HK1129106B

HK1129106B - Cytotoxic agents comprising new tomaymycin derivatives and their therapeutic use

Info

Publication number: HK1129106B
Application number: HK09107327.0A
Authority: HK
Inventors: Laurence Gauzy; Robert Zhao; Yonghong Deng; Wei Li; Hervé Bouchard; Ravi V.J. Chari; Alain Commercon
Original assignee: 赛诺菲-安万特
Priority date: 2006-01-25
Filing date: 2007-01-22
Publication date: 2014-07-11

Description

Cytotoxic agents containing novel tomaymycin derivatives and their therapeutic use

Technical Field

The present invention relates to novel cytotoxic agents and their therapeutic use. More particularly, the present invention relates to novel cytotoxic agents containing tomaymycin derivatives and their therapeutic use. These novel cytotoxic drugs have therapeutic utility due to the targeted delivery of tomaymycin derivatives to specific cell populations through their chemical attachment to cell binding drugs.

Background

Numerous reports have been published on attempts to specifically target tumor cells using monoclonal Antibody drug conjugates (Sela et al, in Immuno-conjugates, 189-216(C.Vogel, ed.1987); Ghose et al, in Targeted Drugs 1-22(E.Goldberg, ed.1983); diene et al, in anti-body media Delivery systems, 1-23(J.Rodwell, ed.1988); Pietesz et al, in anti-body media Delivery systems, 25-53(J.Rodwell, caned.1988); Bumol et al, in anti-body media Delivery systems, 55-79(J.Rodwell, ed.1988); G.A.Pietesz & K.Krauer, 2, J.drug Delivery, 183. blend, 55-79(J.Rodwell, 1988); Seal. Delivery systems, 31-9, 31. Va et al, Chamber et al., 31. Delivery systems, 31. Va et al.; adjuvant, 5. Chamber. Va et al., 5, et al., 5, et al. All references and patents listed herein are incorporated by reference herein.

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been conjugated to various murine monoclonal antibodies. In some cases, the drug molecule is linked to the antibody molecule by an intermediate carrier molecule, such as serum albumin (Garnett et al, 46, Cancer Res.2407-2412 (1986); Ohkawa et al 23, Cancer Immunol.Immunother.81-86 (1986); Endo et al 47 Cancer Res.1076-1080(1980)), dextran (Hurwitz et al, 2 appl.biochem.25-35 (1980); Manabi et al 34 biochem.Pharmacol.289-291 (1985); Dillman et al 46 Cancer Res.4886-4891 (1986); Shoval et al, 85, Proc. Natl.Acad.Sci, 8276-8280(1988)), or polyglutamic acid (ukada et al, uk73, J.Natl.721.721.721, 721 J.1984, 1984, 19814, 1985), Tsab.

Various types of linker technologies have been applied to the preparation of such immunoconjugates, both cleavable and non-cleavable linkers being investigated, however, in most cases, only the full cytotoxic effect of the drug is observed, even though the drug molecule may be released from the conjugate in an unmodified form at the targeted site.

Cleavable linkers that have been used to prepare antibody drug conjugates are cis-aconitic acid-based acid labile linkers that utilize the acidic environment of different intracellular compartments, such as endosomes and lysosomes encountered in receptor mediated endocytosis. Shen and Ryser describe such methods for preparing conjugates of daunorubicin and macromolecular carriers (102 biochem. biophysis. res. commun., 1048-1054 (1981)). The same technique was used by Yang and Reisfeld to bind daunorubicin to an anti-melanoma antibody (80 j. natl. cane. inst.1154-1159 (1988)). Dillman et al also used acid-labile linkers in a similar manner to prepare conjugates of daunorubicin and anti-T cell antibodies (48 Cancer Res.6097-6102 (1988)).

Another approach explored by Trouet et al involves linking daunorubicin to antibodies via a peptide arm (79 Proc. Natl. Acad. Sci.626-629 (1982)). This is based on the premise that free drug can be released from e.g. the conjugate by the action of lysosomal peptidases.

However, in vitro cytotoxicity assays show that antibody drug conjugates rarely achieve the same cytotoxic potency as the free, unattached drug. This suggests that the mechanism of drug molecule release from antibodies may be very inefficient, and that in the immunotoxin region, the conjugates formed between the monoclonal antibodies and the catalytic circulating protein toxins via disulfide bridges appear to be more cytotoxic than those comprising other linkers. See Lambert et al, 260 J.biol.chem.12035-12041 (1985); lambert et al, in Immunotoxins 175-; ghetie et al, 48, Cancer Res.2610-2617 (1988). This is attributed to the high intracellular concentration of glutathione, which facilitates efficient cleavage of disulfide bonds between the antibody molecule and the toxin. Nevertheless, there are only a few reported examples of the use of disulfide bridges for the preparation of conjugates between drugs and macromolecules. Shen et al (260, j. biol. chem.10905-10908(1985)) describe the conversion of methotrexate to mercaptoethylamide derivatives, followed by disulfide bonding to poly-D-lysine. Another report describes the preparation of trisulfide conjugates containing the toxin drug calicheamicin and an antibody (Hinman et al, 53 Cancer Res.3336-3342 (1993)).

One reason for the lack of disulfide linkage to antibody drug conjugates is the unavailability of cytotoxic drugs with sulfur-containing groups that are readily available for linking the drug to the antibody via a disulfide bridge, and furthermore, chemical modification of existing drugs without reducing their cytotoxic potency is difficult.

Another major drawback of existing antibody drug conjugates is the inability to deliver sufficient concentrations of drug to a target site due to the relatively modest cytotoxicity of a limited number of targeted antigens and cancerostatic drugs, such as methotrexate, daunorubicin, and vincristine. To achieve significant cytotoxicity, the attachment of a large number of drug molecules, either directly to the antibody or via a polymeric carrier molecule, is required. However, such heavily modified antibodies often show impaired binding to the target antigen and are rapidly cleared from the blood in vivo.

Despite the above difficulties, useful cytotoxic drugs containing a cell binding moiety and a cytotoxic Drug moiety, known as maytansinoids, have been reported (U.S. Pat. No.5,208,020, U.S. Pat. No.5,416,064, and R.V.J.Chari, 31 Advanced Drug Delivery Reviews89-104 (1998)). Similarly, useful cytotoxic drugs containing cell binding moieties and analogs and derivatives of the potent anti-tumor antibody CC-1065 have also been reported (USP 5,475,092, USP5,585,499 and USP 6,756,397).

The tomaymycin derivative is pyrrolo [1, 4 ]]Benzodiazepine(PBD), a known class of compounds that exert their biological properties through N2 covalently bound to guanine in the minor groove of DNA. PBDs include many minor groove binders such as amphenicol, neothramycin and DC-81, however, tomaymycin anticancer activity is limited due to its non-specific toxicity to normal cells. There is therefore a need to increase the therapeutic activity of tomaymycin compounds and to reduce the non-specific toxic effects. The present inventors have shown that this need is met by targeted delivery of a tomaymycin compound by linking the compound to a cell-binding drug. In addition, there is a need to develop thatch which is soluble and stable in aqueous solutionsA derivative of a mycin. In addition, tomaymycin is not sufficiently effective for use as a conjugate of a cell-binding drug.

In recent years, a few novel PBD derivatives and their anticancer activity in a latent phase model have been disclosed (WO 00/12508 and WO 2005/085260). However, initial human clinical trials have shown that such compounds are highly toxic based on the very low doses that can be administered to humans (i.puzanov, proc.aacr-NCl-EORTC International Conference, philiadelphia, USA2005, Abstract # B117). Thus, there is a need to provide alternative derivatives that are more effective and/or that can bind to cell binding drugs.

Therefore, there is a great need for methods of treating diseases with tomaymycin derivatives, wherein the side effects are reduced without affecting their cytotoxicity.

Summary of The Invention

As described in the first embodiment, it is an object of the present invention to provide tomaymycin derivatives which are highly toxic, but still effective in the treatment of a number of diseases.

It is another object of the present invention to provide a novel tomaymycin, optionally linked or linked to a cell binding agent.

In a second embodiment, the present invention provides a therapeutic composition comprising:

(A) an effective amount of one or more tomaymycin derivatives, optionally linked or linked to a cell binding agent, and

(B) a pharmaceutically acceptable carrier, diluent or excipient.

In a third embodiment, the invention provides a method of killing a selected cell population comprising contacting a cell of interest or a tissue comprising a cell of interest with a cytotoxic amount of a cytotoxic drug comprising one or more tomaymycin derivatives, optionally attached or linked to a cell binding drug.

Detailed Description

The present invention is based on the synthesis of novel tomaymycin derivatives that maintain high cytotoxicity and are efficiently linked to cell binding drugs. The prior art has shown that linking highly cytotoxic drugs to antibodies with cleavable linkages, such as disulfide bonds, ensures the release of the fully active drug in the cell, the conjugate being cytotoxic in an antigen-specific manner (U.S. Pat. No. 6,340,701; U.S. Pat. No. 6,372,738; U.S. Pat. No. 6,436,931). However, the prior art shows that it is extremely difficult to modify existing drugs without reducing their cytotoxic effects, and the present invention solves this problem by modifying the disclosed tomaymycin derivatives with chemical groups. As a result, the disclosed novel tomaymycin derivatives protect, and in some cases even enhance, the cytotoxic efficacy of the tomaymycin derivatives. The cell-binding drug-tomaymycin derivative complex allows the cytotoxic effect of the tomaymycin derivative to act exclusively in a targeted manner on unwanted cells, thus avoiding side effects due to damage to non-target healthy cells. Thus, the present invention provides a medicament for the elimination of killed or lysed diseased or abnormal cells, such as tumor cells (especially solid tumor cells).

The cytotoxic agents of the present invention comprise one or more tomaymycin derivatives optionally attached or already attached to a cell binding agent via a linking group. The linking group is a moiety that is covalently attached to the tomaymycin derivative by conventional methods. In a preferred embodiment, the chemical group may be covalently bound to the tomaymycin derivative via a disulfide bond.

The tomaymycin derivatives used in the present invention have the formula (I) shown below:

wherein

- - -represents an optional single bond;

represents a single bond or a double bond;

the premise is thatU and U ', equal OR different when representing a single bond, represent H, W and W', equal OR different, are selected from the group consisting of OH, ethers, such as-OR, esters (such as acetate), such as-OCOR, carbonates, such as-OCOOR, carbamates, such as-OCONRR ', cyclic carbamates, N10 and C11 are cyclic moieties, ureas, such as-NRCONRR', thiocarbamates, such as-OCNHR, cyclic thiocarbamates, N10 and C11 are cyclic moieties, -SH, sulfides, such as-SR, sulfoxides, such as-SOR, sulfones, such as-SOOR, sulfonates, such as-SO₃ ^-Sulfonamides, e.g. -NRSOOR, amines, e.g. -NRR ', optionally cyclized amines, N10 and C11 being part of a ring, hydroxylamine derivatives, e.g. -NROR', amides, e.g. -NRCOR, azidos, e.g. -N₃Cyano, halogen, trialkyl or triarylphosphonium, amino acid derived groups; preferably W and W' are the same or different and are OH, OMe, OEt, NHCONH₂、SMe；

And whenWhen represents a double bond, U and U 'are absent, and W' represent H;

r1, R2, R1 ' and R2 ' are the same or different and are each selected from halides or optionally substituted by one or more Hal, CN, NRR ', CF₃OR, aryl, Het, S (O)_qR-substituted alkyl, or R1 and R2 and R1 ' and R2 ' together form a double bond comprising the groups ═ B and ═ B ', respectively.

Preferably, R1 and R2 and R1 ' and R2 ' together form a double bond comprising the groups ═ B and ═ B ', respectively.

B and B 'are the same or different and are each selected from the group consisting of optionally substituted by one or more of Hal, CN, NRR', CF₃OR, aryl, Het, S (O)_qR-substituted alkenyl or B and B' represent an oxygen atom.

Preferably, B ═ B'.

More preferably, B ═ CH₂Or ═ CH-CH₃。

X, X' are the same or different and are each selected from one or more of-O-, -NR-, - (C ═ O) -, -S (O)_q-。

Preferably, X ═ X'.

More preferably, X ═ X' ═ O.

A. A 'are identical or different and are each selected from alkyl or alkenyl optionally containing one O, N or S atom, optionally substituted by one or more Hal, CN, NRR', CF₃、OR、S(O)_qR, aryl, Het, alkyl and alkenyl.

Preferably a ═ a'.

More preferably, a ═ a' ═ straight chain unsubstituted alkyl groups.

Y, Y' are the same or different and are each selected from H, OR;

y ═ Y' is preferred.

More preferably, Y ═ O alkyl, and still more preferably methoxy.

T is-NR-, -O-, -S (O)_q-, or 4-to 10-membered aryl, cycloalkyl, heterocycle or heteroaryl, each optionally substituted by one or more Hal, CN, NRR', CF₃、R、OR、S(O)_qR, and/or linker substituted or branched alkyl, optionally substituted by one or more Hal, CN, NRR', CF₃、OR、S(O)_qR and/or a linker, or by one or more Hal, CN, NRR', CF₃、OR、S(O)_qR and/or linkerSubstituted straight chain alkyl.

Preferably T is a 4-10 membered aryl or heteroaryl group, more preferably phenyl or pyridyl, optionally substituted by one or more linkers.

The linker comprises a linking group, suitable linking groups are known in the art and include thiol, sulfide, disulfide, thioether, acid labile, photolabile, peptidase and esterase labile groups. Disulfide groups and thioether groups are preferred.

When the linking group is a thiol-, sulfide (or so-called thioether-S-) or disulfide (-S-S-) group, the side chain carrying the thiol, sulfide or disulfide group may be straight or branched, aromatic or heterocyclic. Suitable side chains can be readily determined by one of ordinary skill in the art.

Preferably, the linker is of the formula:

-G-D-(Z)P-S-Z’

wherein G is a single or double bond, -O-, -S-, or-NR-;

d is a single bond or-E-, -E-NR-F-, -E-O-F-, -E-NR-CO-F-, -E-CO-, -CO-E-, -E-CO-F, -E-S-F-, -E-NR-C-S-, -E-NR-CS-F-;

wherein E and F are the same or different and are independently selected from the group consisting of straight or branched- (OCH)₂CH₂)_iAlkyl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_i-alkyl-, - (OCH)₂CH₂)_i-、-(OCH₂CH₂)_iCycloalkyl (OCH)₂CH₂)_j、-(OCH₂CH₂)_iHeterocyclyl (OCH)₂CH₂)_j、-(OCH₂CH₂)_iAryl (OCH)₂CH₂)_j-、-(OCH₂CH₂)_iHeteroaryl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_iAlkyl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_i-alkyl- (OCH)₂CH₂)_iCycloalkyl (OCH)₂CH₂)_j-alkyl- (OCH)₂CH₂)_iHeterocyclyl (OCH)₂CH₂)_j-alkyl- (OCH)₂CH₂)_iAryl (OCH)₂CH₂)_j-alkyl- (OCH)₂CH₂)_iHeteroaryl (OCH)₂CH₂)_j-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-;

wherein i and j, which are the same or different, are integers selected from 0, 1-2000;

z is linear or branched-alkyl-;

p is 0 or 1;

z' represents H, a thiol protecting group, e.g. COR, R₂₀Or SR₂₀Wherein R is₂₀Represents H, methyl, alkyl, optionally substituted cycloalkyl, aryl, heteroaryl or heterocycle, with the proviso that when Z' is H, the compound is in equilibrium with the corresponding compound formed by intramolecular cyclization from the addition of a thiol group-SH onto the imine bond-NH ═ of one of the PBDs.

n, n', equal or different, are 0 or 1.

q is 0, 1 or 2.

R, R 'are the same or different and are each selected from H, alkyl, aryl, each optionally Hal, CN, NRR', CF₃、R、OR、S(O)_qR, aryl, Het;

or a pharmaceutically acceptable salt, hydrate or hydrate salt thereof, or a polymorphic crystal structure of these compounds or an optical isomer, racemate, diastereomer or enantiomer thereof.

The present invention relates to the following preferred embodiments or any combination thereof:

-G is a single bond or-O-or-NR-;

-G is-O-;

-D is a single bond or-E-, -E-NR-CO-, -ECO-, -CO-E-;

-D is-E-, -E-NR-CO-;

-D is-E-NR-CO-;

e is a linear or branched-alkyl-, - (OCH)₂CH₂)_i-or alkyl-heterocyclyl;

-E is a linear or branched-alkyl-;

-Z is- (CH)₂)₂-C(CH₃)₂-；

-p is 0 or 1;

z' is H or SR₂₀Wherein R is₂₀Represents an alkyl, aryl, heterocyclyl or heteroaryl group;

z' is H or SR₂₀Wherein R is₂₀Represents an alkyl group;

specific examples of thiol-, sulfide-, or disulfide-containing linkers include

-(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_y(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(NR₁₉CO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(OCO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(CO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(CONR₁₉)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t-phenyl-CO (CR)₁₅R₁₆)_uSZ’、-(CR₁₃R₁₄)_t-furyl-CO (CR)₁₅R₁₆)_uSZ’、

-(CR₁₃R₁₄)_t-oxazolyl-CO (CR)₁₅R₁₆)_uSZ’、-(CR₁₃R₁₄)_tthiazolyl-CO (CR)₁₅R₁₆)_uSZ’、

-(CR₁₃R₁₄)_t-thienyl-CO (CR)₁₅R₁₆)_uSZ’、-(CR₁₃R₁₄)_timidazolyl-CO (CR)₁₅R₁₆)_uSZ’、

-(CR₁₃R₁₄)_t-morpholino-CO (CR)₁₅R₁₆)_uSZ’、-(CR₁₃R₁₄)_t-piperazinyl-CO (CR)₁₅R₁₆)_uSZ’、

-(CR₁₃R₁₄)_t-N-methylpiperazino-CO (CR)₁₅R₁₆)_uSZ’、

-(CR₁₃R₁₄)_t-phenyl-QSZ', (CR)₁₃R₁₄)_t-furyl-QSZ', (CR)₁₃R₁₄)_t-oxazolyl-QSZ

-(CR₁₃R₁₄)_t-thiazolyl-QSZ', - (CR)₁₃R₁₄)_t-thienyl-QSZ', (CR)₁₃R₁₄)_t-imidazolyl radical

-QSZ’、-(CR₁₃R₁₄)_t-morpholino-QSZ', (CR)₁₃R₁₄)_t-piperazinyl-QSZ', - (CR)₁₃R₁₄)_t-N-methylpiperazinyl-QSZ', or

-O(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-O(CR₁₃R₁₄)_t(NR₁₉CO)(CR₁₅R₁₆)_u(O CH₂CH₂)_ySZ’、

-O(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_t(OCH₂CH₂)_ySZ’、

-O-phenyl-QSZ ', -O-furyl-QSZ ', -O-oxazolyl-QSZ ', -O-thiazolyl-QSZ ', -O-thienyl-QSZ ', -O-imidazolyl-QSZ ', -O-morpholino-QSZ ', -O-piperazinyl-QSZ ', -O-N-methylpiperazinyl-QSZ ',

-OCO(CR₁₃R₁₄)_t(NR₁₉CO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-OCO(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-OCONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-OCO-phenyl-QSZ ', -OCO-furyl-QSZ ', -OCO-oxazolyl-QSZ ', -OCO-thiazolyl-QSZ ', -OCO-thienyl-QSZ ', -OCO-imidazolyl-QSZ ', -OCO-morpholino-QSZ ', -OCO-piperazinyl-QSZ ', -OCO-N-methylpiperazinyl-QSZ ', or

-CO(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-CO(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-CONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-CO-phenyl-QSZ ', -CO-furyl-QSZ', -CO-oxazolyl-QSZ ', -CO-thiazolyl-QSZ', -CO-thienyl-QSZ ', -CO-imidazolyl-QSZ', -CO-morpholino-QSZ ', -CO-piperazinyl-QSZ', -CO-piperidino-QSZ ', -CO-N-methylpiperazinyl-QSZ'; and/or pharmaceutically acceptable salts thereof,

-NR₁₉(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-NR₁₉CO(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-NR₁₉(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_t(OCH₂CH₂)_ySZ’、

-NR₁₉CO(C R₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_t(OCH₂CH₂)_ySZ’、

-NR₁₉CONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-NR₁₉CONR₁₂(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_t(OCH₂CH₂)_ySZ’、

-NR₁₉CO-phenyl-QSZ', -NR₁₉CO-furyl-QSZ', -NR₁₉CO-oxazolyl-QSZ

-NR₁₉CO-thiazolyl-QSZ', -NR₁₉CO-thienyl-QSZ', -NR₁₉CO-imidazolyl-QSZ

-NR₁₉CO-morpholino-QSZ', -NR₁₉CO-piperazinyl-QSZ', -NR₁₉CO-piperidino-QSZ', -NR₁₉CO-N-methylpiperazinyl-QSZ',

-NR₁₉-phenyl-QSZ', -NR₁₉-furyl-QSZ', -NR₁₉-oxazolyl-QSZ', -NR₁₉-thiazolyl-QSZ', -NR₁₉-thienyl-QSZ', -NR₁₉-imidazolyl-QSZ', -NR₁₉-morpholino-QSZ

-NR₁₉-piperazinyl-QSZ', -NR₁₉-piperidino-QSZ', -NR₁₉-N-methylpiperazinyl-QSZ

-NR₁₉CO-NR₁₂-phenyl-QSZ', -NR₁₉CO-NR₁₂-oxazolyl-QSZ', -NR₁₉CO-NR₁₂-thiazolyl-QSZ', -NR₁₉CO-NR₁₂-thienyl-QSZ', -NR₁₉CO-NR₁₂-piperidino-QSZ

-S(O)_q(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-S(O)_q(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_t(OCH₂CH₂)_ySZ’、

-SCONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-SCO-morpholino-QSZ ', -SCO-piperazinyl-QSZ', -SCO-piperidino-QSZ 'and-SCO-N-methylpiperazinyl-QSZ', wherein:

z' is H, a thiol protecting group, e.g. COR, R₂₀' or SR₂₀', wherein R₂₀' represents H, alkyl, aryl, heterocycle or heteroaryl;

wherein Q is a direct link or a linear or branched alkyl group having 1 to 10 carbon atoms or a polyethylene glycol spacer having 2 to 20 repeating ethyleneoxy units;

R₁₉and R₁₂Are identical or different and are a straight-chain, branched or cyclic alkyl radical having from 1 to 10 carbon atoms or a simple or substituted aryl or heterocyclic radical, and R furthermore₁₂May be H;

R₁₃、R₁₄、R₁₅and R₁₆Are identical or different and are H or a linear or branched alkyl radical having from 1 to 4 carbon atoms;

R₁₇and R₁₈Is H or alkyl;

u is an integer of 1 to 10, and may be 0;

t is an integer of 1 to 10, and may be 0;

y is an integer of 1 to 20, and may be 0.

When the compound of formula (I) is in ionic form (e.g. a sulfonate salt), a counterion (e.g. Na) may be present⁺Or K⁺)。

According to a preferred aspect, the compounds of the invention are compounds of formula (I) wherein T is optionally substituted by one or more Hal, CN, NRR', CF₃、R、OR、S(O)_qR and/or linker-substituted aryl A, A ', X, X', U, U ', W, W', m ', n' -, - -, or,Is as defined above.

According to another preferred embodiment, the compounds of the invention are selected from the following:

8, 8' - [1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [ 5-methoxy-1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [1, 5-pentanediylbis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8，8' - [1, 4-butanediyl bis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [ 3-methyl-1, 5-pentanediylbis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [2, 6-pyridinediyl (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-pyridinediyl bis- (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [5- (3-aminopropoxy) -1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-benzenediyl bis- (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - {5- [3- (4-methyl-4-methyldithio (sulfonyl) -pentanoylamino) propoxy]-1, 3-benzenediylbis (methyleneoxy) } -bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]

8, 8' - [ 5-acetylthiomethyl-1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

Bis- {2- [ (S) -2-methylene-7-methoxy-5-oxo-1, 3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-8-yloxy]-ethyl } -carbamic acid tert-butyl ester

8, 8' - [3- (2-acetylthioethyl) -1, 5-pentanediylbis (oxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [5- (N-4-methyldithio-4, 4-dimethylbutyryl) -amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (2- (4-mercapto-4-methyl) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -benzene-3, 5-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -yl-7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (4- (4-methyl-4-methyldithio) -pentanoylamino-butoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl)]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl)]-propyl) -benzene-3, 5-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy)]-ethoxy } -ethoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxyPhenyl-3, 5-dimethyl-dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy)]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (2- [ methyl- (2-methyl-2-methyldithio-propyl) -amino)]-ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (3- [ methyl- (4-methyl-4-methyldithio-pentanoyl) -amino)]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (3- [ methyl- (2-methyl-2-methyldithio-propyl) -amino)]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (4-methyl-4-methyldithio) -pentanoylamino) -benzene-3, 5-dimethyl]-dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

As well as the corresponding mercapto derivatives thereof,

Preferred compounds are the following:

x, X ', A, A', Y, Y ', T, n' are as defined above.

When used above or below:

alk represents an alkyl, alkenyl or alkynyl group.

"alkyl" means an aliphatic hydrocarbon group which may be straight or branched chain, having 1 to 20 carbon atoms in the chain or a ring having 3 to 10 carbon atoms, with preferred alkyl groups having 1 to 12 carbon atoms in the chain. "branched" means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, and cyclohexyl.

"alkenyl" means an aliphatic hydrocarbon group containing a carbon-carbon double bond, which may be straight or branched, having from 2 to 15 carbon atoms in the chain, with preferred alkenyl groups having from 2 to 12 carbon atoms in the chain, and more preferably about 2 to 4 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, nonenyl, decenyl.

"alkynyl" means an aliphatic hydrocarbon group containing a carbon-carbon triple bond, which may be straight or branched chain, having 2 to 15 carbon atoms in the chain, with alkynyl groups having 2 to 12 carbon atoms in the chain being preferred; more preferably about 2 to 4 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, octynyl and decynyl.

"halogen atom" means a fluorine, chlorine, bromine or iodine atom; fluorine and chlorine atoms are preferred.

"aryl" means an aromatic monocyclic or polycyclic hydrocarbon ring system of 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, exemplary aromatics including phenyl or naphthyl.

"Het" refers to a heterocycle or heteroaryl.

The term "heterocycle" or "heterocyclyl" as used herein refers to a saturated, partially unsaturated or unsaturated, non-aromatic stable 3-14, preferably 5-10 membered mono-, di-or polycyclic ring wherein at least one member of the ring is a heteroatom. Typical heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, selenium and phosphorus atoms, with preferred heteroatoms being oxygen, nitrogen and sulfur.

Suitable heterocycles are also disclosed in the handbook of Chemistry and Physics, 76 th edition, CRCPress, Inc., 1995-1996, pages 2-25 to 2-26, the contents of which are incorporated herein by reference.

Preferred non-aromatic heterocyclic groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyridinyl, tetrahydroindenyl (pyrindinyl), dihydrothiopyranyl, azepanyl, and fused ring systems fused to phenyl.

The term "heteroaryl" or heteroaromatic ring as used herein refers to a 5-14 membered, preferably 5-10 membered, heteroaromatic mono-, di-or polycyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2, 4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzopyrimidinyl, isoxazolyl, pyridyl-N-oxide, and fused systems formed by fusing with phenyl.

"alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocycle" and the like also refer to the corresponding "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene", "heterocyclylene", and the like, which are formed by the removal of two hydrogen atoms.

The term "linkable to a cell-binding drug" as used herein means that the tomaymycin derivative comprises at least one linking group or a precursor thereof, which is suitable for bonding said derivative to the cell-binding drug; preferred linking groups are thiols, sulfides or disulfides or precursors thereof.

As used herein, the term "linked to a cell-binding drug" means that the conjugate molecule comprises at least one tomaymycin derivative bonded to the cell-binding drug via a suitable linking group or precursor thereof, preferably a thiol or disulfide linkage or precursor thereof.

As used herein, a "precursor" of a given group refers to any group that can produce the group by any deprotection, chemical modification, or coupling reaction.

The term "patient" as used herein refers to an animal, such as a valuable animal for feeding, companion or preservation purposes, or preferably a human or child, that has or is likely to have one or more of the diseases and symptoms described herein.

As used herein, "therapeutically effective amount" refers to an amount of a compound of the present invention that is effective for preventing, reducing, eliminating, treating, or managing the diseases and conditions described herein. The term "controlling" refers to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and symptoms described herein, but need not be shown to completely eliminate all disease and symptom syndromes, and is meant to include prophylactic treatment.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, excipients, compositions or dosage forms which are, within the scope of sound clinical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem complications commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the compounds in which the parent compound is modified by making acidic or basic salts thereof. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, the conventional non-toxic salts include salts obtained from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, gluconic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, maleic acid, lactic acid, and the like. Other adduct salts include ammonium salts such as tromethamine, meglumine, epolamine, and the like, metal salts such as sodium, potassium, calcium, zinc, or magnesium.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts is given in Remington's Pharmaceutical Sciences, 17^thed., mack publishing Company, Easton, PA, 1985, p.1418, the contents of which are incorporated herein by reference.

Compounds of formula (I) having geometric and stereoisomers are also part of the invention.

The N-10, C-11 double bond of tomaymycin derivatives of formula (I) is known to be readily converted in a reversible manner to the corresponding imine adduct in the presence of water, alcohols, thiols, primary or secondary amines, urea and other nucleophiles. The process is reversible and the corresponding tomaymycin derivative is easily regenerated in the presence of a dehydrating agent in an aprotic organic solvent under vacuum or at high temperature (z. tozuka, 36, j. antibiotics, 276 (1983)).

Accordingly, the present invention also provides reversible derivatives of tomaymycin derivatives of general formula (II):

wherein A, X, Y, n, T, A ', X ', Y ', n ', R1, R2, R1 ', R2 ' are as defined for formula (I), W and W ', which are identical OR different, are each chosen from the group OH, ethers, such as-OR, esters, such as acetates, such as-OCOR, -COOR, carbonates, such as-OCOOR, carbamates, such as-OCONRR ', cyclic carbamates, N10 and C11 are cyclic moieties, ureas, e.g., -NRCONRR', thiocarbamates, e.g., -OCSHNR, cyclic thiocarbamates, N10 and C11 are cyclic moieties, -SH, sulfides, e.g., -SR, sulfoxides, e.g., -SOR, sulfones, e.g., -SOOR, sulfonates, e.g., -SO₃ ^-Sulfonamides, e.g. -NRSOOR, amines, e.g. -NRR ', optionally cyclized amines, N10 and C11 being part of a ring, hydroxylamine derivatives, e.g. -NROR ', amides, e.g. -NRCOR, -NRCONRR ', azidos, e.g. -N₃Cyano, halogen, trialkyl or triarylphosphonium, amino acid derived groups; preferably W and W' are the same or different and are OH, OMe, OEt, NHCONH₂、SMe。

Thus, the compound of formula (II) may be considered a solvate, including water when the solvent is water; these solvates are particularly useful.

According to another further object, the invention also relates to a process for the preparation of the compounds of formula (I).

The compounds and methods of the present invention can be prepared in a number of ways known to those skilled in the art. The compounds can be synthesized, for example, by application or by employing the methods described below or methods modified as desired by the skilled artisan. Suitable modifications and alternatives are apparent and known to those skilled in the art or are readily available from the scientific literature.

This method can be found in particular in R.C. Larock, Comprehensive organic transformations, Wiley-VCH Publishers, 1999.

It will be apparent that the compounds of the invention may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms. Thus, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is known in the art how to prepare and isolate these optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse phase and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis, from chiral starting materials or by intended synthesis of the chiral center of interest.

The compounds of the present invention can be prepared by a variety of synthetic routes, with reagents and starting materials being commercially available or readily synthesized by techniques known to those skilled in the art. All substituents, unless otherwise specified, are as defined above.

In the reactions described below, it is necessary to protect reactive functional groups, such as hydroxyl, amino, imino, thiol or carboxyl groups, which are required in the final product, in order to avoid their unwanted participation in the reaction. Conventional protecting groups may be used according to standard practice, see for example t.w.greene and p.g.m.wutsin protective groups in Organic Chemistry, 3^rded., John Wiley and Sons, 1999; J.F.W.mcOmie in protective groups in Organic Chemistry, Plenum Press, 1973.

Some of the reactions may be carried out in the presence of a base, the nature of the base used in this reaction is not particularly limited, and any base conventionally used in such reactions may be used as well, as long as it does not adversely affect the rest of the molecule. Examples of suitable bases include: sodium hydroxide, potassium carbonate, triethylamine, alkali metal hydrides such as sodium hydride and potassium hydride; alkyl lithium compounds such as methyl lithium and butyl lithium; and alkali metal alkoxides such as sodium methoxide and sodium ethoxide.

The reaction is usually carried out in a synthesis solvent, and various solvents can be used as long as they do not adversely affect the reaction or the reagents contained therein. Examples of suitable solvents include: hydrocarbons, which may be aromatic, aliphatic or cycloaliphatic, such as hexane, cyclohexane, benzene, toluene and xylene; amides such as dimethylformamide; alcohols such as ethanol and methanol, and ethers such as diethyl ether and tetrahydrofuran.

The reaction can be carried out over a wide range of temperatures, and in general we have found that it is convenient to carry out the reaction at a temperature of from-20 ℃ to 150 ℃ (more preferably from about room temperature to 100 ℃). The time required for the reaction may also vary widely, depending on a number of factors, mainly the reaction temperature and the nature of the reagents. However, if the reaction is carried out under the preferred conditions described above, a period of from 3 to 20 hours is generally sufficient.

The compound produced can be recovered from the reaction mixture by a conventional method. For example, the compound can be recovered by distilling off the solvent from the reaction mixture, or if necessary, after distilling off the solvent from the reaction mixture, pouring the residue into water, extracting with a water-immiscible organic solvent, and distilling off the solvent from the extract. Furthermore, if desired, the product can be further purified by various known methods, such as recrystallization, reprecipitation or various chromatographic techniques, mainly column chromatography or preparative thin layer chromatography.

The process for the preparation of the compounds of formula (I) according to the invention is a further object of the invention.

According to a first aspect, the process for the preparation of a compound of formula (I) comprises the step of deprotecting the corresponding compound of formula (III):

wherein Y, Y ', X, A, A', X ', n', W, W ', U, U', - - -, R1, R2, R1 ', R2'),As defined in formula (I), T' corresponds to T, wherein the functional group has been protected.

Preferably the SH function is protected, preferably the protecting group is acetyl, benzoyl, mesyl, methylthio, pyridylthio, nitropyridinylthio, Triisopropylsilyl (TIPS). Typically the deprotection step is carried out using typical conditions, for example a base for the removal of acetyl, benzoyl and methanesulfonyl protecting groups, a reducing agent such as dithiothreitol or tris (2-carboxyethyl) phosphine (TCEP) for the cleavage of methylthio protecting groups, or it is known to remove TIPS by reacting a compound with ammonium fluoride.

The compounds of formula (II) can be obtained by coupling the corresponding compounds of formulae (IV), (IV') and (V):

wherein Y, Y ', A, A', n ', T', W, W ', U, U' - - - -,r1, R2, R1 ', R2' are as defined in formula (III) and Lg is a leaving group, e.g. halogen, OMs, OTs, OPPh₃ ⁺(intermediate formed in the Mitsunobu reaction).

The compounds of formulae (IV) and (IV') are generally known and are described, for example, in patent applications WO 00/12508, WW00/12507, WO 2005/040170, WO2005/085260, or are commercially available, and/or are obtained synthetically in general (M.mori et al, 42 Tetrahedron, 3793-.

The compounds of formula (V) are obtainable from the corresponding compounds of formula (VI):

HO-An-T’-A’n’-OH(VI)

wherein A, A ', n ', T ' are as defined in formula (III).

The reaction is usually carried out in PPh₃And CHal₄In the presence of a base such as triethylamine or potassium hydroxide, preferably triethylamine, or by reaction with chlorosulfonic acid in the presence of a base such as triethylamine or potassium hydroxide.

The compounds of formula (VI) can be obtained from the corresponding compounds of formula (VII):

HO-An-T”-A’n’-OH(VII)

wherein A, A ', n ' are as defined for formula (III) and T ' is a precursor group for T. A precursor group for T refers to any group that can be deprotected, chemically modified or coupled to yield T. Preferably T is obtained by coupling T ' with a complementary moiety, wherein T ' and the complementary moiety comprise functional groups reactive with each other, e.g.T ' comprises an amine functional group and the complementary moiety comprises an acid functional group.

Representative examples of such reactions are:

the reaction is generally carried out in the presence of N-hydroxysuccinimide and HOBT.

The compounds of formula (VII) may be obtained commercially or prepared by adapting and applying known methods or according to the examples.

An exemplary, non-limiting scheme for this embodiment of the process of the invention is given below:

according to a second aspect, the compounds of formula (I) are obtainable from the corresponding compounds of formula (III'):

wherein Y, Y ', X, A, A', X ', n', W, W ', U, U' - - - -,r1, R2, R1 ', R2' are as defined in formula (I), T "is optional for TA protected precursor group.

A precursor group for T refers to any group that can be chemically modified or coupled to produce T. Preferably T is obtained by coupling T ' with a complementary moiety, wherein T ' and the complementary moiety comprise functional groups reactive with each other, e.g.T ' comprises an amine functional group and the complementary moiety comprises an acid functional group.

Typically this reaction is carried out in the presence of N-hydroxysuccinimide and HOBT.

The compounds of formula (III ') can be obtained by coupling the corresponding compounds of formulae (IV), (IV ') and (V '):

wherein Y, Y ', A, A ', n ', W, W ', U, U ' - -, -or,R1, R2, R1 ', R2' are as defined in formula (III '), T' is an optionally protected precursor group of T, Lg is a leaving group, e.g. halogen or OMs, OTs or OPPh₃ ⁺(intermediate formed in the Mitsunobu reaction).

The compounds of formulae (IV) and (IV') are generally known, obtained by overall synthesis (M.mori et al, 42 Tetrahedron, 3793-.

The compounds of formula (V') are obtainable from the corresponding compounds of formula (VII):

HO-An-T”-A’n’-OH(VI)

wherein A, A ', n ', T ' are as defined for formula (I) and T ' is an optionally protected precursor group for T '.

The reaction is usually carried out in PPh₃And CHal₄In the presence of oxygen.

The compounds of formula (VII) may be obtained commercially or prepared by applying or applying known methods or according to the examples.

According to a third aspect, the process for the preparation of the compound of formula (I) comprises the step of cyclising the corresponding compound of formula (VIII):

wherein Y, Y ', X, A, A', X ', n', R1, R2, R1 ', R2' and T are as defined in formula (I). The reaction is usually carried out in a reagent such as sodium hyposulfite (Na)₂S₂O₄) In the presence of a suitable solvent, for example a mixture of THF and water, followed by addition of methanol and AcCl.

The compounds of formula (VIII) are obtainable from the corresponding compounds of formula (IX):

wherein Y, Y ', A, A ', n ', R1, R2, R1 ', R2 ', T are as defined in formula (I). Typically the reaction is carried out in the presence of a reagent, such as DIBAL-H, in a suitable solvent, such as toluene.

The compounds of formula (IX) can be obtained by coupling the corresponding compounds of formulae (X) and (XI):

wherein Y, Y ', A, A ', n ', R1, R2, R1 ', R2 ', T are as defined in formula (I).

Typically the reaction is carried out by adding a reagent, such as oxalyl chloride, to (X) in a suitable solvent, such as DMF, followed by addition of (XI) in a suitable solvent, such as THF.

Representative protocols are given as follows:

the above reaction is carried out by the skilled person by using or applying the methods illustrated in the following examples.

Furthermore, the process of the invention comprises an additional step of isolating the compounds of formula (I) and (II), which can be carried out by the skilled person by any known conventional method, for example a recovery method as described above.

The starting product is commercially available or may be obtained by utilizing or applying any known method or method described in the examples.

The synthesis can also be carried out in a one-pot process with multi-component reactions.

According to another aspect, the present invention relates to a conjugate molecule comprising at least one tomaymycin derivative covalently bonded to a cell-binding drug via a linking group. The conjugates contain one or more tomaymycin derivatives of the invention, which exhibit a linker, such as-S-or-S-, that covalently links the cell binding agent to the tomaymycin derivative of the invention. According to a preferred aspect, the tomaymycin derivative is of formula (I'):

wherein

- - -represents an optional single bond;

represents a single bond or a double bond;

And whenWhen represents a double bond, U and U 'are absent, and W' represent H;

Preferably, R1 and R2 and R1 ' and R2 ' together form a double bond comprising the groups ═ B and ═ B ', respectively. B and B 'are the same or different and are each selected from the group consisting of optionally substituted by one or more of Hal, CN, NRR', CF₃OR, aryl, Het, S (O)_qR-substituted alkenyl or B and B' represent an oxygen atom.

Preferably, B ═ B'.

More preferably, B ═ CH₂Or ═ CH-CH₃。

Preferably, X ═ X'.

More preferably, X ═ X' ═ O.

Preferably a ═ a'.

More preferably, a ═ a' ═ straight chain unsubstituted alkyl groups.

Y, Y' are the same or different and are each selected from H, OR;

y ═ Y' is preferred.

More preferably, Y ═ O alkyl, and still more preferably methoxy.

T is-alkyl-, -NR-, -O-, -S (O)_q-, or 4-to 10-membered aryl, cycloalkyl, heterocycle or heteroaryl, each optionally substituted by one or more Hal, CN, NRR', CF₃、R、OR、S(O)_qR, and is substituted with one or more linkers.

Preferably T is a 4-10 membered aryl or heteroaryl group substituted with one or more linkers, more preferably phenyl or pyridyl.

When the linking group is a thiol-, sulfide or disulfide-containing group, the side chain carrying the thiol or disulfide group may be straight or branched, aromatic or heterocyclic. Suitable side chains can be readily determined by one of ordinary skill in the art. Preferably, the linker is of the formula:

-G-D-(Z)P-S-Z’

wherein G is a single or double bond, -O-, -S-, or-NR-;

wherein E and F are the same or different and are independently selected from the group consisting of straight or branched- (OCH)₂CH₂)_iAlkyl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_i-alkyl-, - (OCH)₂CH₂)_i-、-(OCH₂CH₂)_iCycloalkyl (OCH)₂CH₂)_j、-(OCH₂CH₂)_iHeterocyclyl (OCH)₂CH₂)_j、-(OCH₂CH₂)_iAryl (OCH)₂CH₂)_j-、-(OCH₂CH₂)_iHeteroaryl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_iAlkyl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_i-alkyl- (OCH)₂CH₂)_iCycloalkyl (OCH)₂CH₂)_j-alkyl- (OCH)₂CH₂)_iHeterocyclyl (OCH)₂CH₂)_j-, -alkyl- (OCH)₂CH₂)_iAryl (OCH)₂CH₂)_jAlkyl radicalRadical- (OCH)₂CH₂)_iHeteroaryl (OCH)₂CH₂)_j-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-;

z is linear or branched-alkyl-;

p is 0 or 1;

n, n ', equal or different, is 0 or 1, m ═ m ' and n ═ n '.

q is 0, 1 or 2.

or a pharmaceutically acceptable salt, hydrate or hydrate salt thereof, or a polymorphic crystal structure of these compounds or an optical isomer, racemate, diastereomer or enantiomer thereof, said derivative being covalently bonded to the cell-binding drug via said linker.

The linker is preferably linked to the cell-bound drug via a functional group active towards thiol, sulfide or disulfide bonds.

-G is a single bond or-O-or-NR-;

-G is-O-;

-D is a single bond or-E-, -E-NR-CO-, -E-CO-, -CO-E-;

-D is-E-, -E-NR-CO-, -CO-E-, -E-CO-;

-D is-E-NR-CO-;

e is a linear or branched-alkyl-, - (OCH)₂CH₂)_i-or alkyl-heterocyclyl;

-E is a linear or branched-alkyl-;

-Z is- (CH)₂)₂-C(CH₃)₂-；

-p is 0 or 1;

z' is H or SR₂₀Wherein R is₂₀Represents an alkyl group;

specific examples of thiol-, sulfide-, or disulfide-containing linkers include

-(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(NR₁₉CO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(OCO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(CO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t(CONR₁₉)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-(CR₁₃R₁₄)_t-N-methylpiperazino-CO (CR)₁₅R₁₆)_uSZ’、

-(CR₁₃R₁₄)_t-thiazolyl-QSZ', - (CR)₁₃R₁₄)_t-thienyl-QSZ', (CR)₁₃R₁₄)_t-imidazolyl-QSZ', - (CR)₁₃R₁₄)_t-morpholino-QSZ', (CR)₁₃R₁₄)_t-piperazinyl-QSZ', - (CR)₁₃R₁₄)_t-N-methylpiperazinyl-QSZ', or

-O(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-O(CR₁₃R₁₄)_t(NR₁₉CO)(CR₁₅R₁₆)_u(O CH₂CH₂)_ySZ’、

-O-phenyl-QSZ ', -O-furyl-QSZ ', -O-oxazolyl-QSZ ', -O-thiazolyl-QSZ

-O-thienyl-QSZ ', -O-imidazolyl-QSZ ', -O-morpholino-QSZ ', -O-piperazinyl-QSZ

-O-N-methylpiperazinyl-QSZ',

-OCO(CR₁₃R₁₄)_t(NR₁₉CO)(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-OCONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-CO(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-CO(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_y(OCH₂CH₂)_ySZ’、

-CONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-NR₁₉(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-NR₁₉CO(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-NR₁₉CO(CR₁₃R₁₄)_t(CR₁₇＝CR₁₈)(CR₁₅R₁₆)_t(OCH₂CH₂)_ySZ’、

-NR₁₉CO-phenyl-QSZ', -NR₁₉CO-furyl-QSZ', -NR₁₉CO-oxazolyl-QSZ

-S(O)_q(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

-SCONR₁₂(CR₁₃R₁₄)_t(CR₁₅R₁₆)_u(OCH₂CH₂)_ySZ’、

R₁₇and R₁₈Is H or alkyl;

u is an integer of 1 to 10, and may be 0;

t is an integer of 1 to 10, and may be 0;

y is an integer of 1 to 20, and may be 0.

For this purpose, representative compounds of formula (I') are:

8, 8' - {5- [3- (4-methyl-4-methyldithio-pentanoylamino) propoxy]-1, 3-benzenediylbis (methyleneoxy) } -bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (4- (4- (4-methyl-4-methyldisulphenyl) -pentan)Amido-butoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

8, 8' - [ (1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]

As well as the corresponding mercapto derivatives thereof,

The cell-bound drug can be of any kind, including peptidic and non-peptidic. Typically they may be antibodies (especially monoclonal antibodies) or antibody fragments containing at least one binding site, lymphokines, hormones, growth factors, nutrient-delivery molecules (e.g. transferrin) or any other cell-binding molecule or matrix. More specific examples of cell-binding drugs that can be used include monoclonal antibodies; a chimeric antibody; a humanized antibody; a fully human antibody; a single chain antibody; antibody fragments, e.g. Fab, Fab ', F (ab')₂And F_v{ Parham, 131 J.Immunol.2895-2902 (1983); spring et al, 113 J.Immunol.470-478 (1974); nisonoff et al, 89 Arch.biochem.Biophys.230-244 (1960); an interferon; a peptide; lymphokines such as IL-2, IL-3, IL-4, IL-6; hormones, such as insulin, TRH (thyroid stimulating hormone), MSH (melanotropin), steroid hormones, such as androgens and estrogens; growth factors and colony stimulating factors, such as EGF, TGF α, insulin-like growth factor (IGF-I, IGF-II) G-CSF, m-CSF and GM-CSF { Burgess, 5 Immunology Today 155 (1984) }; vitamins such as folic acid and transferrin { O' Keefe et al, 260 J.biol.chem.932-937(1985) }.

The term "cell binding agent" herein also includes modified cell binding agents wherein the cell binding agent is modified by a modifying agent to improve the reactivity of the cell binding agent towards the linking group of the linker of the tomaymycin derivative. The modifiers include N-sulfosuccinimidyl-4- (5-nitro-2-pyridinedithio-butyrate (SSNPB), succinimidyl 4- [ N-maleimido ] cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide ester of 4- (2-pyridyldithio) butanoic acid (SPDB), and those discussed below.

Monoclonal antibody technology enables the production of extremely selective cell-bound drugs in the form of specific monoclonal antibodies, and is particularly known in the art as a technology for producing monoclonal antibodies by immunizing mice, rats, hamsters or any other animal with antigens of interest, such as intact target cells, antigens isolated from target cells, whole viruses, attenuated whole viruses and viral proteins, such as viral capsid proteins.

The selection of the appropriate cell binding agent is a matter of choice in number, depending on the particular cell colony targeted, but monoclonal antibodies are generally preferred if one is available.

For example, monoclonal antibody MY9 is a murine IgG₁An antibody which specifically binds to the CD33 antigen { J.D. Griffin et al 8 Leukemia Res., 521(1984) }, which may be used if the target cells express CD33 as an acute myeloid Leukemia (ALM) disease. Similarly, monoclonal antibody anti-B4 is a murine IgG₁It binds to the CD19 antigen of B cells { Nadler et al, 131J. Immunol.244-250(1983) }, and can be used if the target cell is a B cell or a disease cell expressing this antigen, for example in non-Hodgkin's lymphoma or chronic lymphoblastic leukemia. As described above, MY9 and anti-B4 antibodies may be murine, chimeric, humanized, or fully human.

In addition, GM-CSF, which binds to myeloid cells, can be used as a cell-binding agent for disease cells of acute myeloid leukemia. IL-2 bound to activated T-cells can be used for the prevention of transplant rejection, for the treatment and prevention of graft versus host disease, for the treatment of acute T-cell leukemia. MSH binding to melanocytes is used to treat melanoma.

The conjugate molecules of the invention can be formed by any technique, and the tomaymycin derivatives of the invention can be linked to antibodies or other cell-binding drugs via acid-labile or photolabile linkers. The derivative may be fused to a peptide with a suitable sequence and subsequently linked to a cell-bound drug to produce a peptidase labile linker. Conjugates can be prepared to contain a primary hydroxyl group, which can be succinylated and then linked to a cell-binding agent to produce a conjugate, which can be cleaved by intracellular esterases to release the free derivative. Preferably, the derivative is synthesized to comprise a free or protected thiol group, followed by one or more disulfide-containing or thiol-derivatives each covalently linked to the cell-bound drug via a disulfide bond or a thioether bond.

A number of binding methods are taught in USP5,416,064 and USP5,475,092. Tomaymycin derivatives can be modified to produce free amino groups and subsequently linked to antibodies or other cell binding drugs via acid-labile or photolabile linkers. Tomaymycin derivatives bearing a free amino or carboxyl group can be fused to a peptide and subsequently linked to a cell binding agent to produce a peptidase labile linker. The tomaymycin derivative with a free hydroxyl group on the linker can be succinylated and linked to a cell-bound drug to produce a conjugate that can be cleaved by intracellular esterases to release the free drug. Most preferably, the tomaymycin derivative is treated to generate free or protected thiol groups, and the disulfide-or thiol-containing tomaymycin dimer is subsequently linked to the cell binding agent via a disulfide bond.

Representative conjugates of the present invention are antibody-tomaymycin derivatives, antibody fragment-tomaymycin derivative Epidermal Growth Factor (EGF) -tomaymycin derivatives, Melanotropin (MSH) -tomaymycin derivatives, Thyrotropin (TSH) -tomaymycin derivatives, estrogen analog-tomaymycin derivatives, androgen analog-tomaymycin derivatives, and folate-tomaymycin derivatives.

Tomaymycin derivative conjugates of antibodies, antibody fragments, protein or peptide hormones, protein or peptide growth factors and other proteins are prepared by known methods in the same manner. For example, peptides and antibodies can be conjugated with crosslinking agents, such as N-succinimidyl 3- (2-pyridyldithio) propionate, N-succinimidyl 4- (2-pyridyldithio) valerate (SPP), 4-succinimidyl-oxycarbonyl-alpha-methyl-alpha- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl-3- (2-pyridyldithio) butyrate (SDPB), succinimidyl pyridyldithiopropionate (SPDP), N-hydroxysuccinimidyl 4- (2-pyridyldithio) butyrate (SPDB), succinimidyl 4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate (SMCC), N-Thiosuccinimidyl-3- { -2- (5-nitro-pyridyldithio) butyrate (SSNPB), 2-iminosulfane or by modification of S-acetylsuccinic anhydride by known methods. See Carlsson et al, 173 biochem.J.723-737 (1978); blattler et al, 24biochem.1517-1524 (1985); lambert et al, 22 biochem.3913-3920 (1983); klotz et al, 96 arch.biochem.biophysis.605 (1962); and Liu et al, 18 biochem.690(1979), Blakey and Thurpe, 1 Antibody, Immunoconjugates & Radiopharmaceuticals, 1-16(1988), Worrell et al, 1Anti-Cancer Drug Design 179-184 (1986). The thus-derived cell-binding agent containing a free or protected thiol is then reacted with a disulfide-or thiol-containing tomaymycin derivative to produce a conjugate. The conjugate can be purified by HPLC or gel filtration.

Preferably, the monoclonal antibody-or cell-binding drug-tomaymycin derivative conjugate is a substance that is linked via a disulfide bond as discussed above, which is capable of releasing the tomaymycin derivative. The cell binding agents are prepared by known methods, such as monoclonal antibodies modified with succinimidyl pyridyl-dithiopropionate (SPDP) (Carlsson et al, 173 biochem. J.723-737 (1978)). The resulting thiopyridyl group is then replaced by a thiol-containing tomaymycin derivative to produce a disulfide linked conjugate. Alternatively, in the case of aryl dithio-tomaymycin derivatives, the formation of the cell binding conjugate is achieved by direct displacement of the aryl thiol of the tomaymycin derivative with the thiol group previously introduced into the antibody molecule. Conjugates containing 1-10 tomaymycin derivatives linked via disulfide bridges are readily prepared by either method.

More specifically, a dithio-nitropyridine-modified antibody solution having a concentration of 2.5mg/ml in 0.05M potassium phosphate buffer containing 2mM EDTA, pH 7.5 was treated with a thiol-containing tomaymycin derivative (1.3molar eq/dithiopyridyl), and the release of thio-nitropyridine from the modified antibody was completed in about 16 hours as measured by spectrophotometry at 325 nm. The purified antibody-tomaymycin derivative conjugate was subjected to gel filtration through Sephadexg-25 or Sephacryl S300 column to remove unreacted drug and other low molecular substances. The amount of tomaymycin derivative bound per antibody molecule can be determined by measuring the absorbance ratio at 230nm and 275nm, by which method an average of 1-10 tomaymycin derivative molecules/antibody molecule can be disulfide-linked.

The binding effect on antigen expressing cells based on binding affinity can be measured using the method previously described by Liu et al, 93 Proc.Natl.Acad.Sci 8618-. Cytotoxicity of tomaymycin derivatives and antibody conjugates thereof on cell lines can be measured by reverse extrapolation of the cell propagation curve as described by Goldmacher et al, 135J. Immunol.3648-3651 (1985). Cytotoxicity of these compounds against adherent cell lines can be determined by clonogenic assays as described by Goldmacher et al, 102J. CellBiol.1312-1319 (1986).

Representative conjugates of the invention are of tomaymycin derivatives in combination with antibodies, antibody fragments, Epidermal Growth Factor (EGF), Melanotropin (MSH), Thyrotropin (TSH), estrogens, estrogen analogs, androgens, androgen analogs.

Representative examples of methods for preparing various conjugates of derivatives and cell-binding drugs are described below.

Disulfide linker: example (b)For example, monoclonal antibody MY9 is murine IgG₁An antibody which specifically binds to the CD33 antigen { J.D. Griffin et al 8 Leukemia Res., 521(1984) }, which may be used if the target cells express CD33 as an acute myeloid Leukemia (ALM) disease. Similarly, monoclonal antibody anti-B4 is a murine IgG₁It binds to the CD19 antigen of B cells { Nadler et al, 131J. Immunol.244-250(1983) }, and can be used if the target cell is a B cell or a disease cell expressing this antigen, for example in non-Hodgkin's lymphoma or chronic lymphoblastic leukemia.

Antibodies or other cell binding drugs were modified with N-succinimidyl-3-pyridyldithiopropionate as described above { j.carlsson, h.drevin & r.axen, biochem.j., 173: 723(1978) } introduction of an average of 4 pyridyldithio groups per antibody molecule, reaction of the modified molecule with thiol-containing derivatives to generate disulfide-linked conjugates.

Alternatively, conjugates may be prepared using and/or adapted for use in the methods disclosed in WO2004/103272, the teachings of which are incorporated herein by reference.

Thioether linker: thiol-containing derivatives of the invention can be linked to antibodies and other cell-bound drugs via thioether chains as described above (U.S. Pat. No.5,208,020). Antibodies or other cell binding agents may be modified with commercially available compounds, such as N-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amido-hexanoate), which is a "long chain" analogue of SMCC (LC-SMCC). These cross-linking agents form non-cleavable linkages derived from maleimido moieties.

Crosslinkers containing haloacetyl moieties include N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBPA). These cross-linkers form non-cleavable linkages derived from haloacetyl moieties.

Acid labile linkers: amino-containing derivatives of the invention can be as described above { W.A. Blattler et al, Biochemistry 24, 1517-1524 (1985); U.S. Pat. Nos. 4,542,225, 4,569,789, 4,618,492, 4,764,368 are linked to antibodies and other cell binding drugs via acid labile linkers.

Likewise, derivatives of the invention containing a hydrazido group may be linked to the carbohydrate moiety of antibodies and other cell-binding drugs via acid-labile hydrazone linkers [ examples of hydrazone linkers are described in b.c. laguzza et al, j.med.chem., 32, 548-; R.S. greenfield et al, Cancer Res., 50, 6600-.

Photolabile linker: the amino group-containing derivatives of the present invention can be prepared by { P.Senter et al, Photochemistry and Photobiology, 42, 231-; the photolabile linker of U.S. Pat. No.4,625,014 was linked to antibodies and other cell-binding drugs.

Peptidase labile linkers: the amine group-containing derivatives of the present invention may also be linked to a cell-binding drug via a peptide spacer. As shown above, the short peptide spacer between the drug and the macromolecular protein carrier is stable in serum, but is readily hydrolyzed by intracellular peptidases { A.Trouet et al, Proc.Natl. Acad.Sci., 79, 626-629(1982) }. Amino group-containing derivatives can be condensed with a condensing agent, such as 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide-HCl (EDC-HCl), with a peptide to give a peptide derivative, which can be linked to a cell-binding drug.

Esterase labile linkers: derivatization according to the invention with hydroxyl groupsThe conjugate can be succinylated with succinic anhydride and subsequently linked to a cell-bound drug to generate a conjugate which can be cleaved by intracellular esterases to release the free drug { see, e.g., E.Aboud-Pirak et al, Biochem Pharmacol., 38, 641-648(1989) }.

Conjugates prepared by the above methods can be purified using standard chromatographic techniques such as size exclusion, adsorption chromatography, including, but not limited to, ion exchange, hydrophobic interaction chromatography, affinity chromatography, ceramic hydroxyapatite or Porapak based chromatography, or by HPLC. Purification by dialysis or filtration may also be employed.

The conjugate of the monoclonal antibody or cell binding drug and the derivative of the present invention is preferably a substance linked via a disulfide bond as described above. The cell binding agents are prepared by known methods, for example, by modifying monoclonal antibodies { Carlsson et al, 173 biochem. J.723-737(1978) } with succinimidyl-pyridyl-dithiopropionate (SPDP). The resulting thiopyridyl group is then replaced by a thiol-containing derivative to produce a disulfide-linked conjugate. Conjugates containing 1-10 derivatives linked via disulfide bridges are readily prepared by this method. The incorporation by this method is fully described in US5,585,499, which is incorporated herein by reference.

According to a preferred aspect of the invention, the cell binding agent is an antibody, especially a monoclonal antibody.

According to another preferred aspect of the invention, the cell binding agent is an antigen-specific antibody fragment, such as FV, Fab 'or F (ab')₂。

According to a further object, the invention also relates to a pharmaceutical composition comprising a conjugate molecule according to the invention or a compound of formula (I) as defined above and a pharmaceutically acceptable carrier.

According to other objects, the present invention also relates to a method of killing or inhibiting the growth of cells, preferably a selected cell population, comprising contacting the target cells or a tissue containing the target cells with an effective amount of a pharmaceutical composition of the present invention.

The cell population selected is cancerous and/or propagating cells.

According to other objects, the present invention also relates to a method of treatment, preferably selective treatment of cancer, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of the present invention.

According to the present invention, "selectively treating cancer" refers to killing cancer and/or proliferating cells without substantially killing normal and/or non-proliferating cells.

According to other objects, the present invention also relates to the use of a conjugate molecule of the invention as defined above or a compound of formula (I) for the preparation of a medicament for the treatment of cancer.

The method for inhibiting the growth of a selected cell population may be carried out in vitro, in vivo or in vitro (exvivo).

Examples of in vitro uses include treating a cell culture to kill all cells except those that do not express the desired antigen or to kill variants that express undesired antigens.

Conditions for non-clinical in vitro use are readily determined by the skilled artisan.

Examples of ex vivo uses include the treatment of autologous bone marrow to kill diseased or malignant cells prior to transplantation into the same patient: bone marrow is treated prior to transplantation to kill competent T cells, preventing Graft Versus Host Disease (GVHD).

Clinical in vitro processing to remove tumor cells or lymphocytes from bone marrow prior to autologous transplantation for cancer therapy or to treat autoimmune diseases, or to remove T cells and other lymphocytes from allogeneic bone marrow or tissues for prevention of GVHD prior to transplantation can be performed as follows. Bone marrow is collected from a patient or other individual and subsequently incubated at about 37 ℃ for about 30 minutes to about 48 hours in a medium containing serum to which a cytotoxic drug of the present invention is added at a concentration ranging from about 10 μ M to 1 pM. The exact time of concentration and incubation time (dose) are readily determined by one skilled in the art. After culturing, the bone marrow cells are washed with serum-containing medium and returned to the patient by intravenous infusion according to known methods. Bone marrow cells treated during separate chemotherapy or whole body radiation therapy during the time the patient receives other treatments, such as extraction of bone marrow and reinfusion of the treated cells, are frozen in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the cell binding drug of the invention will be supplied as a solution that is tested for sterility and for endotoxin content. A suitable regimen for administering the conjugate is as follows, where the conjugate is administered as an in vivo bolus of 50-400mL of normal salt of human serum albumin (e.g., 0.5-1mL of a concentrated solution of human serum albumin, 100mg/mL) as an in vivo bolus dose administered weekly for 6 weeks. The dose will be about 50 μ g-10mg/kg, i.v., of body weight per week (10 μ g-100mg/kg per injection). At 6 weeks post-treatment, the patient may receive a second course of treatment, the specific clinical regimen being related to route of administration, excipients, diluents, dosage, time, etc., and may be determined by the skilled artisan based on clinical condition.

Examples of medical conditions that may be treated according to the in vivo or in vitro methods of killing selected cell populations include all types of malignancies, including, for example, lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphoid cancers; melanoma; autoimmune diseases such as systemic lupus, 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; viral infections, such as CMV infection, HIV infection, AIDS, and the like; bacterial infection; and parasitic infections such as giardiasis, amebiasis, schistosomiasis and other diseases as determined by one of skill in the art.

The determination of those patients in need of treatment of the diseases and conditions described herein is well within the abilities and knowledge of those skilled in the art. A veterinarian or clinician in the art can readily determine the patient in need of such treatment by employing clinical testing, physical examination, medical/home history, or biological and diagnostic tests.

A therapeutically effective amount can be readily determined by the attending diagnostician as one skilled in the art by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining a therapeutically effective amount, the attending diagnostician takes into account a number of factors, including, but not limited to, patient type, weight, age, and general health; the specific diseases involved; the degree of illness or severity of the disease; the response of the individual patient; the specific compound administered; a mode of administration; bioavailability characteristics of the administered formulation; the selected dosage regimen; use with drug therapy; and other related circumstances.

The amount of a compound or conjugate of formula (I) required to achieve a desired biological effect will vary depending on a number of factors, including the chemical identity (e.g., hydrophobicity) of the compound employed, the potency of the compound, the type of disease, the type of patient, the disease state of the patient, the route of administration, the bioavailability of the compound by the chosen route, all factors indicating the desired dose, delivery and therapy of administration.

By "pharmaceutical" or "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human, as desired.

As used herein, "pharmaceutically acceptable excipient" includes any carrier, diluent, adjuvant, or excipient, such as preservative or antioxidant, filler, disintegrant, wetting agent, emulsifier, suspending agent, solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and the like. The use of such media and agents for the pharmaceutically active substances is known in the art and is contemplated for use in therapeutic compositions, except in any conventional media or agents that are incompatible with the active ingredient. Adjunct active ingredients may also be added to the compositions as appropriate therapeutic combinations.

In the context of the invention, the term "treating" or "treatment" as used herein refers to reversing, slowing, inhibiting the progression or preventing of the disease or condition to which the term applies or one or more symptoms of the disease or condition.

According to the present invention, the term "patient" or "patient in need of treatment" refers to an animal or human being, preferably a human, affected or likely to be affected by the pathological conditions referred to herein.

In general, the compounds of the invention may be provided in an aqueous physiological buffer solution containing 0.1-10% w/v of the compound for parenteral administration, typically in a dosage range of 1. mu.g/kg-0.1 g/kg body weight per day, preferably in a dosage range of 0.01mg/kg-10mg/kg body weight per day or equivalent in children. The preferred dosage of drug administered will likewise depend on variables such as the type and extent of progression of the disease or condition, the overall health status of the particular patient, the relative biological potency of the selected compound, the formulation of the compound, the route of administration (intravenous, intramuscular, etc.), the pharmacokinetic properties of the compound over the chosen route of delivery, and the rate (bolus or continuous infusion) and schedule (number of repetitions over a period of time) of administration.

The compounds of the present invention can also be administered in unit dosage form, wherein "unit dose" refers to a single dose capable of being administered to a patient, which can be readily handled and packaged, maintained as a physically and chemically stable unit dose, containing the active compound per se, or as a pharmaceutically acceptable composition as described herein below. Similarly, a typical total daily dosage range is from 0.01 to 100mg/kg body weight, and as a general guideline, a unit dose for use in humans is from 1mg to 3000mg per day. Preferably, the unit dose is in the range of 1-500mg administered 1-6 times a day, more preferably 10-500 mg once a day. The compounds provided herein can be formulated into pharmaceutical compositions by mixing with one or more pharmaceutically acceptable excipients. The unit dosage composition can be prepared for oral administration, particularly in the form of a tablet, simple capsule or soft gel capsule; or intranasally, especially in the form of a powder, nasal drops or aerosol; or dermally, e.g. topical ointments, lotions, gels or sprays, or transdermally.

The compositions may conveniently be administered in unit dosage form and may be prepared by any method known in the art of pharmacy, for example Remington: the Science and Practice of Pharmacy, 20^th ed；gennaro，A.R.，Ed.；Lippincott Williams &Wilkins: philadelphia, PA, 2000.

Preferred formulations include pharmaceutical compositions wherein the compounds of the invention are formulated for oral or parenteral administration.

For oral administration, tablets, pills, powders, capsules, lozenges, and the like, may contain one or more of any of the following ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose or gum tragacanth; diluents, such as starch or lactose; disintegrants, such as starch and cellulose derivatives; lubricants, such as magnesium stearate; slip agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or a flavoring agent, such as peppermint oil or methyl salicylate. Capsules can be in the form of hard or soft capsules, which are generally prepared from gel beads, optionally mixed with a plasticizer, and starch capsules. In addition, the dosage unit form may contain various other materials which modify the physical form of the dosage unit, such as coatings of sugar, shellac, or enteric agents. Other oral dosage form syrups or elixirs may contain sweetening agents, preserving agents, dyes, colouring agents and flavouring agents. Furthermore, the active compounds can be incorporated into fast dissolving, modified release or delayed release formulations and formulations, wherein the delayed release formulation is preferably bimodal. Preferred tablets contain lactose, corn starch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, or talc in any combination.

Liquid preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions or emulsions. The liquid composition may also include binders, buffers, preservatives, chelating agents, sweeteners, flavorants, colorants, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and organic esters, such as ethyl oleate. The aqueous carrier includes a mixture of alcohol and water, a buffer medium, and saline. Especially biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compound. Intravenous excipients may include liquid and nutritional supplements, electrolyte supplements, such as Ringer's glucose-based substances, and the like. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer microparticles, osmotic pumps, implantable infusion systems, and liposomes.

Other modes of administration include formulations for inhalation, which include means for dry powders, aerosols or droplets. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops or as gels for intranasal application. Formulations for buccal administration include, for example, lozenges or pastilles, and may also include a flavor base, such as sucrose or acacia, and other excipients, such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit dose suppositories which contain a solid base carrier, for example cocoa butter, or contain a salicylate. Formulations for topical application to the skin take the form of ointments, lotions, pastes, gels, sprays, aerosols or oils. Carriers that may be used include petroleum jelly, lanolin, polyethylene glycols, alcohols or combinations thereof. Formulations suitable for transdermal administration may be presented as discrete patches, either as lipophilic emulsions or buffered aqueous solutions dissolved and/or dispersed in a polymer or binder.

Drawings

FIG. 1a shows the in vitro potency of huB 4-SPDB-example 16 compound on antigen negative BJAB cells and antigen negative MOLT-4 cells.

FIG. 1b shows the in vitro potency of huB 4-SMCC-example 16 compound on antigen negative BJAB cells and antigen negative MOLT-4 cells.

FIG. 1c shows the in vitro potency of the free compound of example 16 on BJAB and MOLT-4 cells.

FIG. 2a shows the in vitro potency of huB 4-SPDB-example 17 compound on antigen negative BJAB cells and antigen negative MOLT-4 cells.

FIG. 2b shows the in vitro potency of huB 4-SMCC-example 17 compound on antigen negative BJAB cells and antigen negative MOLT-4 cells.

FIG. 2c shows the in vitro potency of the free compound of example 17 on BJAB and MOLT-4 cells.

FIG. 3a shows the in vitro potency of huMy 9-6-SPDB-example 16 compound on antigen-positive HL60/GC cells and antigen-negative Ramos cells.

FIG. 3b shows the in vitro potency of the free compound of example 16 on HL60/GC cells and Ramos cells.

The invention is further illustrated by, but not limited to, the following description of the examples.

Experimental part

Method A1: high pressure liquid chromatography-Mass Spectrometry (LCMS)

Analysis was performed in an Agilent 1100 series HPLC with a THERMOHypersilgoldC183 μm column (50X 3mm) using Micromass MassLynx software, with a gradient elution with a mixture of (A) acetonitrile and (B) water/0.1% formic acid at a flow rate of 0.8 mL/min (gradient: 5% A: 95% B rising to 95% A: 5% B over 5 min, 95% A: 5% B0.5 min, 95% A: 5% B falling to 5% A: 95% B over 1 min, 5% A: 95% B0.5 min); Waters-Micromass Platform I, Platform II or ZQ spectrophotometer with Electrospray (Positive and negative ionization); a tandem Diode Array (190-; auxiliary detector Sedere (France) model SEDEX 65 adaptive Light Scattering (ELS) detector.

Method A2: high pressure liquid chromatography-Mass Spectrometry (LCMS)

Analysis was performed in a Waters alliance HPLC with a WATERS Xbridge C183,5 μm column (100X 3mm) using Micromass MassLynx software, with a gradient elution using a mixture of (A) methanol and (B) water/0.1% formic acid at a flow rate of 1.1 mL/min (gradient: 5% A: 95% B rising to 95% A: 5% B over 10 min, 95% A: 5% B falling to 5% A: 95% B over 1 min, 5% A: 95% B2 min); Waters-Micromass Platform II spectrophotometer with Electrospray (Positive and negative ionization); tandem DiodeArray (190-; secondary detector Sedere (France) Model SEDEX 85 adaptive Light Scattering (ELS) detector.

Method A3: high pressure liquid chromatography-Mass Spectrometry (LCMS)

Analysis was performed in an Agilent 1100 series HPLC with an Xbridge C182.5 μm column (50X 3mm) using Micromass MassLynx software, with a gradient elution with a mixture of (A) acetonitrile and (B) water/0.1% formic acid at a flow rate of 1.1 mL/min (gradient: 5% A: 95% B rising to 100% A, over 5 min, 100% A0.5 min, 100% A falling to 5% A: 95% B over 1 min, 5% A: 95% B0.5 min); Waters-Micromass ZQ spectrophotometer with Electrospray; in series with a Diode Array (210 and 254 nm). The method B comprises the following steps: high Pressure Liquid Chromatography (HPLC) purification

HPLC purification was performed using a Macherey Nagel Nuclear C18 gradient 5. mu.M column (21X 100mm, Cat. No. 762101) eluting with a mixture of (A) acetonitrile and (B) water at a flow rate of 15 mL/min (method B1) or 20 mL/min (method B2) (gradient: 5% A: 95% B5 min, 5% A: 95% B rising to 100% A over 20 min, 100% A8 min, 100% A to 5% A: 95% B over 1 min, 5% A: 95% B11 min).

The method C comprises the following steps: electron Ionization (EI) mass spectrometry

EI mass spectrometry was performed using a Finnigan SSQ 7000 mass spectrometer (type El: 70eV, ion source temperature 150 ℃ C., direct input).

The method D comprises the following steps: chemical Ionization (CI) mass spectrometry

CI mass spectra were recorded on a Finnigan SSQ 7000 mass spectrometer (ammonia).

The method E comprises the following steps:¹h Nuclear Magnetic Resonance (NMR) spectroscopy

1HNMR spectra were recorded with Bruker Avance Drx-500, Bruker Avance Drx-400, or Bruker Avance DRX-300.

Method F: high Pressure Liquid Chromatography (HPLC) purification

HPLC purification was carried out using a Macherey Nagel VP 250/40mm NUCLEOTURRgRAVITY 100-10C 18 (Cat. No. 762250) using (A) acetonitrile and (B) water/HCOONH at a flow rate of 70 mL/min₄ 0,01m/NH₄OH pH9-10 (gradient: 10% A: 90% B for 3 minutes, 10% A: 90% B rising to 95% A: 5% B for more than 37 minutes, 95% A: 5% B for 8 minutes, 95% A: 5% B to 10% A: 90% B for more than 1 minute, 10% A: 90% B for 1 minute).

Method G1: high pressure liquid chromatography-Mass Spectrometry (LCMS)

Analysis was performed on an Acquity UPLC with Acquity UPLC BEH C181,7 μm column (2.1 × 100mm) using Micromass MassLynx software, with gradient elution at a flow rate of 1.1 mL/min with a mixture of (a) acetonitrile and (B) water/0.1% formic acid (gradient: 5% a: 95% B rising to 95% a: 5% B over 4.7 min, 95% a: 5% B falling to 5% a: 95% B over 0.5 min, 5% a: 95% B0.8 min); quattro Premier spectrometer with Electrospray; in series Diode Array (210-400 nm).

Method G2: high pressure liquid chromatography-Mass Spectrometry (LCMS)

Analysis was performed on Acquity UPLC with an Acquity UPLC BEH C181,7 μm column (2.1X 100mm) using Micromass MassLynx software, eluting with a gradient of a mixture of (A) acetonitrile and (B) water/0.1% formic acid at a flow rate of 0.6 mL/min (gradient: 5% A: 95% B rising to 95% A: 5% B over 10 min, 95% A: 5% B falling to 5% A: 95% B over 1 min, 5% A: 95% B2 min); quattro Premier spectrometer with Electrospray; in series Diode Array (210-400 nm).

Method H: high Pressure Liquid Chromatography (HPLC) purification method

HPLC purification was carried out using Varian HPLC with a Kromasil 16. mu. m C18 column (250X 21.2mm, PN A0490-250X 212, Lot. No. DT0259, SN 9772196) eluting with a mixture of (A) water and (B) acetonitrile at a flow rate of 20 mL/min. The collection time was 80 seconds. Mass spectra of the compounds were obtained on a Bruker Esquire 3000 apparatus and NMR spectra were recorded on a Bruker Avance spectrometer operating at 400 MHz.

Gradient of elution column:

1.8, 8' - [5- (N-4-methyldithio-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Purification of

55% A: 45% B8 min, 55% A: 45% B to 50% A: 50% B over 14 min, 50% A: 50% B to 10% A: 90% B over 4 min, 10% A: 90% B5 min, 10% A: 90% B to 55% A: 45% B over 1 min, 55% A: 45% B3 min.

2.8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Purification of

60% A: 40% B for 4 minutes, 60% A: 40% B to 55% A: 45% B for more than 5 minutes, 55% A: 45% B for 4 minutes, 55% A: 45% B to 50% A: 50% B for more than 13 minutes, 50% A: 50% B to 10% A: 90% B for more than 10 seconds, 10% A: 90% B for 5 minutes, 10% A: 90% B to 60% A: 40% B for more than 10 seconds, 60% A: 40% B for 3 minutes.

3.8, 8' - [5- (N-methyl-N- (2-methyldithio-212-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Purification of

60% A: 40% B8 min, 60% A: 40% B to 45% A: 55% B over 16 min, 45% A: 55% B to 10% A: 90% B over 2 min, 10% A: 90% B5 min, 10% A: 90% B to 60% A: 40% B over 1 min, 60% A: 40% B3 min.

4.8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Purification of

65% A: 35% B to 60% A: 40% B for more than 8 minutes, 60% A: 40% B to 50% A: 50% B for more than 19 minutes, 50% A: 50% B to 10% A: 90% B for more than 10 seconds, 10% A: 90% B for 5 minutes, 10% A: 90% B to 65% A: 35% B for more than 10 seconds, 65% A: 35% B for 3 minutes.

Example 1: 8, 8' - [1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

Potassium carbonate (22.8mg), α' -dibromo-m-xylene (7.3mg), and potassium iodide (9.1mg) were added to a stirred solution of pre-tomaymycin (15mg) in dimethylformamide (0.5 mL). The reaction was stirred at 30 ℃ for 20 h, the solid filtered off and washed twice with dimethylformamide (0.2mL) and then discarded. To the combined dimethylformamide solutions was added water (0.4mL) and the resulting solution was injected for HPLC purification according to method B1. The appropriate fractions were combined and concentrated by centrifugal evaporation in a Joua Model RC10.10 apparatus to give 8, 8' - [1, 3-benzenediyl-bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]White powder (3.33 mg):

LC/MS (method A1, Platform II): ES: m/z 647MH⁺

Retention time 3.53 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：1,75(d，J＝7,0Hz，6H)；2,96(m，4H)；3,89(m，2H)；3,96(s，6H)；4,27(s b，4H)；5,17(d，J＝12,5Hz，2H)；5,23(d，J＝12,5Hz，2H)；5,60(m，2H)；6,85(s，2H)；7,36-7,43(m，3H)；7, 51(s b，1H)；7,53(s，2H)；7,63(d，J＝4,5Hz，2H)。

Example 2: 8, 8' - [ 5-methoxy-1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

According to the preparation of 8, 8' - [1, 3-benzenediyl-bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Example 1 preparation of 8, 8' - [ 5-methoxy-1, 3-benzenediyl bis (methyleneoxy) starting from 1, 3-di-bromomethyl-5-methoxy-benzene]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: 677MH m/z⁺

Retention time 4.17 min

¹H N.M.R.(300MHz，CDCl₃-d1，δppm)：1,75(d，J＝7,0Hz，6H)；2,96(m，4H)；3,81(s，3H)；3,89(m，2H)；3,96(s，6H)；4,26(s b，4H)；5,14(d，J＝12,5Hz，2H)；5,21(d，J＝12,5Hz，2H)；5,60(m，2H)；6,82(s，2H)；6,95(s b，2H)；7,07(s b，1H)；7,53(s，2H)；7,63(d，J＝4,5Hz，2H)。

1, 3-di-bromomethyl-5-methoxy-benzene can be prepared as follows:

carbon tetrabromide (663mg) was added to a stirred solution of 1-bromomethyl-3-hydroxymethyl-5-methoxy-benzene (420mg) in anhydrous dichloromethane (10mL) under argon, the resulting solution was cooled at 0 ℃ and a solution of triphenylphosphine (500mg) in anhydrous dichloromethane (10mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 hours and then concentrated in vacuo to a residue. The residue was purified by silica gel chromatography (Merck SuperVario Flash 30g column, Si6015-40 μm, eluting with dichloromethane/heptane 40: 60) to give 1, 3-di-bromomethyl-5-methoxy-benzene (170 mg):

el (method C): 292 m/z⁺

m/z＝213[M-Br]⁺

m/z＝134[213-Br]⁺

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：3,77(s，3H)；4,65(s，4H)；6,98(s b，2H)；7,10(s b，1H)。

1-bromomethyl-3-hydroxymethyl-5-methoxy-benzene can be prepared as follows:

carbon tetrabromide (3.47g) was added to a stirred solution of 1, 3-dihydroxymethyl-5-methoxy-benzene (800mg) in anhydrous dichloromethane (16mL) under argon, the resulting solution was cooled at 0 deg.C, and a solution of triphenylphosphine (2.68g) in anhydrous dichloromethane (16mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 hours and then concentrated in vacuo to a residue. The residue was purified by silica gel chromatography (Merck SuperVarioFlash 90g column, Si6015-40 μm, eluting with methanol/dichloromethane 4: 96) to give 1-bromomethyl-3-hydroxymethyl-5-methoxy-benzene (420 mg):

EI (method C): 230 m/z⁺

m/z＝151[M-Br]⁺

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：3,75(s，3H)；4,46(d b，J＝5,5Hz，2H)；4,65(s，2H)；5,22(t b，J＝5,5Hz，1H)；6,83(s b，1H)；6.88(s b，1H)；6,97(s b，1H)。

Example 3: 8, 8' - [1, 5-pentanediylbis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

Potassium carbonate (22.8mg) and 1, 5-diiodopentane (8.2. mu.L) were added to a stirred solution of pre-tomaymycin (15mg) in dimethylformamide (0.5mL), the reaction was stirred at room temperature for 20 hours and an additional portion of potassium carbonate (8mg) was added. The reaction was stirred at room temperature for an additional 20 hours.

The solid was filtered off, the dimethylformamide solution was purified by HPLC according to method B2 syringe, the appropriate fractions were combined and concentrated by centrifugal evaporation on a Joua Model RC10.10 apparatus to give 8, 8' - [1, 5-pentanediylbis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]White powder (4 mg):

LC/MS (method A2): ES: 613MH m/z⁺

Retention time 9.04 min

¹H N.M.R.(500MHz，CDCl₃-d1, δ ppm): 1,66(m partially masked, 2H); 1,75(d b, J ═ 7,0Hz, 6H); 1,96(m, 4H); 2,97(d b, J ═ 7,0Hz, 4H); 3.89(m, 2H); 3,94(s, 6H); 4,06(m, 2H); 4,13(m, 2H); 4,26(s b, 4H); 5,60(m, 2H); 6,80(s, 2H); 7,50(s, 2H); 7,66(d, J ═ 4,5Hz, 2H).

Example 4: 8, 8' - [1, 4-butanediyl bis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

According to the preparation of 8, 8' - [1, 5-pentanediylbis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Example 3 preparation of 8, 8' - [1, 4-butanediyl bis (oxy) starting from 1, 4-diiodobutane]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: 599MH m/z⁺

m/z＝318,5(M+H+K)²⁺/2

Retention time 3.23 min

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：1,75(d b，J＝7,0Hz，6H)；2,10(m，4H)；2,98(d b，J＝7,0Hz，4H)；3,90(m，2H)；3,93(s，6H)；4,11(m，2H)；4,20(m，2H)；4,27(s b，4H)；5,60(m，2H)；6,82(s，2H)；7,50(s，2H)；7,66(d，J＝4,5Hz，2H)。

Example 5: 8, 8' - [ 3-methyl-1, 5-pentanediylbis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

According to the preparation of 8, 8' - [1, 3-benzenediyl-bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Example 1 preparation of 8, 8' - [ 3-methyl-1, 5-pentadiyl di (oxy) from 1, 5-dibromo-3-methylpentane]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: 627MH for m/z⁺

Retention time 3.92 minutes

Example 6: 8, 8' - [2, 6-pyridinediyl bis (oxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

According to the preparation of 8, 8' - [1, 3-benzenediyl-bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Example 1 preparation of 8, 8' - [2, 6-pyridinediyl bis (oxy) starting from 2, 6-di-bromomethyl-pyridine]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, ZQ): ES: 648MH m/z⁺

Retention time 3.21 min

¹H N.M.R.(400MHz，CDCl₃-d1，δppm)：1,75(d b，J＝6,5Hz，6H)；2,94-2,99(m，4H)；3,90(m，2H)；3,99(s，6H)；4,27(s b，4H)；5,32(s，4H)；5,60(m，2H)；6,86(s，2H)；7,48(d，J＝8,0Hz，2H)；7,56(s，2H)；7,64(d，J＝4,5Hz，2H)；7,74(t，J＝8,0Hz，1H)。

Example 7: 8, 8' - [4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-pyridinediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a stirred solution of pre-tomaymycin (30mg) in dimethylformamide (0.5mL) were added potassium carbonate (45.7mg), a solution of 4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-bis (toluenesulfonyloxymethyl) -pyridine (41mg) in dimethylformamide (0.5mL), and potassium iodide (18.3 mg). The reaction was stirred at 30 ℃ for 20 h, and the solid was filtered off and washed with dimethylformamide (0.2 mL). To the combined dimethylformamide solutions was added water (0.5mL) and formic acid was added until the precipitate completely disappeared. The resulting solution was purified by HPLC according to method B1 injection, the appropriate fractions were combined and concentrated by centrifugal evaporation using a Jouanmodel RC10.10. apparatus to give 8, 8' - [4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-pyridinediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](8.3mg)。

LC/MS (method A1, Platform I): ES: 857MH m/z⁺+2H₂O

m/z＝839MH⁺+H₂O

m/z＝821MH⁺

m/z＝721[M-C₅O₂H₈]+H⁺

Retention time 3.67 min

¹H N.M.R.(500MHz，CD3CO2D-d4，δppm)：1,41(s，9H)；1,71(d，J＝6,5Hz，6H)；2,08(m，2H)；2,95(m，4H)；3,34(m，2H)；3,90(s，6H)；4,06(m，2H)；4,18(m，2H)；4,24-4,36(m，4H)；4,43(t，J＝6,0Hz，2H)；5,50(s b，4H)；5,61(m，2H)；6,80-7,70(m very b，2H)；6,95(s b，2H)；7,48-7,58(m，4H)。

4- (3-tert-Butoxycarbonylaminopropoxy) -2, 6-bis (tosyloxymethyl) -pyridine can be prepared as follows:

to a pre-cooled (0 ℃ C.) solution of 4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-bis (hydroxymethyl) -pyridine (76mg) in dichloromethane (0.7mL) was added a solution of potassium hydroxide (30mg) in water (0.3 mL). Tosyl chloride (93.7mg) was added and the resulting heterogeneous mixture was vigorously shaken for 1 hour, followed by washing with dichloromethane and water into a separatory funnel. The layers were separated, the aqueous layer was extracted three times with dichloromethane, the combined organic solutions were dried over magnesium sulfate and concentrated in vacuo to a residue. The residue was purified by silica gel chromatography (Interchrom Purifash 10g column, SiOH15-35 μm) eluting with a gradient of a mixture of heptane (A) and ethyl acetate (B) (gradient: 90% A: 10% B rising to 50% A: 50% B) to give 4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-bis (tosyloxymethyl) -pyridine (56 mg):

LC/MS (method A1, Platform I): ES: 621MH m/z⁺

Retention time 4.90 minutes

4- (3-tert-Butoxycarbonylaminopropoxy) -2, 6-bis (hydroxymethyl) -pyridine can be prepared as follows:

to a solution of diethyl 4- (3-tert-butoxycarbonylaminopropoxy) -pyridine-2, 6-dicarboxylate (150mg) in anhydrous ethanol (5mL) were added sodium borohydride (43mg) and calcium chloride (128 mg). After stirring for 4 hours, the evolution of hydrogen was stopped and the reaction was stopped with water. The solvent was evaporated under reduced pressure and the residue was washed with dichloromethane and water into a separatory funnel. The layers were separated, the aqueous layer was extracted three times with dichloromethane, the combined organic solutions were dried over magnesium sulfate and concentrated in vacuo to give 4- (3-tert-butoxycarbonylaminopropoxy) -2, 6-bis (hydroxymethyl) -pyridine (80 mg):

LC/MS (method A1, ZQ): 313MH ESm/z⁺

Retention time 1.90 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：1,37(s，9H)；1,84(m，2H)；3,08(q，J＝6,5Hz，2H)；4,05(t，J＝6,5Hz12H)；4,45(d，J＝6,0Hz，4H)；5,32(t，J＝6,0Hz，2H)；6,84(s，2H)；6,90(t b，J＝6,5Hz，1H)。

Diethyl 4- (3-tert-butoxycarbonylaminopropoxy) -pyridine-2, 6-dicarboxylate can be prepared as follows:

to anhydrous dimethylformamide (2mL) was dissolved diethyl chelidonate (Scrimin, P.; Technila, P.; Tonellato, U.; Vendrame, T.J. org.chem.1989, 54, 5988) (150mg), and 3- (tert-butoxycarbonyl-amino) -propyl bromide (164mg) and potassium carbonate (130mg) were added. The resulting mixture was stirred at 70 ℃ for 15 hours, quenched with saturated aqueous ammonium chloride, and then washed with ethyl acetate into a separatory funnel. The layers were separated, the aqueous layer was extracted 3 times with ethyl acetate, and the combined organic solutions were dried over magnesium sulfate and concentrated in vacuo to a residue. The residue was purified by silica gel chromatography (Merck SuperVarioFlash 30g column, Si6015-40 μm) eluting with a gradient of a mixture of heptane (A) and ethyl acetate (B) (gradient: 60% A: 40% B rising to 50% A: 50% B) to give diethyl 4- (3-tert-butoxycarbonylaminopropoxy) -pyridine-2, 6-dicarboxylate (150 mg):

cl (method D): m/z-397 MH⁺

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：1,34(t，J＝7,0Hz，6H)；1,36(s，9H)；1,86(m，2H)；3,10(q，J＝6,5Hz，2H)；4,21(t，J＝6,5Hz，2H)；4,37(q，J＝7,0Hz，4H)；6,89(m b，1H)；7,71(s，2H)。

Example 8: 8, 8' - [5- (3-aminopropoxy) -1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a stirred solution of pre-tomaymycin (21mg) in dimethylformamide (0.7mL) were added potassium carbonate (32mg), 1- (3-allyloxycarbonylamino-propoxy) -3, 5-bis- (bromomethyl) -benzene (16.2mg), and potassium iodide (12.8 mg). The reaction was stirred at 30 ℃ for 20 h, the solid filtered off and washed twice with dimethylformamide (0.2mL) and then discarded. Water (0.5mL) was added to the combined dimethylformamide solutions, and the resulting precipitate was filtered off, washed with water, and dried by centrifugal evaporation using a Joean model RC10.10.

To the crude compound (27mg) dissolved in dimethylformamide (0.8mL) were added tetrakis (triphenylphosphine) palladium (2mg), triphenylphosphine (0.9mg) and pyrrolidine (5.6. mu.L), and after stirring at 30 ℃ for 15 hours, tetrakis (triphenylphosphine) palladium (2mg), triphenylphosphine (1mg) and pyrrolidine (2.8. mu.L) were added, and the reaction mixture was stirred at room temperature for another 15 hours. Water (0.4mL) was added to the dimethylformamide solution, and formic acid was added until the precipitate completely disappeared. The resulting solution was purified by HPLC according to method B1 for injection, the appropriate fractions were collected and concentrated by centrifugal evaporation on a Joean Model RC10.10 apparatus to give 8, 8' - [5- (3-aminopropoxy) propyloxy ] ethyl acetate) -1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](0.2mg)。

LC/MS (method A1, Platform II) ES: 800MH m/z⁺

Retention time 2.84 minutes

1- (3-allyloxycarbonylamino-propoxy) -3, 5-bis- (bromomethyl) -benzene can be prepared as follows:

to a suspension of 1- (3-allyloxycarbonylamino-propoxy) -3, 5-bis- (hydroxymethyl) -benzene (70mg) in dichloromethane (3mL) was added a solution of carbon tetrabromide (248mg) and triphenylphosphine (199mg) in dichloromethane (2 mL). After refluxing for 3 hours, the reaction mixture was purified by silica gel chromatography (Merck SuperVarioFlash 25g column, Si6015-40 μm) eluting with dichloromethane to give 1- (3-allyloxycarbonylamino-propoxy) -3, 5-bis- (bromomethyl) -benzene (52 mg):

LC/MS (method A1, Platform II): 420MH ESm/z⁺

Retention time 450 minutes

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：1,85(m，2H)；3,15(q，J＝6,5Hz，2H)；3,99(t，J＝6,5Hz，2H)；4,46(d b，J＝5,5Hz，2H)；4,65(s，4H)；5,16(d b，J＝11,0Hz，1H)；5,26(d b，J＝17,5Hz，1H)；5,90(m，1H)；6,96(d，J＝1,5Hz，2H)；7,09(t，J＝1,5Hz，1H)；7,29(t b，J＝6,5Hz，1H)。

1- (3-allyloxycarbonylamino-propoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared as follows:

5- (3-phthalimido-propoxy) -1, 3- (hydroxymethyl) -benzene (1.45g) was dissolved in a mixture of dichloromethane and ethanol (25mL, 25: 75). Hydrazine hydrate (0.62mL) was added and the reaction mixture was refluxed for 1 hour, the solvent was removed in vacuo and the residue was dissolved in dichloromethane. The insoluble residue was filtered off and purified by chromatography on silica gel (Merck SuperVarioPrep 70g column, Si6015-40 μm) eluting with methanol/dichloromethane, 20: 80 followed by ammonium hydroxide/methanol/dichloromethane, 0.5: 25: 75 to give 1- (3-amino-propoxy) -3, 5-bis- (hydroxymethyl) -benzene (1g) suitable for further conversion.

A sample of 1- (3-amino-propoxy) -3, 5-bis- (hydroxymethyl) -benzene (100mg) was dissolved in methanol (5mL), and a solution of sodium carbonate (120mg) in water (5mL) and allyl chloroformate (42 μ L) were added to the cooled solution (0 ℃). After stirring for 30 minutes at 0 ℃, the reaction mixture was stirred for a further 15 hours at room temperature. The solvent was removed in vacuo, the residue washed with ethyl acetate and water into a separatory funnel, the layers were separated, the aqueous layer was extracted twice with ethyl acetate and concentrated in vacuo to give 5- (3-allyloxycarbonylamino-propoxy) -2, 6-bis- (hydroxymethyl) benzene (75 mg):

el (method C): 295 m/z⁺

m/z＝142(M-C₈H₉O₃)+

m/z＝41C₃H₅ ⁺

¹H N.M.R.(300MHz，DMSO-d₆δ ppm): 1,85(m, 2H); 3,14(q, J ═ 6,5Hz, 2H); 3,96(t, J ═ 6,5Hz, 2H); 4,41-4,48(m, 6H); 5,11(t partial mask, J ═ 5,5Hz, 2H); 5,16(qd, J ═ 1,5et 10,5Hz, 1H); 5,26(qd, J ═ 1,5et 17,0Hz,1H)；5,90(m，1H)；6,72(s b，2H)；6,83(s b，1H)；7,26(t b，J＝6,5Hz，1H)。

5- (3-phthalimido-propoxy) -1, 3- (hydroxymethyl) -benzene may be prepared as follows:

3, 5-bis-hydroxymethylphenol (Felder, D.; Gutierrez Nava, m.; del PilarCarreon, M.; Eckert, J.F.; Luccisano, M.; Schall, C.; Masson, P.; Gallani, J.L.; Heinrich, B.; Guillon, D.; Nierengarten, J.F.Helv. Chimica Acta 2002, 85, 288) (2.35g), N- (3-bromopropyl) -phthalimide (4.49g), and potassium carbonate (10.53g) were mixed in acetonitrile (25mL) and refluxed for 12 hours. The reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure, the residue was redissolved in dichloromethane and the insoluble residue was filtered off. The filtrate was washed with water, dried over magnesium sulfate and concentrated in vacuo to give a residue. The residue was purified by silica gel chromatography (Merck SuperVarioPrep 90g column, Si6015-40 μm) eluting with methanol/dichloromethane 4: 96 to give 5- (3-phthalimido-propoxy) -1, 3- (hydroxymethyl) -benzene (1,45 g):

LC/MS (method A1, Platform II): ESm/z 342MH⁺

m/z＝324(MH⁺-H₂O)

Retention time 2.90 minutes

¹H N.M.R.(300MHz，DMSO-d₆，δppm)：2,05(m，2H)；3,76(t，J＝6,5Hz，2H)；3,99(t，J＝6,5Hz，2H)；4,40(d，J＝5,5Hz，4H)；5,09(t，J＝5,5Hz，2H)；6,59(s b，2H)；6,82(s b，1H)；7,80-7,90(m，4H)。

Examples9: 8, 8' - [5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a cooled (0 ℃ C.) solution of 5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-bis- (hydroxymethyl) -benzene (50mg) and triethylamine (113. mu.L) in dichloromethane (2mL) was added methanesulfonyl chloride (26. mu.L). After 30 minutes, the reaction mixture was washed twice with water, and the resulting dichloromethane solution was dried over magnesium sulfate and concentrated in vacuo to give a viscous oil (50.3 mg).

A solution of pre-tomaymycin (15mg) in dimethylformamide (0.5mL) was added to the crude compound (13mg), potassium carbonate (23mg) and potassium iodide (9mg), and the reaction mixture was stirred at 30 ℃ for 20 hours. An additional sample of crude compound (6mg) was added and the reaction mixture was stirred at 30 ℃ for an additional 20 hours. The solid was filtered off, washed with dimethylformamide (0.2mL) and then discarded. To the combined dimethylformamide solutions were added water (0.4mL), one drop of formic acid, and additional water (1.5 mL).

A sample of the resulting suspension (2mL) was filtered and the resulting solid was dried in vacuo to give 8, 8' - [5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](3.1mg)：

LC/MS (method A1, Platform II): ES: 818MH m/z⁺

Retention time 4.11 min

5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-bis- (hydroxymethyl) -benzene was prepared as follows:

to a cooled solution (-5 ℃ C.) of diethyl 5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-dicarboxylate (100mg) in tetrahydrofuran (2mL) was slowly added a 1M solution of lithium aluminum hydride in diethyl ether (0.55 mL). After the addition was complete, sodium sulfate decahydrate was added until gas evolution ceased. The solid was filtered off, washed twice with ethyl acetate and the appropriate organic solution was concentrated in vacuo to give 5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1, 3-bis- (hydroxymethyl) -benzene (66.8mg) as a viscous oil: cl (method D): 327mNH for m/z₄ ⁺

m/z＝310MH⁺

m/z＝271(MNH₄ ⁺-C₄H₈)

¹H N.M.R.(300MHz，DMSO-d₆δ ppm): 1,37(s b, 9H); 1,75(m, 2H); 2,45-2,54 (mshaden, 2H); 2,77(s, 3H); 3,18(t, J ═ 7,0Hz, 2H); 4,45(d, J ═ 5,5Hz, 4H); 5,08(t, J ═ 5,5Hz, 2H); 7,00(s b, 2H); 7,08(s b, 1H).

5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -benzene-1, 3-dicarboxylic acid dibasic ester was prepared as follows:

to a solution of diethyl 5- (N-methyl-3-tert-butoxycarbonylaminopropyn-1-yl) -benzene-1, 3-dicarboxylate (890mg) in methanol (10mL) was added palladium 10%/carbon (89mg), and the solution was stirred at room temperature for 18 hours under a hydrogen atmosphere (1 bar). The solid was filtered off and the solvent was removed in vacuo to give diethyl 5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -benzene-1, 3-dicarboxylate (767mg) as a yellow oil:

EI (method C): m/z is 365M⁺

m/z＝309(M-C₄H₈)⁺

m/z＝265(m/z＝309-CO₂)⁺

m/z＝57C₄H₉ ⁺

m/z＝44C₂H₆N⁺

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：1,32(s b，9H)；1,79(m，2H)；2,70(t，J＝7,0Hz，2H)；2,76(s，3H)；3,16(m，2H)；3,87(s，6H)；8,06(d，J＝2,0Hz，2H)；8,32(t，J＝2,0Hz，1H)。

Diethyl 5- (N-methyl-3-tert-butoxycarbonylaminopropyn-1-yl) -benzene-1, 3-dicarboxylate was prepared as follows:

dimethyl 5-trifluoromethylsulfonyloxy-isophthalate (Bodwell, g.J.; Fleming, J.J.; Mannion, M.R.; Miller, D.O.J.Org.Chem.2000, 65(17), 5360) (1g) was dissolved in 2mL of acetonitrile and N-methyl-N-tert-butoxycarbonyl-propynylamine (Bradbury, B.J.; Baumgold, J.; Jacobsen, K.A.J.Med.Chem.1990, 33(2), 741) (643mg), bis (triphenylphosphine) palladium chloride (205mg), copper iodide (56mg) and triethylamine (591mg) were added. The resulting mixture was stirred at room temperature for 15 hours, the solvent was removed by evaporation under reduced pressure, and the residue was subsequently washed with ethyl acetate and water into a separatory funnel. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic solutions were washed with saturated aqueous sodium chloride, dried over magnesium sulfate and concentrated in vacuo to give a residue. The residue was chromatographed on silica gel (Biotage FLASH 40+ M100 g column, SiOH 32-63 μ M, eluting with ethyl acetate/heptane, 20: 80) to give 5- (N-methyl-3-tert-butoxycarbonylaminopropyn-1-yl) -benzene-1, 3-dicarboxylic acid diethyl ester (896 mg):

¹H N.M.R.(300MHz，DMSO-d₆，δppm)：1,43(s，9H)；2,90(s，3H)；3,90(s，6H)；4,30(s，2H)；8，16(d，J＝1,5Hz，2H)；8,42(t，J＝1，5Hz，1H)。

example 10: 8, 8' - {5- [3- (4-methyl-4-methyldithio-pentanoylamino) -propoxy]-1, 3-benzenediylbis (methyleneoxy) } -bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a cooled (0 ℃ C.) solution of 1- [3- (4-methyl-4-methyldisulfanyl-pentanoylamino) -propoxy ] -3, 5-bis- (hydroxymethyl) -benzene (45mg) and triethylamine (49[ mu ] L) in dichloromethane (1.5mL) was added methanesulfonyl chloride (19. mu.L). After 30 minutes, the reaction mixture was washed twice with water, and the resulting dichloromethane solution was dried over magnesium sulfate and concentrated in vacuo to give a viscous oil (39 mg).

To a solution of pre-tomaymycin (26mg) in dimethylformamide (0.9mL) were added potassium carbonate (40mg), potassium iodide (16mg) and a crude compound sample (31mg), and the reaction mixture was stirred at 30 ℃ for 20 hours. An additional sample of crude compound (6mg) was added and the reaction mixture was stirred at 30 ℃ for an additional 20 hours. Filtering off the solid with dimethylformamide(0.3mL) and discarded. Water (1.6mL) was added to the combined dimethylformamide solutions and the resulting solid was filtered, washed with water and dried in vacuo to give a residue. The residue was chromatographed on silica gel (Interchrom Purifash 2g column, SiOH15-35 μm, eluting with dichloromethane/methanol, 95: 5) and then again on silica gel (Chromabond OH 2g column, eluting 45 μm) to give 8, 8' - {5- [3- (4-methyl-4-methyldithio-pentanoylamino) -propoxy ] -5]-1, 3-benzenediylbis (methyleneoxy) } -bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones](0.2mg)：

LC/MS (method A1, Platform I): ES: 896MH⁺

Retention time 4.09 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：1,29(s，6H)；1,75(d，J＝6,5Hz，6H)；1,92-2,03(m，4H)；2,28(m，2H)；2,39(s，3H)；2,97(m，4H)；3,45(q，J＝6,0Hz，2H)；3,89(q，J＝5,5Hz，2H)；3,96(s，6H)；4,04(t，J＝5,0Hz，2H)；4,27(s b，4H)；5,14(d，J＝12,5Hz，2H)；5,20(d，J＝12,5Hz，2H)；5,60(m，2H)；5,84(t b，J＝6,0Hz，1H)；6,83(s，2H)；6,94(s，2H)；7,09(s，1H)；7,53(s，2H)；7,64(d，J＝5,0Hz，2H)。

1- [3- (4-methyl-4-methyldisulfanyl-pentanoylamino) -propoxy ] -3, 5-bis- (hydroxymethyl) -benzene

To a solution of 1- (3-amino-propoxy) -3, 5-bis- (hydroxymethyl) -benzene (50mg) in dimethylformamide (1mL) were added 4-methyl-4-methyldithio-pentanoic acid (44mg), N' -diisopropylcarbodiimide (35mL) and 1-hydroxybenzotriazole hydrate (5.8 mg). After 15 hours at room temperature, water was added to the reaction mixture and the aqueous solution was extracted twice with ethyl acetate. The combined organic solutions were washed with saturated aqueous sodium chloride, dried over magnesium sulfate and concentrated in vacuo to give a residue. The residue was purified by silica gel chromatography (Interchrom Purifash 5g column, SiOH15-35 μm), eluting with methanol/dichloromethane, 5: 95, to give 1- [3- (4-methyl-4-methyldithio-pentanoylamino) -propoxy ] -3, 5-bis- (hydroxymethyl) -benzene (48 mg):

LC/MS (method A1, ZQ): ESm/z 388MH⁺

Retention time 3.03 min

¹H N.M.R.(300MHz，DMSO-d₆，δppm)：1,24(s，6H)；1,76-1,87(m，4H)；2,16(m，2H)；2,40(s，3H)；3,19(q，J＝6,5Hz，2H)；3,95(t，J＝6,5Hz，2H)；4,44(d b，J＝5,5Hz，4H)；5,12(d，J＝5,5Hz，2H)；6,73(s b，2H)；6,83(s b，1H)；7,92(t b，J＝5,5Hz，1H)。

Example 11: preparation of starting products and/or intermediates

To a solution of 4-benzyloxy-5-methoxy-2-nitrobenzoic acid (4.8g, 16mmol) in anhydrous dichloromethane (80mL) and THF (5mL) was added oxalyl chloride (2.8mL, 32mmol) and DMF (30. mu.L, 0.38mmol) at room temperature. A large amount of bubbles formed after addition of DMF and the mixture was stirred overnight, followed by removal of the solvent by rotary evaporation under vacuum. The residue was co-evaporated by adding anhydrous dichloromethane less than once to give acetyl chloride as a yellow solid.

To 4-methylene-L-proline methyl ester, compound A (3.95g, 15.5mmol) in anhydrous mediumTo a solution in THF (80mL) was added triethylamine (6.7mL, 48mmol) at 0 deg.C, and after 2 minutes, the above acetyl chloride in anhydrous THF (80mL) was added rapidly via catheter at the same temperature over 10 minutes. The resulting yellow cloudy solution was stirred at 0-5 ℃ for 30 minutes and then at room temperature for 4 hours. The reaction solution was diluted with ethyl acetate and water, the aqueous layer was extracted twice with ethyl acetate, and the combined organic layers were washed with 2% hydrochloric acid solution and brine, and dried over anhydrous magnesium sulfate. Filtration and removal of the solvent, and purification of the residue by flash chromatography (hexane/AcOEt, 1: 1, 1: 1.5) to give (2S) -4- (methylene) -1- [ 5-methoxy-2-nitro-4- (phenylmethoxy) -benzoyl]Methyl-2-pyrrolidinecarboxylate as a yellow solid (5.6g, y 85%).¹H NMR(400Hz，CDCl₃): the compounds show a pair of distinct rotamers. δ 7.76(s, 0.7H), 7.73(s, 0.3H), 7.43-7.29(m, 5H), 6.83(s, 0.7H), 6.80(s, 0.3H), 5.17(s, 1.4H), 5.16(s, 0.6H), 5.10-4.89(m, 2.7H), 4.57(d, J ═ 16Hz, 0.3H), 4.19-4.12(m, 0.7H), 3.95-3.77(m, 6.3H), 3.57(s, 1H), 3.06-2.96(m, 1H), 2.73-2.62(m, 1H);¹³C NMR(400Hz，CDCl₃)：171.8，171.7，166.5，166.2，154.9，154.4，148.25，148.18，141.7，141.3，137.19，137.13，135.3，135.2，128.7，128.43，128.41，127.5，127.3，109.5，109.1，109.0，108.9，71.3，60.6，58.1，56.7，56.5，52.39，52.37，52.0，50.2，37.1，35.5；

MS(ESI)：m/z 449.3(M+Na)⁺。

to a solution of the ester (3.15g, 7.39mmol) in dry dichloromethane (9mL) and toluene (27mL) was added dibal-H (15mL, 1.0m toluene solution) dropwise via syringe pump over 30 min at-78 deg.C, the mixture was stirred for 2H at-78 deg.C, TLC (hexane/AcOEt, 1: 1.5) showed consumption of the starting material. The reaction was stopped at-78 deg.C with methanol (0.3mL, 7.4mmol) and 5% hydrochloric acid (20 m)L), followed by AcOEt (50 mL). The dry ice/acetone bath was removed and the mixture was warmed to 0 ℃ and stirred for 15 minutes. The aqueous layer was extracted twice with AcOEt and the combined organic layers were washed with cold 5% hydrochloric acid and brine and dried over anhydrous sodium sulfate. Filtration through celite, removal of solvent, and purification of the residue by flash chromatography (hexane/AcOEt, 1: 1, 1: 1.5, 1: 2, 1: 3, 1: 5, 100% AcOEt) to give: (2S) -4- (methylene) -1- [ 5-methoxy-2-nitro-4- (phenylmethoxy) benzoyl]-2-pyrrolidinecarboxaldehyde as a fluffy yellow solid (2.69g, y 92%).¹H NMR(400Hz，CDCl₃): the compounds show a pair of distinct rotamers. δ 9.75(s, 0.7H), 9.32(s, 0.3H), 7.76(s, 0.7H), 7.69(s, 0.3H), 7.45-7.31(m, 5H), 6.85(s, 0.7H), 6.80(s, 0.3H), 5.19-4.82(m, 4.7H), 4.56(d, J ═ 16Hz, 0.3H), 4.14-3.79(m, 5H), 2.99-2.68(m, 2H);¹³C NMR(400Hz，CDCl3)：198.3，197.1，167.1，155.0，154.6，148.4，141.3，140.4，137.2，135.2，128.7，128.5，128.4，127.5，126.9，126.6，109.8，109.4，109.3，109.2，109.1.71.3，66.6，64.3，56.7，56.6，52.2，50.6，33.2，31.9；MS(ESI)：m/z419.2(M+Na)⁺。

method 1: to (1)2S) -4- (methylene) -1- [ 5-methoxy-2-nitro-4- (phenylmethoxy) benzoyl]-2-Pyrrolidinecarboxaldehyde (1.0 equiv.) in THF/H₂To the solution in O (v/v, 1.7: 1, 0.03m) sodium sulphate (5-8 equivalents) was added portionwise over 2 minutes at room temperature, the mixture was stirred for a further 6-20 hours, and TLC (hexane/AcOEt 1: 2 and CH) was used₂Cl₂MeOH 5: 1). After the aldehyde was almost consumed, the reaction was stopped with methanol (approximately the same volume as THF used). The solvent was removed by rotary evaporation under vacuum (temperature < 40 ℃) and the residue solid was left to dry completely in high vacuum. The solid was suspended in dry methanol (0.03m) and AcCl (8-10 equivalents) was added dropwise at room temperature. Under stirring 15After minutes, the cloudy solution was filtered and the solid was washed with dry methanol. The clear yellow filtrate was stirred at room temperature for 1-2 hours and quenched with saturated sodium bicarbonate. After most of the methanol was removed by rotary evaporation, the residue was diluted with dichloromethane and water. The aqueous layer was extracted with AcOEt, the combined organic layers were dried over anhydrous sodium sulfate and filtered, the solvent was removed, and the residue was purified by flash chromatography (hexane/AcOEt, 1: 3, 1: 5) to give (11aS) -7-methoxy-2-methylene-8- (phenylmethoxy) -1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketone, 70-85% yield. The NMR spectrum was consistent with that reported in the literature. Ms (esi): m/z 371.2(M + Na)⁺. MS (ESI, using CH)₃OH)：m/z 403.3(M+CH₃OH+Na)⁺. MS (ESI, using H)₂O)：m/z 389.2(M+H₂O+Na)⁺。

Method 2: to (1)2S) -4- (methylene) -1- [ 5-methoxy-2-nitro-4- (phenylmethoxy) benzoyl]-2-Pyrrolidinecarboxaldehyde (1.0 equiv.) in THF/H₂To a solution in O (v/v, 3.2: 1, 0.03m) was added portionwise sodium hyposulphate (6-8 equiv) followed by sodium bisulphate (0.5-1.0 equiv) over 2 minutes at room temperature. The mixture was stirred for a further 12-20 hours and TLC (hexane/AcOEt 1: 2 and CH) was used₂Cl₂MeOH 5: 1). In the intermediate [ ms (esi): 459.0(M-H)^-]After almost complete consumption, the reaction was quenched with saturated sodium bicarbonate to pH 5-6. The solvent was removed by rotary evaporation under vacuum (temperature < 40 ℃) and the residue solid was left to dry completely in high vacuum. The solid was suspended in dry methanol (0.03m) and AcCl (8-10 equivalents) was added dropwise at room temperature. After stirring for 15 minutes, the cloudy solution was filtered and the solid was washed with anhydrous methanol. The clear yellow filtrate was stirred at room temperature for 1-2 hours and quenched with saturated sodium bicarbonate. After most of the methanol was removed by rotary evaporation, the residue was diluted with dichloromethane and water. The aqueous layer was extracted with AcOEt, the combined organic layers were dried over anhydrous sodium sulfate and filtered, the solvent was removed, and the residue was purified by flash chromatography (hexane/Ac OEt, 1: C)3, 1: 5) to obtain (11aS) -7-methoxy-2-methylene-8- (phenylmethoxy) -1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketone, 65-80% yield.

To a solution of the starting material (98mg, 0.28mmol) in anhydrous dichloromethane (2mL) at room temperature was added a solution of freshly mixed methanesulfonic acid (2mL) in anhydrous dichloromethane (4 mL). The mixture was stirred at room temperature for 1.5 hours, poured onto ice (. about.30 g), and washed with saturated NaHCO₃Stop and dilute with dichloromethane. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried over anhydrous sodium sulfate, filtered and the solvent removed. Flash chromatography of the residue (CH)₂Cl₂MeOH, 15: 1) to give the product as a yellow solid (29 mg). The aqueous layer was stirred at room temperature overnight, extracted with dichloromethane and AcOEt in that order, the combined dichloromethane and AcOEt were dried over anhydrous sodium sulfate, filtered and the solvent removed to give (11aS) -8-hydroxy-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-one (25 mg). The total yield was 74%.¹H NMR(400Hz，CDCl₃)：δ7.65(d，J＝4.8Hz，1H)，7.48(s，1H)16.87(s，1H)，6.35(bs，1H)，5.17(t，J＝1.6Hz，1H)，5.14(7，J＝1.6Hz，1H)，4.26(s，2H)，3.93(s，3H)，3.87-3.83(m，1H)，3.12-3.05(m，1H)，2.91(d，J＝16Hz，1H)；MS(ESI)：m/z281.0(M+Na)⁺. MS (ESI, water): m/z 258.9(M + H)⁺，m/z 299.1(M+H₂O+Na)⁺。

Example 12: 8, 8' - [ 5-acetylsulfanylmethyl-1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

Diethyl azodicarboxylate (2.2m in toluene, 791. mu.L, 1.7mmol) was added dropwise to a solution of triphenylphosphine (577mg, 2.2mmol) in anhydrous THF (5mL) at 0 deg.C, and after stirring for 50 minutes at 0 deg.C, 1,3, 5-tris (hydroxymethyl) benzene (269mg, 1.6 mmol) was added dropwise, prepared by reacting trimethyl 1,3, 5-benzenetricarboxylic acid with lithium aluminum hydride in THF, co-evaporating with anhydrous benzene, drying under high vacuum for several hours before use) and a solution of thioacetic acid (108. mu.L, 1.45mmol) in anhydrous THF (4 mL). After 1 hour, the ice-water bath was removed and the reaction was stirred at room temperature for 15 hours. The solvent was removed by rotary evaporation in vacuo and the residue was purified by flash chromatography to give 5-acetylthiomethyl-1, 3-bis (hydroxymethyl) -benzene as an anhydrous solid (110 mg).

¹H NMR(400Hz，CDCl₃)：δ7.13-6.99(m，3H)14.45(apt，J＝20.4Hz，4H)，3.98(apt，J＝20.4Hz，4H)，3.73(bs，2H)，2.24(apt，J＝20.4Hz，3H)；

MS(ESI)：m/z 249.0(M+Na)⁺。

To a solution of triphenylphosphine (28mg, 0.1mmol) in anhydrous THF (0.3mL) was added dropwise diisopropyl azodicarboxylate (19. mu.L, 0.09mmol) at 0 deg.C, and after stirring at 0 deg.C for 35 minutes, (11aS) -8-hydroxyYl-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketone, compound 5, (18mg, 0.07mmol, coevaporated with anhydrous benzene before use, dried under high vacuum for several hours) in anhydrous THF (0.2 mL). The mixture was stirred for 10 minutes, then 5-acetylthiomethyl-1, 3-bis (hydroxymethyl) -benzene in dry THF (0.2mL), compound 6, (6.6mg, 0.03 mmol) was added, co-evaporated with dry benzene before use, dried under high vacuum for several hours. The reaction mixture was stirred at 0 ℃ for 35 minutes, the ice-water bath was removed and the solution was stirred at room temperature for 21 hours. The solvent was removed by rotary evaporation in vacuo and the residue was purified by flash chromatography to give the crude product which was further purified by preparative HPLC (C18 column, CH)₃CN/H₂O) to yield 0.7mg of 8, 8' - [ 5-acetylsulfanylmethyl-1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Compound 7. MS (ESI, using H)₂O)：m/z 765.3(M+2H₂O+Na)⁺，747.3(M+H₂O+Na)⁺，729.2(M+Na)⁺，707.3(M+H)⁺，663.2(M-Ac)^-。

Example 13Bis- {2- [ (S) -2-methylene-7-methoxy-5-oxo-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-8-yloxy]-ethyl } -carbamic acid tert-butyl ester

Scheme 1

Compound 1 thionyl chloride (5.6mL, 76.3mmol) was added dropwise to anhydrous methanol (76mL) at-20 deg.C, followed by trans-4-hydroxy-L-proline (5.0g, 38.1 mmol). The resulting mixture was allowed to warm to room temperature and stirred for 20 hours. The solvent was removed under reduced pressure and the residue was further dried under high vacuum to give trans-4-hydroxy-L-proline methyl ester 1as a white solid:¹HNMR(300MHz，DMSO-d₆)δ 2.18-2.23(m，2H)，3.06(m，1H)13.32-3.36(m，2H)，3.76(s，3H)，4.42(br.s，1H)，4.48(dd，J＝5.4，8.1Hz，1H)，5.56(br.s，1H)；EIMSm/z 146([M]⁺+1)。

compound 2 to a solution of trans-4-hydroxy-L-proline methyl ester 1(4.48g, 30.9mmol) and sodium bicarbonate (1.56g, 18.5mmol) in anhydrous DMF (42mL) under argon at 0 deg.C was added (BOC)₂Solution of O in DMF (20 mL). After stirring overnight at room temperature, the reaction was stopped by addition of 100mL of water at 0 deg.C and extracted with EtOAc (4X 80 mL). The combined organic layers were washed with brine (100mL), dried (magnesium sulfate), filtered and concentrated by rotary evaporation. The residue was purified by flash chromatography (silica gel, 1: 1 hexanes/EtOAc) to afford N-BOC protected 4-hydroxy-L-proline methyl ester 2 as a colorless oil:

¹HNMR(300MHz，CDCl₃rotamers) δ 1.38 and 1.43(2 x s, 9H), 2.04-2.07(m, 1H), 2.23-2.27(m, 2H), 3.54-3.63(m, 2H), 3.70(s, 3H), 4.34-4.38(m, 1H), 4.46(br.s, 1H).

Compound 3 (Francomanfre, Jean-Marc Kern, and Jean-Francois Biellmann J. org. chem.1992, 57, 2060-. N-BOC protected 4-hydroxy-L-proline methyl ester Compound 2(3.24g, 13.2mmol) was dissolved in CH₂Cl₂(132mL), cool to 0 ℃. Pyridine and Dess-Martin periodinane were added and stirring was continued until TLC showed no SM remaining. Reaction mixture with CH₂Cl₂Diluting with 10% aqueous Na₂S₂O₃(3X 50mL), 1N aqueous HCl (50mL), saturated aqueous NaHCO₃Washed (50mL) with brine (50mL), dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash chromatography (silica gel, 7: 3 hexanes/EtOAc) to afford N-BOC protected methyl 4-oxo-L-proline 3 as a pale yellow oil:¹HNMR(300MHz，CDCl₃rotamers) delta 1.44(s, 9H), 2.53-2.57(m, 1H), 2.85-2.96(m, 1H), 3.72 and 3.74(2 x s, 3H), 3.85-3.87(m, 2H), 4.67-4.77(m, 1H).

Compound 4 (Kuei-Ying Lin, Markmatteucci US 5414077) A solution of potassium tert-butoxide (2.51g, 22.3mmol) in anhydrous THF (40mL) was added to a suspension of methyltriphenylphosphonium bromide (7.99g, 22.3mmol) in THF (40mL) at 0 ℃. The resulting yellow suspension of the ylide was stirred at 0 ℃ for 2h, followed by addition of a solution of N-BOC protected 4-oxo-L-proline methyl ester 3(2.72g, 11.2mmol) in THF (32 mL). After stirring at room temperature for 1 hour, the reaction mixture was diluted with EtOAc (100mL), washed with water (80mL), brine (80mL), dried (magnesium sulfate), and concentrated. Flash chromatography of the residue (silica, 9: 1 hexanes/EtOAc) afforded N-BOC protected 4-methylene-L-proline methyl ester, compound 4, as a colorless oil:¹HNMR(300MHz，CDCl₃rotamers) δ 1.40 and 1.45(2 x s, 9H), 2.58-2.62(m, 1H), 2.88-2.98(m, 1H), 3.69 and 3.70(2 x s, 3H), 4.03-4.06(m, 2H), 4.36-4.49(m, 1H), 4.97-4.99(m, 2H); EIMSm/z 264([ M ]]⁺+Na)。

Compound 5N-BOC protected 4-methylene-L-proline methyl ester, Compound 4, (0.8g, 3.31mmol) was dissolved in CH2Cl2(6.5mL) and cooled to 0 ℃. Trifluoroacetic acid (6.5mL) was added dropwise to CH₂Cl₂(6.5mL), and the resulting mixture was stirred at room temperature for 1.5 hours. After removal of the volatile solvents by rotary evaporator, the brown residue was dissolved in 10mL of water and washed with Et2O (3X 5 mL). The aqueous solution was concentrated and it was further dried under high vacuum to give 4-methylene-L-proline methyl ester 5TFA salt:¹HNMR(300MHz，CDCl₃rotamers) δ 2.83-2.87(m, 1H), 3.05-3.11(m, 1H), 3.80 and 3.81(2 x s, 3H), 4.00-4.10(m, 2H), 4.55(dd, J ═ 5.7, 5.7Hz, 1H), 5.17-5.21(m, 2H);¹³CNMR(534.0，49.1，53.8，59.3，111.7，137.6，169.8；EIMSm/z 142([M]⁺+1)。

compound 6 (Kamal, A.; Et al J.med. chem.2002, 45, 4679-Buff 4688) diethanolamine (3.57g, 34mmol) was dissolved in methanol (20mL), treated with Et3N (4.7mL, 34mmol) and ethyl trifluoroacetate (4.90g, 34mmol) at room temperature for 20 hours, followed by the addition of another 1mL CF₃And (4) COOEt. After another 20 hours, the volatile solvent was removed under high vacuum to give N-trifluoroacetyl-diethanolamine 6 as a pale yellow oil which was used without further purification.

Compound 7. diethyl azodicarboxylate (7.66g, 44mmol) was added dropwise to a stirred solution of methyl vanilate (7.30g, 40.1mmol) and triphenylphosphine (15.67g, 59.7mmol) in anhydrous THF (57mL) at 0 ℃, the resulting mixture was stirred at this temperature for 1 hour, followed by addition of a solution of N-trifluoroacetyl-diethanolamine 6(7.30g, 40.1mmol) in anhydrous THF (20 mL). After stirring overnight at room temperature, the reaction was quenched with water (100mL) and Et₂O (3X 80mL) extraction, pooled Et₂The O layer was washed with brine (100mL), dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 8: 2 to 7: 3 hexanes/EtOAc) to give N-trifluoroacetyl-N, N-bis [2- (4-methoxycarbonyl-2-methoxy-phenoxy) ethyl]Amine, compound 7, white solid:

¹HNMR(300MHz，CDCl₃) δ 3.81(s, 3H), 3.84(s, 3H), 3.87(s, 3H), 3.88(s, 3H), 4.04-4.08(m, 4H), 4.28-4.32(m, 4H), 6.84 and 6.85(2 x d, J ═ 6.3Hz, 2H), 7.50 and 7.51(2 x d, J ═ 1.5Hz, 2H), 7.61(dd, J ═ 1.5, 6.3Hz, 2H).

Compound 8 solid Cu (NO) at 0 deg.C₃)₂.xH₂O (2.33g, 12.41mmol) was added to N-trifluoroacetyl-N, N-bis [2- (4-methoxycarbonyl-2-methoxy-phenoxy) ethyl]Amine 7(2.62g, 4.96mmol) in a stirred solution of acetic anhydride (50 mL). The reaction mixture was stirred at 0 ℃ for 1 hour, at room temperature for 2 hours, and then poured into 200mL of ice water. Stirring was continued for an additional 1 hour. ObtainedThe yellow precipitate was collected by filtration and purified by flash chromatography (silica gel, 6: 4 hexane/EtOAc) to give N-trifluoroacetyl-N, N-bis [2- (4-methoxycarbonyl-2-methoxy-5-nitro-phenoxy) ethyl]Amine, compound 8 light yellow solid:¹HNMR(300MHz，CDCl₃) δ 3.86(s, 3H), 3.88(s, 3H), 3.886(s, 3H), 3.890(s, 3H), 4.04-4.09(m, 4H), 4.30-4.35(m, 4H), 6.98 and 6.99(2 x s, 2H), 7.37 and 7.40(2 x s, 2H).

The compound 9. N-trifluoroacetyl-N, N-bis [2- (4-methoxycarbonyl-2-methoxy-5-nitro-phenoxy) ethyl]A solution of amine 8(2.58g, 4.16mmol) in THF-MeOH (1: 2, 48mL) at room temperature with 10% aqueous K₂CO₃(16mL) for 12 hours. After removal of volatiles using a rotary evaporator, the residue was diluted with 100mL of water and extracted with EtOAc (3X 100 mL). The combined EtOAc layers were washed with brine (100mL), dried (magnesium sulfate) and concentrated to give N, N-bis [2- (4-methoxycarbonyl-2-methoxy-5-nitro-phenoxy) ethyl]Amine, compound 9 as yellow solid, which was used without further purification:¹HNMR(300MHz，CDCl₃)δ3.17(t，J＝3.9Hz，4H)，3.89(s，6H)，3.93(s，6H)，4.19(t，J＝3.9Hz，4H)，7.05(s，2H)，7.47(s，2H)。

compound 10 is prepared by reacting N, N-bis [2- (4-methoxycarbonyl-2-methoxy-5-nitro-phenoxy) ethyl]Amine 9 (crude, 4.16mmol) and NaHCO₃(210mg, 2.50mmol) was suspended in THF at 0 deg.C with (BOC)₂O (999mg, 4.58mmol) and stirring was continued at room temperature for 3 hours. After THF was removed, the residue was partitioned between water and EtOAc (100/100mL), and the aqueous layer was further extracted with EtOAc (2X 50 mL). The combined layers were washed with brine (80mL), dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 6: 4 hexane/EtOAc) to give N-tert-butoxycarbonyl-N, N-bis [2- (4-methoxycarbonyl-2-methoxy-5-nitro-phenoxy) ethyl]Amine, compound 10 light yellow solid:¹HNMR(300MHz，CDCl₃) δ 1.44(s, 9H), 3.77(m, 4H), 3.83, 3.86 and 3.87(3 x s, 12H), 4.20 and 4.26(2 x t, J ═ 3.9Hz, 4H), 6.97 and 6.99(2 x s, 2H), 7.36 and 7.40(2 x s, 2H)H)；EIMS m/z 646([M]⁺+Na)。

Compound 11. preparation of N-tert-butoxycarbonyl-N, N-bis [2- (4-methoxycarbonyl-2-methoxy-5-nitro-phenoxy) ethyl]Amine, Compound 10(2.11g, 3.39mmol) suspended in THF-MeOH-H₂O (3: 1,65 mL), treated with 1m aqueous LiOH (14mL) at room temperature for 3 hours. After removal of the volatile solvent, the residue was diluted with water (25 mL). The resulting aqueous solution was acidified with concentrated HCl to a pH of about 1. The precipitated N-tert-butoxycarbonyl-N, N-bis [2- (4-carboxy-2-methoxy-5-nitro-phenoxy) ethyl group was collected by filtration]Amine, compound 11, washed with water and further dried under high vacuum:¹HNMR(300MHz，CDCl₃rotamers) δ 1.39(s, 9H), 3.70(m, 4H), 3.88 and 3.89(2 x s, 6H), 4.29(m, 4H), 7.29 and 7.31(2 x s, 2H), 7.63(s, 2H), 13.60(br.s, 2H);¹³CNMR δ 27.8, 46.2 and 46.6, 56.3 and 56.4, 67.2, 79.2, 107.9 and 108.0, 111.3, 141.4 and 141.5, 149.1, 151.7, 154.5, 165.9; HRMSm/z C₂₅H₂₉N₃O₁₄Calculated Na 618.1547, found 618.1552([ M ]]⁺+Na)。

Compound 12. addition of catalytic amount of DMF (2 drops) to N-tert-butoxycarbonyl-N, N-bis [2- (4-carboxy-2-methoxy-5-nitro-phenoxy) ethyl]A solution of amine 11(194mg, 0.33mmol) and oxalyl chloride (72.7. mu.L, 0.81mmol) in dry THF (6.5mL) was added and the resulting mixture was stirred at room temperature overnight. Excess THF and oxalyl chloride were removed by rotary evaporator, the acetyl chloride was resuspended in fresh THF (4mL) and added dropwise to 4-methylene-L-proline methyl ester 5(206.7mg, 0.81mmol), Et under an argon atmosphere at 0 deg.C₃N (0.19mL, 1.39mmol) and H₂O (0.4mL) in THF (1 mL). The reaction mixture was warmed to room temperature and stirring continued for 2 hours. After THF was removed, the residue was partitioned between water and EtOAc (10/10mL), and the aqueous layer was further extracted with EtOAc (2X 8 mL). The combined organic layers were washed with brine (10mL), dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 2: 8 hexane/EtOAc) to give bis {2- [ 5-methoxy-2-nitro-4- [ (S) -4-methylene-2-methoxycarbonyl-1-pyrrolidinyl-)Carbonyl radical]-phenoxy group]-ethyl } -carbamic acid tert-butyl ester 12 pale yellow oil (rotamer):¹HNMR(300MHz，CDCl₃rotamers) delta 1.46(s, 9H), 2.68-2.75(m, 2H), 2.99-3.10(m, 2H), 3.60-4.28(m, 24H), 4.56-5.12(m, 6H), 6.78-6.83(m, 2H), 7.63-7.71(m, 2H); EIMSm/z 864([ M ]]⁺+Na)。

Compound 13 bis {2- [ 5-methoxy-2-nitro-4- [ (S) -4-methylene-2-methoxycarbonyl-1-pyrrolidinylcarbonyl ] at-78 ℃ under an argon atmosphere]Phenoxy radical]-Ethyl } -carbamic acid tert-butyl ester 12(100mg, 0.12mmol) in a vigorously stirred solution of dry toluene (2.4mL) DIBAL-H (480. mu.L of 1M toluene solution) was added dropwise. After the mixture was stirred for an additional 45 minutes, excess reagent was decomposed by the addition of 5 drops of methanol followed by the addition of 5% HCl (4 mL). The resulting mixture was warmed to 0 deg.C, the layers were separated and the aqueous layer was further treated with CH₂Cl₂(3X 3 mL). The combined organic layers were washed with brine, dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 95: 5 CHCl)₃MeOH) to give bis {2- [ 5-methoxy-2-nitro-4- [ (S) -4-methylene-2-formyl-1-pyrrolidinylcarbonyl group]Phenoxy radical]-Ethyl } -carbamic acid tert-butyl ester 13 as pale yellow oil (84mg, 91%).

Compound 14. Di {2- [ 5-methoxy-2-nitro-4- [ (S) -4-methylene-2-formyl-1-pyrrolidinylcarbonyl ] at room temperature]Phenoxy radical]-Ethyl } -carbamic acid tert-butyl ester 13(180mg, 0.23mmol), Na₂S₂O₄(1.84mmol, 8 equiv.), 3.5mL THF and 2.2mLH₂O stirred for 20 hours. The solvent was removed under high vacuum, the residue was resuspended in MeOH (30mL), and AcCl was added dropwise until the pH was about 2. The resulting mixture was stirred at room temperature for 1 hour, and the reaction was worked up by removing most of the MeOH, followed by dilution with EtOAc (25 mL). EtOAc solution with saturated aqueous NaHCO₃Washed with brine, dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 97: 3 CHCl)₃MeOH) to give bis- {2- [ (S) -2-methylene-7-methoxy-5-oxo-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c)][1，4]Benzodiazepine-8-yl]-ethyl } -carbamic acid tert-butyl ester 14 as a white solid (86mg, 50%).

Example 14: (11aS) -7- (5-Bromopentyloxy) -2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones

Scheme 2

Compound 15 to a solution of vanillic acid methyl ester (9.109g, 50mmol) in acetone (200mL) was added K₂CO₃(27.64g, 200mmol) and 1, 5-dibromopentane (20.4mL, 150mmol), and the resulting mixture is heated at reflux. After 6 hours, TLC showed no starting material remaining. The mixture was cooled to room temperature and filtered to remove solids. The filtrate was concentrated and purified by flash chromatography (silica gel, 8: 2 hexanes/EtOAc) to afford 4- (5-bromopentyloxy) -3-methoxy-benzoic acid methyl ester 15 as a white solid (13.65g, 82%):¹HNMR(300MHz，CDCl₃) δ 1.60-1.66(m, 2H), 1.85-1.97(m, 4H), 3.42(t, J ═ 5.0Hz, 2H), 3.87(s, 3H), 3.89(s, 3H), 4.06(t, J ═ 5.0Hz, 2H), 6.85(d, J ═ 6.3Hz, 1H), 7.52(d, J ═ 1.5Hz, 1H), 7.63(dd, J ═ 6.3, 1.5Hz, 1H); EIMSm/z 353 and 355([ M ]]⁺+Na)。

Compound 16. solid Cu (NO) at 0 deg.C₃)₂.xH₂O (3.64g, 19.42mmol) was added to a stirred solution of 4- (5-bromopentyloxy) -3-methoxy-benzoic acid methyl ester 15(5.36g, 16.18mmol) in acetic anhydride (81 mL). The reaction mixture was stirred at 0 ℃ for 1 hour, at room temperature for 2 hours, and then poured into 200mL of ice water. Filtering to collect the yellow precipitateStarch, wash with water. Further drying under high vacuum gave 4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoic acid methyl ester compound 16 as a pale yellow solid (5.98g) which was used directly in the next step:

¹HNMR(300MHz，CDCl₃) δ 1.59-1.70(m, 2H), 1.85-1.98(m, 4H)13.43(t, J ═ 5.1Hz, 2H), 3.89(s, 3H), 3.94(s, 3H), 4.08(t, J ═ 4.8Hz, 2H), 7.05(s, 1H), 7.42(s, 1H). EIMSm/z 398 and 400([ M ]]⁺+Na)。

Compound 17 methyl 4- (5-Bromopentyloxy) -5-methoxy-2-nitro-benzoate 16(5.98g, 15.9mmol) was suspended in THF-MeOH-H₂O (3: 1, 157mL) was treated with 1M aqueous LiOH (31mL) at room temperature for 5 hours. After removal of the volatile solvent, the residue was diluted with water (70 mL). The resulting aqueous solution was acidified to pH-2 with concentrated hydrochloric acid, the precipitated 4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoic acid 17 was collected by filtration, washed with water and further dried under high vacuum (5.47 g):¹HNMR(300MHz，CDCl₃) δ 1.64-1.68(m, 2H), 1.87-1.98(m, 4H), 3.43(t, J ═ 4.8Hz, 2H), 4.08(s, 3H), 4.10(t, J ═ 5.1Hz, 2H), 7.21(s, 1H), 7.35(s, 1H), 13.60(br.s, 1H); EIMSm/z 384 and 386([ M ]]⁺+Na)。

Compound 18. A catalytic amount of DMF (2 drops) was added to a solution of 4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoic acid 17(270mg, 0.74mmol) and oxalyl chloride (80. mu.L, 0.89mmol) in dry THF (7.5mL) and the resulting mixture was stirred at room temperature overnight. Excess THF and oxalyl chloride were removed by rotary evaporator, the acetyl chloride resuspended in fresh THF (6mL) and added dropwise to 4-methylene-L-proline methyl ester 5(228mg, 0.89mmol), Et, under an argon atmosphere at 0 deg.C₃N (0.32mL, 2.31mmol) and H₂O (0.15mL) in THF (1.5 mL). The reaction mixture was allowed to warm to room temperature and stirring was continued for 4h, after removal of THF, the residue was partitioned between water and EtOAc (20/20 mL). The aqueous layer was further extracted with EtOAc (3X 10mL), and the combined organic layers were washed with brine (20mL), dried (magnesium sulfate) and concentrated. Flash chromatography of the residue (silica gel, 6:)4 Hexane/EtOAc) to yield 1- [4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoyl]-4-methylene-L-proline methyl ester 18 light yellow oil (rotamer):

¹HNMR(300MHz，CDCl₃rotamers) δ 1.62-1.68(m, 2H), 1.86-1.98(m, 4H), 2.64-2.75(m, 1H), 2.99-3.08(m, 1H), 3.43(t, J ═ 5.1Hz, 2H), 3.59-3.96(m, 7H), 4.05-4.21(m, 3H), 4.57-4.61 and 4.90-5.12(m, 3H), 6.80 and 6.83(2s, 1H), 7.64 and 7.67(2s, 1H); EIMSm/z 507 and 509([ M ]]⁺+Na)。

Compound 19. preparation of 1- [4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoyl]-4-methylene-L-proline methyl ester 18(61mg, 0.12mmol) in dry toluene-CH₂Cl₂DIBAL-H (188. mu.L of 1M in toluene) was added dropwise to a vigorously stirred solution (3: 1, 2.5mL) at-78 ℃ under an argon atmosphere. After the mixture was stirred for an additional 45 minutes, excess reagent was decomposed by the addition of 5 drops of methanol followed by the addition of 5% HCl (2 mL). The resulting mixture was warmed to 0 deg.C, the layers were separated and the aqueous layer was further treated with CH₂Cl₂(3X 3 mL). The combined organic layers were washed with brine, dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 1: 1 hexane/EtOAc) to give (S) -1- [4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoyl]-4-methylene-2-pyrrolidinecarboxaldehyde 19 as a pale yellow oil (44mg, 80%).

Compound 20 (S) -1- [4- (5-bromopentyloxy) -5-methoxy-2-nitro-benzoyl at room temperature]-4-methylene-2-pyrrolidinecarboxaldehyde 19(43.7mg, 0.096mmol), Na₂S₂O₄(0.58mmol, 6equiv), 1.5mL THF and 0.9mLH₂The mixture of O was stirred for 18 hours. The solvent was removed under high vacuum, the residue was resuspended in MeOH (6mL), and AcCl was added dropwise until pH 2. The resulting mixture was stirred at room temperature for 1 hour, and the reaction was purified by removing most of the MeOH, followed by treatment with dilute EtOAc (20 mL). EtOAc solution with saturated aqueous NaHCO₃Washed with brine, dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (silica gel, 98: 2 CHCl)₃Purification with MeOH to give (11aS) -7- (5-)Bromopentyloxy) -2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-Ketone 20 pale yellow oil (31mg, 74%):

¹HNMR(300MHz，CDCl₃) δ 1.58-1.66(m, 2H), 1.84-1.99(m, 4H), 2.91-2.95(m, 1H), 3.08-3.14(m, 1H), 3.42(t, J ═ 5.1Hz, 2H), 3.93(s, 3H), 3.87-4.27(m, 5H), 5.15(br s, 1H), 5.18(br s, 1H), 6.84(s, 1H), 7.49(s, 1H), 7.71(d, J ═ 4.0Hz, 1H); EIMSm/z 429 and 431([ M ]]⁺+Na)。

Compound 20 can then be coupled with the PBD moiety prepared as in example 11 to form a compound of the invention.

Example 15: 8, 8' - [3- (2-acetylsulfanylmethyl) -1, 5-pentanediylbis (oxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]

Diethyl azodicarboxylate (2.2M in toluene, 2.4mL, 5.4mmol) was added dropwise to a solution of triphenylphosphine (1.77g, 6.8mmol) in anhydrous THF (15mL) at 0 deg.C, and after stirring for 55 minutes at 0 deg.C, a solution of 3- (2-hydroxyethyl) pentane-1, 5-diol (740mg, 5mmol) and thioacetic acid (335. mu.L, 4.5mmol) in anhydrous THF (7mL) was added dropwise. After 1 hour the ice water bath was removed and the reaction stirred at room temperature for 16 hours and the solvent was removed by rotary evaporation under vacuum. Flash chromatography of the residue (CHCl)₃MeOH, 20: 1, 15: 1, 10: 1, 4: 1) to give 3- (2-ethyl-2)Acylthioethyl) pentane-1, 5-diol as a white solid (350mg), and the starting triol (406mg) was recovered.¹HNMR(400Hz，CDCl₃)：δ3.65-3.63(m，4H)，3.45(bs，2H)，2.84-2.80(m，2H)，2.28(s，3H)，1.73-1.67(m，1H)，1.55-1.49(m，6H)。MS(ESI)：m/z 229.0(M+Na)⁺。

To a solution of triphenylphosphine (53mg, 0.2mmol) in anhydrous THF (0.4mL) was added dropwise diisopropyl azodicarboxylate (36. mu.L, 0.17mmol) at 0 ℃. After stirring for 25 minutes at 0 deg.C, (11aS) -8-hydroxy-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ] was added][1，4]Benzodiazepine-5-ketone, PBD monomer, (38mg, 0.14mmol, coevaporated with dry benzene before use, dried under high vacuum for several hours) in dry THF (0.2 mL). The mixture was stirred for 10 minutes, then a solution of thioacetate compound (12mg, 0.058mmol, co-evaporated with dry benzene before use, dried under high vacuum for several hours) in dry THF (0.2mL) was added. The reaction mixture was stirred at 0 ℃ for 35 minutes, the ice-water bath was removed and the solution was stirred at room temperature for 12 hours. The solvent was removed by vacuum rotary evaporation and the residue was purified by flash chromatography (CHCl)₃MeOH, 100: 1, 50: 1, 25: 1, 20: 1) to give 8, 8' - [3- (2-acetylsulfanylmethyl) -1, 5-pentanediylbis (oxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](19mg，y＝47％)。¹H NMR(400Hz，CDCl₃)：δ7.66(d，J＝4.4Hz，2H)，7.46(s，2H)，6.78(s，2H)，5.17(s，2H)，5.14(s，2H)，4.26(s，4H)，4.17-4.09(m，4H)，3.88(s，6H)，3.87-3.83(m，2H) 3.13-3.06(m, 2H), 2.94-2.90(m, 4H), 2.29(s, 3H) 11.95 (bs, 3H), 1.68(bs, 4H). MS (ESI, water): m/z 745.3(M + 2H)₂O+Na)⁺，727.3(M+H₂O+Na)⁺，709.2(M+Na)⁺。

Example 16: 8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a suspension of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 52mg, 0.18mmol) in water (0.3mL) was added dropwise saturated sodium bicarbonate (. about.0.6 mL) to adjust the pH to 6-7, followed by addition of a phosphate buffer (pH 7.0, 10mM, Na)₂HPO₄/H₃PO₄0.5 mL). The TCEP solution thus obtained is added to 8, 8' - [5- (N-4-methyldithio-4, 4-dimethylbutyryl) -amino-1, 3-benzenediyl-bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](29mg, 0.036mmol) in methanol (2.2mL) was stirred at room temperature for 3 hours. The reaction was stopped with phosphate buffer (pH 6.5) and extracted with dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and the solvent removed under reduced pressure to give a white solid. The solid was dissolved in dichloromethane/MeOH (2: 1) and evaporated again. Dichloromethane was added and evaporated, the residue dried under high vacuum and purified by reverse phase C18 column { CH₃CN/H₂O, dissolving the solid in CH before column packing₃CN/H₂O (3: 1, 2mL), stirred for 30 min } to give 8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](11mg) white solid (method H).

MS(ESI)：m/z＝786.3MNa⁺

m/z＝804.2MNa⁺+H₂O。

8, 8' - [5- (N-4-methyldithio-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a solution of 5- (N-4-methyldithio-4, 4-dimethylbutanoyl) -amino-1, 3-bis- (methylsulfonyloxymethyl) -benzene (89mg, 0.18mmol) dissolved in anhydrous DMF (2mL) at room temperature was added 8-hydroxy-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-one (93mg, 0.36mmol), potassium carbonate powder (100mg, 0.72mmol), potassium iodide powder (15mg, 0.09mmol), and tetrabutylammonium iodide (13mg, 0.036 mmol). After the mixture was stirred for 1 hour, a second portion of 8-hydroxy-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ] was added][1，4]Benzodiazepine-5-one (22mg, 0.085 mmol). The solution was stirred for 3 hours at room temperature and the reaction was stopped with water and diluted with dichloromethane. The organic layer was separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The filtrate was filtered and evaporated under reduced pressure, followed by high vacuum evaporation to remove residual DMF. The residue was subsequently subjected to a reverse phase C18 Column (CH)₃CN/H₂O) purification to give 8, 8' - [5- (N-4-methyldithio-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](29mg) white solid (method H).

MS(ESI)：m/z＝832.2MNa⁺

m/z＝850.2MNa⁺+H₂O

¹H NMR(400Hz，CDCl₃-d1δppm)：7.67(bs，2H)，7.58-7.26(m，5H)，6.82(s，2H)，5.21-5.14(m，8H)，4.30(s，4H)14.02-3.88(m，9H)，3.16-3.10(m，2H)，2.97-2.93(m，2H)，2.45-2.40(m，5H)，2.09-2.03(m，2H)，1.34(s，6H)。

5- (N-4-Methyldithio-4, 4-dimethylbutyryl) amino-1, 3-bis- (methylsulfonoxymethyl) -benzene can be prepared as follows:

to a suspension of 5- (N-4-methyldithio-4, 4-dimethylbutyryl) -amino-1, 3-bis- (hydroxymethyl) -benzene (329mg, 1.0mmol) in dry dichloromethane (7mL) was added triethylamine (348. mu.L, 2.5mmol) dropwise followed by methanesulfonyl chloride (193. mu.L, 2.5mmol) dropwise at-2 ℃ over 10 min. The solution was stirred at 0 ℃ for a further 10 minutes, quenched with an ice-water mixture, the mixture was extracted with cold dichloromethane and the combined dichloromethane layers were washed with cold water and dried over anhydrous sodium sulphate. Filtration, rotary evaporation of the filtrate in vacuo and flash purification of the residue on a short silica gel column (dichloromethane/hexane/ethyl acetate, 1: 2: 4) afforded 5- (N-4-methyldithio-4, 4-dimethylbutyryl) amino-1, 3-bis- (methylsulfonyloxymethyl) -benzene as a colorless oil (410 mg).

MS(ESI)：m/z＝508.0MNa⁺

m/z＝483.9M-H

m/z＝519.9M-H+2H₂O

5- (N-4-Methyldithio-4, 4-dimethylbutyryl) -amino-1, 3-bis- (hydroxymethyl) -benzene can be prepared as follows:

to a solution of 5-nitro-m-xylene- α, α' -diol (890mg, 5.4mmol) in methanol (50mL) was added Pd/C (10%, 287 mg). Introducing hydrogen and the mixture under pressure (H)₂5-8psi) at room temperature for 5 hours. The solution was filtered through celite and the filtrate was evaporated by vacuum rotary evaporation to give 5-amino-m-xylene- α, α' -diol, which was dissolved in THF (10mL)/DMF (15 mL). 4-Methyldithio-4, 4-dimethylbutyric acid (1.05g, 5.4mmol) dissolved in THF (5ml) was added at room temperature followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.1g, 10.8mmol) and 4-dimethylaminopyridine (66mg, 0.54 mmol). The resulting mixture was stirred at room temperature overnight, quenched with 10% ammonium chloride, extracted with ethyl acetate, washed and dried. Filtering, removing the solvent by rotary evaporation in vacuo, and subjecting the residue to flash Chromatography (CH)₂Cl₂MeOH, 15: 1, 10: 1, 7: 1) to obtain 5- (N-4-methyldithio-4, 4-dimethylbutyryl) -Amino-1, 3-bis- (hydroxymethyl) -benzene as a white solid (729 mg).

MS(ESI)：m/z＝352.1MNa⁺

¹H NMR(400Hz，CDCl₃-d1，δppm)：7.47(s，2H)，7.10(s，1H)，4.58(s，4H)，2.52-2.47(m，2H)，2.42(s，3H)，2.02-1.98(m，2H)，1.34(s，6H)。

Example 17: 8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a suspension of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 64mg, 0.22mmol) in water (0.05mL) was added dropwise saturated sodium bicarbonate (. about.0.7 mL) to adjust the pH to 6-7, followed by addition of a phosphate buffer (pH 7.0, 10mM, Na)₂HPO₄/H₃PO₄0.8 mL). The TCEP solution thus obtained is added to 8, 8' - [5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]A mixture of (35mg, 0.045mmol) in methanol (5mL) was stirred at room temperature for 4.5 hours. The reaction was stopped with phosphate buffer (pH 6.5) and extracted with dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and reduced pressureThe solvent was removed to give a white solid. The solid was dissolved in dichloromethane/MeOH (2: 1) and evaporated again. Dichloromethane was added and evaporated, the residue dried under high vacuum and purified by reverse phase C18 column { CH₃CN/H₂O, dissolving the solid in CH before column packing₃CN/H₂O (3: 1, 2mL), stirred for 30 min } to give 8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](10.5mg, y ═ 32%) white solid (method H).

MS(ESI)：m/z＝758.2MNa⁺

m/z＝776.2MNa⁺+H₂O

m/z＝794.3MNa⁺+2H₂O

8, 8' - [5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a solution of 5- (N-methyl-N- (2-methyldisulphenyl-2, 2-dimethylethyl) amino-1, 3-bis- (methylsulfonoxymethyl) -benzene (105mg, 0.23mmol) in anhydrous DMF (2.5mL) at room temperature was added 8-hydroxy-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-one (119mg, 0.46mmol), potassium carbonate powder (127mg, 0.92mmol), potassium iodide powder (20mg, 0.12mmol), and tetrabutylammonium iodide (17mg, 0.046 mmol). After the mixture was stirred for 1.5 hours, a second portion of 8-hydroxy-7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ] was added][1，4]Benzodiazepine-5-one (23mg, 0.089 mmol). The solution was stirred for 4.5 hours at room temperature and the reaction was stopped with water and diluted with dichloromethane. The organic layer was separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and the filtrate was evaporated under reduced pressure, followed by high vacuum evaporation to remove residual DMF. Resuspending the residue in CH₃CN/H₂O (10: 1), and filtering. The filtrate was evaporated and the crude product was purified by reverse phase C18 Column (CH)₃CN/H₂O) purification to give 8, 8' - [5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](39mg) white solid (method H).

MS(ESI)：m/z＝804.2MNa⁺

m/z＝822.2MNa⁺+H₂O

m/z＝840.2MNa⁺+2H₂O

¹H NMR(400Hz，CDCl₃-d1，δppm)：7.64(bs，2H)，7.50(s，2H)，6.83-6.76(m，5H)，5.18-5.10(m，8H)，4.27(s，4H)，3.95(s，6H)，3.90-3.84(m，3H)，3.51(s，2H)，3.13-3.07(m，2H)，3.01(s，3H)，2.94-2.90(m，2H)12.41(s，3H)，1.30(s，6H)。

5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-bis- (methylsulfonoxymethyl) -benzene can be prepared as follows:

to a solution of 5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-bis- (hydroxymethyl) -benzene (135mg, 0.45mmol) in anhydrous dichloromethane (3mL) was added triethylamine (153. mu.L, 1.1mmol) dropwise followed by methanesulfonyl chloride (87. mu.L, 1.1mmol) dropwise at 0 ℃ for an additional 30 minutes, quenched with ice/water, the mixture extracted with cold dichloromethane, the combined dichloromethane layers were washed with cold water, dried over anhydrous sodium sulfate, filtered, the filtrate was rotary evaporated under vacuum, the residue was purified by TLC plate (hexane/ethyl acetate, 1: 1.5) to give 5- (N-methyl-N- (2-methyldithio-2, 2-Dimethylethyl) amino-1, 3-bis- (methylsulfonoxymethyl) -benzene as a colorless oil (105 mg).

MS(ESI)：m/z＝480.0MNa⁺

5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-bis- (hydroxymethyl) -benzene can be prepared as follows:

to a solution of 5- (N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-bis- (hydroxymethyl) -benzene (226mg, 0.78mmol) in DMF (4mL) was added iodomethane (149. mu.L, 2.4mmol) followed by potassium carbonate powder, after stirring at room temperature for 4 days, the mixture was quenched with saturated ammonium chloride, diluted with dichloromethane, the organic layer was separated, the aqueous layer was extracted with dichloromethane, the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and the residue was purified by flash chromatography (hexane/ethyl acetate, 1: 2) to give 5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-bis- (hydroxymethyl) -benzene as a colorless foam (152 mg).

MS(ESI)：m/z＝324.1MNa⁺

¹H NMR(400Hz，CDCl₃-d1，δppm)：6.70(s，2H)，6.66(s，1H)，4.06(s，4H)，3.54(s，2H)，3.04(s，3H)，2.46(s，3H)，1.37(s，6H)。

5- (N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-bis- (hydroxymethyl) -benzene can be prepared as follows:

to a solution of 5-amino-1, 3-bis-hydroxymethyl-benzene (765mg, 5mmol) in anhydrous ethanol (25mL) was added 2- (methyldisulphanyl) -isobutyraldehyde (751mg, 5 mmol). After stirring at room temperature for 4 hours, the solution was cooled to 0 ℃ with an ice/water bath and sodium borohydride (220mg, 5.8mmol) was added. The mixture was stirred at 0 ℃ for 1 hour and then quenched with cold 5% hydrochloric acid and diluted with dichloromethane. Saturated sodium bicarbonate was added to make the solution slightly basic. The organic layer was separated, the aqueous layer was extracted with dichloromethane, and the combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and the filtrate was evaporated under reduced pressure. Flash chromatography of the residue (CH)₂Cl₂MeOH, 15: 1, 10: 1) to give 5- (N- (2-methyldisulphenyl-2, 2-dimethylethyl) amino-1, 3-bis- (hydroxymethyl) -benzene as a colorless oil (832 mg). Ms (esi): 310.0MNa⁺

Example 18：

8, 8' - [ (4- (2- (4-mercapto-4-methyl) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to 8, 8' - [ (4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones](60mg) to a solution in methanol (1.6mL) was added a solution of DMF (0.2mL) and tris (2-carboxyethyl) phosphine hydrochloride (48mg) in 0.2mL of water. The reaction mixture was stirred at room temperature for 18 hours and the solvent was removed in vacuo to give a residue which was purified by silica gel chromatography (Merck SuperVario Flash 10g column, Si6015-40 μm) eluting with a methanol (A)/dichloromethane/acetonitrile 9: 1(B) gradient (gradient: 100% B down to 10% A: 90% B) to give 8, 8' - [ (4- (2- (4-mercapto-4-methyl) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy group)]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones](17 mg): LC/MS (method A1, ZQ): ES: 855 MH/z⁺+H₂O

LC/MS (method A1, ZQ): ES: 855 MH/z⁺+H₂O

m/z＝837MH⁺

Retention time 3.70 min

8, 8' - [ (4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -yl-7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyri-dinePyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a cooled (0 ℃ C.) solution of 4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine (360mg) and triethylamine (810. mu.L) in dichloromethane (4mL) was added a solution of methanesulfonyl chloride (298. mu.L) in dichloromethane (4 mL). After 2 hours, water was added, the layers were separated and the aqueous layer was extracted twice with dichloromethane. The combined organic solutions were dried over sodium sulfate and concentrated in vacuo to give a residue. The residue was purified by chromatography on silica gel (Interchrom Purifash 20g column, SiOH15-35 μm) using a gradient elution with a mixture of methanol (A)/dichloromethane (B) (gradient: 100% B down to 3% A: 97% B) to give 280mg of the methanesulfonate compound.

To a solution of pre-tomaymycin (120mg) in dimethylformamide (4mL) were added potassium carbonate (244mg), potassium iodide (147mg) and a mesylate compound (137 mg). The reaction mixture was stirred at 30 ℃ for 20 h, water (20mL) was added, the resulting solid was filtered off, washed with water and dried in vacuo to give a residue. The residue was purified by chromatography on silica gel (Interchrom Purifash 20g column, SiOH15-35 μm), eluting with a gradient of a mixture of methanol (A)/dichloromethane (B) (gradient: 100% B down to 6% A: 94% B) to give the crude compound, which was dissolved in water/acetonitrile 1: 1 and subsequently concentrated in vacuo to give 8, 8' - [ (4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy-l]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones](120mg)：

LC/MS (method A1, ZQ): ES: 919 m/z MH⁺+2H₂O

m/z＝901MH⁺+H₂O

m/z＝883MH⁺

Retention time 3.82 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,28(s，6H)；1,75(d，J＝6,5Hz，6H)；1,94(m，2H)；2,29(m，2H)；2,39(s，3H)；2,97(m，4H)；3,50-4,20(m，6H)；4,00(s，6H)；4,27(s，4H)；5,27(m，4H)；5,61(m，2H)；5,87(t b，J＝5,5Hz，1H)；6,83(s，2H)；6,97(s，2H)；7,55(s，2H)；7,64(d，J＝4,5Hz，2H)。

4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine can be prepared as follows:

to a solution of 4- (2-tert-butoxycarbonylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine (656mg) in dioxane (3.5mL) was added a solution of 4N dioxane hydrochloride (5.5 mL). After 4h at room temperature, the reaction mixture was concentrated in vacuo to give a residue (603 mg).

To a solution of the above residue (150mg) in dimethylformamide (3.5mL) were added triethylamine (267. mu.L), 4-methyl-4-methyldithio-pentanoic acid (149mg), N' -diisopropylcarbodiimide (119. mu.L) and 1-hydroxybenzotriazole hydrate (49 mg). After 15 hours at room temperature, water was added to the reaction mixture, and the aqueous solution was extracted three times with ethyl acetate. The combined organic solutions were washed with saturated aqueous sodium chloride, dried over magnesium sulfate and concentrated in vacuo to give a residue. The residue was purified by chromatography on silica gel (Interchrom Purifash 20g column, SiOH15-35 μm) using a gradient elution with a mixture of methanol (A)/dichloromethane (B) (gradient: 100% B down to 10% A: 90% B) to give 4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine (81 mg):

LC/MS (method A3): 375MH ESm/z⁺

m/z＝156C₇H₁₀NO₃ ⁺

Retention time 2.2 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,22(s，6H)；1,79(m，2H)；2,19(m，2H)；2,39(s，3H)；3,43(q，J＝5,5Hz，2H)；4,06(t，J＝5,5Hz，2H)；4,45(d b，J＝6,0Hz，4H)；5,33(d，J＝6,0Hz，2H)；6,84(s，2H)；8,13(t b，J＝5,5Hz，1H)。

4- (2-tert-Butoxycarbonylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (2-tert-Butoxycarbonylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine can be prepared according to the process for preparing 4- (3-tert-butoxycarbonylamino-propoxy) -2, 6-bis- (hydroxymethyl) pyridine starting from diethyl 4- (2-tert-butoxycarbonylamino-ethoxy) -pyridine-2, 6-dicarboxylate.

LC/MS (method A3): 299MH of ES m/z⁺

m/z＝156C₇H₁₀NO₃ ⁺

Retention time 1.7 min

¹H N.M.R.(300MHz，DMSO-d₆δ ppm): δ 1,39(s, 9H); 3,32(m partially masked, 2H); 4,03(t, J ═ 6,0Hz, 2H); 4,46(d, J ═ 6),0Hz，4H)；5,30(t，J＝6,0Hz，2H)；6,83(s，2H)；7,00(t b，J＝6,0Hz，1H)。

4- (2-tert-Butoxycarbonylamino-ethoxy) -pyridine-2, 6-dicarboxylic acid diethyl ester

According to the method for producing diethyl 4- (2-tert-butoxycarbonylamino-propoxy) -pyridine-2, 6-dicarboxylate, diethyl 4- (2-tert-butoxycarbonylamino-ethoxy) -pyridine-2, 6-dicarboxylate can be produced from 2-tert-butoxycarbonylamino-ethyl bromide.

LC/MS (method A3): ES m/z 383MH⁺

m/z＝240C₁₁H₁₄NO₅ ⁺

Retention time 3.6 min

¹H N.M.R.(300MHz，DMSO-d₆δ ppm): δ 1,23(t, J7, 0Hz, 6H); 1,37(s, 9H); 3,33(m partially masked, 2H); 4,22(t, J ═ 5,5Hz, 2H); 4,38(q, J ═ 7,0Hz, 4H); 7,01(t b, J ═ 5,5Hz, 1H); 7,71(s, 2H).

Example 19：

According to 8, 8' - [ (4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy-group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The process for the preparation of 8, 8' - [ (1- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -benzene-3, 5-dimethyl) -dioxy-benzene starting from 1- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform I): ES: 882MH m/z⁺

Retention time 4.13 min

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：1,30(s，6H)；1,75(d，J＝6,5Hz，6H)；1,96(m，2H)；2,31(m，2H)；2,40(s，3H)；2,97(m，4H)；3,66(m，2H)；3,89(m，2H)；3,97(s，6H)；4,04(t，J＝5,5Hz，2H)；4,27(s b，4H)；5,14(d，J＝12,5Hz，2H)；5,19(d，J＝12,5Hz，2H)；5,61(m，2H)；5,93(t，J＝6,0Hz，1H)；6,82(s，2H)；6,94(s b，2H)；7,09(s b，1H)；7,53(s，2H)；7,64(d，J＝4,5Hz，2H)。

1- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene

1- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared from 1- (2-tert-butoxycarbonylamino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene according to the preparation of 4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine:

¹H N.M.R.(400MHz，CDCl₃-d1，δppm)：δ＝1,22(s，6H)；1,80(m，2H)；2,20(m，2H)；2,39(s，3H)；3,40(q，J＝6,0Hz，2H)；3,95(t，J＝6,0Hz，2H)；4,44(d，J＝6,0Hz，4H)；5,11(t b，J＝6,0Hz，2H)；6,73(s b，2H)；6,84(s b，1H)；8,09(t b，1H)。

1- (2-tert-Butoxycarbonylamino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene

1- (2-tert-butoxycarbonylamino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene was prepared from 2-tert-butoxycarbonylamino-ethyl bromide according to the preparation method of 5- (3-phthalimido-propoxy) -1, 3-bis- (hydroxymethyl) -benzene:

LC/MS (method A3): ES m/z 298MH⁺

m/z＝242(M+2H-tBu)⁺

m/z＝224(m/z＝242-H₂O)⁺

m/z＝206(m/z＝224-H₂O)⁺

m/z＝162(m/z＝206-CO₂)⁺

Retention time 2.7 min

¹H N.M.R.(500MHz，DMSO-d₆，δppm)：δ＝1,39(s，9H)；3,28(q，J＝6,0Hz，2H)；3,91(t，J＝6,0Hz，2H)；4,44(d，J＝6,0Hz，4H)；5,14(t，J＝6,0Hz，2H)；6,72([epsilon]b，2H)；6,83(s b，1H)；7,01(t b，J＝7,0Hz，1H)。

Example 20:

8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

to a cooled (0 ℃ C.) solution of 4- (3- (4-methyl-4-methyldisulfanyl) -pentanoylamino-propoxy) -2, 6-bis- (hydroxymethyl) -pyridine (55mg) and triethylamine (99. mu.L) in dichloromethane (2mL) was added methanesulfonyl chloride (36. mu.L). After 30 minutes, water was added, the layers were separated and the aqueous layer was extracted twice with dichloromethane. The combined organic solutions were dried over magnesium sulfate and concentrated in vacuo to give a residue (95 mg).

To a solution of pre-tomaymycin (50mg) in dimethylformamide (0.75mL) were added potassium carbonate (114mg), a solution of the above residue (55mg) in dimethylformamide (1mL), and potassium iodide (46 mg). The reaction was stirred at 30 ℃ for 20 hours, and the solid was filtered off and washed with dimethylformamide. To the combined dimethylformamide solution was added water and formic acid was added until the precipitate completely disappeared. The resulting solution was purified by HPLC according to method B injection. The appropriate fractions were combined and concentrated by centrifugal evaporation using a Joua Model RC10.10. apparatus to give 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy]Bis [ (S) -2-ylidene ]Ethyl- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones](13.5mg)。

LC/MS (method A3): ES m/z 897MH⁺

m/z＝664(M-C₁₀H₂₀NOS₂+2H)⁺

m/z＝234C₁₀H₂₀NOS₂ ⁺

Retention time 3.7 minutes

¹H N.M.R.(500MHz，CD₃COOD-d₄δ ppm): δ 1,25(s, 6H); 1,60-2,20(m partially masked, 10H); 2,35(m, 5H); 2,80-4,44(m, 14H); 3,91(s, 6H); 5,40(s, 4H); 5,62(m, 2H); 6,83-7,95(m, 8H).

4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -2, 6-bis- (hydroxymethyl) -pyridine can be prepared according to the preparation method of 4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine using 4- (3-tert-butoxycarbonylamino-propoxy) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ESm/z-389 MH⁺

m/z＝234(M-C₇H₈NO₃)⁺

m/z＝156C₇H₁₀NO₃ ⁺

Retention time 2.3 min

Thiol (SH) derivatives derived from the compounds of example 20 are in protective equilibrium with compounds of the formula:

example 21:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]Preparation of 8, 8' - [ (4- (4- (4-methyl-4-methyldithio) -pentanoylamino-butoxy) -pyridine-2, 6-dimethyl) -dioxy-e from 4- (4-methyl-4-methyldithio) -pentanoylamino-butoxy) -2, 6-bis- (hydroxymethyl) -pyridine]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: 947MH m/z⁺+2H₂O

m/z＝929MH⁺+H₂O

m/z＝911MH⁺

Retention time 3.74 minutes

¹H N.M.R.(500MHz，CD₃COOD-d₄，δppm)：δ＝1,26(s，6H)；1,59-1,76(m，8H)；1,81-1,94(m，4H)；2,35(m，5H)；2,80-4,40(m，14H)；3,92(s，6H)；5,39(m，4H)；5,50(m，2H)；6,72-7,95(m，8H)。

4- (4- (4-methyl-4-methyldithio) -pentanoylamino-butoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (4- (4-methyl-4-methyldithio) -pentanoylamino-butoxy) -2, 6-bis- (hydroxymethyl) -pyridine can be prepared according to the preparation method of 4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine using 4- (4-tert-butoxycarbonylamino-butoxy) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ES m/z 403MH⁺

m/z＝248(M-C₇H₈NO₃)⁺

m/z＝156C₇H₁₀NO₃ ⁺

Retention time 2.3 min

4- (4-tert-Butoxycarbonylamino-butoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (4-tert-Butoxycarbonylamino-butoxy) -2, 6-bis- (hydroxymethyl) -pyridine was prepared from diethyl 4- (4-tert-butoxycarbonylamino-butoxy) -pyridine-2, 6-dicarboxylate according to the preparation method of 4- (3-tert-butoxycarbonylamino-propoxy) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ES m/z 411MH⁺

m/z＝355(M+2H-tBu)⁺

m/z＝240C₁₁H₁₄NO₅ ⁺

Retention time 3.9 min

4- (4-tert-Butoxycarbonylamino-butoxy) -pyridine-2, 6-dicarboxylic acid diethyl ester

According to the preparation of diethyl 4- (3-tert-butoxycarbonylamino-propoxy) -pyridine-2, 6-dicarboxylate, 4- (4-tert-butoxycarbonylamino-butoxy) -pyridine-2, 6-dicarboxylate was prepared from 4-tert-butoxycarbonylamino-butyl methanesulfonate (Cazenave Gassiot, A.; Charton, J.; Girault-Mizzi, S.; Gilleron, P.; Debreu-Fontaine, M-A.; Sergheraert, C.; Melnyk, P.Bioorg.Med.Chem.Chem.Lett.2005, 15(21), 4828):

LC/MS (method A3): ESm/z 327MH⁺

m/z＝271(M+2H-tBu)⁺

m/z＝156C₇H₁₀NO₃ ⁺

Retention time 2.0 min

Example 22:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The preparation of (4) is carried out using 4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl]Preparation of 8, 8' - [ (4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl) -2, 6-bis- (hydroxymethyl) -pyridine]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: 950MH m/z⁺

Retention time 3.32 min

¹H N.M.R.(500MHz，CDCl₃，δppm)：δ＝1,32(s，6H)；1,76(d，J＝6,5Hz，6H)；1,80(m，2H)；1,94(m，2H)；2,27-2,46(m，11H)；2,67(m，2H)；2,97(m，4H)；3,40-3,70(m，4H)；3,90(m，2H)；4,00(s，6H)；4,27(s b，4H)；5,29(s b，4H)；5,60(q b，J＝6,5Hz，2H)；6,86(s，2H)；7,30(s，2H)；7,56(s，2H)；7,65(d，J＝4,5Hz，2H)。

4- (3- [4- (4-methyl-4-methyldisulfanyl-pentanoyl) -piperazin-1-yl ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine may be prepared as follows:

to a solution of 4- (3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl) -2, 6-bis- (hydroxymethyl) -pyridine (610mg) in dioxane (10mL) was added a 4N dioxane hydrochloride (2.5mL) solution. After 4h at room temperature, the reaction mixture was concentrated in vacuo to give a residue (650 mg).

To a solution of the above residue (160mg) in dimethylformamide (2.5mL) were added N, N-diisopropylethylamine (181. mu.L), 4-methyl-4-methyldithio-pentanoic acid (158mg), N' -diisopropylcarbodiimide (88. mu.L), and 1-hydroxybenzotriazole hydrate (15 mg). After 15 hours at room temperature, the solid was filtered off and the dimethylformamide solution was subjected to injection HPLC according to method F. The appropriate fractions were combined, lyophilized and concentrated to give 4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine (105 mg):

LC/MS (method A3): 442MH ESm/z⁺

m/z＝266(M+2H-C₇H₁₃OS₂)⁺

Retention time 2.3 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,27(s，6H)；1,70-1,83(m，4H)；2,25-2,39(m，8H)；2,40(s，3H)；2,63(m，2H)；3,43(m，4H)；4,49(s，4H)；5,28(m b，2H)；7,18(s，2H)。

4- (3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl) -2, 6-bis- (hydroxymethyl) -pyridine

4- (3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl) -2, 6-bis- (hydroxymethyl) -pyridine was prepared according to the preparation method of 4- (3-tert-butoxycarbonylamino-propoxy) -2, 6-bis- (hydroxymethyl) pyridine using diethyl 4- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl ] -pyridine-2, 6-dicarboxylate:

LC/MS (method A3): 366MH when ES m/z⁺

m/z＝310(M+2H-tBu)⁺

m/z＝266(M+2H-CO₂tBu)⁺

Retention time 0.5 min

¹H N.M.R.(400MHz，DMSO-d₆δ ppm): δ 1,39(s, 9H); 1,73(m, 2H); 2,29(m, 6H); 2,61(m, 2H); 3,30(m partially masked, 4H); 4,49(d, J ═ 6,0Hz, 4H); 5,30(t b, J ═ 6,0Hz, 2H); 7,18(s, 2H).

4- [3- (4-tert-Butoxycarbonyl-piperazin-1-yl) -propyl ] -pyridine-2, 6-dicarboxylic acid diethyl ester

According to the preparation method of 5- (3-tert-butoxycarbonyl-N-methyl-amino-propyl) -benzene-1, 3-dicarboxylic acid diethyl ester, 4- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -prop-1-ynyl ] -pyridine-2, 6-dicarboxylic acid diethyl ester was used to prepare 4- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl ] -pyridine-2, 6-dicarboxylic acid diethyl ester:

LC/MS (method A3): ES m/z 450MH⁺

m/z＝394(M+2H-tBu)⁺

m/z＝350(M+2H-CO₂tBu)⁺

Retention time 2.4 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,35(t，J＝7,0Hz，6H)；1,39(s，9H)；1,79(m，2H)；2,27(m，6H)；2,80(m，2H)；3,29(m，4H)；4,39(q，J＝7,0Hz，4H)；8,12(s，2H)。

4- [3- (4-tert-Butoxycarbonyl-piperazin-1-yl) -prop-1-ynyl ] -pyridine-2, 6-dicarboxylic acid diethyl ester

4- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -prop-1-ynyl ] -pyridine-2, 6-dicarboxylic acid diethyl ester was prepared from 4-trifluoromethylsulfonyloxy-pyridine-2, 6-dicarboxylic acid diethyl ester and tert-butyl-4-propargyl-piperazine-1-carboxylate (Zheng, H.; Weiner, L.M.; Bar-Am, O.; Epsztejn, S.; Cabantchik, Z.I.; Warshawsky, A.; Youdim, M.B.H.; and Fridkin, M.Bioorg.Med.Chem.2005, 3, 773) according to the preparation method of 5- (3-tert-butoxycarbonyl-N-methyl-amino-propyn-1-yl) -benzene-1, 3-dicarboxylic acid diethyl ester.

El (method C) M/z 445M⁺

m/z＝388(M-C₄H₉)⁺

m/z＝344(m/z＝388-CO₂)⁺

m/z＝57C₄H₉ ⁺

¹H N.M.R.(400MHz，CDCl₃-d1，δppm)：δ＝1,46(t，J＝7,0Hz，6H)；1,48(s，9H)；2,60(m b，4H)；3,52(m b，4H)；3,62(s b，2H)；4,49(q，J＝7,0Hz，4H)；8,23(s，2H)。

Diethyl 4-trifluoromethanesulfonyloxy-pyridine-2, 6-dicarboxylate can be prepared as follows:

trifluoromethanesulfonyl chloride (2.6mL) was added dropwise to a cooled (0 ℃) solution of diethyl chelidamate (Chaubet, F.; Nguyen van duong, M.; Gref, A.; Courtieu, J.; Crumbliss, A.L.; Gaudemer, A.tetrahedron Lett.1990, 31(40), 5729-5732) (3.5g) in pyridine (35 mL). The reaction was then stirred at room temperature for 3 hours, water and ethyl acetate were added. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic solutions were dried over magnesium sulfate and concentrated in vacuo to a residue. The residue was purified by silica gel chromatography (Merck SuperVarioPrep 90g column, Si6015-40 μm) eluting with dichloromethane to give diethyl 4-trifluoromethylsulfonyloxy-pyridine-2, 6-dicarboxylate (4.2 g).

LC/MS (method A1, Platform I): 372MH ESm/z⁺

Retention time 4.38 min

Example 23:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The preparation of (1) from 1- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl]As the (E) -propyl) -3, 5-bis- (hydroxymethyl) -benzene, 8' - [ (1- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl) -can be used]-propyl) -benzene-3, 5-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A3): 949MH ES m/z⁺

m/z＝475,3(M+2H)²⁺/2

Retention time 3.3 min

1- (3- [4- (4-methyl-4-methyldisulphur-pentanoyl) -piperazin-1-yl ] -propyl) -3, 5-bis- (hydroxymethyl) -benzene

According to the preparation method of 4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine, 1- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl ] -propyl) -3, 5-bis- (hydroxymethyl) -benzene can be prepared, starting from 1- (3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl) -3, 5-bis- (hydroxymethyl) -benzene:

LC/MS (method A3): ES m/z 441MH⁺

Retention time 2.4 min

¹H N.M.R.(400MHz DMSO-d₆，δppm)：δ＝1,26(s，6H)；1,71(m，2H)；1,79(m，2H)；2,22-2,40(m，8H)；2,39(s，3H)；2,58(m，2H)；3,44(m，4H)；4,45(d，J＝6,0Hz，4H)；5,09(t，J＝6,0Hz，2H)；7,00(s b，2H)；7,08(s b，1H)。

1- (3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl) -3, 5-bis- (hydroxymethyl) -benzene

1- (3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl) -3, 5-bis- (hydroxymethyl) -benzene was prepared starting from 5- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl ] -phthalic acid dimethyl ester according to the preparation method of 5- (3-tert-butoxycarbonyl-N-methyl-amino-propyl) -1, 3-bis- (hydroxymethyl) -benzene:

LC/MS (method A3): ES m/z 365MH⁺

m/z＝309(M+2H-tBu)⁺

Retention time 2.0 min

¹H N.M.R.(300MHz，DMSO-d₆δ ppm): δ 1,39(s, 9H); 1,71(m, 2H); 2,28(m, 6H); 2,57(m, 2H); 3,29(m partially masked, 4H); 4,44(d, J ═ 6,0Hz, 4H); 5,08(t, J ═ 6,0Hz, 2H); 7,00(s b, 2H); 7,08(s b, 1H).

5- [3- (4-tert-Butoxycarbonyl-piperazin-1-yl) -propyl ] -phthalic acid dimethyl ester

According to the preparation method of diethyl 5- (3-tert-butoxycarbonyl-N-methyl-amino-propyl) -benzene-1, 3-dicarboxylate, dimethyl 5- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl-1-ynyl ] -phthalate was prepared starting from 5- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -propyl ] -phthalate:

LC/MS (method A3): ES m/z 421MH⁺

m/z＝365(M+2H-tBu)⁺

m/z＝321(M+2H-CO₂tBu)⁺

Retention time 2.7 min

¹H N.M.R.(400MHz，DMSO-d₆δ ppm): δ 1,39(s, 9H); 1,74(m, 2H); 2,28(m, 6H); 2,75(m, 2H); 3,30(m partially masked, 4H); 3,89(s, 6H); 8,08(d, J ═ 2,0Hz, 2H); 8,32(t, J ═ 2,0Hz, 1H).

5- [3- (4-tert-Butoxycarbonyl-piperazin-1-yl) -prop-1-ynyl ] -phthalic acid dimethyl ester

According to the preparation method of diethyl 5- (3-tert-butoxycarbonyl-N-methyl-amino-prop-1-ynyl) -benzene-1, 3-dicarboxylate, starting from tert-butyl-4-propargyl-piperazine-1-carboxylate, 5- [3- (4-tert-butoxycarbonyl-piperazin-1-yl) -prop-1-ynyl ] -phthalic acid dimethyl ester was prepared:

LC/MS (method A3): 417MH ESm/z⁺

m/z＝361(M+2H-tBu)⁺

m/z＝317(M+2H-CO₂tBu)⁺

Retention time 3.1 min

¹H N.M.R.(400MHz，DMSO-d₆δ ppm): δ 1,40(s, 9H); 2,50(m mask, 4H); 3,35(m, 4H); 3,61(s, 2H); 3,90(s, 6H); 8,14(s b, 2H); 8,40(s b, 1H).

Example 24:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The process for the preparation of (1) from 4- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy]Preparation of 8, 8' - [ (4- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2, 6-bis- (hydroxymethyl) -pyridine]-ethoxy radical} -ethoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A3): 971MH for ES m/z⁺

m/z＝486,3(M+2H)²⁺/2

Retention time 3.60 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,28(s，6H)；1,76(d，J＝6,5Hz，6H)；1,94(m，2H)；2,26(m，2H)；2,40(s，3H)；2,98(m，4H)；3,36-3,95(m，12H)；4,00(s，6H)；4,18(m，2H)；4,28(s b，4H)；5,27(m，4H)；5,61(m，2H)；5,97(m b，1H)；6,84(s，2H)；7,01(s b，2H)；7,56(s，2H)；7,64(d，J＝4,5Hz，2H)。

4- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine

Preparation of 4- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine starting from 4- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine according to the preparation of 4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ESm/z 463MH⁺

Retention time 2.3 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,22(s，6H)；1,79(m，2H)；2,15(m，2H)；2,39(s，3H)；3,18(q，J＝6,0Hz，2H)；3,40(t，J＝6,0Hz，2H)；3,52(m，2H)；3,60(m，2H)；3,76(m，2H)；4,18(m，2H)；4,45(s，4H)；5,32(m b，2H)；6,85(s，2H)；7,90(t b，J＝6,0Hz，1H)。

4- (2- {2- [ 2-tert-Butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine was prepared starting from 4- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -pyridine-2, 6-dicarboxylic acid diethyl ester according to 4- (3-tert-butoxycarbonylamino-propoxy-) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ESm/z 387MH⁺

m/z＝331(M+2H-tBu)⁺

Retention time 2.0 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,37(s，9H)；3,07(q，J＝6,0Hz，2H)；3,39(t，J＝6,0Hz，2H)；3,51(m，2H)；3,59(m，2H)；3,73(m，2H)；4,18(m，2H)；4,45(d，J＝6,0Hz，4H)；5,31(t，J＝6,0Hz，2H)；6,73(t b，J＝6,0Hz，1H)；6,85(s，2H)。

Diethyl 4- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -pyridine-2, 6-dicarboxylate was prepared as follows:

to 4- (2- {2- [ 2-azido-ethoxy ] -ethyl]-ethoxy } -ethoxy) -pyridine-2, 6-dicarboxylic acid diethyl ester (Roy, b.c.; santos, m.; mallik, s.; a solution of Campglia, A.D.J.org.chem.2003, 68(10), 3999) (900mg) in ethyl acetate (18mL) was added di-tert-butyl pyrocarbonate (545mg) and 10% palladium on carbon (73 mg). The solution was stirred at room temperature under an atmosphere of hydrogen (2bar) for 18 hours, the solid was filtered off and the solvent was removed in vacuo to give a residue. The residue was chromatographed on silica gel (Analogix Super Flash SiO)₂SF25-34g) was purified, eluting with a mixture of dichloromethane (a) and methanol (B) in a gradient (gradient: 100% A decreased to 97.5% A: 2.5% B) to give 4- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy)]-ethoxy } -ethoxy) -pyridine-2, 6-dicarboxylic acid diethyl ester (760 mg):

LC/MS (method A3): ESm/z 471MH⁺

m/z＝415(M+2H-tBu)⁺

m/z＝371(M+2H-CO₂tBu)⁺

Retention time 3.6 min

¹H N.M.R.(300MHz1 DMSO-d₆δ ppm): δ 1,32(t, J7, 0Hz, 6H); 1,36(s, 9H); 3,04(q, J ═ 6,0Hz, 2H); 3,38(t, J ═ 6,0Hz, 2H); 3,51(m, 2H); 3,59(m, 2H); 3,79(m, 2H); 4,25(m mask, 2H); 4,29(q, J ═ 7,0Hz, 4H); 6,70(t b, J ═ 6,0Hz, 1H); 7,73(s, 2H).

Example 25:

8, 8' - [ (1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -yl-7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyri-dinePyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy-) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The process for the preparation of (1) - (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy)]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene, 8' - [ (1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -was prepared]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: m/z 1102MH +

Retention time 4.49 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,30(s，6H)；1,75(d，J＝6，5Hz，6H)；1,95(m，2H)；2,28(m，2H)；2,41(s，3H)；2,97(m，4H)；3，33-3,96(m，24H)；3,96(s，6H)；4，12(m，2H)；4,27(s b，4H)；5,07-5,24(m，4H)；5,60(m，2H)；6,21(m b，1H)；6,81(s，2H)；6,96(s，2H)；7,07(s，1H)；7,51(s，2H)；7,65(d，J＝4,5Hz，2H)。

1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene

1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldisulfanyl-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -1- (2- {2- [2- (4-methyl-4-methyldisulfanyl-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -was prepared according to the method of preparation of 1- (3- (4-methyl-4-methyldisulfanyl) -pentanoylamino-propoxy-) -3, 5-bis- (hydroxymethyl) -benzene starting from 1- (2- {2- [2- (2- {2- [ 2-amino-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene ) -3, 5-bis- (hydroxymethyl) -benzene:

LC/MS (method G1): ESm/z-594 MH +

Retention time 1.9 min.

1- (2- {2- [2- (2- {2- [ 2-amino-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared as follows:

to 1- (2- {2- [2- (2- {2- [ 2-azido-ethoxy) -ethanol]-ethoxy } -ethoxy) -ethoxy]-Ethyloxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene (540mg) in THF (5.5mL) was added triphenylphosphine (320mg) and water (22. mu.L), the solution was stirred at room temperature for 18 h, and the solvent was removed in vacuo to a residue. The residue is purified by chromatography on silica gel (Merck SuperVarioFlash 30g column, Si6015-40 μm) with dichloromethane/methanol 95: 5 and subsequently dichloromethane/methanol/NH₄OH 75: 25: 2.5 to obtainTo 1- (2- {2- [2- (2- {2- [ 2-amino-ethoxy)]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene (430 mg):

LC/MS (method A3): ES: 418 MH/z⁺

Retention time 1.3and 1.7 minutes

1- (2- {2- [2- (2- {2- [ 2-azido-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared as follows:

to 2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethanol]-ethoxy } -ethoxy) -ethoxy]To a cooled (0 ℃ C.) solution of ethanol (1.6g) and triethylamine (1.45mL) in dichloromethane (40mL) was added methanesulfonyl chloride (617. mu.L), after 1 hour, water was added. The layers were separated and the organic layer was dried over magnesium sulfate and concentrated in vacuo to give a residue (1.83 g). A residue sample (1.4g), 3, 5-dihydroxymethylphenol (510mg), and potassium carbonate (686mg) were combined in dimethylformamide (8mL) and heated to 70 ℃ for 15 hours. The reaction mixture was cooled to room temperature, water was then added, and the aqueous solution was extracted three times with ethyl acetate. The combined organic solutions were washed with saturated aqueous sodium chloride, dried over magnesium sulfate and concentrated in vacuo to a residue. The residue was chromatographed on silica gel (Merck SuperVarioPrep 70g column, Si6015-40 μm) using a mixture of methanol (A)/dichloromethane (B) (gradient: 2% A: 98% B to 10% A: 90% B) to give 1- (2- {2- [2- (2- {2- [ 2-azido-ethoxy-)]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene (540 mg): cl (method D): m/z 461 MNH₄ ⁺

2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethanol was prepared as follows:

to a solution of 2- [2- (2- {2- [2- (2-tosyloxy-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethanol (Loiseau, f.a.; Hii, k.k.; Hill, a.m.j.org.chem.2004, 69, 639) (4.5g) in dichloromethane (30mL) was added sodium azide (0.89 g). The solution was stirred at 70 ℃ for 18 hours, followed by removal of the solvent in vacuo to give a residue. Dichloromethane was added and the resulting precipitate was filtered off. The organic layer was concentrated in vacuo to give 2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethanol (3.1 g):

cl (method D): 325MNH₄ ⁺

Example 26：

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy-) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]From 1- (2- {2- [2- (4-methyl-4-methyl)Dithio-pentanoylamino) -ethoxy]-ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene, 8' - [ (1- (2- {2- [2- (4-methyl-4-methyldisulphur-pentanoylamino) -ethoxy ] -is prepared]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform II): ES: 970MH of m/z⁺

Retention time 3.89 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,27(s，6H)；1,75(d，J＝6,5Hz，6H)；1,94(m，2H)；2,26(m，2H)；2,40(s，3H)；2,98(m，4H)；3,35-3,92(m，12H)；3,96(s，6H)；4,15(m，2H)；4,28(s b，4H)；5,10-5,23(m，4H)；5,60(qb，J＝6,5Hz，2H)；6,05(t b，J＝6,0Hz，1H)；6,81(s，2H)；6,98(s，2H)；7,09(s，1H)；7,53(s，2H)；7,64(d，J＝4,5Hz，2H)。

1- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene

1- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared according to the preparation method of 1- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy-) -3, 5-bis- (hydroxymethyl) -benzene starting from 1- (2- {2- [ 2-amino-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene:

LC/MS (method A3): ES: 462MH m/z⁺

m/z＝444(M+H-H₂O)⁺

Retention time 3.0 min

1- (2- {2- [ 2-amino-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene

1- (2- {2- [ 2-amino-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared according to the method of preparation of 1- (2- {2- [2- (2- {2- [ 2-amino-ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethoxy } -3, 5-bis- (hydroxymethyl) -benzene starting from 1- (2- {2- [ 2-azido-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene:

LC/MS (method A3): ES: 286MH m/z⁺

m/z＝268(M+H-H₂O)⁺

Retention time 0.8 min

1- (2- {2- [ 2-azido-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene

1- (2- {2- [ 2-azido-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared according to the method for the preparation of 1- (2- {2- [2- (2- {2- [ 2-azido-ethoxy ] -ethoxy } -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene starting from 2- [2- (2-azido-ethoxy) -ethoxy ] -ethanol (Roy, B.C; Santos, M.; Malik, S.; Camtiglia, A.D.J.org.Chem.2003, 68(10), 3999):

cl (method D) m/z 329MNH₄ ⁺。

Example 27:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy-) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The process for the preparation of (1) from 4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine, 8' - [ (4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -is prepared]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS(Method a 3): ES: 1103MH m/z⁺

m/z＝552(M+2H)²⁺/2

Retention time 3.7 minutes

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,29(s，6H)；1,75(d，J＝6,5Hz，6H)；1,94(m，2H)；2,28(m，2H)；2,40(s，3H)；2,97(m，4H)；3,33-3,95(m，24H)；3,99(s，6H)；4,18(m，2H)；4,28(s b，4H)；5,27(m，4H)；5,60(m，2H)；6,19(m b，1H)；6,82(s，2H)；7,00(s b，2H)；7,55(s，2H)；7,64(d，J＝4,5Hz，2H)。

4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine can be prepared according to the process for the preparation of 4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine starting from 4- (2- {2- [2- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethoxy } -2, 6-bis- (hydroxymethyl) -pyridine Yl) -ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ES: m/z 596MH⁺

Retention time 2.4 min

4- (2- {2- [2- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine

4- (2- {2- [2- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -2 was prepared according to the preparation of 4- (3-tert-butoxycarbonylamino-propoxy-) -2, 6-bis- (hydroxymethyl) pyridine starting from 4- {2- [2- (2- {2- [2- (2-tert-butoxycarbonylamino-ethoxy) -ethoxy ] -ethoxy } -pyridine-2, 6-dicarboxylic acid diethyl ester, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ES: 519MH of m/z⁺

Retention time 2.2 min

4- {2- [2- (2- {2- [2- (2-tert-Butoxycarbonylamino-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -pyridine-2, 6-dicarboxylic acid diethyl ester

4- {2- [2- (2- {2- [2- (2-tert-butoxycarbonylamino-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -pyridine-2, 6-dicarboxylic acid diethyl ester was prepared according to the preparation method of diethyl 4- (2- {2- [ 2-tert-butoxycarbonylamino-ethoxy ] -ethoxy } -ethoxy) -pyridine-2, 6-dicarboxylate starting from 4- {2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethoxy } -pyridine-2, 6-dicarboxylic acid diethyl ester, 6-dicarboxylic acid diethyl ester:

LC/MS (method A3): ES: 603MH m/z⁺

m/z＝271(M+2H-CO₂tBu)⁺

Retention time 3.6 min

4- {2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethoxy } -pyridine-2, 6-dicarboxylic acid diethyl ester was prepared as follows:

to a solution of diglylamine diethyl ester (1.03g) in dimethylformamide (10mL) were added methanesulfonic acid 2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -ethoxy ] -ethyl ester (1.82g) and potassium carbonate (893 mg). The resulting mixture was heated at 70 ℃ for 15 hours and then cooled to room temperature. Water was added, and the aqueous solution was extracted 3 times with ethyl acetate. The combined organic solutions were washed with saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and concentrated in vacuo to a residue. The residue was purified by silica gel chromatography (Merck SuperVarioPrep 70g column, Si6015-40 μm) eluting with a mixture of methanol (A)/dichloromethane (B) (gradient: 3% A: 97% B to 5% A: 95% B) to give 4- {2- [2- (2- {2- [2- (2-azido-ethoxy) -ethoxy ] -ethoxy } -pyridine-2, 6-dicarboxylic acid diethyl ester (2.19 g):

LC/MS (method A3): ES: m/z 529MH⁺

Retention time 3.4 min

Example 28:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy-) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]By the preparation of 1- (2- [ methyl- (2-methyl-2-methyldithio-propyl) -amino]-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene, from which 8, 8' - [ (1- (2- [ methyl- (2-methyl-2-methyldithio-propyl) -amino]-ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform I): ES: 872MH of m/z⁺+H₂O

m/z＝854MH⁺

Retention time 3.40 min

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,31(s，6H)；1,75(d，J＝6,5Hz，6H)；2,39(s，3H)；2,45(s，3H)；2,62(s，2H)；2,92(t，J＝6,5Hz，2H)；2,97(m，4H)；3,89(m，2H)；3,96(s，6H)；4,06(t，J＝6,5Hz，2H)；4,26(s b，4H)；5,12(d，J＝12,5Hz，2H)；5,19(d，J＝12,5Hz，2H)；5,60(q b，J＝6,5Hz，2H)；6,82(s，2H)；6,95(s b，2H)；7,06(s b，1H)；7,52(s，2H)；7,64(d，J＝4,5Hz，2H)。

1- (2- [ methyl- (2-methyl-2-methyldithio-propyl) -amino ] -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared as follows:

to a cooled (5 ℃) suspension of 1- (2- (2-methyl-2-methyldisulphanyl-propyl) -amino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene (280mg) in formaldehyde (228 μ L) was added formic acid (319 μ L) and the resulting mixture was heated at 100 ℃ for 1h 15 min and subsequently cooled to room temperature. Water and ice were added followed by aqueous sodium hydroxide until pH 12. The resulting aqueous solution was extracted 3 times with ethyl acetate, and the combined organic solutions were dried over magnesium sulfate and concentrated in vacuo to a residue. The residue was chromatographed on silica gel (Merck SuperVarioFlash 25g column, Si6015-40 μm) using a methanol (A)/dichloromethane (B) mixture (gradient: 2% A: 98% B to 5% A: 95% B) to give 1- (2- [ methyl- (2-methyl-2-methyldithio-propyl) -amino ] -ethoxy) -3, 5-bis- (hydroxymethyl) -benzene (210 mg):

LC/MS (method A3): ES: 346MH⁺

m/z＝212(M+2H-C₅H₁₁S₂)⁺

m/z＝135C₅H₁₁S₂ ⁺

Retention time 2.1 min

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,27(s，6H)；2,39(s，3H)；2,40(s，3H)；2,60(s，2H)；2,85(t，J＝6,0Hz，2H)；4,02(t，J＝6,0Hz，2H)；4,44(d，J＝6,0Hz，4H)；5,10(t，J＝6,0Hz，2H)；6,72(s b，2H)；6,82(s b，1H)。

1- (2- (2-methyl-2-methyldithio-propyl) -amino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene can be prepared as follows:

to a suspension of 1- (2-amino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene hydrochloride (900mg) in tetrahydrofuran (4.5mL) was added triethylamine (1.07 mL). After stirring for 15 minutes, 2- (methyldithio) isobutyraldehyde (530. mu.L) and titanium isopropoxide (1.42mg) were added, and the resulting mixture was stirred at room temperature for 2 hours. Ethanol (9mL) and sodium cyanoborohydride (242mg) were added to the solution, and the new mixture was stirred at room temperature for 18 hours. The solid was filtered off and the filtrate was concentrated in vacuo to a residue. The residue was diluted with ethyl acetate and the resulting solid was filtered off. The organic solution was then washed with water and saturated aqueous sodium chloride, dried over magnesium sulphate and concentrated in vacuo to a new residue which was purified by silica gel chromatography (Merck SuperVarioFlash 30g column, Si6015-40 μm), eluting with a methanol (A)/dichloromethane (B) mixture, (gradient: 4% A: 96% B to 10% A: 90% B) to give 1- (2- (2-methyl-2-methyldithio-propyl) -amino-ethoxy) -3, 5-bis (hydroxymethyl) -benzene (290 mg):

¹H N.M.R.(400MHz，DMSO-d₆，δppm)：δ＝1,27(s，6H)；1,82(m b，1H)；2,39(s，3H)；2,67(s，2H)；2,91(t，J＝6,0Hz，2H)；4,00(t，J＝6,0Hz，2H)；4,43(d，J＝6,0Hz，4H)；5,10(t，J＝6,0Hz，2H)；6,73(s b，2H)；6,83(s b，1H)。

example 29:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy-) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]By the preparation of 4- (3- [ methyl- (4-methyl-4-methyldithio-pentanoyl) -amino]Preparation of 8, 8' - [ (4- (3- [ methyl- (4-methyl-4-methyldithio-pentanoyl) -amino) -2, 6-bis- (hydroxymethyl) -pyridine]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,32(s，6H)；1,75(d，J＝6,5Hz，6H)；1,94(m，4H)；2,20-4,30(m，18H)；4,00(s，6H)；4,27(s b，4H)；5,21-5,68(m，6H)；6,80-7,70(m，8H)。

4- (3- [ methyl- (4-methyl-4-methyldisulfanyl-pentanoyl) -amino ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine

4- (3- [ methyl- (4-methyl-4-methyldithio-pentanoyl) -amino ] -propyl) -2, 6-bis- (hydroxymethyl) -pyridine can be prepared according to the preparation method of 4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -2, 6-bis- (hydroxymethyl) -pyridine using 4- (3- (tert-butoxycarbonyl-methyl-amino) -propyl) -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A3): ES: m/z 387MH⁺

Retention time 2.5 min

4- (3- (tert-butoxycarbonyl-methyl-amino) -propyl) -2, 6-bis- (hydroxymethyl) -pyridine

4- (3- (tert-butoxycarbonyl-methyl-amino) -propyl) -2, 6-bis- (hydroxymethyl) -pyridine was prepared according to the procedure for the preparation of 4- (3-tert-butoxycarbonylamino-propoxy-) -2, 6-bis- (hydroxymethyl) pyridine using diethyl 4- (3- (tert-butoxycarbonyl-methyl-amino) -propyl) -pyridine-2, 6-bis-dicarboxylate:

cl (method D) m/z 311MH⁺

4- (3- (tert-Butoxycarbonyl-methyl-amino) -propyl) -pyridine-2, 6-dicarboxylic acid diethyl ester

Diethyl 4- (3- (tert-butoxycarbonyl-methyl-amino) -propyl) -pyridine-2, 6-dicarboxylate was prepared according to the preparation of diethyl 5- (3-tert-butoxycarbonyl-N-methyl-amino-propyl) -benzene-1, 3-dicarboxylate using diethyl 4- [3- (tert-butoxycarbonyl-methyl-amino) -prop-1-ynyl ] -pyridine-2, 6-dicarboxylate:

LC/MS (method G2): ES: 395MH m/z⁺

m/z＝339(M+2H-tBu)⁺

Retention time 7.5 min

4- [3- (tert-Butoxycarbonyl-methyl-amino) -prop-1-ynyl ] -pyridine-2, 6-dicarboxylic acid diethyl ester

Diethyl 4- [3- (tert-butoxycarbonyl-methyl-amino) -prop-1-ynyl ] -pyridine-2, 6-dicarboxylate can be prepared from tert-butoxycarbonyl-N-methyl-propargylamine according to the preparation of diethyl 5- (3-tert-butoxycarbonyl-N-methyl-amino-propyn-1-yl) -benzene-1, 3-dicarboxylate.

Cl (method D) m/z 391MH⁺。

Example 30:

According to 8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]By the preparation of 4- {3- [ methyl- (2-methyl-2-methyldithio-propyl) -amino]Preparation of 8, 8' - [ (4- (3- [ methyl- (2-methyl-2-methyldithio-propyl) -amino ] -propyl } -2, 6-bis- (hydroxymethyl) -pyridine]-propyl radical) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

¹H N.M.R(500MHz，CDCl₃-d1, δ ppm): δ — 1,28(s, 6H); 1,65(m partially masked, 2H); 1,75(d, J ═ 6,5Hz, 6H); 2,29(s, 3H); 2,38(s, 3H); 2,46(m, 4H); 2,65(m, 2H); 2,97(m, 4H); 3,89(m, 2H); 4,00(s, 6H); 4,27(s b, 4H); 5,29(s, 4H); 5,60(q b, J ═ 6,5Hz, 2H); 6,86(s, 2H); 7,30(s, 2H); 7,55(s, 2H); 7,64(d, J ═ 4,5Hz, 2H).

4- {3- [ methyl- (2-methyl-2-methyldithio-propyl) -amino ] -propyl } -2, 6-bis- (hydroxymethyl) -pyridine

P-34409-034-3

4- {3- [ methyl- (2-methyl-2-methyldithio-propyl) -amino ] -propyl } -2, 6-bis- (hydroxymethyl) -pyridine may be prepared according to the method of preparation of 1- (2- (2-methyl-2-methyldithio-propyl) -amino-ethoxy) -3, 5-bis- (hydroxymethyl) -benzene using 4- {3- [ methyl-amino ] -propyl } -2, 6-bis- (hydroxymethyl) -pyridine:

LC/MS (method A1, Platform II): 345MH ESm/z⁺

Retention time 1.15 min

Example 31:

8, 8' - [ (1- (4-methyl-4-methyldithio) -pentanoylamino) -benzene-3, 5-dimethyl]-dioxy group]-bis [ (S) -2-ethylidene- (E) -yl-7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]Can be prepared as follows:

according to 8, 8' - [ (4- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy-group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]The process for preparing 8, 8' - [ (1- (4-methyl-4-methyldithio) -pentanoylamino) -benzene-3, 5-dimethyl-benzene from (1- (4-methyl-4-methyldithio) -pentanoylamino) -3, 5-bis- (hydroxymethyl) -benzene]-dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]：

LC/MS (method A1, Platform I): ES: 838MH⁺

Retention time 4.11 min

¹H N.M.R.(500MHz，CDCl₃-d1，δppm)：δ＝1,33(s，6H)；1,76(d，J＝6,5Hz，6H)；2,03(m，2H)；2,43(s，3H)；2,46(m，2H)；2,97(m，4H)；3,89(m，2H)；3,95(s，6H)；4,27(s b，4H)；5,14(d，J＝12,5Hz，2H)；5,19(d，J＝12,5Hz，2H)；5,60(q，J＝6,5Hz，2H)；6,81(s，2H)；from 7,20to 7,60(m，6H)；7,64(d，J＝4,5Hz，2H)。

The corresponding thiol derivatives of the compounds of examples 19-31 can be prepared as described in example 18.

Example A: general methods for conjugate preparation

An anti-B4 antibody that binds to CD19 antigen preferentially expressed on the surface of human lymphoma cells was selected for binding to tomaymycin derivatives.

In a first step, the antibody was reacted with the modifier N-sulfosuccinimidyl 5-nitro-2-pyridyldithiobutyrate (SSNPB) to introduce a nitropyridinyldithio group, and a huC242 antibody solution (65.6mL) at a concentration of 8mg/mL in an aqueous buffer (pH 6.5) containing 0.05M potassium phosphate, 0.05M sodium chloride and 2mM ethylenediaminetetraacetic acid (EDTA) was treated with a solution of SSNPB in Dimethylacetamide (DMA) in 8-fold molar excess. The reaction mixture was stirred at room temperature for 90 minutes, and then packed in a Sephadex G25 gel filtration column (50 mM. times.35.5 mM, column) equilibrated in advance in an aqueous buffer solution (pH 7.5) containing 0.05M potassium phosphate, 0.05M sodium chloride and 2mM EDTA. The modified antibody-containing fractions were collected and pooled to give the product. A small aliquot of the modified antibody was treated with dithiothreitol to cleave the nitropyridinyl disulfide and the liberated nitropyridine-2-thione was analyzed spectrophotometrically (for compound ε)_323nm＝4,299M^-1 cm^-1，ε_280nm＝565M^-1cm^-1To the antibody epsilon_280nm＝217,560M^-1cm^-1). Each antibody molecule is typically linked to an average of 4-6 nitro-pyridyl disulfide molecules.

The modified antibody was diluted to 2.5mg/mL in the above buffer pH 7.5 and treated with a solution of tomaymycin derivative in DMA such that the final concentration of DMA in the buffer was 20%. The conjugate mixture was stirred at room temperature for 16 hours, and the reaction mixture was purified by passing through a Sephacryl S300 gel filtration column (50 mm. times.42 cm, column) equilibrated beforehand at pH6.5 in Phosphate Buffered Saline (PBS). Fractions containing monomeric antibody-tomaymycin derivative conjugate were pooled and dialyzed against PBS buffer. The final conjugate was analyzed spectrophotometrically using extinction coefficients, which were measured individually for each tomaymycin derivative.

SPDB-PBD and SMCC-PBD conjugates of the compounds of the invention

huB 4-SPDB-Compound of example 16

Example A1: an anti-B4 antibody that binds to CD19 antigen preferentially expressed on the surface of human lymphoma cells was selected for binding to tomaymycin derivatives.

The antibody was first modified with 4- (2-pyridyldithio) butanoic acid N-hydroxysuccinimide ester (SPDB) to introduce pyridyldithio groups. A4.5-fold molar excess of SPDB (0.25. mu. mol, 81.1. mu. L) in Dimethylacetamide (DMA) (50. mu.L) was added to a solution of huB4 (8mg, 0.055. mu. mol) in an aqueous buffer (pH 6.5) (0.95mL) containing 50mM potassium phosphate, 50mM sodium chloride and 2mM ethylenediaminetetraacetic acid (EDTA) to a final protein concentration of 8mg/mL containing 5% DMA in buffer. The modification was rotated at room temperature for 90 minutes and purified using a Sephadex G25 gel filtration column equilibrated in an aqueous buffer (pH 7.5) containing 50mM potassium phosphate, 50mM sodium chloride and 2mM EDTA. A small aliquot of the modified antibody was treated with Dithiothreitol (DTT) to cleave the pyridyl disulfide group, and the modified antibody and liberated pyridinethiol were analyzed spectrophotometrically (for liberated pyridinethiol. epsilon.)_343nm＝8,080M^-1 cm^-1Para-modified pyridine dithio epsilon_280nm＝5100M^-1cm^-1To the antibody epsilon_280nm＝222,960M^-1 cm^-1). Each antibody molecule is typically linked to an average of 3.36 pyridyl disulfide molecules.

The modified antibody (2.29mg, 0.016. mu. mol) was diluted in the above-mentioned pH 7.5 buffer (732.8. mu.L) and DMA (91.6. mu.L, 10% v/v) and subsequently treated with 8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](example 16 Compound) (0.157. mu. mol, 0.12mg) in DMA (91.6. mu.l, 10% v/v). The final protein concentration was 2.5mg/mL, containing 20% DMA in buffer. The binding process was spun overnight at room temperature and then clarified by settling (13,200RPM for 4 minutes). The supernatant was then purified using a Sephadex G25 gel filtration column equilibrated in phosphate saline (PBS) buffer, pH 6.5. The purified conjugate was dialyzed against 4 buffer exchanges into PBS pH6.5 buffer (approximately 1: 650 dilution). The conjugate was clarified by 0.22 μm syringe filter for spectrophotometric analysis (for PBD ε)_280nm＝7743M^-1cm^-1，ε_318nm＝9137M^-1cm^-1To the antibody epsilon_280nm＝222,960M^-1 cm^-1). An average of 1.76PBD molecules (example 16 compound) were attached per antibody molecule.

huB 4-SMCC-Compound of example 16

Example A2: an anti-B4 antibody that binds to CD19 antigen preferentially expressed on the surface of human lymphoma cells was selected for binding to tomaymycin derivatives.

In the first step, the antibody is reacted with the modifier succinimidyl 4- [ maleimidomethyl]Cyclohexane-1-carboxylate (SMCC) to introduce maleimide groups. A solution of huB4 antibody at a concentration of 6mg/mL (1mL) in an aqueous buffer (pH 6.7) containing 0.05M potassium phosphate, 0.05M sodium chloride and 2mM ethylenediaminetetraacetic acid (EDTA) was treated with a 6.5-fold molar excess of SMCC in Dimethylacetamide (DMA). The reaction mixture was stirred at room temperature for 90 minutes and then packed in a Sephadex G25 gel filtration column (NAP 10) which had previously been loaded with 0.10MN- (2-hydroxyethyl) -piperazine-N' -2-ethanesulfonic acid (H)EPES) in aqueous buffer at pH 8.0. The modified antibody-containing fractions were collected and pooled to give the product. A small aliquot of the modified antibody was treated with beta-mercaptoethanol for 10 minutes, followed by the addition of 5, 5' -dithiobis-2-nitrobenzoic acid (DTNB) to detect residual thiols (ε for 5-thio-2-nitrobenzoic acid (TNB))_412nm＝14,150M^-1cm^-1To the antibody epsilon_280nm＝222,960M^-1cm^-1). The amount of thiol consumed in the reaction of the antibody with the maleimide (compared to the control without antibody) is equal to the molar amount of maleimide attached to the antibody (subtraction Ellman's test). Each antibody is attached to about 3.2 active maleimide groups.

The modified antibody (3mg, 0.021 μmol) was diluted to 3.0mg/mL in pH 8.0HEPES buffer and subsequently treated with a solution of the compound of example 16 in DMA (5mM) such that the final concentration of DMA in the buffer was 15%. 3molar equivalents of 8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl-bis (methyleneoxy) are added per linker]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]Compound of example 16) (9.6 eq/antibody, 0.202 μmol, 149 μ g). The combined mixture was stirred at room temperature for 20 hours, and the reaction mixture was purified by passing through a G25 column (NAP 5) equilibrated in 0.05 potassium phosphate (KPi), 0.05M NaCl, 0.002M EDTA buffer solution (pH 6.7). Fractions containing huB 4-example 16 compound conjugate were pooled and dialyzed against Kpi buffer using 3 exchanges (24 h). The final conjugate (2.2mg, 2.3mg/ml) was analyzed spectrophotometrically using extinction coefficient and the compound of example 16 (. epsilon.) (_318nm＝9,137M^-1cm^-1，ε_280nm＝7,734M^-1cm^-1) And B4 antibody (. epsilon.)_280nm＝222,960M^-1cm^-1). An average of 2.8PBD molecules (example 16 compound) were attached per molecule antibody.

huB 4-SPDB-Compound of example 17

Example A3: an anti-B4 antibody that binds to CD19 antigen preferentially expressed on the surface of human lymphoma cells was selected for binding to tomaymycin derivatives.

The antibody was first modified with 4- (2-pyridyldithio) butanoic acid N-hydroxysuccinimide ester (SPDB) to introduce pyridyldithio groups. A4.5-fold molar excess of SPDB (0.124. mu. mol, 40.6. mu.g) in DMA (25. mu.L) was added to a solution of huB4 (4mg, 0.028. mu. mol) in aqueous buffer (pH 6.5) (0.475mL) containing 50mM potassium phosphate, 50mM sodium chloride and 2mM EDTA to a final protein concentration of 8mg/mL containing 5% DMA in buffer. The modification was rotated at room temperature for 90 minutes and purified using a Sephadex G25 gel filtration column equilibrated in an aqueous buffer (pH 7.5) containing 50mM potassium phosphate, 50mM sodium chloride and 2mM EDTA. A small aliquot of the modified antibody was treated with DTT to cleave the pyridyl disulfide group, and the modified antibody and liberated pyridinethiol were analyzed spectrophotometrically (for liberated pyridinethiol. epsilon.)_343nm＝8,080M^-1cm^-1Para-modified pyridine dithio group ε_280nm＝5100M^-1cm^-1To the antibody epsilon_280nm＝222,960M^-1cm^-1). An average of 3.31 pyridyl disulfide molecules per antibody molecule were attached.

The modified antibody (3.06mg, 0.021. mu. mol) was diluted in the above pH 7.5 buffer (0.976mL) and DMA (122. mu.L, 10% v/v) followed by 8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ] - [][1，4]Benzodiazepine-5-ketones](example 17 Compound) (0.209. mu. mol, 0.154mg) in DMA (122. mu.l, 10% v/v). The final protein concentration was 2.5mg/mL, containing 20% DMA in buffer. The bonding process is rotated at room temperatureNight, then clear by settling (13,200RPM for 4 minutes). The supernatant was then purified by Sephadex G25 gel filtration column equilibrated in PBS buffer, pH 6.5. The purified conjugate was dialyzed against 3 buffer exchanges into PBS pH6.5 buffer (approximately 1: 1200 dilution). The conjugate was clarified by 0.22 μm syringe filter for spectrophotometric analysis (for PBD ε)_280nm＝10736M^-1cm^-1，ε_318nm＝12053M^-1cm^-1To the antibody epsilon_280nm＝222,960M^-1 cm^-1). An average of 3.05PBD molecules (example 17 compound) were attached per antibody molecule.

huB 4-SMCC-Compound of example 17

Example A4: an anti-B4 antibody that binds to CD19 antigen preferentially expressed on the surface of human lymphoma cells was selected for binding to tomaymycin derivatives.

In the first step, the antibody is reacted with the modifier succinimidyl 4- [ maleimidomethyl]Cyclohexane-1-carboxylate (SMCC) to introduce maleimide groups. A solution of huB4 antibody at a concentration of 6mg/mL (1mL) in an aqueous buffer (pH 6.7) containing 0.05M potassium phosphate, 0.05M sodium chloride and 2mM ethylenediaminetetraacetic acid (EDTA) was treated with a 7-fold molar excess of a solution of SMCC in Dimethylacetamide (DMA). The reaction mixture was stirred at room temperature for 90 minutes and then loaded on a Sephadex G25 gel filtration column (NAP 10) equilibrated beforehand in an aqueous buffer containing 0.10MN- (2-hydroxyethyl) -piperazine-N' -2-ethanesulfonic acid (HEPES) pH 8.0. The modified antibody-containing fractions were collected and pooled to give the product. A small aliquot of the modified antibody was treated with beta-mercaptoethanol for 10 minutes, followed by the addition of 5, 5' -dithiobis-2-nitrobenzoic acid (DTNB) to detect residual thiols (ε for 5-thio-2-nitrobenzoic acid (TNB))_412nm＝14,150M^-1 cm^-1To the antibody epsilon_280nm＝222,960M^-1cm^-1). The amount of thiol consumed in the reaction of antibody with maleimide (compared to control without antibody)) Equal to the molar amount of maleimide attached to the antibody (subtractive Ellman's assay). Each antibody is attached to about 3.7 active maleimide groups.

The modified antibody (4.4mg, 0.03 μmol) was diluted to 8.7mg/mL in pH 8.0HEPES buffer, and subsequently treated with a solution of the compound of example 17 in DMA (5.4mM) such that the final concentration of DMA in the buffer was 20%. 2 molar equivalents of 8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ] are added per linker][1，4]Benzodiazepine-5-ketones]Compound of example 17 (7.4 eq./antibody, 0.222 μmol, 164 μ g). The combined mixture was stirred at room temperature for 20 hours, and the reaction mixture was purified by passing through a G25 column (NAP 5) equilibrated in 0.05 potassium phosphate (KPi), 0.05M NaCl, 0.002M EDTA buffer solution (pH 6.7). Fractions containing huB 4-example 17 compound conjugate were pooled and dialyzed against Kpi buffer using 3 exchanges (24 h). The final conjugate (2.25mg, 2.25mg/ml) was analyzed spectrophotometrically using extinction coefficient and the compound of example 17 (. epsilon.) (_318nm＝12,053M^-1cm^-1，ε_280nm＝10,736M^-1cm^-1) And B4 antibody (. epsilon.)_280nm＝222,960M^-1cm^-1). An average of 2.8PBD molecules (example 17 compound) were attached per molecule antibody.

huMy 9-6-SPDB-Compound of example 16

Example A5: the antibody was first modified with 4- (2-pyridyldithio) butanoic acid N-hydroxysuccinimide ester (SPDB) to introduce pyridyldithio groups. A4.5-fold molar excess of SPDB (0.246. mu. mol, 80.1. mu.g) in DMA (50. mu.L) was added to a solution of humY9-6 (8mg, 0.055. mu. mol) in aqueous buffer (pH 6.5) (0.950mL) containing 50mM potassium phosphate, 50mM sodium chloride and 2mM EDTA, to obtain the final proteinThe plasmid concentration was 8mg/mL and contained 5% DMA in buffer. The modification was rotated at room temperature for 90 minutes and purified by Sephadex G25 gel filtration column equilibrated in aqueous buffer (pH 8.5) containing 50mM potassium phosphate, 50mM sodium chloride and 2mM EDTA. A small aliquot of the modified antibody was treated with DTT to cleave the pyridyl disulfide group, and the modified antibody and liberated pyridinethiol were analyzed spectrophotometrically (for liberated pyridinethiol. epsilon.)_343nm＝8,080M^-1cm^-1Para-modified pyridine dithio group ε_280nm＝5100M^-1cm^-1To the antibody epsilon_280nm＝206,460M^-1cm^-1). An average of 3.32 pyridyl disulfide molecules per antibody molecule were attached.

The modified antibody (3.05mg, 0.0208. mu. mol) was diluted in the above-mentioned pH 8.5 buffer (976. mu.L) and DMA (122. mu.L, 10% v/v) and subsequently treated with 8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones](example 16 Compound) (0.207. mu. mol, 0.158mg) in DMA (122. mu.l, 10% v/v). The final protein concentration was 2.5mg/mL, containing 20% DMA in buffer. The binding process was spun overnight at room temperature and then clarified by settling (13,200RPM for 4 minutes). The supernatant was then purified using a Sephadex G25 gel filtration column equilibrated in PBS buffer, pH 6.5. The purified conjugate was dialyzed against 3 buffer exchanges into PBS pH6.5 buffer (approximately 1: 600 dilution). The conjugate was clarified by 0.22 μm syringe filter for spectrophotometric analysis (for PBD ε)_280nm＝7743M^-1 cm^-1，ε_318nm＝9137M^-1cm^-1To the antibody epsilon_280nm＝206,460M^-1cm^-1). An average of 2.58PBD molecules (example 16 compound) were attached per antibody molecule.

Example B: binding assay

The relative binding affinities of the anti-B4 antibody and its tomaymycin conjugate to antigens expressed on Ramos cells were determined using a fluorescence-based assay. Antibody-tomaymycin conjugates and simple antibodies at 10^-7The concentration of M was added to a 96-well round bottom plate and titrated with 3-fold serial dilutions to replicate each concentration. Ramos cells were added at 50000 cells per well to each well containing different antibody or conjugate concentrations and control wells and the plates were incubated on ice for 3 hours. After the incubation period, the cells in the plate were washed, a fluorescein-labeled secondary antibody conjugated to human IgG, such as anti-B4, was added, and the plate was incubated on ice for 1 hour. After the incubation period, the plates were washed again and the cells were fixed with 1% formaldehyde/PBS solution. The fluorescence in each well of the plate was read with a Becton dickinson facscalibur fluorescence analyzer. The data are plotted as a percentage of the maximum fluorescence obtained at the highest concentration of antibody or conjugate.

Example C: in vitro potency and specificity of tomaymycin derivatives or tomaymycin derivative conjugates. General experimental methods used:

adding tomaymycin derivative or tomaymycin derivative conjugate samples to 96-well flat-bottomed tissue culture plates using 1 × 10^-12M to 3X 10^-7Serial dilution solution titrations of M. Antigen positive and antigen negative tumor cells were added to the wells such that there were three replicates for each drug concentration per cell line, plates at 5% CO₂The culture was carried out at 37 ℃ for 4 days in an atmosphere.

At the end of the incubation period, 20[ mu ]1 tetrazolium reagent WST-8(2- (2-methoxy-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-dithiophenyl) -2-tetrazolium monosodium salt) was added to each well and the plates were recovered in the incubator for 2 hours. The absorbance of each well of the plate was then measured at 450nm using a Molecular Devices plate reader. The proportion of cells surviving at each tomaymycin derivative or conjugate concentration is plotted.

Compound (I)	A549(IC₅₀)	KB(IC₅₀)	MCF7(IC₅₀)
				Example 5	7×10^-11M	1×10^-11M	1×10^-11M
Example 1	＜10^-12M	＜10^-12M	＜10^-12M
				Example 2	＜10^-12M	＜10^-12M	＜10^-12M

The compounds of the invention and conjugates were tested for specific cytotoxicity against MOLT-4 and BJAB or HL60/GC and Ramos cell lines and the results are illustrated in FIGS. 1a-c, 2a-c and 3 a-b.

FIG. 1a shows the in vitro potency of huB 4-SPDB-example 16 compound on antigen positive BJAB cells and antigen negative MOLT-4 cells.

FIG. 1b shows the in vitro potency of huB 4-SMCC-example 16 compound on antigen positive BJAB cells and antigen negative MOLT-4 cells.

FIG. 2a shows the in vitro potency of huB 4-SPDB-example 17 compound on antigen positive BJAB cells and antigen negative MOLT-4 cells.

FIG. 2b shows the in vitro potency of huB 4-SMCC-example 17 compound on antigen positive BJAB cells and antigen negative MOLT-4 cells.

FIG. 3b shows the in vitro potency of the free compound of example 16 on HL60/GC and Ramos cells.

Example D: in vivo efficacy of tomaymycin derivatives or tomaymycin derivative conjugates

The assay can be carried out by using and/or applying the experimental methods described in WO2004/103272, using huB4 as antibody and suitable antigen positive cell lines, such as Ramos and RajiiBurkitt's lymphoma cell lines.

Claims

1. A compound of formula (I):

wherein

- - -represents an optional single bond;

represents a double bond;

u and U 'are absent, W and W' represent H;

r1 and R2 and R1 ' and R2 ' together form a double bond comprising the groups ═ B and ═ B ', respectively; wherein

B and B' are the same or different and are each selected from the group consisting of optionally substituted one or more (C)₁-C₁₂) Alkyl-substituted alkenyl;

A. a' are the same or different and are independently selected from (C)₁-C₁₂) An alkyl group;

y, Y' are the same or different and are independently selected from O (C)₁-C₁₂) An alkyl group;

t is phenyl or pyridyl, said phenyl or pyridyl being substituted with one or more linkers;

n, n', equal or different, is 0 or 1;

r is selected from H and (C)₁-C₁₂) An alkyl group;

wherein the linker comprises a thiol-, sulfide-, or disulfide-containing substituent and is of the formula:

-G-D-(Z)_p-S-Z’

wherein

G is a single bond, -O-or-NR-;

d is a single bond or-E-, -E-NR-CO-, -E-CO-, -CO-E-;

e is selected from straight or branched- (C)₁-C₁₂) Alkyl-, - (OCH)₂CH₂)_i-or (C)₁-C₁₂) Alkyl-piperazinyl;

z is linear or branched- (C)₁-C₁₂) Alkyl-;

p is 0 or 1;

z' represents H or SR₂₀Wherein R is₂₀Represents H, methyl or (C)₁-C₁₂) Alkyl, with the proviso that when Z' is H, the compound reacts with pyrrolo [1, 4 ] through intramolecular cyclization]BenzodiazepineThe imine bond-NH-of one of the moieties is balanced by the corresponding compound formed by addition of a thiol group-SH.

2. The compound of claim 1, wherein B ═ B' ═ CH₂Or CH-CH₃。

3. The compound of claim 1 or 2 which is:

4. a compound according to any preceding claim, wherein n ═ n' ═ 1.

5. A compound according to any preceding claim, wherein a ═ a'.

6. A compound according to any preceding claim, wherein a ═ a' ═ straight chain unsubstituted (C)₁-C₁₂) An alkyl group.

7. A compound according to any preceding claim, wherein Y ═ Y'.

8. A compound according to any preceding claim, wherein Y ═ Y' ═ O (C)₁-C₁₂) An alkyl group.

9. The compound of claim 7, wherein Y ═ Y' ═ OMe.

10. A compound according to any one of the preceding claims wherein G is-O-.

11. A compound according to any one of the preceding claims wherein D is-E-NR-CO-.

12. A compound according to any preceding claim, wherein Z is- (CH)₂)₂-C(CH₃)₂-。

13. A compound according to any one of the preceding claims wherein p is 1.

14. A compound selected from the group consisting of:

8, 8' - {5- [3- (4-methyl-4-methyldithio-pentanoylamino) propoxy]-1, 3-benzenediylbis (methyleneoxy) } -bis [ (S) -2-ethylidene- (E) -yl-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ 5-acetylthiomethyl-1, 3-benzenediyl bis (methyleneoxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]；

8, 8' - [3- (2-acetylthioethyl) -1, 5-pentanediylbis (oxy)]-bis [ (S) -2-methylene-7-methoxy-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]；

8, 8' - [5- (N-4-mercapto-4, 4-dimethylbutyryl) amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]；

8, 8' - [5- (N-4-methyldithio-4, 4-dimethylbutyryl) -amino-1, 3-benzenediyl bis (methyleneoxy)]-bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]；

8, 8' - [5- (N-methyl-N- (2-mercapto-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]；

8, 8' - [5- (N-methyl-N- (2-methyldithio-2, 2-dimethylethyl) amino-1, 3-benzenediyl (methyleneoxy)) -bis [ 7-methoxy-2-methylene-1, 2,3, 11 a-tetrahydro-5H-pyrrolo [2, 1-c ]][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (2- (4-mercapto-4-methyl) -pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (2- (4-methyl-4-methyldithio) pentanoylamino-ethoxy) -pyridine-2, 6-dimethyl) dioxy group]-bis [ (S) -2-ethylidene- (E) -yl-7-dimethoxy-1, 2,3, 11 a-tetrahydropyridinePyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (1- (2- (4-methyl-4-methyldithio) -pentanoylamino-ethoxy) -benzene-3, 5-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (3- (4-methyl-4-methyldithio) -pentanoylamino-propoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (4- (4-methyl-4-methyldithio) -pentanoylamino-butoxy) -pyridine-2, 6-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl)]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (1- (3- [4- (4-methyl-4-methyldithio-pentanoyl) -piperazin-1-yl)]-propyl) -benzene-3, 5-dimethyl) -dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy)]-ethoxy } -ethoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine5-ketones]；

8, 8' - [ (1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (1- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy)]-ethoxy } -ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentanoylamino) -ethoxy ] -2]-ethoxy } -ethoxy) -ethoxy]-ethoxy } -ethoxy) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (1- (2- [ methyl- (2-methyl-2-methyldithio-propyl) -amino)]-ethoxy) -benzene-3, 5-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -yl-7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (3- [ methyl- (4-methyl-4-methyldithio-pentanoyl) -amino)]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (4- (3- [ methyl- (2-methyl-2-methyldithio-propyl) -amino)]-propyl) -pyridine-2, 6-dimethyl) -dioxy]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

8, 8' - [ (1- (4-methyl-4-methyldithio) -pentanoylamino) -benzene-3, 5-dimethyl]-dioxy group]-bis [ (S) -2-ethylidene- (E) -7-dimethoxy-1, 2,3, 11 a-tetrahydro-pyrrolo [2, 1-c)][1，4]Benzodiazepine-5-ketones]；

Or a pharmaceutically acceptable salt thereof.

15. A conjugate comprising one or more compounds according to any one of claims 1 to 14 covalently linked to a cell-binding drug via a linking group of a linker,

wherein the linking group is a thiol, sulfide or disulfide group.

16. The conjugate according to claim 15, wherein the cell-bound drug has been modified with a modifying agent to improve the reactivity of the cell-bound drug towards the linking group of the linker.

17. The conjugate of claim 15 or 16, wherein the cell-binding drug is selected from the group consisting of an antibody or antibody fragment containing at least one binding site, lymphokine, hormone, growth factor, trophic delivery molecule.

18. The conjugate according to any one of claims 15-17, wherein the cell-binding drug is selected from the group consisting of a monoclonal antibody; a chimeric antibody; a humanized antibody; a fully human antibody; a single chain antibody; an antibody fragment; an interferon; a peptide; a lymphokine; hormones, steroid hormones; growth factors and colony stimulating factors; and (3) vitamins.

19. A method of preparing a conjugate as claimed in any one of claims 15 to 18 comprising the step of reacting a compound as defined in any one of claims 1 to 14 wherein T contains a sulphide, disulphide or thiol group or a precursor thereof, with a cell-bound drug containing a functional group reactive towards a disulphide or thiol, thereby linking the compound and the cell-bound drug together via a sulphide or disulphide bond.

20. A method of making a conjugate, wherein a cell-bound drug containing a free or protected thiol is reacted with the disulfide or thiol containing compound of any one of claims 1-14, wherein the cell-bound drug is a peptide or antibody modified by a crosslinking agent.

21. The method of claim 20, wherein the crosslinking agent is N-succinimidyl 3- (2-pyridyldithio) propionate, N-succinimidyl 4- (2-pyridyldithio) valerate, 4-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyldithio) -toluene, N-succinimidyl-3- (2-pyridyldithio) butyrate, succinimidyl pyridyldithio propionate, 4- (2-pyridyldithio) butanoic acid N-hydroxysuccinimidyl ester, succinimidyl 4- [ N-maleylaminomethyl ] cyclohexane-1-carboxylate, N-thiosuccinimidyl-3- { -2- (5-nitro-pyridyldithio) cyclohexane-1-carboxylate, N-succinimidyl-3 { -2- (2-pyridyldithio) pentanoate, or a mixture thereof Yl) butyrate, 2-iminosulfane or S-acetylsuccinic anhydride.

22. The method of claim 20, wherein:

-the thiopyridyl group of a monoclonal antibody modified with succinimidylpyridyl-dithiopropionate by displacement with a thiol-containing compound of any one of claims 1-14 to produce a disulfide linked conjugate;

-the aryl thiol group of the compound of any one of claims 1-14 is replaced by a thiol group previously introduced into the antibody molecule to produce a disulfide linked conjugate.

23. The method of claim 20 wherein the thiol-containing compound of any one of claims 1-14 is linked via a thioether chain to antibodies and other cell-bound drugs modified with N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate, N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amido-hexanoate), N-succinimidyl-4- (iodoacetyl) -aminobenzoate, N-succinimidyl iodoacetate, N-succinimidyl bromoacetate or N-succinimidyl 3- (bromoacetamido) propionate.

24. The method of any one of claims 20-23, wherein the conjugate can be purified using standard chromatographic techniques or by dialysis or filtration.

25. The method of claim 24, wherein the chromatographic technique is HPLC, size exclusion chromatography, adsorption chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography or ceramic hydroxyapatite-based chromatography.

26. A pharmaceutical composition comprising a conjugate molecule as defined in any one of claims 15 to 18 or a compound as defined in any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

27. Use of an effective amount of a conjugate molecule as defined in any one of claims 15 to 18 or a compound as defined in any one of claims 1 to 14 for the manufacture of a medicament for the treatment of cancer.

28. Use of a conjugate molecule as defined in any one of claims 15 to 18 in the manufacture of an anti-cancer medicament.

29. Use of a compound as defined in any one of claims 1 to 14 in the manufacture of an anti-cancer medicament.