mixture as mobile phase at 50cm by 250cm, 10 micron Diazem^TMAnd (5) purifying on a CN column. The flow rate was 118 mL/min. The desired product 4c eluted at a retention time of 11 minutes and the undesired enantiomer 4d had a retention time of 19 minutes. The fractions containing the desired product were combined and the solvent removed in vacuo to yield 12.0mg of product 4c (36% yield).¹H NMR(CDCl₃): δ 0.80(3H, s), 1.19-1.23(1H, m), 1.28-1.36(9H, m), 1.42-1.46(1H, m), 1.53-1.63(2H, m), 1.64(3H, s), 1.80-1.89(1H, m), 1.90-2.09(1H, m), 2.18(1H, dd, J ═ 3Hz and 14Hz), 2.32(3H, s), 2.33-2.42(1H, m), 2.49-2.62(2H, m), 2.88(3H, s), 3.04(1H, d, J ═ 9Hz), 3.11(1H, d, J ═ 11Hz), 3.23(3H, s), 3.35(3H, s), 3.49(1H, d, J ═ 9Hz), 3.11(1H, d, J ═ 11Hz), 3.23(3H, s), 3.35(3H, s), 3.49J ═ 9H, 1H, d, 3.9 Hz, 3.9H, 3.79 (1H, J ═ 9Hz), 1H, d, 3.9H, J ═ 9Hz, 1H, 3.9H, J ═ 9H, 1, j7 Hz), 5.66(1H, dd J9 Hz and 15Hz), 6.21(1H, s), 6.42(1H, dd, J11 Hz and 15Hz), 6.65(1H, d, J1.5 Hz), 6.73(1H, d, J11 Hz), 6.81(1H, d, J1.5 Hz). MS (M + Na)⁺) Calculated values: 834.3, found: 834.3.

N^2’-deacetyl-N^2’- (4-mercapto-1-oxopentyl) -maytansine (L-DM3, 4 a): L-DM3-SMe (4c, 12mg, 0.015mmol) was dissolved in 1.0ml of a 1: 1 mixture of ethyl acetate: methanol. A solution of dithiothreitol (18mg, 0.117mmol) in 0.50ml of 50mM phosphate buffer, pH7.5, was added. The reaction solution was magnetically stirred under argon atmosphere for 3 hours, then 1mL of 200mM phosphate buffer, pH6.0, was added and the mixture was extracted three times with 2mL of ethyl acetate. Combining the organic layersWashed with 1mL of a saturated sodium chloride solution, and then dried over 1g of anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was taken up in a minimum volume of ethyl acetate and a mixture of hexane, 2-propanol and ethyl acetate as mobile phase at a ratio of 70: 8: 22 at 50cm x250cm, 10 micron Diazem^TMAnd (5) purifying on a CN column. The flow rate was 22 mL/min. The desired product eluted at a retention time of 10 minutes. The fractions containing the purified product were combined and the solvent removed in vacuo to yield 11mg of product 4a (97% yield).¹H NMR(CDCl₃): δ 0.80(3H, s), 1.19-1.23(1H, m), 1.28-1.36(9H, m), 1.42-1.46(1H, m), 1.53-1.63(2H, m), 1.64(3H, s), 1.80-1.89(1H, m), 1.90-2.09(1H, m), 2.18(1H, dd, J ═ 3Hz and 14Hz), 2.33-2.42(1H, m), 2.49-2.62(2H, m), 2.88(3H, s), 3.04(1H, d, J ═ 9Hz), 3.11(1H, d, J ═ 11Hz), 3.23(3H, s), 3.35(3H, s), 3.49(1H, d, J ═ 9, 3.63(1H, d, J ═ 11Hz), 3.7 (1H, J ═ 12H, J ═ 11Hz), 3.5 (1H, 5, J ═ 9H, d, J ═ 9Hz, 3.9H, J ═ 9, 3.9, 3H, J ═ 9, 3.9, dd J ═ 9Hz and 15Hz), 6.21(1H, s), 6.42(1H, dd, J ═ 11Hz and 15Hz), 6.65(1H, d, J ═ 1.5Hz), 6.73(1H, d, J ═ 11Hz), 6.81(1H, d, J ═ 1.5 Hz). MS: (M + Na)⁺) Calculated values: 788.3, found: 788.3.

example 3

Synthesis of maytansinoids 4g, 4h (FIG. 3c)

R-1, 3-di-O-p-toluenesulfonyl-butane (17): a solution of R- (-) -1, 3-butanediol (16, 2.00g, 22.22mmol) dissolved in a mixture of anhydrous pyridine (40mL) and anhydrous toluene (60mL) was treated with p-toluenesulfonyl chloride (12.70g, 66.84mmol) at 0 deg.C under argon. After stirring for 5 minutes at 0 ℃ and stirring for 2 hours at room temperature, the mixture is evaporated in vacuo, redissolved in ethyl acetate and washed with 0.1M aqueous NaHCO₃Washing was followed by saturated NaCl. With MgSO₄The organic layer was dried, filtered and the solvent was evaporated. Purifying by chromatography on silica gel with 1: 2 (v/v) ethyl acetate/hexaneElution gave 6.51g (74%) of the title product 17. R_f0.40 (1: 1 EtOAc/hexanes);¹H NMR(CDCl₃)7.76(dd，4H，J＝1.0，8.0Hz)，7.35(dt，4H，J＝0.4，8.0+8.0Hz)，4.70(m，1H)，4.03(m，1H)，3.94(m，1H)，2.46(s，6H)，1.92(m，2H)，1.26(d，3H，J＝6.3Hz)；¹³C NMR145.17，133.00，130.11，128.12，127.91，76.28，66.21，36.08，21.86，21.06；MS：420.99(M+Na)⁺，421.93(M+1+Na)⁺。

s-4-0-ethylxanthogen-valeronitrile (S-4-O-ethylxanthonic-pentanenitrile) (18): a solution of R-1, 3-di-O-p-toluenesulfonyl-butane (17, 4.80g, 12.06mmol) in anhydrous DMSO (50mL) was treated with NaCN (0.65). After stirring at room temperature for 18 hours under argon, the reaction mixture was diluted with ethyl acetate and successively with cold 1.0M NaH, pH7.5₂PO₄Water and 1.0M NaH at pH4.0₂PO₄And (6) washing. The organic layer was separated and MgSO₄Drying, filtration and evaporation gave 2.63g of crude product R-3-O-p-toluenesulfonyl-valeronitrile. MS275.80(M + Na)⁺，276.75(M+1+Na)⁺. The product was used directly without further purification.

To a solution of the crude product R-3-O-p-toluenesulfonyl-valeronitrile (2.63g) in ethanol (15ml) was added ethanol (50ml) containing potassium O-xanthogenate (4.55 g). After stirring overnight under argon, the mixture was concentrated, diluted with ethyl acetate and filtered through a short silica column. The eluate was concentrated and purified by silica gel chromatography eluting with 1: 4 (v/v) EtOAc/hexane to give 1.54g of the title product 18 (63%, 2 steps). R_f0.40 (1: 4 EtOAc/hexanes).¹H NMR(CDCl₃)4.67(dd，2H，J＝7.1，14.2Hz)，3.86(ddd，1H，J＝7.0，14.0，21.9Hz)，2.50(t，2H J＝7.3+7.6Hz)，2.06(m，2H)，1.44(m，6H)；¹³C NMR213.04，119.16，70.28，44.57，32.10，20.20，15.21，13.93；MS：226.51(M+Na)⁺，242.51(M+K)⁺。

S- (+) -4-methyldiThio-pentanoic acid (19): to a solution containing a mixture of S-4-O-ethylxanthogen-valeronitrile (18, 1.95g (9.61mmol)) in ethanol (10ml) and water (150ml) was added 5.0g NaOH. The reaction mixture was refluxed overnight under argon. The mixture was cooled to room temperature, diluted with water (150ml) and extracted with 1: 1 EtOAc/hexanes (2X100 ml). By H₃PO₄The aqueous layer was acidified to pH2.5-3.0 and extracted with EtOAc (6X75 ml). The organic layers were combined and MgSO₄Drying, filtration and evaporation to dryness gave the crude product S-4-mercaptopentanoic acid. The crude product was used directly in the next step without further purification.

To the crude S-4-mercaptopentanoic acid (1.2g) was dissolved ethanol (50ml) and 0.5M NaH over a period of 45 minutes at 0 deg.C₂PO₃To a solution of the mixture at pH7.0(75mL) was added dropwise 5 anhydrous TNF (5mL) containing methylmercaptan sulfonate (1.47g, 11.65 mmol). After stirring at 0 ℃ for 30 minutes under argon, the mixture was concentrated and extracted with dichloromethane (2 × 50 ml). By H₃PO₄The aqueous layer was acidified to pH2.5-3.0 and extracted with EtOAc (4X100 ml). The organic layers were combined and MgSO₄Drying, filtering and evaporating. The residue was purified by silica gel chromatography, eluting with (1: 100: 400 HOAc/EtOAc/hexane), to give 1.43g (83%) of the title product 19. R_f0.32 (HOAc/EtOAc/hexane, 1: 100: 400);¹H NMR(CDCl₃)2.91(ddd，1H，J＝6.8，13.7，20.5Hz)，2.53(t，2H，J＝7.7+7.4Hz)，2.42(s，3H)，1.94(m，2H)，1.36(d，3H，J＝6.8Hz)；¹³C NMR179.18，45.35，31.58，30.73，24.70，21.05；MS：202.92(M+Na)⁺，203.91 (M+1+Na)⁺；[α]＝41.35(c＝2，CH₃OH)。

N-methyl-N- [4- (S) -methyldithio-I-oxopentyl radical]-S-alanine (15 a): s- (+) -4- (methyldithio) -pentanoic acid (19) is converted to N-hydroxysuccinimide ester 20 using the procedure described above for compound 14. Reaction with N-methyl-L-alanine by the method described above for Compound 15 gave 15a (62% yield). H¹NMRδ1.36(3H，d，J＝7)，1.42(3H，d，J＝7)，1.93-1.98(2H, m), 2.40(3H, s), 2.50-2.53(2H, m), 2.90-2.95(1H, m), 2.99(3H, s), 5.14(1H, q, J ═ 7), MS: calculated (M + Na): 288.1, found: 288.1.

N^2’-deacetyl-N^2’- (4- (S) -methyldithio-1-oxopentyl) -maytansine (DM3-SMe, 4g, h): maytansinol (11) and 15a were linked using dichloromethane containing DCC and zinc chloride as in the synthesis of 4c above. A mixture of 2 diastereomers with an N-methyl-S-alanyl moiety (4g, S, S) and an N-methyl-R-alanyl moiety (4h, R, S) was obtained. The diastereoisomers were separated by HPLC on a Kromasil cyanogen column (4.6 mm. times.250 250mm) using an isocratic elution with hexane: ethyl acetate: 2-propanol (68: 24: 8, v/v/v) at a flow rate of 1 mL/min. Under these conditions, 4g of isomer (S, S) eluted at 24.5 minutes. Mass spectrum: m/z834.2(M + Na)⁺. At 34.6 minutes, the other isomer, 4H (R, S), was well separated and eluted. MS: m/z834.2(M + Na)⁺。

Example 4

Synthesis of maytansinoids 4k, l (FIG. 3d)

S-1, 3-di-O-p-toluenesulfonyl-butane 22: a solution of S- (-) -1, 3-butanediol (21, 2.00g (22.22mmol)) in a mixture of anhydrous pyridine (40ml) and anhydrous toluene (60ml) was treated with p-toluenesulfonyl chloride (12.70g, 66.84mmol) at 0 ℃ under an argon atmosphere. After stirring at 0 ℃ for 5 minutes, at room temperature for 2 hours, the mixture is evaporated in vacuo. The residue was redissolved in ethyl acetate and washed with 0.1M aqueous NaHCO₃And a saturated NaCl wash. Separating the organic layer with MgSO₄Dried, filtered and evaporated. The residue was purified by silica gel chromatography, eluting with 1: 2 ethyl acetate/hexane to give 6.25g (71%) of the title product 22. R_f0.40 (1: 1 EtOAc/hexanes);¹H NMR(CDCl₃)7.76(dd，4H，J＝1.0，8.0Hz)，7.35(dt，4H，J＝0.4，8.0+8.0Hz)，4.70(m，1H)，4.03(m，1H)，3.94(m，1H)，2.46(s，6H)，1.92(m，2H)，1.26(d，3H，J＝6.3Hz)；¹³C NMR145.17，133.00，130.11，128.12，127.91，76.28，66.21，36.08，21.86，21.06；MS：420.99(M+Na)⁺。

R-4-O-ethylxanthogen-valeronitrile (23): a solution of S-1, 3-di-O-p-toluenesulfonyl-butane (22, 6.25g (15.70mmol)) in 60 anhydrous DMSO (50mL) was treated with NaCN (0.85 g). The reaction mixture was stirred at room temperature for 18 hours under argon atmosphere. The reaction mixture was then diluted with ethyl acetate and successively with cold 1.0M NaH, pH7.5₂PO₄Water and 1.0M NaH at pH4.0₂PO₄And (6) washing. With MgSO₄The organic layer was dried, filtered and evaporated to give 3.62g of crude S-3-O-p-toluenesulfonyl-valeronitrile. The product was used directly without further purification.

To a solution of the crude product S-3-O-p-toluenesulfonyl-valeronitrile (3.62g) in ethanol (50ml) was added ethanol (100ml) containing potassium ethyl O-xanthate (5.72 g). After stirring overnight under argon, the mixture was concentrated, diluted with ethyl acetate and filtered through a short silica gel column. The eluate was concentrated and the residue was purified by silica gel chromatography eluting with 1: 4 EtOAc/hexane to give 2.0g of the title product 23 (62%, 2 steps). R_f0.40 (1: 4 EtOAc/hexanes).¹H NMR(CDCl₃)4.67(dd，2H，J＝7.1，14.2Hz)，3.86(ddd，1H，J＝7.0，14.0，21.9Hz)，2.50(t，2H J＝7.3+7.6Hz)，2.06(m，2H)，1.44(m，6H)；¹³C NMR213.04，119.16，70.28，44.57，32.10，20.20，15.21，13.93；MS：226.51(M+Na)⁺，242.51(M+K)⁺。

R- (-) -4-methyldithio-pentanoic acid (24): a solution containing a mixture of R-4-O-ethylxanthogen-valeronitrile (23, 2.0g (9.85mmol)) in ethanol (10ml) and 200ml of water was treated with NaOH (6.0 g). The reaction mixture was refluxed overnight under argon. The mixture was diluted with water (150ml) and extracted with 1: 1 EtOAc/hexane (2X100 ml). By H₃PO₄The aqueous layer was acidified to pH2.5-3.0 and extracted with EtOAc (6X75 ml). The organic layers were combined and MgSO₄Drying and filteringAnd evaporated to dryness to give the crude product R-4-mercaptopentanoic acid. The crude product was used directly in the next step without further purification.

To ethanol (50ml) containing 1.60g of crude R-4-mercaptopentanoic acid and 0.5M NaH at 0 ℃ over a period of 45 minutes₂PO₄To a solution of the mixture at pH7.0(75mL) was added dropwise anhydrous TNF (7mL) containing methylmercaptan sulfonate (1.96g, 15.53 mmol). The reaction mixture was stirred at 0 ℃ for 30 minutes under argon atmosphere and then at room temperature for 2 hours. The mixture was concentrated and extracted with dichloromethane (2 × 50 ml). By H₃PO₄The aqueous layer was acidified to pH2.5-3.0 and extracted with EtOAc (4X100 ml). The organic layers were combined and MgSO₄Drying, filtering and evaporating. The residue was purified by silica gel chromatography, eluting with (1: 100: 400 HOAc/EtOAc/hexane), to give 1.65g (93%) of the title product 24. R_f0.32 (HOAc/EtOAc/hexane, 1: 100: 400);¹H NMR(CDCl₃)2.91(ddd，1H，J＝ 6.8，13.7，20.4Hz)，2.53(t，2H，J＝7.7+7.4Hz)，2.42(s，3H)，1.96(m，2H)，1.36(d，3H，J＝6.8Hz)；¹³C NMR179.46，45.67，31.91，31.07，25.02，21.36；MS：202.9(M+Na)⁺，203.9(M+1+Na)⁺；[α]＝-39.16(c＝2，CH₃OH)。

N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine (15 b): r- (+) -4- (methyldithio) -pentanoic acid (24) is converted to N-hydroxysuccinimide ester 25 using the procedure described above for compound 14. Reaction with N-methyl-L-alanine by the method described above for Compound 15 gives 15 b. MS: m/z (M + Na): calculated values: 288.1, found: 288.1.

N^2'-deacetyl-N^2’- (4- (R) -methyldithio-1-oxopentyl) -maytansine (DM3-SMe, 4k, l): maytansinol (11) and 15b were linked using DCC and zinc chloride dissolved in dichloromethane as described for the synthesis of 4c above. A mixture of 2 diastereomers with an N-methyl-S-alanyl moiety (4k, S, R) and an N-methyl-R-alanyl moiety (41, R, R) was obtained. Using a mixture of hexane:ethyl acetate: 2-propanol (68: 24: 8, v/v/v) was eluted isocratically and the diastereomers were separated by HPLC on a Kromasil cyanogen column (4.6mm X250mm) at a flow rate of 1 mL/min. Under these conditions, isomer 4k (S, R) eluted at 23.9 minutes. Mass spectrum: m/z834.2(M + Na)⁺. At 33.7 minutes, the other isomer 41(R, R) peak was well separated and eluted. MS: m/z834.2(M + Na)⁺。

Example 5a

In vitro cytotoxicity of maytansinoids and antibody-maytansinoid conjugates

The KB (ATCC CCl-17) cell line is of human epithelial origin. The SK-BR-3(ATCC HTB-30) cell line was established from human breast adenocarcinoma. Human colon tumor cell lines COLO205(ATCC CCL-222) and HT-29(ATCC HTB38), human melanoma cell line A-375(ATCC CRL1619), human Burkitt lymphoma cell line Ramos (ATCC CRL-1596) and human myeloid leukemia cell line HL60(ATCC CCL-240) were all obtained from ATCC in Maryland. The cell lines were grown in Dulbecco's modified Eagles Medium (DMEM, Biowhittaker, Walkersville, Md.) with L-glutamine, supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and 50. mu.g/ml gentamicin sulfate (Life technologies, Rockville, Md.). In the presence of 6% CO₂In a humid atmosphere at 36-37.5 deg.C.

The cytotoxicity studies carried out employ the clonogenic assay (clonogenic assay). Test cell lines were plated in 6-well dishes at a fixed number of 1000 cells per well. Cells were incubated with various concentrations (0 to 3nm) of various maytansinoids (free or conjugated to antibodies) for 72 hours. Then, the medium was aspirated from the plate and fresh medium was placed. Cultures were allowed to grow and form colonies, which were maintained for 7-10 days after plating. The cultures were then fixed and stained with 0.2% crystal violet in 10% formalin/PBS and the number of colonies counted. Plating efficiency of non-treated cells (medium alone) was determined by dividing the number of colonies counted by the number of cells plated. The survival score of the drug-exposed cells was determined by dividing the number of colonies in the drug-exposed wells by the number of colonies in the control wells.

FIG. 4 shows the results of in vitro cytotoxicity assays of the novel maytansinoids of the invention. For the cell lines tested, SK-BR-3 and A-375, the novel maytansinoids 4c, e with sterically hindered disulfide bonds were highly cytotoxic, IC₅₀The range of values is from 7x10^-12M to 2.5x10^-11And M. Thus, introduction of alkyl substituents on the carbon bearing the disulfide moiety maintains high cytotoxicity. The sterically hindered thiol containing maytansinoids 4a of the present invention are 30 to 50 times more potent than the corresponding non-hindered maytansinoids 1 described previously. Thus, the introduction of alkyl substituents on the carbon bearing the thiol moiety greatly increases efficacy.

The results of in vitro testing of the antibody conjugates of the maytansinoids of the invention are shown in FIGS. 4c and 4 d. Linking two new maytansinoids, 4a or 4b, to huC242 antibodies against human colon tumors resulted in antigen-specific killing of target cells. Thus, these conjugates were highly effective against antigen-positive COLO205 cells, IC_5oThe range of values is from 1.1x10^-11M to 1.3x10^-11And M. In contrast, these conjugates were 100 to 250-fold less cytotoxic to antigen-negative a-375 cells, indicating that the novel maytansinoids of the invention produce conjugates possessing sterically hindered disulfide bonds that exhibit high target-specific cytotoxicity.

Example 5b

Preparation of cytotoxic conjugates of huC242 antibodies with maytansinoids 4a or 4b (method A, FIGS. 5a, b)

A solution of huC242 antibody (8mg/ml) in pH6.5 aqueous buffer (50mM potassium phosphate, 50mM sodium chloride, 2mM disodium ethylenediaminetetraacetate) was mixed with 7 to 10-fold molar excess of SPDP [ succinimidyl-3- (2-pyrido) to obtain a mixturePyridyldithio) -propionate, 3a]Or with N-succinimidyl-4- (2-pyridyldithio) -butyrate (SPDB, 3 b). The reaction mixture was purified by passing through a Sephadex G25 gel filtration column. Using the known extinction coefficient epsilon of the antibody_280nm＝217,560M^-1cm^-1The antibody concentration was determined spectrophotometrically.

The modified antibody was diluted to 2.5mg/ml with aqueous buffer (50mM potassium phosphate, 50mM sodium chloride, 2mM disodium ethylenediaminetetraacetate) of pH6.5, and then treated with 1.5 to 2.5-fold molar excess of DM3 or DM4 in dimethylethylamine (final concentration of DMA was 3% v/v). The reaction mixture was incubated at room temperature for 18 hours. The reaction mixture was purified by Sephadex G25 gel filtration column. Using the known extinction coefficient epsilon of the antibody_280nm＝217,560M^-1cm^-1And ε_252m＝80,062M^-1cm^-1(ii) a Known extinction coefficients ε of DM3 or DM4_280nm＝5,700M^-1cm^-And ε_252nm＝26,790M^-1cm^-1The concentration of the conjugate was determined spectrophotometrically. The resulting conjugate is monomeric and has an average of 3.2-3.5 DM3 or DM4 attached per antibody molecule.

Example 5c

Preparation of cytotoxic conjugates of huC242 antibody with maytansinoids 4a or 4B (method B, FIG. 5c)

A solution of the huC242 antibody (8mg/m1) in an aqueous buffer solution (50mM potassium phosphate, 50mM sodium chloride, 2mM disodium ethylenediaminetetraacetate) at pH6.5 was mixed with 7 to 10-fold molar excess of SMCC [ succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate, 26%]Incubate for 2 hours. The reaction mixture was purified by Sephadex G25 gel filtration column. Using the known extinction coefficient ε of the antibody_280nm＝217,560M^-1cm^-1The antibody concentration was determined spectrophotometrically.

With an aqueous buffer (50mM potassium phosphate, 50mM sodium chloride, 2mM ethylene glycol) pH6.5Ethylenediaminetetraacetic acid disodium salt) to 2.5mg/ml, then treated with 1.5 to 2.5-fold molar excess of DM3 or DM4 in dimethylethylamine (final concentration of DMA is 3% v/v). The reaction mixture was incubated at room temperature for 18 hours. The reaction mixture was purified by Sephadex G25 gel filtration column. Using a known extinction coefficient (. epsilon. for antibodies)_280nm＝217,560M^-1cm^-1And ε_252nm＝80,062M^-1cm^-1(ii) a For DM3 or DM4,. epsilon_280nm＝5,700M^-1cm^-1And ε_252nm＝26,790M^-1cm^-1) The concentration of the conjugate was determined spectrophotometrically. The resulting conjugate is monomeric and has an average of 3.2-3.5 DM3 or DM4 attached per antibody molecule.

Example 5d

Preparation of cytotoxic conjugates of huC242 antibody with maytansinoids 4a or 4b (method C, FIG. 5d)

A solution of huC242 antibody (8mg/ml) in pH6.5 aqueous buffer (50mM potassium phosphate, 50mM sodium chloride, 2mM disodium ethylenediaminetetraacetate) was mixed with 7-to 10-fold molar excess of SIAB [ N-succinimidyl 4- (iodoacetyl) -aminobenzoate, 27]Incubate for 2 hours. The reaction mixture was purified by Sephadex G25 gel filtration column. Using the known extinction coefficient ε of the antibody_280nm＝217,560M^-1cm^-1The antibody concentration was determined spectrophotometrically.

Example 6

Efficacy of huC 242-maytansinoid conjugates against HT-29 xenografts in vivo

Five week old female SCID mice (20 animals) were inoculated subcutaneously on the right side with HT-29 human colon cancer cells (1.5X 10)⁶Cells/mouse) dissolved in 0.1ml of serum-free medium. The tumors were allowed to grow for 11 days to an average volume of 100mm³. Then, the animals were randomly assignedFour groups (five animals per group). The first group received the huC242-DM1 conjugate (DM1 dose was 75 μ g/kg, qd x5) administered intravenously. The second group received the huC242-DM3 conjugate (DM3 dose was 75 μ g/kg, qd x5) administered intravenously. The third group received the intravenous huC242-DM4 conjugate (DM4 at a dose of 75 μ g/kg, qd x5), the fourth group of animals served as a control group and received PBS, and the treatment protocol was the same as in groups 1-3.

Tumor volume was measured twice weekly using the formula: tumor volume is calculated as 1/2 (length x width x height). Body weights of the animals were also measured twice a week. The results are shown in fig. 7. Tumors in the mouse control group grew to approximately 1000mm at day 35³The volume of (a). Treatment with huC242-DM1 resulted in a delay in tumor growth of 18 days, whereas conjugates made with the maytansinoids 4a and 4b of the invention were significantly more effective, delaying tumor growth to 28 days and 36 days, respectively.

Example 7

Efficacy of huC 242-maytansinoid conjugates against COLO205 xenografts in vivo

Five-week-old female SCID mice (20 animals) were inoculated subcutaneously on the right side with COLO205 human colon cancer cells (1.5X 10)⁶Cells/mouse) dissolved in 0.1ml of serum-free medium. The tumors were allowed to grow for 11 days to an average volume of 100mm³. The animals were then randomized into four groups (five animals per group). The first group received the huC242-DM1 conjugate (DM1 dose was 75 μ g/kg, qd x5) administered intravenously. The second group received the huC242-DM3 conjugate (DM3 dose was 75 μ g/kg, qd x5) administered intravenously. The third group received the intravenous huC242-DM4 conjugate (DM4 dose was 75 μ g/kg, qd x5), while the fourth group received PBS as a control group, with the same treatment protocol as in groups 1-3.

Tumor volume was measured twice weekly using the formula: tumor volume is calculated as 1/2 (length x width x height). Body weights of the animals were also measured twice a week. The results are shown in fig. 8. MouseTumors in the control group grew to approximately 900mm at day 24³The volume of (a). Treatment with huC242-DM1 resulted in a 20 day delay in tumor growth, whereas conjugates made with maytansinoid 4a of the invention were significantly more effective, resulting in complete tumor regression lasting 45 days. Conjugates made with maytansinoid 4b of the invention were even more effective, resulting in recovery in all treated animals.

Example 8

Effectiveness of MY 9-6-maytansinoid conjugates against HL-60 xenografts in vivo

Five-week-old female SCID mice (20 animals) were inoculated subcutaneously on the right side with HL-60 human myeloid leukemia cells (1.5X 10)⁶Cells/mouse) dissolved in 0.1ml of serum-free medium. The tumors were allowed to grow for 12 days to an average volume of 100mm³. The animals were then randomized into four groups (five animals per group). The first group received an intravenous administration of MY9-6-DM1 conjugate (DM1 at a dose of 200. mu.g/kg, qd x 5). The second group received an intravenous dose of MY9-6-DM3 conjugate (DM3 at a dose of 200. mu.g/kg, qd x 5). The third group received an intravenous administration of MY9-6-DM4 conjugate (DM4 at a dose of 200. mu.g/kg, qd x5), while the fourth group of animals received PBS as a control group, the treatment protocol being identical to that in groups 1-3.

Tumor volume was measured twice weekly using the formula: tumor volume is calculated as 1/2 (length x width x height). Body weights of the animals were also measured twice a week. The results are shown in fig. 9. Tumors in the mouse control group grew rapidly to approximately 1600mm at 21 days³The volume of (a). Treatment with MY9-6-DM1 resulted in a delay in tumor growth of approximately 5 days, whereas conjugates made with the maytansinoids 4a and 4b of the invention were significantly more effective, delaying tumor growth for greater than 20 days.

Example 9

Preparation of cytotoxic conjugates of the huMy9-6 antibody with maytansinoids DM4(4b)

A solution of humY9-6 antibody at a concentration of 8mg/ml was mixed with a 6.5-fold molar excess of SSNPB [ thiosuccinimide 4- (5 '-nitro-2' -pyridyldithio) butyrate ] in a pH6.550mM potassium phosphate solution containing 2mM ethylenediaminetetraacetic acid (buffer A) and 5% ethanol]And (4) incubation. The modified antibody was purified by Sephadex G25 gel filtration column equilibrated with buffer A, and the concentration of the purified antibody was determined spectrophotometrically using the extinction coefficient of the antibody at 280 nm. The modified antibody was diluted to 4.9mg/ml with buffer A and incubated with 1.7-fold molar excess of DM4 for 18 hours at room temperature, DM4 was added to the reaction mixture as a stock solution in dimethylethylamine (final concentration of dimethylacetamide is 3% v/v). The antibody-drug conjugate was purified through Sephadex G25 column equilibrated with PBS at pH 6.5. Using the known extinction coefficients of the antibody and DM4 (for the antibody,. epsilon.)_280nm＝206,460M^-1cm^-1，ε_252nm＝72,261M^-1cm^-1(ii) a For DM4,. epsilon_280nm＝5,700M^-1cm^-1，ε_252nm＝26,790M^-1cm^-1) The concentration of the conjugate was determined spectrophotometrically. The resulting antibody-drug conjugate had an average of 3.6 molecules of DM4 per antibody molecule. Biochemical analysis demonstrated that the antibody retained greater than 94% monomer and had binding affinity after conjugation, as compared to unmodified antibody, as determined by flow cytometry. The amount of drug not covalently attached to the antibody (free drug) was determined by HPLC analysis and was found to be less than 1% of all attached drugs.

Example 10

In vitro selectivity and Effect of huMy9-6-DM4 conjugates

The cytotoxicity of huMy9-6-DM4 on CD33 expressing cells (HL-60) and CD33 negative Namalwa cells was tested using a colony formation assay, in which cell killing activity was determined by quantifying the number of colonies that could grow after treatment. For CD 33-positive HL-60 human tumor cells, humY9-6-DM4 showed potent cell killing activity in vitro (FIG. 10). No significant cytotoxicity was observed for CD33 negative human Namalwa cells, suggesting that CD 33-dependent cytotoxicity was due to the specific targeting of the anti-CD 33 antibody huMy9-6 of the conjugate.

Example 11

In vivo Effect of huMy9-6-DM4 conjugate against HL60 human tumor xenografts in SCID mice

The in vivo effect of huMy9-6-DM4 was determined in SCID mice bearing human HL-60 tumor xenografts. Injecting HL-60 cells subcutaneously to allow the tumor to grow to an average volume of 100mm³. huMy9-6-DM4 was delivered intravenously once daily for 5 days at the doses indicated in figure 11. The dose is expressed in micrograms of DM4 in the conjugate, corresponding to an antibody dose of approximately 67 μ g per μ g of DM 4. Tumor volume was measured as an indicator of the efficacy of the treatment and body weight of the mice was monitored to indicate toxicity caused by the treatment. At doses that caused little toxicity, huMy9-6-DM4 induced a prolonged delay in tumor growth of human HL-60 cell xenografts (fig. 11). The effect of huMy9-6-DM4 was also compared to the effect of huMy9-6-DM 1. Unexpectedly, it was found that huMy9-6-DM4 was more effective than huMy9-6-DM 1. Whereas huMy9-6-DM4 maintained Complete Remission (CR) in animals for almost 60 days, animals treated with huMy9-6-DM1 relapsed after approximately 20 days of CR.

Example 12

Preparation of cytotoxic conjugates of huB4 antibodies with maytansinoids DM4(4b)

A20 mg/ml solution of huB4 antibody was mixed with an 8-fold molar excess of SSNPB [ sulfosuccinimide 4- (5 '-nitro-2' -pyridyldithio ] pyridine ] in a 50mM potassium phosphate buffer pH6.5 containing 2mM ethylenediaminetetraacetic acid (buffer A) and 5% dimethylacetamide) Butyric ester]And (4) incubation. The modified antibody was purified by Sephadex G25 gel filtration column equilibrated with buffer A and the extinction coefficient of the antibody at 280nm (199,560M)^-1cm^-1) The concentration of the purified antibody was determined spectrophotometrically. The modified antibody was diluted to 8mg/ml with buffer A and incubated with 1.7-fold molar excess of DM4 for 3 hours at room temperature, DM4 was added to the reaction mixture as a stock solution in dimethylacetamide (final concentration of dimethylacetamide is 3% v/v). The antibody-drug conjugate was purified through Sephadex G25 column and Sephadex S300 column equilibrated with PBS buffer solution of pH 6.5. Using antibodies (. epsilon.)_280nm＝199,560M^-1cm^-1；ε_252nm＝67,850M^-1cm^-1) And DM4(ε_280nm＝5,700M^-1cm^-1，ε_252nm＝26,790M^-1cm^-1) The concentration of the conjugate was determined spectrophotometrically, using the known extinction coefficient. In the resulting antibody-drug conjugate, there were an average of 4.0 molecules of DM4 per antibody molecule. Biochemical analysis demonstrated that more than 98% of the antibody remained monomeric and had binding affinity after conjugation, as compared to unmodified antibody, which could be determined using flow cytometry. The amount of drug not covalently attached to the antibody (free drug) was determined by HPLC analysis, which was about 2% of the total attached drug.

Example 13

In vitro selectivity and Effect of huB4-DM4 conjugates

Cytotoxicity of huB4-DM4 on CD-19 expressing cells (Ramos) and a CD-19 negative cell line (Colo205) was tested using an MTT-based assay, in which cell killing activity was determined by quantifying the number of surviving cells remaining after treatment with the conjugate. The number of surviving cells was determined spectrophotometrically after incubation of the cells with the vital dye MTT. HuB4-DM4 showed potent cell killing activity against CD-19 positive Ramos human tumor cells in vitro (FIG. 12). No significant toxicity was observed for CD-19 negative cells, suggesting that CD-19 dependent cytotoxicity was due to the specific targeting effect of the anti-CD 19 antibody huB 4.

Example 14

In SCID mice, in vivo Effect of huB4-DM4 conjugate against Ramos human tumor xenografts

The in vivo effect of huB4-DM4 was determined using SCID mice bearing established human Ramos tumor xenografts. Injecting Ramos cells subcutaneously to allow tumor growth to an average volume of 100mm³. The huB4-DM4 conjugate was delivered intravenously in a single injection at the doses indicated in figure 13 a. The dose is expressed as micrograms of DM4 in the conjugate, which corresponds to an antibody dose of about 44 μ g of antibody per μ g of DM 4. Tumor volume was measured as an indicator of treatment efficacy and mouse body weight was monitored to indicate toxicity caused by treatment. At doses above 50 μ g/kg, huB4-DM4 caused complete regression of tumors in all animals. In the 100 μ g/kg treatment group, the animals maintained a status of no detectable disease for approximately 35 days, and in the two highest dose groups this status was maintained for more than 55 days. These treatments caused little if any change in body weight of the treated animals if the toxicity was judged by such change (FIG. 13 b).

Claims

1. A compound represented by formula 4:

wherein:

y represents

(CR₇R₈)₁(CR₅R₆)_mCR₁R₂SZ, wherein:

a)R₁、R₅、R₆、R₇and R₈Is H; r₂Is methyl, 1 is 1, m is 1, and Z is H;

b)R₁、R₅、R₆、R₇and R₈Is H; r₂Is methyl, 1 is 1, m is 1, and Z is SCH₃；

c)R₅、R₆、R₇And R₈Is H; r₁And R₂Is methyl, 1 is 1, m is 1, and Z is H; or

d)R₅、R₆、R₇And R₈Is H; r₁And R₂Is methyl, 1 is 1, m is 1, and Z is SCH₃。

2. A compound represented by formula 4:

wherein:

y represents

(CR₇R₈)₁(CR₅R₆)_m(CR₃R₄)_nCR₁R₂SZ, wherein:

R₁and R₂Each independently is a linear alkyl group having 1 to 10 carbon atoms, a branched alkyl group having 3 to 10 carbon atoms, and further, R is₂May be H;

R₃、R₄、R₅、R₆、R₇and R₈Each of which is independently H, linear alkyl or alkenyl having from l to 10 carbon atoms, or branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl substituted with at least one alkyl having 1-4 carbon atoms, alkoxy, halo, or nitro, or a 3-to 10-membered heteroaryl having one or two heteroatoms selected from N, O or S, or a 3-to 10-membered heterocycloalkyl having one or two heteroatoms selected from N, O or S;

1. each of m and n is independently an integer from 1 to 5, further n may be 0; and

z is H, SR or-COR, wherein R is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, or a simple aryl group or an aryl group substituted with at least one alkyl group containing 1-4 carbon atoms, or an alkoxy, halogen or nitro group, or a 3 to 10 membered heterocyclic aryl group having one or two heteroatoms selected from N, O or S, or a 3 to 10 membered heterocyclic alkyl group having one or two heteroatoms selected from N, O or S.

3. The compound of claim 2, wherein:

R₃、R₄、R₅、R₆、R₇and R₈Each is independently H, a linear alkyl group having l to 10 carbon atoms, a branched alkyl group having 3 to 10 carbon atoms; and the number of the first and second electrodes,

r is a linear alkyl group having 1 to 10 carbon atoms, a branched alkyl group having 3 to 10 carbon atoms.

4. The compound of claim 2, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, 1 and m are each 1, n is 0, and Z is H.

5. The compound of claim 2, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are l, n is 0, Z is H.

6. The compound of claim 2, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, n is 0, Z is-SCH₃。

7. The compound of claim 2, wherein R₁And R₂Is methyl, R₅、R₆、R₇、R₈Are each H, 1 and m are l, n is 0, Z is-SCH₃。

8. A maytansinoid-cell-binding agent conjugate, wherein the maytansinoid is represented by formula 4₁The method comprises the following steps:

wherein:

Y₁is represented by (CR)₇R₈)₁(CR₅R₆)_m(CR₃R₄)_nCR₁R₂S-, wherein:

R₃、R₄、R₅、R₆、R₇and R₈Each of which is independently H, a linear alkyl or alkenyl group having l to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, phenyl, substituted phenyl substituted with at least one alkyl group having 1-4 carbon atoms, alkoxy, halogen or nitro, or a 3 to 10 membered heteroaryl group having one or two heteroatoms selected from N, O or S or a 3 to 10 membered heterocycloalkyl group having one or two heteroatoms selected from N, O or S;

1. each of m and n is independently an integer from 1 to 5, and further n may be 0.

9. The maytansinoid-cell-binding agent conjugate of claim 8, wherein R₃、R₄、R₅、R₆、R₇And R₈Each of which is independently H, a linear alkane having from l to 10 carbon atomsA branched alkyl group having 3 to 10 carbon atoms.

10. The maytansinoid-cell-binding agent conjugate of claim 8 or 9, wherein the cell-binding agent comprises at least one antibody binding site, and wherein the antibody is a humanized or resurfaced antibody of murine antibody B4 that specifically binds to the CD19 antigen.

11. The maytansinoid-cell-binding agent conjugate of claim 8 or 9, wherein the cell-binding agent comprises at least one antibody binding site, and wherein the antibody is MY9, anti-B4, or C242.

12. A maytansinoid-cell-binding agent conjugate, wherein the cell-binding agent is an antibody and the maytansinoid-cell-binding agent conjugate is represented by the formula:

wherein R is H or methyl; the antibody comprises at least one antibody binding site; and w is l-10.

13. The maytansinoid-cell-binding agent conjugate of claim 12, wherein R is methyl.

14. The maytansinoid-cell-binding agent conjugate of claim 12 or 13, wherein the antibody is a humanized or resurfaced antibody of murine antibody B4 that specifically binds to CD19 antigen.

15. The maytansinoid-cell-binding agent conjugate of claim 12 or 13, wherein the antibody comprises at least one binding site for MY9, anti-B4, or C242.

16. The maytansinoid-cell-binding agent conjugate of claim 15, wherein the antibody is MY9, anti-B4, or C242.

17. The maytansinoid-cell-binding agent conjugate of claim 15, wherein the antibody is a humanized or resurfaced MY9 that specifically binds to CD33 antigen, a humanized or resurfaced anti-B4 antibody that specifically binds to CD19 antigen, or a humanized or resurfaced C242 antibody that specifically binds to CanAg antigen.

18. A maytansinoid-cell-binding agent conjugate comprising at least one maytansinoid linked to a cell-binding agent, wherein the maytansinoid is

19. The maytansinoid-cell-binding agent conjugate of claim 18, wherein the maytansinoid is DM 4.

20. The maytansinoid-cell-binding agent conjugate of claim 18 or 19, wherein the cell-binding agent comprises at least one antibody binding site, and wherein the antibody is a humanized or resurfaced antibody of murine antibody B4 that specifically binds to the CD19 antigen.

21. The maytansinoid-cell-binding agent conjugate of claim 18 or 19, wherein the cell-binding agent comprises at least one antibody binding site, and wherein the antibody is MY9, anti-B4, or C242.

22. A pharmaceutical composition comprising an effective amount of the maytansinoid-cell-binding agent conjugate of any one of claims 8 to 21, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

23. Use of a conjugate according to any one of claims 8 to 21, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a tumour.

N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -S-alanine, N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -R-alanine or racemic N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -alanine.

25. A process for the manufacture of N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -S-alanine, N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -R-alanine or racemic N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -alanine comprising:

(1) reacting the isobutenylsulfide (5) with the anion of acetonitrile to give 4-mercapto-4-methyl-valeronitrile (6);

(2) hydrolyzing 4-mercapto-4-methyl-valeronitrile (6) to obtain 4-mercapto-4-methyl pentanoic acid (7);

(3) converting 4-mercapto-4-methylpentanoic acid (7) to 4-methyl-4- (methyldithio) pentanoic acid (8) by reaction with methylmercaptanesulfonate;

(4) converting 4-methyl-4- (methyldithio) pentanoic acid (8) to 4-methyl (methyldithio) pentanoic acid N-hydroxysuccinimide ester (9); and

(5) reacting N-hydroxysuccinimide 4-methyl (methyldithio) valerate (9) with N-methyl-L-alanine, N-methyl-D-alanine or racemic N-methylalanine to give said N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -S-alanine, N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -R-alanine or racemic N-methyl-N- (4-methyl-4-methyldithio-1-oxopentyl) -alanine.

A mixture of N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -S-alanine and N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine.

27. A method of making a mixture of N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -S-alanine and N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine comprising:

(1) reacting 4-mercaptopentanoic acid (12) with methyl methanethiol sulfonate to give 4-methyldithio-pentanoic acid (13);

(2) converting 4-methyldithio-pentanoic acid (13) to 4-methyldithio-pentanoic acid N-hydroxysuccinimide ester (14); and

(3) reacting N-hydroxysuccinimide 4-methyldithio-pentanoate (14) with N-methyl-L-alanine to obtain said mixture of N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -S-alanine and N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine.

A mixture of N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -R-alanine and N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -R-alanine.

29. A process for making a mixture of N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -R-alanine and N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -R-alanine comprising:

(3) reacting N-hydroxysuccinimide 4-methyldithio-pentanoate (14) with N-methyl-D-alanine to obtain said mixture of N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -R-alanine and N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -R-alanine.

N-methyl-N- [ 4-methyldithio-1-oxopentyl ] -alanine, N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -alanine or N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -alanine.

31. A process for the manufacture of N-methyl-N- [ 4-methyldithio-1-oxopentyl ] -alanine, N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -alanine or N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -alanine, which process comprises:

(l) Reacting 4-mercaptopentanoic acid (12) with methyl methanethiol sulfonate to give 4-methyldithio-pentanoic acid (13);

(2) converting 4-methyldithio-pentanoic acid (13) to 4-methyldithio-pentanoic acid N-hydroxysuccinimide ester (14);

(3) reacting 4-methyldithio-pentanoic acid N-hydroxysuccinimide ester (14) with racemic N-methylalanine to give said N-methyl-N- [ 4-methyldithio-1-oxopentyl ] -alanine.

N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -S-alanine.

33. A process for the manufacture of N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -S-alanine (15a) comprising:

(1) converting (R) -1, 3-butanediol to (S) -4- (methyldithio) pentanoic acid (19);

(2) converting (S) -4- (methyldithio) pentanoic acid (19) to its (S) -4- (methyldithio) pentanoic acid N-hydroxysuccinimide ester (20); and

(3) reacting (S) -4- (methyldithio) pentanoic acid N-hydroxysuccinimide ester (20) with N-methyl-L-alanine to give said N-methyl-N- [4- (S) -methyldithio-1-oxopentyl ] -S-alanine.

N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine.

35. A process for the manufacture of N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine (15b) comprising:

(1) converting (S) -1, 3-butanediol to (R) -4- (methyldithio) pentanoic acid (24);

(2) converting (R) -4- (methyldithio) pentanoic acid (24) to its (R) -4- (methyldithio) pentanoic acid hydroxysuccinimide ester (25); and

(3) reacting (R) -4- (methyldithio) pentanoic acid hydroxysuccinimide ester (25) with N-methyl-L-alanine to give said N-methyl-N- [4- (R) -methyldithio-1-oxopentyl ] -S-alanine.

36. A pharmaceutical composition comprising an effective amount of a compound of any one of claims 1-7 and 24, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

37. The pharmaceutical composition of claim 36, further comprising an antibody.

38. Use of a conjugate according to any one of claims 8 to 21, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inducing cell death in a selected cell population.

39. The use of claim 38, wherein the medicament is for the treatment of a malignant tumor.

40. The use of claim 39, wherein the malignancy is selected from breast cancer, prostate cancer, ovarian cancer, lung cancer, colon cancer, kidney cancer, pancreatic cancer, and lymphoid organ cancer.

41. The use of claim 40, wherein the lymphoid organ cancer is non-Hodgkin's lymphoma or chronic lymphoblastic leukemia.

42. The use of claim 40, wherein the lymphoid organ cancer is non-Hodgkin's lymphoma.