CZ20003037A3 - Anticancer agents - Google Patents

Abstract

Antitumor substances of general formula I or II, in which the individual general symbols have the meaning given in these general formulas, are compounds with very good effect against malignant tumors, including solid tumors and tumors resistant to the action of commonly used chemotherapeutic agents. The solution also includes a pharmaceutical composition containing the said substances.

Description

Technical field

The invention relates to a group of antitumor substances and pharmaceutical compositions containing them.

State of the art

Some malignant tumors, which are mainly treated with chemotherapy to prolong life, have already achieved significant success. These include Burkitt lymphoma, acute lymphocytic leukemia and Hodgkin sarcoma, and another 10 to 15 types of tumors. These tumors were described, for example, in the publication by A. Golden et al., Eur. J. Cancer, 17, 129, 1981 in Table 1. However, these tumors, in which the life span of patients and the quality of life during this period have been significantly extended, represent only a small fraction of the various types of malignant tumors. In addition, there are only a very small number of substances that are truly highly effective against solid tumors, such as ovarian, breast, lung, etc. These substances include cyclophosphamide, adriamycin, 5-FU, hexamethylmelamine, etc. This means that patients with many types of malignant tumors remain at a significant risk of relapse and increased mortality.

In the event of relapse, some patients may be able to achieve remission again with a different treatment. However, in this case, it is usually necessary to use high doses of the original chemotherapeutic agent or additional agents must be used in addition, since the tumor develops at least partial resistance to the agents used. Current research shows that resistance can develop simultaneously against several active agents, including those that have not been used in the patient before. The development of resistant tumors may be a function of the tumor mass • ·

and is the main cause of unsuccessful treatment. In order to overcome this resistance, chemotherapy can be used in high doses, or together with irradiation and allogeneic or autologous bone marrow transplantation. When using high doses of chemical substances, it is possible to use the original substance or administer other substances in addition. It is obvious that when developing new substances, new substances should be discovered that do not cross-resistant with mdr phenotypes, otherwise there cannot be an increase in the success of treatment and an easier treatment in previously treated patients.

The antitumor activity of a new group of natural substances, called illudins, was investigated in vitro according to the publication Kelner M. et al., Cancer Res., 47, 3186, 1987. Illudin M was purified and subjected to in vivo activity tests at the National Cancer Institute Division of Cancer Treatment NCI DCT. It was shown that illudin M statistically significantly increases the life span in rats with Dunning leukemia, but has a significantly lower therapeutic index in the case of solid tumors. The high toxicity of the listed substances did not allow any tests on humans. Recently, synthetic analogues of illudin with promising antitumor activity have been developed, including analogues that were described in US 5439936 and 5523490.

It is clear that despite significant progress, there is still a need to find new chemotherapeutic agents capable of inhibiting tumor growth, especially in the case of solid tumors. At the same time, new agents should have a therapeutic index such that they can be used in vivo treatment.

The essence of the invention

The invention consists of antitumor substances of general formula I « · • ·

where

R 1 represents a hydrogen atom, a hydroxy group, a mercapto group, an amino group, a halogen atom, a carboxy group, a nitro group or a -(CH ₂ ) n -(X) -(Y) group.

n is an integer from 0 to 4,

X represents an oxy group -O-, a thio group -S-, -N(R _a )- or is absent,

Y represents C3-C6cycloalkyl, aryl, heteroaryl, a residue of a saccharide, amino acid, peptide or a 1 to 15-membered branched or unbranched hydrocarbon chain, optionally containing 1, 2 or 3 non-peroxide oxy groups, thio groups or -N(R _a )- groups, wherein this chain is optionally substituted on the carbon atom by 1, 2 or 3 substituents from the group oxo group =O, hydroxy group, carboxy group, halogen atom, mercapto group, nitro group, -N(R b ) _c , C3-C6cycloalkyl, aryl, heteroaryl, a residue of a saccharide, amino acid or peptide and at the same time the chain is saturated or unsaturated,

R ₂ represents a carboxy group, C1-C6alkanoyl, C1-C6alkoxycarbonyl, halogen C1-C6alkyl, -C(=O)NRdRe, a residue of a saccharide, amino acid, peptide or C1-C6alkyl, substituted with 1 or 2 hydroxy groups, C1-C6alkoxy groups,

C1-C6alkanoyloxy groups, carboxy groups, residues of amino acids, peptides or carbohydrates or groups -C(=O)NRdR _e >

R ₃ represents a hydrogen atom, a C1-C6alkyl, a C1-C6alkoxy group,

C1-C6alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy,

R ₄ represents a hydrogen atom or a C1-C6alkyl, R ₅ represents a hydroxy group, a C1-C6alkoxy group or a C1-C6alkanoyloxy group or R ₄ and R ₅ together form an ethylenedioxy group,

R ₆ represents a hydrogen atom, a carboxy group, a C1-C6alkanoyl, a C1-C6alkoxycarbonyl, a halogenoC1-C6alkyl, -C(=O)NR _f R _g , a residue of a saccharide, amino acid or peptide or a C1-C6alkyl, optionally substituted with 1 to 2 substituents from the group of hydroxy group, C1-C6alkoxy group, C1-C6alkanoyloxy group, a carboxy group, a residue of an amino acid, peptide or saccharide or a group of -C(=O)NR _f R _g ,

R _a represents a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl and

Rb, Rc, Rd, Re, Rf3 Rg independently represent a hydrogen atom, a C1-C6alkyl, a C1-C6alkanoyl, phenyl or benzyl or form Rb and _Rc or _Rd and _Re or Rf and _Rg together with the nitrogen atom to which a pyrrolidine, piperidine or morpholine ring is attached, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group in the meaning of Y or _R3 is optionally substituted with 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy, C1-C6alkanoyl, C1-C6alkanoyloxy, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, haloC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N(Rh)(Rj), where Rh and Rj independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof.

The invention also includes those compounds of general formula I in which R1 represents -(CH2) _n- (X)-(O, where n represents an integer

number 0 to 4, X represents an oxy group, a thio group, -N(R _a )- or is absent, Y represents an amino acid residue, protected by 1 or 2 protecting groups, a peptide residue or a one to 15-membered branched or unbranched hydrocarbon chain, optionally containing 1, 2 or 3 non-peroxide oxy groups, thio groups or -N(R _a )- groups, this chain is substituted by 1, 2 or 3 peptide residues and is saturated or unsaturated,

R ₂ represents a hydrogen atom or C1-C6alkyl, R ₃ represents a hydrogen atom, C1-C6alkyl, C1-C6alkoxy, C1-C6alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R 4 represents a hydrogen atom or C1-C6alkyl and R 5 represents a hydroxy, C1-C6alkoxy or C1-C6alkanoyloxy group or R 4 and R ₅ together form an ethylenedioxy group, Rg represents a hydrogen atom, carboxy, C1-C6alkanoyl,

C1-C6alkoxycarbonyl, halogenC1-C6alkyl, -C(=O)NRfR _g , residue of a saccharide, amino acid or peptide or C1-C6alkyl, optionally substituted with 1 to 2 substituents from the group hydroxy group, C1-C6alkoxy group, C1-C6alkanoyloxy group, carboxy group, residue of an amino acid, peptide or saccharide or the group -C(=O)NR _f R _g ,

R _a represents a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl and

Rb, Rc, Rd, Re, _Rf and _Rg independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form Rb and _Rc or Rd and _Re or Rf and _Rg together with the nitrogen atom to which a pyrrolidine, piperidine or morpholine ring is attached, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group in the meaning of Y or _R3 is optionally substituted with 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy, C1-C6alkanoyl, C1-C6alkanoyloxy, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, haloC1-C6alkyl, hydroxy, halogen, carboxy, mercapto,

nitro group or -N(Rh)(Rj), where R _h and Rj independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. A preferred meaning for Y is C1-C6alkyl, substituted with a peptide residue.

The invention also includes compounds of the general formula I, in which Ri represents a hydrogen atom, a hydroxy group, a mercapto group, an amino group, a halogen atom, a carboxy group, a nitro group or -(CH ₂ ) _n -(X)-(Y), n represents an integer from 0 to 4, X represents an oxy group, a thio group, -N(R _a )- or is absent, Y represents a C3-C6cycloalkyl, aryl, heteroaryl, a residue of a saccharide, amino acid or peptide or a 1 to 15-membered branched or unbranched carbon chain, optionally containing 1, 2 or 3 non-peroxide oxy groups, thio groups or -N(R _a )- groups, the chain is optionally substituted with 1, 2 or 3 groups from the group oxo group, hydroxy group, carboxy group, halogen atom, mercapto group, nitro group,

-N(R _b )(R _c ), C3-C6cycloalkyl, aryl, heteroaryl, residue of a saccharide, amino acid or peptide, the chain may be saturated or unsaturated, R ₂ represents a hydrogen atom or C1-C6alkyl, R ₃ represents a hydrogen atom, C1-C6alkyl, C1-C6alkoxy, C1-C6alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C1-C6alkyl, R 5 represents a hydroxy group, C1-C6alkoxy or C1-C6alkanoyloxy or R ₄ and R 5 together form an ethylenedioxy group, R 6 represents a carboxy group, C1-C6alkanoyl, C1-C6alkoxycarbonyl, -C(=O)NR _f R _g , residue of a saccharide, amino acid or peptide or C1-C6alkyl, substituted by 1 or 2 substituents from the group C1-C6 alkoxy group,

C1-C6alkanoyloxy group, carboxy group, amino acid, peptide or carbohydrate residue or -C(=O)NRfR _g , R _a represents a hydrogen atom, ·· ·· ► * * | · · ·· • ·

C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl and R _b , R _c , R d , Re , R f and R g independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form R _b and R _c , or R _d and _Re or R f and R _g together with the nitrogen atom to which a pyrrolidine, piperidine or morpholine ring is attached, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group in the meaning of Y or R ₃ is optionally substituted with 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy, C1-C6alkanoyl,

C1-6alkanoyloxy, C1-6alkoxycarbonyl, hydroxyC1-6alkyl, haloC1-6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N(Rh)(Rj), where R _h and Rj independently represent hydrogen, C1-6alkyl, C1-6alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts of these substances.

The invention also relates to dimeric compounds which are formed by two compounds of formula I, connected by a linker. This group may be, for example, an alkyl-type or ester-type group. Examples include the groups -(CH ₂ ) p O-(CH ₂ ) q -, -(CH ₂ ) _r - and -CH ₂ -S-CH ₂ C(O)-O-(CH ₂ ) ₂ -OC(O)CH ₂ -S-CH ₂ -, where p and q are integers from 1 to 8 and r is an integer from 1 to 16, preferably from 1 to 8. It is clear that other groups of similar length could also be used. Two compounds of general formula I can be connected, for example, by independently replacing one of the symbols Ri, R ₃ , R 4 or R ₅ in each of these compounds with a bifunctional linking group. In the case where the symbol R1 is replaced, this group is preferably the group -CH ₂ -O-CH ₂ - or -CH ₂ -S-CH ₂ C(O)-O-(CH ₂ ) ₂ -OC(O)CH ₂ -S-CH ₂ -.

The invention also relates to compounds of general formula I where R1 represents -(CH ₂ ) _n -(X)-(O, ⁿ represents an integer from 1 to 4, preferably 1, X represents an oxy group, a thio group or a group -N(R _a )-, preferably « · « ♦ * · • ·· · thio group, Y represents a 2 to 15-membered branched or unbranched carbon chain, optionally containing 1, 2 or 3 non-peroxide oxy groups, thio groups or groups -N(R _a )-, the chain is substituted on carbon atom 1, 2 or 3, substituents from the group oxo group, carboxy group, mercapto group, -N(R _b )(R _c ), C3-C6cycloalkyl, aryl, heteroaryl, a residue of a saccharide, amino group or peptide and is saturated or unsaturated, preferably Y represents a 2 to 6-membered branched or unbranched carbon chain, substituted on carbon atom 1, 2 or 3 with substituents from the group oxo group, heteroaryl, amino acid residue or peptide residue, R2 represents a hydrogen atom or C1-C6alkyl, _R3 represents a hydrogen atom, C1-C6alkyl, C1-C6alkoxy group, C1-C6alkylthio group, aryl, heteroaryl, aryloxy group or heteroaryloxy group, _R4 represents a hydrogen atom or C1-C6alkyl and _R5 represents a hydroxy group, C1-C6alkoxy group or C1-C6alkanoyloxy group or _R4 and _R5 together form an ethylenedioxy group, _R6 represents a hydrogen atom, carboxy group, C1-C6alkanoyl, C1-C6alkoxycarbonyl, halogenC1-C6alkyl, -C(=O)NRfR _g , residue of a saccharide, amino acid or peptide or C1-C6alkyl, optionally substituted with 1 or 2 substituents from the group hydroxy, C1-C6alkoxy, C1-C6alkanoyloxy, carboxy, amino acid, peptide or saccharide residue or -C(=O)NRfR _g , R _a represents a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl and R _b , R _c , Rf and R _g independently represent a hydrogen atom C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form R _b and R _c or R _f and R _g together with the nitrogen atom to which a pyrrolidine, piperidine or morpholine ring is attached, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group in the meaning of Y or R ₃ is optionally substituted with 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6Alkoxy, C1-C6alkanoy, C1-6alkanoyloxy, C1-C6Alkoxycarbonyl, hydroxyC1-C6alkyl, haloC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro, or

-N(Rh)(Rj), where R _h and Rj independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts of these substances.

The invention also relates to compounds of the general formula I

where

R2 represents C1-C6alkyl,

R ₃ represents a hydrogen atom, a C1-C6alkyl, a C1-C6alkoxy group, a C1-C6alkylthio group, aryl, heteroaryl, aryloxy group or heteroaryloxy group,

R4 represents a hydrogen atom or C1-C6alkyl and

R ₅ represents a hydroxy group, a C1-C6 alkoxy group or a C1-C6 alkanoyloxy group or R ₄ and R ₅ together form an ethylenedioxy group,

R ₆ represents a hydrogen atom, a carboxy group, a C1-C6alkanoyl group, a C1-C6alkoxycarbonyl group, a halogenoC1-C6alkyl group, -C(=O)NRfR _g , a residue of a saccharide, amino acid or peptide group or C1-C6alkyl group, optionally substituted with 1 or 2 substituents from the group of hydroxy group, C1-C6alkoxy group, C1-C6alkanoyloxy group, a carboxy group, a residue of an amino acid, peptide or saccharide group or a -C(=O)NR _f R _g group, • · · • « · · · ·♦ ··

R ₇ represents a carboxy group, C1-C6alkanoyl, C1-C6alkoxycarbonyl, halogenoC1-C6alkyl, -C(=O)NR _d R _e , a residue of a saccharide, amino acid or peptide or C1-C6alkyl, optionally substituted with 1 or 2 substituents from the group hydroxy group, C1-C6alkoxy group, C1-C6alkanoyloxy group, carboxy group, a residue of an amino acid, peptide or saccharide or a group -C(=O)NR _d R _e ,

Rd. Re, Rf and Rg independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form _Rd and _Re or Rf and _Rg together with the nitrogen atom to which a pyrrolidine, piperidine or morpholine ring is attached, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted with 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy, C1-C6alkanoyl, C1-C6alkanoyloxy, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, haloC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N(Rh)(Rj), where Rh and Rj independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or together with the nitrogen atom to which they are attached form a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof.

The compounds of the invention are antineoplastic agents, i.e. agents capable of inhibiting the growth of tumor cells in vitro or in vivo in mammals, for example humans or domestic animals, and are particularly effective against solid tumors and tumors resistant to a wide range of active agents. The said agents can therefore inhibit the growth of said cells.

The compounds of the invention can be targeted to a specific tumor by combining them with a reagent capable of binding to an antigen associated with the tumor. This antigen can be located directly in the tumor or in the area of tumor cells. Suitable reagents ·· ·· · · • · ··· • ·

The invention also includes pharmaceutical compositions, e.g., dosage forms containing a single dose of one or more compounds of the invention in combination with a pharmaceutically acceptable diluent or carrier.

The invention also provides compounds of general formulae I or II, their dimers and conjugates, containing these substances and a reagent capable of binding to a tumor-associated antigen, or salts of these substances for use in therapeutic purposes, preferably for the treatment of malignant, e.g. solid tumors, and the use of said substances for the manufacture of pharmaceutical compositions for said purpose.

The invention also provides a method for producing the compounds mentioned and novel intermediates useful in the production of these compounds. Some compounds of the invention can be used for the production of other compounds of the invention.

The invention will be further described in connection with the accompanying drawings.

Overview of images in drawings

Figure 1 shows representative compounds 1 to 9 of the invention and the corresponding intermediates 10 and 11.

9

9

9

9

99 • 9 ··

9 9 · · ··

9 9 9 · 9 9

99

Figure 2 shows representative compounds 13 to 16 of the invention.

Figure 3 shows representative compounds 17 and 18 of the invention.

In the following description, a halogen atom means a fluorine, chlorine, bromine or iodine atom. An alkyl group, an alkoxy group, an alkenyl group and the like may be straight-chain or branched, but in the case of individual radicals such as propyl, this name includes only the straight-chain radical, the branched-chain isomer such as isopropyl being specifically mentioned. Aryl means a phenyl radical or a bicyclic radical with a carbon ring, fused in the ortho position and containing 9 to 10 ring atoms, at least one of these rings being aromatic. Heteroaryl includes residues connected by a carbon atom of a monocyclic aromatic ring containing 5 or 6 atoms in the ring, the residue containing carbon atoms and 1 to 4 non-peroxide oxygen atoms, sulfur or the group N(R _y ), where R _y is absent or represents a hydrogen or oxygen atom, C1-C4alkyl, phenyl or benzyl, it can also be residues of a bicyclic, heterocyclic group of 8 to 10 carbon atoms, condensed in the ortho position and derived in particular from a benzene derivative or a derivative formed by condensation of a propylene, trimethylene or tetramethylene divalent group with this ring.

The term "inhibition" refers to a reduction in the growth rate of tumor cells and/or a reduction in tumor weight. Inhibition also includes complete regression of the tumor. This means that the compounds of the invention may be cytostatic or cytotoxic to tumor cells.

• · • 0 00

000 ·· ··

0 0 • · · • 0 ·

0 0 · • 0 00

The compounds of the invention can be administered to mammals suffering from tumors against which the compounds of the invention are effective. The substances are active in vivo and also in vitro on human tumor cell lines. They are particularly suitable for the treatment of solid tumors for which there are currently insufficient chemotherapeutic agents. The compounds of the invention are therefore effective, for example, against epidermoid and myeloid tumors in acute (AML) or chronic (CML) form. These tumors also include tumors other than small cell tumors, liver tumors, tumors in the neck, kidney, adrenal, stomach, esophagus, oral cavity, mucous membranes and skin tumors as well as lung, ovarian, mammary gland, colon and melanomas including amelanotic subtypes. In addition, the substances mentioned can also be used in the case of endometrial, bladder, pancreatic, lymphoma, Hodgkin's disease, prostate cancer, sarcomas and testicular tumors, as well as in the case of central nervous system tumors such as brain tumors, neuroblastomas and tumors of hematopoietic cells, such as B-cell leukemia/lymphomas, myelomas, T-cell leukemia/lymphomas, small cell leukemia/lymphomas, monocytic leukemia, myelomonocytic leukemia and hairy cell leukemia. These leukemias/lymphomas can be acute (ALL) or chronic (CLL).

The term saccharide is understood to mean monosaccharides, disaccharides, trisaccharides and polysaccharides. This term therefore includes glucose, sucrose, fructose and ribose as well as deoxysugars such as deoxyribose and the like. The saccharide derivatives can be easily prepared by the method described in international patent applications WO 96/34005 and WO 97/03995. The saccharide can be easily linked to the rest of the compound of general formula I or II by an ether bond.

The term amino acid refers to natural amino acid residues, such as Aia, Arg, Asn, Asp, Cys, Glu, Gin, φφ • Φ φφ φ φ φ φ φφφ φ φ φ φφ φ φφ φ · φ · φ φ φ φ • φ • φ φφφ φφφ φ

φ φ

φ

Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val in D- or L-form as well as amino acid residues that do not have a natural origin, such as phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statin, 1,2,3,4-tetraisoquinoline-3-carboxylic acid, penicillamine, ornithine, citrulline, alpha-methylalanine, p-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine and tert.butylglycine. This term also includes natural and unnatural amino acids provided with a conventional protecting group, e.g. an acetyl or benzyloxycarbonyl group on the amino group or provided with a protecting group on the carboxyl group, e.g. a C1-C6 alkyl group, a phenyl or benzyl ester group or an amide group or an alpha-methylbenzylamide group. Other suitable protecting groups for the amino group and the carboxyl group are commonly known and are listed, for example, in the general publication T. W. Greene, Protecting Groups In Organic Synthesis, Wiley: New York 1981. The amino acid may be bound to the residue of the compound of general formula I via its carboxyl group, amino group or in any other way, for example via the sulfur atom of cysteine.

The term peptide refers to a sequence of 2 to 25 amino acid residues as defined above or any peptide residue. The sequence used may be linear or cyclic. A cyclic peptide may be prepared by forming disulfide bridges between two cysteine residues in the sequence. The peptide residue may be linked to a residue of the molecule of formula I by a carboxyl group, an amino group or in any other suitable manner, for example via the sulfur atom of the cysteine. Preferably, the peptide contains 3 to 25 and especially 5 to 21 amino acid residues. Peptide derivatives may be prepared by the methods described in US 4,612,302, US 4,853,371 and US 4,684,620 or by the methods described in the following examples. Specific peptide sequences are illustrated with

0

0« ··

0

0

0

000 000 ·>♦ «

0« «· 0 • ·0 ·

0 0 ·

0 0 · ·· ·· with the amino terminal on the left side and the carboxyl group on the right side.

It has been shown that some peptides can specifically bind to specific tumor-associated antigens in a manner similar to the binding of antibodies to these antigens, as reported, for example, in Arap et al., Science, 1998, 279, 5349, 377-380. In this way, it is possible to preferentially bring into contact a peptide with said ability, i.e. with the ability to bind to specific receptors on tumor cells towards the tumor tissue. Based on this property, a preferred embodiment of the invention are compounds of general formula I or II containing a peptide residue capable of specifically binding to a tumor-associated antigen.

Preferred peptides are, for example, -Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO:1), -Cys-Asp-Gly-Arg-Cys (SEQ ID NO:2), and -Cys-Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO:3).

It is understood that the compounds of the invention which contain a chiral center may exist and be isolated in optically active and racemic forms. Some of the compounds may be polymorphic. It is understood that the invention includes any racemic, optically active, polymorphic or stereoisomeric forms of the compounds of the invention or mixtures thereof having the desired properties described. It is known to prepare optically active forms, for example, by resolution of the racemic form by recrystallization, synthesis from optically active starting materials, chiral synthesis or chromatographic separation using a chiral stationary phase. It will be described below how the antitumor activity of these compounds can be determined using the following described test A or test B or using other known tests. In preferred compounds of formula I, the absolute stereochemistry at the carbon atom bearing the substituents R4 and R5 is (R).

• fl flfl « flfl fl fl fl · · • flfl fl • flfl · flfl flfl • · • fl flfl • flfl • fl · ·· • flfl fl flfl · flfl flfl

The specific values given below for individual radicals or substituents are illustrative only. They do not exclude the use of other radicals within the meaning given above for individual radicals or substituents.

The term C1-C4alkyl means methyl, ethyl, propyl, isopropyl, butyl, isobutyl or sec.butyl, the term C1-C6alkyl means methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec.butyl, pentyl, 3-pentyl, or hexyl, C1-C8alkyl can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec.butyl, pentyl, 3-pentyl, hexyl, heptyl, or octyl, C3-C6cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, C1-C4alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, or sec.butoxy, C1-C6alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec.butoxy group, pentoxy group,

3-pentoxy or hexyloxy, C2-C6alkenyl means vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 3-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,

4-hexenyl, 5-hexenyl or 4-methyl-3-pentenyl, C1-C6alkanoyl can be acetyl, propanoyl or butanoyl, halogen C1-C6alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trichloromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl or pentafluoroethyl, hydroxyC1-C6alkyl can be hydroxymethyl,

1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,

3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,

5-hydroxypentyl, 1-hydroxyhexyl or 6-hydroxyhexyl,

C1-C6alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, ··

-17 ···· .♦·· · · pentoxycarbonyl or hexyloxycarbonyl, C1-C6alkylthio group can be methylthio group, ethylthio group, propylthio group, isopropylthio group, butylthio group, isobutylthio group, pentylthio group or hexylthio group, C1-C6alkanoyloxy group can be formyloxy-, acetoxy-, propanoyloxy-, butanyoloxy-, isobutanoyloxy-, pentanoyloxy-, or hexanoyloxy group, aryl can be phenyl, indenyl or naphthyl and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl or its N-oxide, thienyl, pyrimidinyl or its N-oxide, indolyl, isoquinolyl or its N-oxide or quinolyl or its N-oxide.

Specific meanings for R 1 are hydroxy, halogen, carboxy, aryl, heteroaryl, and a saccharide, amino acid, or peptide residue.

Another specific meaning for fy is the group -(CH ₂ ) _n -(X)-(Y), where n = 0 to 4, X represents an oxy group, a thio group, -N(R _a )- or is absent and Y represents a peptide residue or a C1 -C6 alkyl, substituted by a peptide residue.

Another specific meaning for R 1 is hydrogen, or C 1 -C 6 alkyl, optionally substituted with 1 or 2 substituents selected from the group consisting of hydroxy, halogen, methoxy or ethoxy.

Another specific meaning for Ri is the group -(CH ₂ ) _n -(X)-(Y), where X represents an oxy group, a thio group or a -N(R _a )- group and Y represents CH ₂ OC(O)(C1-C4alkyl), CH ₂ C(O)-(CH ₂ ) ₂ -OC(O)CH ₂ SH, (CH ₂ ) ₂ -O-(CH ₂ ) ₂ halide, (C1-C4alkyl)-O-(C1-C4alkyl), CH ₂ CO ₂ (C1-C4alkyl), CH ₂ CO ₂ H, arylC 1-C5alkyl, a saccharide or amino acid residue or C1-C8alkyl, optionally substituted with 1 or 2 hydroxy groups or halogen atoms, wherein any aryl

9

8 4

4 444 4

4 4 4 9 9

4 4 4 4

99 884 « 88 44

8 9 4 8 8

8 4 4 4

4 9 4 8 8 « · · · ·

448 84 48 or the heteroaryl group in the meaning of Y is optionally substituted with 1 or 2 substituents from the group of hydroxy, halogen, C1-C4alkyl or C1-C4alkoxy.

Another specific meaning for Rj is the group -CH2-(X)-(Y), where X represents an oxy group, a thio group or -N(R _a )- and Y represents a C1 -C8alkyl, optionally substituted with 1 or 2 substituents from the group hydroxy group, a halogen atom, a carboxy group, an oxo group, a mercapto group, a group -N(R b )(R _c ), a C3-C6cycloalkyl, aryl, a heteroaryl, a residue of a saccharide, an amino acid or a peptide, wherein any aryl or heteroaryl group in the meaning of Y is optionally substituted, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy group, C1-C6alkanoyl, C1-C6alkanoyloxy group, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, halogenC1-C6alkyl, hydroxy group, halogen atom, mercapto group, nitro group or -N(Rh)(Rj), where Rh and Rj independently represent a hydrogen atom, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form Rh and Rj together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring.

Another specific meaning for Ri is the group -(CH ₂ ) _n -(X)-(Y), where n = 1 or 2, X represents an oxy group, a thio group or -N(R _a )- and Y represents a C1-C6alkyl or C2-C6alkenyl, optionally substituted with 1 or 2 substituents from the group oxo group, hydroxy group, carboxy group, halogen atom, mercapto group, nitro group, -N(R _b )(R c ), C3-C6cycloalkyl, aryl, heteroaryl, a residue of a saccharide, amino acid or peptide, wherein any aryl or heteroaryl group in the meaning of Y, is optionally substituted with 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy group, C1-C6alkanoyl, C1-C6alkanoyloxy group, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, haloC1-C6alkyl, hydroxy, halogen, mercapto, nitro or -N(Rh)(Rj), where Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl «9 • · • 9 • ·

99«

9

9 9 • 9

99 » 9 9 9

9 9 ·

9 9 9 ·

9 9 9 • 99 99 or benzyl or R _h and R j together with the nitrogen atom to which they are attached form a pyrrolidine, piperidine or morpholine ring.

Another specific meaning for R 1 is -CH ₂ -(cysteine residue, bonded via a sulfur atom)-R _x , where R _x represents an amino acid residue or a peptide containing 2 to 24 amino acid residues.

Another specific meaning for Rf is -CH ₂ -(N-acylcysteine residue, bonded via a sulfur atom)-R _x , where R _x represents an amino acid residue or a peptide containing 2 to 24 amino acid residues.

Another specific meaning for Rt is -CH ₂ -(glutathione, bonded via a sulfur atom).

Another specific meaning for Rt is 3-[(R)-alpha-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl. The ethyl group at position 2 preferably has the (S)-configuration of cysteine.

A preferred value for Rt is hydrogen, methyl, hydroxymethyl, methoxymethyl or acetoxymethyl.

A specific value for R ₂ is hydroxymethyl, methoxymethyl or acetoxymethyl.

Another specific meaning for R ₂ is carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl or -C(=O)NR d R _e .

Another specific meaning for R ₂ is C1-C6alkyl, substituted with 1 or 2 substituents from the group of hydroxy, C1-C6alkoxy, C1-C6alkanoyloxy, carboxy, amino acid, peptide or saccharide residue or -C(=O)NRdR _e · • ·

Another specific meaning for R ₂ is -CH ₂ -(cysteine residue, bonded via a sulfur atom)-R _x , where R _x represents an amino acid residue or peptides containing 2 to 24 amino acid residues.

Another specific meaning for R ₂ is -CH 2 -(N-acylcysteine residue, bonded via a sulfur atom)-R _x , where R _x represents an amino acid residue or peptides containing 2 to 24 amino acid residues.

Another specific meaning for R ₂ is -CH ₂ -(glutathione, bonded via a sulfur atom).

A specific meaning for R ₃ is hydrogen.

A specific meaning for R4 is methyl.

A specific meaning for R5 is hydroxy.

A specific meaning for R ₆ is hydrogen.

Another specific meaning for R 6 is carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl or -C(=O)NR f R _g .

Another specific meaning for Re is C1-C6alkyl, optionally substituted with 1 or 2 substituents from the group C1-C6alkoxy, C1-C6alkanoyloxy, carboxy, amino acid, peptide or saccharide residue or -C(=O)NR _f R _g .

Another specific meaning for R ₆ is -CH ₂ -(cysteine residue, bonded via a sulfur atom)-R _x , where R _x represents an amino acid residue or peptides containing 2 to 24 amino acid residues.

Another specific meaning for R ₆ is -CH ₂ -(residue

N-acylcysteine, bonded via a sulfur atom)-R _x , where R _x represents an amino acid residue or a peptide containing 2 to 24 amino acid residues.

A preferred meaning for R is methyl or hydroxymethyl.

A specific meaning for Ry is C1-C6alkyl, substituted with 1 or 2 substituents from the group of hydroxy, C1-C6alkoxy, C1-C6alkanoyloxy, carboxy, an amino acid, peptide or saccharide residue or -C(=O)NR _d R _e .

A specific meaning for R ₇ is also hydroxymethyl.

A specific meaning for R _x is a peptide containing 4 to 20 amino acid residues.

Another specific meaning for R _x is -Leu-Gly-Phe, -Phe-Leu-Gly, -Leu-Leu-Phe, -Gly-Phe, or -Leu.

Another specific meaning for R _x is -Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO:4), -Asp-Gly-Arg-Cys (SEQ ID NO:5) or -Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO:6).

A specific compound according to the invention is, for example, a compound of general formula I, in which R ₂ represents a C1-C6alkyl substituted by a hydroxy group, a C1-C6alkoxy group, a C1-C6alkanoyloxy group or a carboxy group, R ₃ represents a hydrogen atom,

C1-C6alkoxy, C1-C6alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C1-C6alkyl, R 5 represents a hydroxy group, and R 6 represents C1-C6alkyl, optionally substituted with a hydroxy group, • · oo ···· · · · ·<

C1-C6alkoxy, C1-C6alkanoyloxy or carboxy and pharmaceutically acceptable salts thereof.

Another specific compound according to the invention may be a compound of general formula I, in which Rf represents a hydrogen atom or C1-C6alkyl, optionally substituted by a hydroxy group, a halogen atom, a methoxy group, an ethoxy group or an acetoxy group,

R ₂ represents hydroxymethyl, methoxymethyl or acetoxymethyl, R ₃ represents a hydrogen atom, R ₄ represents methyl, R ₅ represents a hydroxy group and R 6 represents methyl or hydroxymethyl, as well as pharmaceutically acceptable salts thereof.

Another specific compound according to the invention is a compound of general formula I, in which Ri represents -(CH ₂ ) _n -(X)-(Y), n = 0 to 4, X represents an oxy group, a thio group, -N(R _a )-, or is absent, Y represents an amino acid residue with 1 or 2 protecting groups and R ₂ represents a hydrogen atom or C 1 -C 6 alkyl, as well as pharmaceutically acceptable salts thereof.

Another specific compound according to the invention is a compound of general formula I, where R1 represents _-CH2- (N-acylcysteine residue, bonded via a sulfur atom), (S)-2-[(R)-alpha-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl or (R)-2-[(R)-alpha-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl and _R2 represents a hydrogen atom or C1-C6alkyl, as well as pharmaceutically acceptable salts thereof.

A specific compound of general formula I is a compound of the formula

O

9

·· ·· • 9 9 • · · · 9 • · · 9 9

*..··..· or a pharmaceutically acceptable salt thereof.

A specific compound of general formula II is a compound of the formula

or a pharmaceutically acceptable salt thereof.

A preferred compound of the invention is a compound of formula I in which _R4 is hydrogen or C1-C6alkyl and R5 is hydroxy or acetoxy, the absolute stereochemistry at the carbon atom bearing _R4 and R5 being (R), as well as pharmaceutically acceptable salts thereof.

The compounds of the invention can be prepared by the processes described below, in which the substituents in each process have the meanings given above, unless a different specific meaning is explicitly stated for some substituents.

The compounds of the invention, i.e. the compounds of general formula I and II and their salts, can be derived from illudin S, hydroxymethylacylfulvene HMAF (i.e. the compounds of general formula I in which R1 is _CH2OH , _R2 is methyl, _R3 is hydrogen, _R4 is methyl, R5 is OH and R6 is methyl) and fulvene (i.e. the compounds of general formula I in which R1 is hydrogen, _R2 is methyl, _R3 is hydrogen, _R4 is methyl, _R5 is OH and R6 is

44 • 4 4 4

4 4 4

4 4 4

4 4 4

4 4 4 methyl), known processes are used in the preparation of the compounds of the invention, as disclosed, for example, in WO 91/04754 and WO 94/18151.

The compound of formula I in which _R2 is hydroxymethyl can be prepared by oxidation of the corresponding compound of formula I in which _R2 is methyl. The oxidation can be conveniently carried out by the action of selenium dioxide and tert-butyl hydroperoxide under conditions analogous to those described in Example 1.

The compound of formula I, in which R-ι represents hydroxymethyl, can be prepared from the corresponding compound of formula I, in which R-ι represents a hydrogen atom, by the action of paraformaldehyde and sulfuric acid. The reaction proceeds well under the conditions described in Example 2.

A compound of formula I in which _R2 is acetoxymethyl can be obtained by acylation of the corresponding compound of formula I in which _R2 is hydroxymethyl. The acylation can be carried out with acetic anhydride under the conditions given in Example 3.

A compound of formula I in which _R2 is methoxymethyl can be prepared by reacting the corresponding compound of formula I in which _R2 is hydroxymethyl with methyl iodide and silver oxide. The reaction can be carried out under the conditions set out in Example 7.

A compound of formula I in which Rf is methoxymethyl can be prepared by reacting the corresponding compound of formula I in which R1 is hydroxymethyl with methanol and sulfuric acid. The reaction proceeds well under the conditions described in Example 8, part (a).

The compound of formula I, in which Rf represents -CH ₂ -(cysteine residue, bonded via a sulfur atom) can be obtained by reacting the corresponding compound • · ·· • · · · • · « · · ·

• · of formula I, in which R1 is hydroxymethyl, with cysteine. The reaction can be carried out under the conditions given in Example 10.

A compound of formula I in which R1 is _-CH2- (an N-acylcysteine residue bonded via a sulfur atom) can be prepared by reacting the corresponding compound of formula I in which R1 is hydroxymethyl with N-acylcysteine. The reaction can be conveniently carried out under the conditions set forth in Example 11.

The compound of formula I, in which R1 is 2-[(R)-alpha-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl, can be prepared by reacting the corresponding compound of formula I, in which R1 is _-CH2- (N-acylcysteine residue, bonded via a sulfur atom), with alpha-methylbenzylamine. The reaction proceeds well under the conditions given in Example 12.

A compound of formula I in which R1 is _-CH2- (glutathione residue, bonded via a sulfur atom) can be prepared by reacting the corresponding compound of formula I in which R1 is hydroxymethyl with glutathione. The reaction is readily carried out under the conditions given in Example 14.

A compound of formula I in which R1 is _-CH2- (cysteine residue, bonded via a sulfur atom) _-Rx or _CH2- (N-acylcysteine residue, bonded via a sulfur atom) _-Rx can be prepared by reacting the corresponding compound of formula I in which R1 is _-CH2- (cysteine residue, bonded via a sulfur atom) or _CH2- (N-acylcysteine residue, bonded via a sulfur atom) with the appropriate amino acid or peptide corresponding to _Rx . The reaction is readily carried out under the conditions given in Example 15.

• 9 • · 9 · · ♦ · · 9 9 th · ··· ·

9 9 9 9 9 • 999 9

99 999

The compound of general formula II can be prepared from illudin S by the method set forth in Example 17.

The compounds of the invention can also be prepared using the procedures and intermediates described in T. McMorris et al., Tetrahedron, 1997, 53, 44, 14579-14590, T. McMorris et al., J. Org. Chem. 1997, 62, 3015-3018, T. McMorris et al., Chem. Comm. 1997, 315-316, T. McMorris et al., Experientia, 1996, 52, 75-80 and also in US 5,439,942, US 5,439,936, US 5,523,490, US 5,536,176, WO 91/04754, WO 94/18151 and WO 98/03458. Some compounds of general formulas I and II can be used to prepare other compounds of general formula I or II.

In the case where the obtained substances are sufficiently basic or acidic to form stable non-toxic salts with acids or bases, it may be appropriate to administer these substances in the form of salts. Examples of pharmaceutically acceptable salts include addition salts with organic acids forming a physiologically acceptable anion, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, alpha-ketoglutarate and alpha-glycerophosphate. Inorganic salts include hydrochlorides, sulfates, nitrates, hydrogen carbonates and carbonates.

Pharmaceutically acceptable salts may be prepared using conventional methods known in the art, for example by reacting a sufficiently basic compound, for example an amine, with a suitable acid to form a physiologically acceptable anion. In the case of carboxylic acids, alkali metal salts, for example sodium, potassium or lithium salts, or alkaline earth metal salts, for example calcium salts, may be formed.

The compounds of the invention can be formulated into pharmaceutical compositions and administered in this form to mammals, for example, humans, in doses adjusted to the chosen route of administration, for example, orally or parenterally, intravenously, intramuscularly, topically or subcutaneously.

The compounds of the invention can be administered systemically, for example orally in combination with a pharmaceutically acceptable carrier, for example an inert diluent or an absorbable edible carrier. These materials can be administered in hard or soft gelatin capsules, can be compressed into tablets or can be mixed with the patient's diet. In the case of oral administration, the active ingredient is usually mixed with one or more excipients and then used in the form of tablets, buccal tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The said compositions should contain at least 0.1% of the active ingredient. However, the content of the active ingredient in these compositions can be varied and usually ranges from 2 to 60% by weight in individual dosage forms. The amount of the active ingredient should always be such that an effective concentration in the bloodstream is achieved.

Tablets, pills, capsules and the like may contain various excipients, for example binders such as gum tragacanth, acacia, corn starch or gelatin, bulking agents such as calcium hydrogen sulfate, disintegrants such as corn starch, potato starch, alginic acid and the like, lubricants such as magnesium stearate and sweeteners such as sucrose, fructose, lactose or aspartame or also flavouring agents such as peppermint oil or cherry flavouring. In the case where the dosage form used is a capsule, the capsule may contain, in addition to the materials of the above type, a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be used as coatings or to otherwise modify the physical properties of the solid dosage form. For example, tablets, pills or capsules may be provided with ·· ·· • · 9

9 9·· • · ·

9 9 • 9 99 • · ·· 99 ·· ·· · 9 9 9 • · · · · · · · ···· • * · · · 9 ··· 999 · 9 999 with a coating of gelatin, wax, shellac, sugar, and the like. Syrups or elixirs may contain the active ingredient, sucrose or fructose as a sweetener, methyl- and propylparabens as a preservative, and in addition a colorant and a flavoring agent, for example cherry orange flavor. It goes without saying that any material used to produce the dosage form should be pharmaceutically acceptable and substantially non-toxic in the amount used. In addition, the active ingredient may be formulated to provide a sustained or delayed release of the ingredient.

The active ingredients may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active ingredient or its salt may be prepared in water, or in a mixture with a non-toxic wetting agent. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, triacetin and mixtures of these materials, and also in oils. Under normal conditions of storage and use, these compositions will contain preservatives to prevent the growth of microorganisms.

Pharmaceutical dosage forms for administration by injection or infusion include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient and intended for the preparation of sterile solutions or dispersions immediately before use, the active ingredient may optionally be encapsulated in liposomes. In all cases, the resulting dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier may be a solvent or liquid dispersion medium, such as water, ethanol or a polyol, e.g. glycerol, propylene glycol, liquid polyethylene glycols and the like, it is also possible to use vegetable oils, non-toxic glyceryl esters and mixtures of these substances. The fluidity can be adjusted, for example, by forming liposomes and maintaining the desired particle size in the case of dispersion or by using a wetting agent. Preservatives can be used

09 · · 90 ·· • 9 · 9 · 8 8 8 8 9 9

9 9 98 9 9 9 9 9 9

9 9 8 8 9 9 9 9 8 8 9

9 9 9 * 9 9 0 0 9 • 9 99 999 099 Μ 09 various antibacterial and antifungal substances, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be advantageous to add substances to ensure the required osmotic pressure, such as sugars, buffers, or sodium chloride. Prolonged absorption of injectable preparations can be ensured by including substances that delay absorption, such as aluminum monostearate and gelatin.

Sterile injection solutions are prepared by mixing the active substance with the required amount of the appropriate solvent and, if necessary, the above-mentioned excipients, and then sterilizing the mixture by filtration. In the case of sterile powders, the sterile injection solutions are dried in a vacuum and lyophilized, thereby obtaining the active substance in powder form, containing other components that were present in the dried solution.

For topical administration, the compounds of the invention can be administered in neat form in the case of liquids. However, they are usually applied in the form of compositions containing a dermatologically acceptable carrier, solid or liquid.

Solid carriers include finely powdered solids such as talc, clays, microcrystalline cellulose, silicon dioxide, aluminum oxide, and the like. The liquid carrier that can be used may be water, alcohols or glycols, or mixtures of water, alcohol, or glycol, in which the active ingredients can be dissolved or dispersed in effective concentrations, optionally in a mixture with non-toxic wetting agents. For example, fragrances and antimicrobials can be used as auxiliaries. The resulting liquid compositions can be applied using cotton wool, used to impregnate dressings, or applied in spray form, for example in aerosol form.

99 • 9

99 » · · • 9 9 9· • 9 9 • 9 9 ·

99 • · ♦ · 99 • 9 • 9

9

999 999

9 ·

9 9

9 9 • 9 9

Thickeners such as synthetic polymers, fatty acids and their salts and esters, fatty alcohols, modified celluloses or modified inorganic materials may also be used in the liquid carrier to provide spreadable pastes, gels, ointments, soaps and the like for direct application to the skin of the user. Examples of suitable dermatological compositions with which the compound of the invention can be applied to the skin are generally known and have been described, for example, in US 4608392 (Jacquet et al.), US 4992478 (Geria), US 4559157 (Smith et al.) and US 4820508 (Wortzman).

Suitable doses of the compounds of the invention can be determined by correlating their in vitro efficacy with their in vivo efficacy in an animal model, for example in mice or dogs, using known assays developed for illudin analogues and described in US 5,439,936 and US 5,523,409, and an efficacy assay in higher mammals, including children and adult humans, has also been described in US 4,938,949 (Borch et al.).

The therapeutically effective amount of the compounds of the invention will vary with the subject being treated and the tumor being treated. It has been found that relatively high doses of representative compounds of the invention can be administered due to reduced toxicity compared to that of illudin S and M. For example, the maximum tolerated dose of illudin S is approximately 250 micrograms/kg, while compound 2 can be chronically administered at a dose of 25 milligrams/kg without toxicity. A dose of 30 to 112,000 micrograms/kg body weight is particularly suitable for intravenous administration, while an effective dose of 300 to 112,000 micrograms/kg body weight is suitable for intraperitoneal administration. It will be appreciated that the amount of active ingredient used may vary depending on the route of administration. The amount of compound or its salt or derivative used will also vary with the substance used, the route of administration, the nature of the disease being treated, and the age and general condition of the patient; the resulting dose must always be determined by the attending physician.

0* 00

0 0

0 0 0 0

0 0

0 0 0

00

0« 00 00 0· 0*00

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

000 000 00 00

The compounds of the invention are conveniently administered in dosage forms containing a single dose, for example 5 to 1000 mg, preferably 10 to 750 mg and especially 50 to 500 mg.

Ideally, the active substance should be administered to achieve maximum plasma concentrations of the active substance in the range of 0.5 to 75 microM, preferably 1 to 50 and especially 2 to 30 microM. This can be achieved, for example, by intravenous injection of a solution of the active substance with a concentration of 0.05 to 5%, optionally in physiological sodium chloride solution, or by oral administration of 1 to 100 mg of the active substance. The desired concentrations in the bloodstream can be maintained by continuous infusion of the active substance in an amount of 0.01 to 5.0 mg/kg/h or by intermittent infusion with a content of 0.4 to 15 mg/kg of the active substance.

The required dose can be administered all at once or divided throughout the day at appropriate intervals, which can be 2, 3, 4 or more partial doses throughout the day.

The cytotoxic and antitumor properties of active substances can be determined using commonly known pharmacological models or using the following tests A and B.

Test A: In vitro test

In order to determine the cytotoxic effects, different concentrations of the compounds of the invention were added to the culture of MV522 (human lung carcinoma cell line), HL60 (myeloid leukemia cells) and 8392 (B cell leukemia/lymphoma cells). For 48 hours, cell growth and cell viability were determined by trypan blue exclusion. Alternatively, the cells are plated in liquid culture on 96-well plates and exposed to φ φ φ φ • · φ • φ φφφ φ φ • φ · φ φφ φφ

ΦΦ ΦΦ ·· *· ΦΦΦΦ

Φ Φ ΦΦΦΦ

Φ

Φ Φ ΦΦΦΦ

ΦΦΦ ΦΦΦ ΦΦΦ ΦΦ different concentrations of the active substance for 2 hours, then ³ H-thymidine is added for 1 to 2 hours and the material is transferred to filter paper.

The filter papers are added to vials containing scintillation fluid and the radioactivity is measured using a beta-radiation counter.

The following Table 1 shows the results of Test A for representative compounds of the invention. The values are given as mean ± standard deviation, units are nanomoles/L, the term NT means that the test was not performed.

Table 1

2 h, _IC50 (nm/l) Compound MV522 HL60 8392 1 2640 ± 360 NT 37000 ± 2300 2 11300 ±1500 NT NT 3 19600 ± 9700 15600 ± 4600 62000 ± 3600 4 20400 ± 6300 >40000 NT 5 24000 ± 6100 38,400 ± 9000 NT 6 NT NT NT 7 7700 ± 3500 6000 ± 1200 NT 8 >80000 >80000 NT 9 >50000 49800 ± 20000 NT - 13 10000 ±1800 NT NT 14 3050 ± 550 NT NT 15 NT NT NT

Test B: In vivo test

Several representative compounds of the invention were used to perform the in vivo test. The antitumor substance mitomycin C was used as a control substance. The active substances were administered daily intraperitoneally for 5 consecutive days from the 10th day after inoculation. • · · 0 0

0 0 0 0 0

0 000 0

0 0 0 0 0

0 0 0 0

00 000 ·

00

0 0 0

0 0 0

0 0 0 • 0 0 0

00 following days. Animals were monitored for three weeks from the start of treatment. The MTD was reached for the control substance mitomycin C, but not for compounds 1 or 2.

Female BALB/c nu/nu mice, 4 weeks of age and weighing 18 to 22 g, were obtained from Simonsen Inc. (Gilroy CA) and maintained in a pathogen-free athymic mouse colony at the University of California, San Diego CA in HEPA-filtered cages. The animals were given sterilized food and water ad libitum. The animals were housed 5 per plastic cage, ventilated through polyester filters. Personnel handling the animals wore clean sterilized gowns, gloves, face masks, headgear, and boots. All experiments were performed in accordance with the NIH “Guide for Care and Use of Animals” as per the University Institutional Animal Care and Use Committee (Protocol 3-006-2).

The lung carcinoma cell line MV522 was derived according to Kelner et al., Anticancer Res., 15: 867-872 and 873-878, 1995 and maintained in antibiotic-free RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% fetal calf serum and 2 mM glutamine in a humidified carbon dioxide incubator maintained at 37°C.

Mice were randomly divided into groups of five animals in the initial experiments and into groups of 16 to 20 animals to confirm the effectiveness of the analogue. Each animal was marked on the ear and monitored individually throughout the experiment. The mice were injected with 10 million MV522 cells in the shoulder area in a single vaccination. After ten days, the subcutaneous tumors were approximately 3x3 mm in size and the animals were given the active substance. The effect of the active substance on prolonging life was then monitored.

• · · · • · · · • * ··*

9 * 9

9 9 9

9 9 9 '9 • ♦

99

9 9 * • 9 9 9

9 9 9 9

9 9 9

99

Although the MV522 cell line causes death by metastasis in mice, primary tumor growth was also monitored from the first day of treatment at weekly intervals. Tumor size was measured in two perpendicular directions. Tumor weight was determined according to the equation weight = (width) ² x length/2. Relative weight RW was calculated to standardize tumor size variability across experimental groups at the start of treatment using the formula RW = Wt/Wi, where Wi is the tumor weight of a given animal at the start of treatment and Wt is the tumor weight after a certain time since the start of treatment. Animals were sacrificed after a certain time and organs were monitored for the presence of metastases.

Kaplan-Meier survival curves were used for comparison. The relative tumor weights between groups of animals were compared using standard ANOVA followed by Tukey-Kramer multiple comparison test according to Kelner et al., Anticancer Res., 15: 867-872 and 873 and 878, 1995. The values were statistically significant at (p) < 0.05.

Test B data for representative compounds of the invention are shown in Table 2. 8 animals were used in the control group and 4 animals in each experimental group. The values given are relative tumor weight, i.e., Wt/Wi (this value was 1.0 on day 10). The active substances were administered intraperitoneally on days 10, 11, 12, 13 and 14 (QD x 5 days).

• · · · ·· 44

4 9 9 9 9 9 4 4 4 9

9 444 9 9 4 4 4 4 • 9 9 4 9 · 4 4 4 4 4 4

9 4 4 4 9 · « « · ·· 94 444 444 94 49

Table 2

Compound Day 17 Day 24 Day 31 Control (physiological NaCl solution) 4.3 ± 0.7 7.0 ± 1.3 11.8 ± 3.0 1 (14mg/kg) 1.4 ± 0.7 7.0 ± 1.3 11.8 ± 3.0 2 (35mg/kg) 2.1 ± 0.2 3.3 ± 0.3 4.0 ± 0.9 13 (32mg/kg) 2.3 ±0.5 4.1 ± 0.8 4.7 ± 1.5 14 (28mg/kg) 3.2 ± 0.6 4.1 ± 0.8 6.0 ± 1.8

The data in Tables 1 and 2 show that representative compounds of the invention are effective cytotoxic and antitumor agents.

The invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention. In the examples, melting points are given without correction, ¹ H and ¹³ C NMR spectra were measured at 300 and 75 MHz, high-resolution mass spectra were determined at the University of Minnesota Mass Spectrometry Service Laboratory, Chromatography was carried out on Davisil silica gel with a particle size of 230 to 425 mesh (Fisher Scientific) using a mixture of ethyl acetate and hexanes as eluent, analytical thin layer chromatography (TLC) was carried out on Whatman 4420 222 silica gel plates, reactions were routinely monitored by TLC. Illudin S, hydroxymethylacylfulven HMAF and fulven were prepared by the above-mentioned procedures, for example according to international patent applications WO 91/04754 and WO 94/18151.

Examples of embodiments of the invention

Example 1

Compound 1 (a compound of formula I, wherein R1 is hydrogen, _R2 is hydroxymethyl, _R3 is hydrogen, _R4 is methyl, R5 is hydroxy and _R6 is methyl.

• · flfl • flfl · • flfl flfl fl flfl flfl • flfl fl • fl flfl · • fl flfl • flfl · • flfl · • · · · · • flfl ·

To a stirred solution of 6.9 g, 32 mmol of acylfulvene in 7.0 mL of ethyl acetate EtOAc was added 1.75 g, 15.7 mmol of 99% selenium dioxide and 6 mL, 60 mmol of 90% tert-butyl hydroperoxide, density 0.901. The mixture was stirred at room temperature for 24 h and then partitioned between EtOAc and 3 x 3 mL of saturated sodium sulfite, followed by washing with saturated sodium chloride solution. The organic extracts were dried over magnesium sulfate and the crude product was chromatographed to give 922.5 mg of the title compound as a yellow-orange gum in 12% yield and 5.8 g of acylfulvene in 84% yield.

¹ H NMR (CDCIs) : 0.76 (ddd, 1H), 1.14 (ddd, 1H), 1.27 (ddd, 1H), 1.36 (s, 3H), 1.54 (ddd, 1H), 2.03 (s, 3H), 4.02 (s, 1H), 4.56 (s, 2H), 6.67 (s, 1H), 7.29 (s, 1H).

¹³ C NMR (CDCl ₃ ): 197.9, 161.5, 146.0, 140.2, 132.9, 127.0, 119.8,

76.6, 59.9, 37.5, 27.7, 17.0, 14.7, 9.9.

MS m/e 232 (M ⁺ ), UV max. wavelength (EtOH) 327.2 nm (e 7631).

Example 2

Compound 2 (compound of formula I, wherein R-ι is hydroxymethyl, R2 is hydroxymethyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and _R6 is methyl.

36.3 g of paraformaldehyde was added to 275 ml of 1.5 M sulfuric acid solution and 300 ml of Μβ2<30. The mixture was stirred and heated to dissolve the solid. The solution was then cooled to 0 °C and 922.50 mg, 3.98 mmol of compound 1 was added to the solution and the solution was stirred while allowing to warm to room temperature. After 24 h, the orange-yellow mixture was extracted with 2 x 300 ml of EtOAc, the extracts were combined, washed with 30 ml of sodium bicarbonate and then with saturated sodium chloride solution. The organic extract was dried over magnesium sulfate. Removal of the solvent and chromatography of the residue on silica gel using a mixture of ethyl acetate and hexane as eluent

4 44

4 4

4 44«

4 4 • 4 4 4 4 •4 44 444

4 4 4 • 4 4

4 4

4 4

4 4 4 >4 «4 gives 357.3 mg of the resulting product in the form of a dark yellow-orange gum in a yield of 34%.

¹ H NMR (CDCb) : 0-79 (ddd, 1H), 1.16 (ddd, 1H), 1.26 (ddd, 1H), 1.38 (s, 3H), 1.41 (ddd, 1H), 2.20 (s, 3H), 3.90 (s, 1H), 4.63 (s, 2H), 4.74 (s, 2H), 7.25 (s, 1H).

MS m/e 262 (M ⁺ ), UV max. wavelength EtOH 330.0 nm (e 5011).

Example 3

Compound 3 (a compound of formula I, wherein Rf is hydrogen, _R2 is acetoxymethyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and _R6 is methyl.

To a stirred solution of 76.0 mg, 0.328 mmol of compound 1 in 2 mL of acetic anhydride at room temperature was added 126.2 mg, 1.54 mmol of anhydrous sodium acetate. The mixture was stirred at room temperature for 24 h and then filtered to remove NaOAc. The solution was placed under reduced pressure for 4 h, after which the crude product was partitioned between EtOAc and saturated sodium bicarbonate solution, washed with saturated sodium chloride solution and dried over magnesium sulfate. After evaporation, the crude product was chromatographed on silica gel using ethyl acetate-hexane to give 50 mg of the title compound as a yellow gum in 56% yield.

¹ H NMR (CDCb) : 0.77 (ddd, 1H), 1.16 (ddd, 1H), 1.34 (ddd,

1H), 1.38 (s, 3H), 1.58 (ddd, 1H), 2.06 (s, 3H), 2.11 (s, 3H), 4.99 (s, 2H), 6.69 (s, 1H), 7.27 (s, 1H).

¹³ C NMR (CDCb) : 197.9, 170.8, 163.6, 140.5, 140.0, 132.8, 126.7,

121.9, 76.7, 61.3, 37.9, 27.8, 20.9, 17.3, 15.3, 10.3.

• · ··· · · · · · · • · · · * * ······ • · · · 4» 4» «·· · ·· ·· ··· ··· ftt »·

Example 4

Compound 4 (a compound of formula I wherein Rf is acetoxymethyl, _R2 is hydroxymethyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and Rg is methyl)

To a stirred solution of 430 mg, 1.49 mmol of compound 10 in 3 mL of EtOAc was added 186 mg, 1.67 mmol of 99% selenium dioxide followed by 0.40 mL, 4.0 mmol of 90% tert-butyl hydroperoxide with a density of 0.901.

The mixture was stirred at room temperature for 4 days and then partitioned between EtOAc and saturated sodium sulfite (3 x 3 mL), washed with brine, and dried over magnesium sulfate. After evaporation, the crude product was chromatographed to give 8.5 mg of the title compound as a yellow gum (2% yield).

¹ H NMR (CDCl ₃ ) : 0.79 (ddd, 1H), 1.17 (ddd, 1H), 1.39 (s, 3H), 1.43 (ddd, 1H), 1.52 (ddd, 1H), 2.09 (s, 3H), 2.14 (s, 3H), 3.93 (s, 1H), 4.65 (q, 2H), 5.21 (q, 2H), 7.32 (s, 1H).

MS m/e 304 (M ⁺ ), UV max. wavelength EtOH 330.6 nm (e 5950).

The intermediate of formula 10 can be prepared as follows, a. Compound 10

Anhydrous sodium acetate is added to 6 ml of a stirred solution of 1.4 g,

5.7 mmol of HMAF in acetic anhydride at room temperature. After 18 hours, the mixture was filtered and the resulting solution was stored under reduced pressure for 4 hours. The resulting material was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate, then washed with saturated sodium chloride solution and the resulting ethyl acetate solution was dried over magnesium sulfate. The solution was then evaporated and purified by chromatography on silica gel using ethyl acetate/hexane as eluent to give 1.4 g of compound 10 as a yellow-orange gum in 85% yield.

¹ H NMR (CDCl ₃ ) : 0.74 (ddd, 1H), 1.11 (ddd, 1H), 1.36 (s, 3H), 1.51 (ddd, 1H), 2.04 (s, 3H), 2.09 (s, 3H), 2.17 (s, 3H), 3.90 (s, 1H), 5.10 (s, 2H), 7.11 (s, 1H).

Example 5

Compound 5 (a compound of formula I wherein R1 is acetoxymethyl, _R2 is acetoxymethyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and R6 is methyl)

To a stirred solution of 2 mg, 0.0066 mmol of compound 4 in 15 microliters of methylene chloride and 135 microliters of acetic anhydride was added 1 mg, 0.038 mmol of sodium acetate. The reaction mixture was stirred for 4 hours at room temperature, after which the solid was filtered off. The mixture was placed under reduced pressure to remove the acetic anhydride. The crude product was then partitioned between ethyl acetate and saturated sodium bicarbonate solution, washed with saturated sodium chloride solution and dried over magnesium sulfate. After evaporation, the residue was chromatographed on silica gel using ethyl acetate-hexane to give 0.9 mg of the product as a yellow gum in 40% yield. ¹ H NMR (CDCls) : 0.79 (ddd, 1H), 1.15 (ddd, 1H), 1.39 (s, 3H), 1.40 (ddd, 1H), 1.58 (ddd, 1H), 2.09 (s, 3H), 2.093 (s, 3H), 2.10 (s, 3H),

3.89 (s, 1H), 5.08 (s, 2H), 5.17 (s, 2H), 7.25 (s, 1H).

MS m/e 346 (M ⁺ ), UV max. wavelength EtOH 332.1 nm (e 8378).

Example 6

Compound 6 (compound of formula I, wherein R-ι is hydroxymethyl, R ₂ is acetoxymethyl, R ₃ is hydrogen, R ₄ is methyl, R ₅ is hydroxy and R 6 is methyl)

To a stirred solution of 76.0 mg, 0.290 mmol of compound 2 in

To 1.5 ml of acetic anhydride was added 45.3 mg, 0.552 mmol of sodium acetate. The reaction mixture was stirred for 75 minutes at room temperature and then ·· ·· · · · · · · · · · · · · · « ···· ·

A ···· · · ···

4U ·· ·· ·· ·· ·· ·· ·· ·· the solid was filtered off. The mixture was partitioned between ethyl acetate and saturated sodium bicarbonate solution, washed with saturated sodium chloride solution and dried over magnesium sulfate. The extract was then evaporated under reduced pressure and the residue was chromatographed on silica gel using ethyl acetate-hexane to give 6.9 mg of compound 4 as a yellow gum, 10.0 mg of compound 2 as a yellow-orange gum, minor amounts of 3 other products including compound 5 and in addition 10.0 mg of the final product as a yellow gum in 11.3% yield.

¹ H NMR (CDCb) : 0.81 (ddd, 1H), 1.17 (ddd, 1H), 1.38 (s, 3H), 1.60 (ddd, 1H), 2.06 (s, 3H), 2.24 (s, 3H), 3.89 (s, 1H), 4.74 (m, 2H), 5.03 (m, 2H), 7.23 (1H).

MS m/e 304 (M ⁺ ), UV max wavelength EtOH 331.3 nm (e 5921).

Example 7

Compound of formula 7 (compound of formula I, wherein R 1 is hydrogen,

R ₂ is methoxymethyl, R ₃ is hydrogen, R ₄ is methyl, R 5 is hydroxy and R ₆ is methyl)

To a stirred solution of 300 mg, 1.29 mmol of compound 1 in 10 mL of CH ₃ CN was added 1 mL of CH 3 I and 110 mg, 0.475 mmol of Ag ₂ O. The mixture was stirred for 3 days at room temperature and then filtered and evaporated. The residue was chromatographed on silica gel using ethyl acetate-hexane to give 133 mg of compound 1 and additionally 53 mg of the final product as a dark orange gum in 17% yield. ¹ NMR (CDCb) : 0.76 (ddd, 1H), 1.13 (ddd, 1H), 1.34 (ddd, 1H), 1.38 (s, 3H), 2.05 (s, 3H), 3.41 (s, 3H), 3.96 (s, 1H), 4.35 (s, 2H), 6.66 (s,

1H), 7.28 (s, 1H).

¹³ C NMR (CDCb) : 197.8, 161.8, 143.3, 140.2, 133.1, 126.7, 120.9,

76.6, 69.6, 58.3, 37.6, 27.8, 17.1, 14.9, 10.0.

• · • ·

Example 8

Compound 8 (a compound of formula I wherein R1 is methoxymethyl, R2 is hydroxymethyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and _R6 is methyl)

To a stirred solution of 830 mg, 3.19 mmol of compound 11 in 10 mL of ethyl acetate was added 150 mg, 1.35 mmol of 99% selenium dioxide followed by 1 mL, 10.0 mmol of tert-butyl hydroperoxide, density 0.901. The reaction mixture was stirred at room temperature for 5 days and then partitioned between diethyl ether and saturated sodium sulfide, washed with saturated sodium chloride solution and dried over magnesium sulfate. After evaporation, the crude material was chromatographed to give 47.2 mg of the resulting product as a dark orange gum in 5% yield.

¹ H NMR (CDCl ₃ ) : 0.74 (ddd, 1H), 1.08 (ddd, 1H), 1.32 (s, 3H), 1.33 (ddd, 1H), 1.47 (ddd, 1H), 2.08 (s, 3H), 3.14 (brs, 1H), 3.35 (s, 3H),

4.41 (q, 2H), 4.51 (br s, 2H), 7.28 (s, 1H).

a. Compound 11

Compound 11, used as an intermediate in this example, is a compound of formula I in which R1 is methoxymethyl, _R2 is methyl, R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and _R6 is methyl and can be prepared as follows.

To a stirred solution of 320 mg, 1.3 mmol of HMAF in 3 mL of Me ₃ CO were added 3 mL of methanol and 3 mL of 1 M sulfuric acid. The reaction mixture was stirred at room temperature for 24 h and then extracted with diethyl ether. The extracts were combined, washed with saturated sodium bicarbonate and then saturated sodium chloride, and dried over magnesium sulfate. After evaporation, the residue was chromatographed on silica gel using ethyl acetate/hexane to give 290 mg of compound 50 as a dark orange gum in 86% yield.

¹ H NMR (CDCl ₃ ) : 0.62 (ddd, 1H), 0.98 (ddd, 1H), 1.24 (ddd, 1H), 1.27 s, 3H), 1.37 (ddd, 1H), 2.00 (s, 3H), 2.04 (s, 3H), 3.26 (s, 3H), 3.91 (br s, 1H), 4.29 (q, 2H), 7.0 (s, 1H).

¹³ C NMR (CDCb) : 197.6, 159.7, 142.6, 138.8, 134.3, 129.7, 126.6,

75.9, 65.3, 57.3, 37.3, 27.3, 15.7, 13.9, 12.9, 9.1.

Example 9

Compound 9 (a compound of formula I wherein R1 is methoxymethyl, _R2 is methoxymethyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and R6 is methyl)

To a stirred solution of 240 mg, 0.87 mmol of compound 8 in 15 mL of _CH3CN was added 1.5 mL of _CH3I and 150 mg, 0.647 mmol of _Ag2O . The reaction mixture was stirred at room temperature for 48 h, filtered and evaporated. The residue was chromatographed on silica gel using ethyl acetate-hexane to give 25 mg of the title compound as a yellow-orange gum in 10% yield.

¹ H NMR (CDCb) : 0.76 (ddd, 1H), 1.11 (ddd, 1H), 1.37 (s, 3H), 1.40 (ddd, 1H), 1.53 (ddd, 1H), 2.13 (s, 3H), 3.36 (s, 3H), 3.38 (s, 3H), 3.91 (s, 1H), 4.37 (q, 2H), 4.43 (q, 2H), 7.27 (s, 1H).

¹³ C NMR (CDCb) : 198.0, 163.4, 142.8, 139.1, 132.6, 132.0, 127.2,

76.6, 67.2, 65.5, 58.3, 57.8, 38.1, 27.5, 16.3, 14.8, 9.8.

Example 10

Compound 12 [a compound of formula I in which R1 is _-CH2- (cysteine, bonded via a sulfur atom), _R2 is methyl, _R3 is hydrogen, _R4 is methyl, R5 is hydroxy and R6 is methyl]

To a solution of HMAF in a 1:1 mixture of acetone and 1 M sulfuric acid solution, an equivalent amount of cysteine is added. The mixture is stirred overnight at room temperature. A large amount of ethyl acetate is then added and the water is removed with magnesium sulfate. Also added

Φ Φ 4

Φ Φ

Φ Φ solid sodium bicarbonate to neutralize the sulfuric acid. The solution is filtered, evaporated, and the residue is chromatographed to give compound 102 as a yellow gum.

¹ H NMR (CDCb) : 0.78 (m, 1H), 0.89 (m, 1H), 1.06 (m, 1H), 1.31 (s, 3H), 1.43 (m, 1H), 2.15 (s, 3H), 2.21 (s, 3H), 2.91 - 4.02 (m, 8H), 7.04 (s, 1H).

Example 11

Compound 13 [a compound of formula I wherein Rj is -CH ₂ -(N-acylcysteine, bonded to a sulfur atom), R ₂ is methyl, R ₃ is hydrogen, R ₄ is methyl, R ₅ is hydroxy and R 6 is methyl]

To a solution of 36 mg, 0.146 mmol of HMAF in 3 ml of a 1:1 mixture of 1 M sulfuric acid solution and acetone at room temperature is added

22.4 mg, 0.137 mmol of N-acetylcysteine.

The mixture was stirred for 22 hours and then extracted with ethyl acetate. The organic extracts were washed with saturated sodium bicarbonate and then saturated sodium chloride and dried over magnesium sulfate. After evaporation, the crude product was chromatographed using ethyl acetate/hexane with 2-5% acetic acid to give 45.5 mg (85%) of compound 103 as a yellow gum.

¹ H NMR (CDCl 2 ) : 0.72 (m, 1H), 1.09 (m, 1H), 1.23 (m, 1H), 1.36 (s, 3H), 1.47 (m, 1H), 2.07 (s, 3H), 2.10 (s, 3H), 2.13 (s, 3H), 2.97 (m,

1H), 3.14 (m, 1H), 3.82 (dd, 3.82), 4.80 (m, 2H), 6.56 (d, J = 7.2 Hz),

7.10 (s,1H).

MS m/e 391 (M ⁺ ), 373, 229, 185, HRMS for C ₂₀ H ₂₅ NO ₅ S calcd 391.1455, found 391.1452.

Example 12

Compound 14 (compound of formula I in which R1 is (S)-2-[(R)44 • · ·· · · · · ·

-alpha-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl, R ₂ is methyl, R ₃ is hydrogen, R 4 is methyl, R ₅ is hydroxy and R 5 is methyl)

To a solution of 40 mg, 0.102 mmol of compound 13 in 1 mL of methylene chloride was added 20 mg, 0.132 mmol of N-hydroxybenzotriazole, 20 μl, 0.12 mmol of N,N-diisopropylcarbodiimide, and 12 μl, 0.093 mmol of (d)-(+)-alpha-methylbenzylamine. The mixture was stirred at room temperature for 1.5 h and then partitioned between ethyl acetate and water. The organic extract was dried over magnesium sulfate. After evaporation, the crude product was chromatographed to give 33.6 mg of compound 14 as a yellow gum in 73% yield.

¹ H NMR (CDCl3) : 0.70 (m, 1H), 1.07 (m, 1H), 1.29 (m, 1H), 1.35 (s, 3H), 1.48 (d, J = 6.9 Hz, 3H), 1.51 (m, 1H), 1.93 (s, 3H), 2.09 (s, 3H),

2.10 (s, 3H), 2.91 (m, 2H), 3.84 (dd, 2H), 4.61 (m, 1H), 5.03 (m, 1H), 6.64 (d, J = 7.8 Hz, 1H), 7.07 (s, 1H), 7.26 (m, 5H).

¹³ C NMR (CDCb) : 197.5, 170.4, 169.2, 159.7, 142.6, 142.00, 138.3,

134.9, 129.8, 128.7, 127.4, 126.4, 125.9, 77.4, 52.5, 49.3, 37.7, 34.4,

29.8,27.6, 23.1, 22.1, 16.3, 14.3, 13.1, 9.5.

HRMS for _C28H34N2O4S calcd _494.2241 and found 494.2225.

Example 13

Compound 15 (compound of formula I, wherein R1 is (R)-2-[(R)alpha-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl, _R2 is methyl, _R3 is hydrogen, _R4 is methyl, _R5 is hydroxy and _R6 is methyl)

Chromatography from Example 12 also yielded a total of 5.3 mg of compound 15 in the form of a yellow gum in 13% yield, ¹ H NMR (CDCl ₃ ): 0.70 (m, 1H), 1.07 (m, 1H), 1.32 (m, 1H), 1.34 (s, 3H), 1.45 (m, 1H), 1.48 (d, J = 6.9 Hz, 3H), 2.03 (s, 3H), 2.05 (s, 6H),

2.76 (m, 1H), 2.87 (m, 1H), 3.73 (dd, 2H), 4.50 (m, 1H), 5.03 (m, 1H), ·· ·· • 9 ·· ··

9

9 99

6.46 (d, J = 7.5 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 7.05 (s, 1H), 7.31 (m, 5H)

HRMS for C28H34N2O4S calcd 494.2241, found 494.2238.

Example 14

Compound 16 [compound of formula I, wherein Rf is -CH ₂ -(glutathione, bonded via a sulfur atom), R ₂ is methyl, R ₃ is hydrogen,

R ₄ is methyl, R 5 is hydroxy and R ₆ is methyl]

Compound 16 can be prepared by the method of Example 10 with the difference that the cysteine derivative is replaced by a glutathione derivative.

Example 15

General procedure for the preparation of compounds in which R1 is -CH ₂ -(cysteine, bonded via a sulfur atom)-peptide, -CH ₂ -(cysteine, bonded via a sulfur atom)-amino acid, -CH ₂ -(N-acylcysteine, bonded via a sulfur atom)-peptide or -CH ₂ -(N-acylcysteine, bonded via a sulfur atom)-amino acid.

Using solid phase peptide synthesis, the amino acids Leu, Phe and Gly can be combined in a known manner to yield the following tripeptides and dipeptides bound to a solid resin.

-Leu-Leu-Leu -Leu-Leu-Phe -Leu-Leu-Gly -Leu-Phe-Leu -Leu-Phe-Phe -Leu-Phe-Gly -Leu-Gly-Leu -Leu-Gly-Phe -Leu-Gly-Gly -Phe-Leu-Leu -Leu-Leu-Phe -Phe-Leu-Gly -Phe-Phe-Leu -Phe-Phe-Phe -Phe-Phe-Gly -Phe-Gly-Leu -Phe-Gly-Phe -Phe-Gly-Gly -Gly-Leu-Leu -Gly-Leu-Phe -Gly-Leu-Gly -Gly-Phe-Leu -Gly-Phe-Phe -Gly-Phe-Gly -Gly-Gly-Leu -Gly-Gly-Phe -Gly-Gly-Gly

-Leu-Leu -Leu-Phe -Leu-Gly -Phe-Leu -Phe-Phe -Phe-Gly -Gly-Leu -Giy-Phe -Gly-Gly

An acidic resin with the following structure was used as the solid phase for these reactions, where P stands for polystyrene divinylbenzene resin

The first, N-(9-fluorenylmethoxycarbonyl) protected amino acid was coupled to the resin by forming a bond between the C-terminal end and the hydroxyl group of the resin using a mixture of DIPC (diisopropylcarbodiimide) and DMAP (4-dimethylaminopyridine). The percent yield was calculated after coupling and if it was lower than 90%, the coupling was repeated until the percent yield was higher than 90%. Then, the Fmoc protecting group from the first amino acid was removed by treating it with 20% piperidine in 1-methyl-2-pyrrolidinone, NMP. The Kaiser test was used to verify the result of the removal of the protecting group. 3 Kaiser reagents, ninhydrin, phenol and potassium cyanide, were added to the sample to form a light yellowish solution. The mixture was heated for 3 minutes at 100 °C and if the color of the solution changed to dark purple, the Fmoc group could be considered removed. The next amino group was attached by peptide bond formation using a DIPC/HOBT (1-hydroxybenzotriazole) mixture. NMP was used as the solvent for removing the protecting group and dichloromethane was used for washing and drying. Then the Kaiser test was used again, in

9

9 «

9 99

9 9

9 9 ♦ « 9 9 *

9 9 9

9 9 9

9 99 case, if there was no color change, the binding was successful. The binding reaction was repeated with additional amino acids. Compound 13 was bound to the last amino acid or to the peptide using dichloromethane as the solvent. NMP was not used as the solvent for the binding of compound 13 because this substance causes the inversion of the chirality center in cysteine and a racemic mixture is formed. In addition, HOBT was not used in the binding reaction with compound 13 because this substance can also cause the formation of a mixture of products. The peptide was then cleaved from the resin by the action of 10% acetic acid in dichloromethane. The products were subjected to tests in ultraviolet light and by mass spectrometry. HMAF has two absorption maxima in ultraviolet light at wavelengths of 210 and 330 nm, so the presence of the products could be confirmed by the presence of these two bands. Mass spectroscopy was also used to confirm the presence of the desired products.

Using this general procedure, compound 13 was coupled to 36 of the above tripeptides and dipeptides and also to the amino acids Leu, Phe and Gly individually, thereby obtaining 39 compounds of the invention. The N-acetyl group can be removed from these compounds using similar conditions as in Example 11, thereby obtaining a further 39 compounds of the invention. In a compound of general formula I, R _x may therefore preferably be Leu, Phe, Gly or any of the above tripeptides and dipeptides.

Example 16

Compound 17 (compound of formula I wherein R1 is (S)-2-(2-carboxypyrrolidin-1-ylcarbonyl)propylthiomethyl, R2 is methyl, R3 is hydrogen, R4 is methyl, R5 is hydroxy and R6 is methyl)

Compound 17 can be prepared in a similar manner to the compound from Example 10, with the difference that cysteine is replaced by 1-(3• ·

94

4 4 4 • 4 4 4

4 4 4 9 • 4 4 4 ·· * · · • 4 9 94

4 4 4 • 4 9 4

44 • 4 44

-mercapto-2-methyl-1-oxopropyl)-L-proline, i.e. captopril.

Example 17

Compound 18 (a compound of formula II wherein _R2 is methyl, _R3 is hydrogen, _R4 is methyl, R5 is hydroxy, R6 is methyl and R7 is hydroxymethyl.

178.5 mg of illudin S is dissolved in 2.0 ml of acetic anhydride and 96 mg of sodium acetate is added. After 24 hours, a mixture of monoacetate and diacetate is obtained. 171 mg of monoacetate is dissolved in dichloromethane and 695 mg of pyridinium dichromate is added. The mixture is stirred for 21 hours at room temperature. After working up the mixture, 118 mg of dehydroilludin S-monoacetate is obtained, which is dissolved in 5 ml of acetone and 3 ml of 1 M sulfuric acid, after standing for 12 hours, 66 mg of compound 18 is obtained.

¹ H NMR (CDCb) : 0.63 (1H), 1.13 (1H), 1.23 (4H), 1.38 (4H), 2.05 (3H), 3.62 (1H), 3.76 (1H), 6.82 (1H).

Example 18

Representative pharmaceutical dosage forms containing compounds of formula I or II (compound X) and intended for therapeutic or prophylactic use in humans will be presented below.

(i) tablet 1 mg/tablet compound X 100.0 lactose 77.5 polyvinylpyrrolidone 15.0 croscarmellose sodium 12.0 microcrystalline cellulose 92.5 magnesium stearate 3.0

300

99

9 9 9

9 9 9

9 9 9

9 9 9

9 9 9

99 • ·9

9 9 99 • 9 9 9 • 9 9 9

9 9

(ii) tablet 2 mg/tablet compound X 20.0 microcrystalline cellulose 410.0 starch 50.0 sodium starch glycolate 15.0 magnesium stearate L0 500 (iii) capsules mg/capsule compound X 10.0 colloidal silicon dioxide 1.5 lactose 465.5 pregelatinized starch 120.0 magnesium stearate 3.0 - · · · „ 600.0 (iv) injection 1 (1 mg/ml) mg/ml compound X (free) 1.0 sodium hydrogen phosphate 12.0 sodium dihydrogen phosphate 0.7 sodium chloride 1.0 N sodium hydroxide, 4.5 adjusting pH to 7.0 to 7.5 as needed water for injection up to 1 ml

(v) injection 2 (10 mg/ml) mg/ml compound X (free) 10.0 sodium dihydrogen phosphate 0.3 sodium hydrogen phosphate 1.1 polyethylene glycol 400 200.0 0.1 N sodium hydroxide

• · ·· fl flfl • flfl flfl • · · • · · • fl flfl fl · fl • flflfl fl · • fl flfl • · I • · <

adjusting pH to 7.0 - 7.5 as needed water for injection up to 1 ml (vi) aerosol mg/vial compound X 20.0 oleic acid 10.0 trichlorofluoromethane 5,000.0 dichlorodifluoromethane 10,000.0 dichlorotetrafluoroethane 5,000.0

The above types of compositions can be prepared by conventional methods.

The invention has been described in connection with various specific and preferred embodiments, but it is obvious that many variations and modifications could be made which would also fall within the scope of the invention.

Claims (51)

PATENT CLAIMS An antitumor compound of the general formula I wherein R 1 is hydrogen, hydroxy, mercapto, amino, halogen, carboxy, nitro or - (CH 2) n - (X) - (Y), n is an integer from 0 to 4, X is -O-, thio -S-, -N (R _a ) - or absent, Y is C 3 -C 6 cycloalkyl, aryl, heteroaryl, a saccharide, amino acid, peptide residue or a 1 to 15-membered branched or unbranched hydrocarbon chain, optionally containing 1, 2 or 3 non-peroxide oxy, thio or -N (R _a ) - groups, wherein: this chain is optionally substituted on a carbon atom with 1, 2 or 3 substituents from oxo = O, hydroxy, carboxy, halogen, mercapto, nitro, -N (R b) c, C 3 -C 6 cycloalkyl, aryl, heteroaryl, saccharide residue, amino acids or peptides and at the same time the chain is saturated or unsaturated, R ₂ represents a carboxy group, C 1 -C 6 alkanoyl Φ 0 00 0 · 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 • 0 0 0 0 0 · · · 000 000 C 1 -C 6 alkoxycarbonyl, C 1 -C 6 alkyl halogen, -C (= O) NR _d R _e , a saccharide, amino acid, peptide or C 1 -C 6 alkyl residue substituted with 1 or 2 hydroxy groups, C 1 -C 6 alkoxy groups, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate residues or -C (= O) NR _d R _e , R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ is hydrogen or C 1 -C 6 alkyl, R ₅ is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R 5 together form ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a saccharide, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 to 2 hydroxy substituents, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R _g , R _a represents a hydrogen atom, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl a R b, R c, R a, R e, R f and R g independently represent a hydrogen atom, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or forms R _b and R _c or R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, wherein any aryl, heteroaryl , aryloxy or heteroaryloxy in the definition of Y, or R ₃ is optionally substituted with 1, 2 or 3 substituents selected from C1-C6alkyl, C1-C6alkoxy, C1 6 alkanoyl, C 1 -C C6alkanoyloxyskupina, C 1 -C 6 alkoxycarbonyl, hydroxyC1-C6alkyl, halogenC1 -C 6 alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein R _h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl, or together form With a nitrogen atom to which the pyrrolidine, piperidine or morpholine ring, as well as the pharmaceutically acceptable salts thereof, are attached. 2. Antitumor agents of formula I R (I) wherein R 1 is - (CH ₂ ) n - (X) - (Y), wherein n is an integer from 0 to 4, X is oxy, thio, -N (R _a ) - or absent, Y is amino acid residue protected with 1 or 2 protecting groups, peptide residue or a 1 to 15-membered branched or unbranched hydrocarbon chain, optionally containing 1, 2 or 3 non-peroxide oxy, thio or -N (R _a ) - groups, which chain is substituted with 1 , 2 or 3 peptide residues and is saturated or unsaturated, R ₂ is hydrogen or C1-C6 alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy; R ₄ is hydrogen or C1 -C6 alkyl and R ₅ is hydroxy , C 1 -C 6 alkoxy or C 1 -C C6alkanoyloxyskupinu or consisting of R ₄ and R 5 together ethylenedioxy radical, R ₆ represents hydrogen, carboxyl, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a saccharide, amino acid or peptide residue, or C 1 -C 6 alkyl, optionally substituted with 1 to 2 substituents from the hydroxy group, • · · · 9 9 · C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate residues, or -C (= O) NR _f R _g , R _a is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl; R b, R c, R a, R e, R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R b and R _c or R _d and R _e or R f and R _g together with the nitrogen atom; to which a pyrrolidine, piperidine or morpholine ring is bonded. wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group of Y or R ₃ is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 C6alkyl, halogenC1-C6 alkyl, hydroxy, halo, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein R _h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. 3. Antitumor agents of formula I 9 9 99 99 9 9 9 9 Wherein R 1 represents a hydrogen atom, a hydroxy group, a mercapto group, an amino group, a halogen atom, a carboxy group, a nitro group or (CH ₂ ) _n - (X) ) - (Y), ⁿ is an integer from 0 to 4, X is oxy, thio, -N (R _a ) - or absent, Y is C 3 -C 6 cycloalkyl, aryl, heteroaryl, carbohydrate, amino acid or peptide residue or a 1 to 15-membered branched or unbranched carbon chain, optionally containing 1, 2 or 3 non-peroxide oxy, thio or -N (R _a ) - groups, the chain being optionally substituted with 1, 2 or 3 groups selected from oxo, hydroxy, carboxy, halogen, mercapto, nitro, -N (R _b ) (R _c ), C 3 -C 6 cycloalkyl, aryl, heteroaryl, carbohydrate, amino acid or peptide residue, the chain may be saturated or unsaturated, R ₂ is hydrogen or C 1 -C 6 alkyl, R 3 is hydrogen, C 1 - C6 alkyl, C1 6 alkoxy, C1 C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy; R ₄ is hydrogen or C1 -C6 alkyl, _R5 is hydroxy, C 1 -C 6 alkoxy or C 1 -C C6alkanoyloxyskupinu or _R4 and _R5 together form ethylenedioxy, R6 is carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, -C (= O) NR _f R _g, a saccharide residue, amino acid or peptide or a C1-C6alkyl, substituted with 1 or 2 substituents selected from C1-C6alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate residue or -C (= O) NR _f R _g , R _a represents a hydrogen atom, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl and R _b , R _c , Rd, R e, R f and R g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _b and R _c , or R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or a morpholino ring wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group Y or R 3 is optionally substituted with 1, 2 or 3 substituents from the group C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, 9 9 9 9 9 9 · 9 9 · 9 · ·· 99 9 9 9 ·· C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 alkyl, halogenC 1 -C 6 alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (R _h ) (R j), where R _h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. Anti-cancer agents according to claims 1, 2 or 3, wherein R f represents - (CH 2) _n - (X) - (Y), n = 0 to 4, X represents an oxy group, a thio group, -N (R _a ) - or is absent and Y represents a peptide residue or C 1 -C 6 alkyl substituted with a peptide residue. Anti-cancer agents according to claim 1 or 3, wherein R f represents a hydroxy group, a halogen atom, a carboxy group, an aryl, a heteroaryl, a saccharide residue, an amino acid or a peptide. The antitumor agents of claim 1 or 3, wherein R f is hydrogen or C 1 -C 6 alkyl, optionally substituted with 1 or 2 substituents selected from hydroxy, halogen, methoxy or ethoxy. The antitumor agents of claim 1 or 3, wherein R f is - (CH ₂ ) _n - (X) - (Y), n = 1 or 2, X is oxy, thio or -N (R _a ) - and Y is C1-C6 alkyl or C2-C6 alkenyl, optionally substituted with 1 or 2 groups selected from hydroxy, carboxy, halo, mercapto, nitro, N (R b) (R _c) C 3 -C 6 cycloalkyl, aryl, heteroaryl or a radical of saccharide, amino acid, or peptide, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group of Y is optionally substituted with 1, 2 or 3 substituents from the group C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl; 444 4 4 4 5 4 4 4 4 4 4 4 4 4 4 4 4 C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 alkyl, halogenC 1 -C 6 alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (R _h ) (R j), where R _h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, The antitumor agents of claim 1, 2 or 3, wherein R f is CH ₂ (a sulfur-bound cysteine residue) -R _x , wherein R _x is an amino acid or peptide residue of 2 to 24 amino acids. The antitumor agent of claim 8, wherein R _x is -Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO: 4), -Asp-Gly-Arg-Cys (SEQ ID NO: 5) or -Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO: 6). The antitumor agent according to claim 8, wherein R _x is a peptide residue containing 4 to 20 amino acid residues. Anti-cancer agents according to claim 1 or 3, wherein R 1 is -CH ₂ (a cysteine residue linked via a sulfur atom) or -CH ₂ (a Nacylcysteine residue linked via a sulfur atom). Anti-cancer agents according to claim 1, 2 or 3, wherein R-t is 2 - [(R) - alpha-methylbenzylaminocarbonyl] -2 (acylamino) ethylthiomethyl. Anti-cancer agents according to claim 1, 2 or 3, wherein R 1 is -CH ₂ - (a glutathione residue linked via a sulfur atom). • · · · · · · · · · · · · · · · ··· Anti-cancer agents according to claim 1 or 3, wherein R 1 represents a hydrogen atom, methyl, hydroxymethyl, methoxymethyl or acetoxymethyl. The antitumor agents of claim 1, wherein R ₂ is hydroxymethyl, methoxymethyl or acetoxymethyl. The antitumor agents of claim 1, wherein R ₂ is carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl or -C (= O) NR _d R _e . The antitumor agent of claim 1, wherein R ₂ is C 1 -C 6 alkyl, substituted with 1 or 2 substituents selected from hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _d R _e . The antitumor agent according to claim 1, 2 or 3, wherein R ₄ is methyl. Anti-cancer agents according to claim 1, 2 or 3, wherein R 5 is hydroxy. Anti-cancer agents according to claim 1 or 2, wherein R 6 represents a hydrogen atom. Anti-cancer agents according to claim 1, 2 or 3, wherein R 6 is carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl or -C (= O) NR _f R _g . Anti-cancer agents according to claim 1 or 2, wherein R 6 is C 1 -C 6 alkyl, optionally substituted with 1 or 2 hydroxy groups, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R _g . The antitumor agent according to claim 1 or 2, wherein R ₆ is methyl or hydroxymethyl. The antitumor agent of claim 1, wherein R ₂ is C 1 -C 6 alkyl, substituted with hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy or carboxy, R 3 is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, aryl, heteroaryl , aryloxy or heteroaryloxy, R ₄ is hydrogen or C 1 -C 6 alkyl, R 5 is hydroxy, and R ₆ is C 1 -C 6 alkyl, optionally substituted with hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy or carboxy, and pharmaceutically acceptable salts thereof. Anti-cancer agents according to claim 1, wherein R 1 is hydrogen or C 1 -C 6 alkyl optionally substituted by hydroxy, halogen, methoxy, ethoxy or acetoxy, R ₂ is hydroxymethyl, methoxymethyl or acetoxymethyl, R 3 is hydrogen, R ₄ is methyl R ₅ is hydroxy and R ₅ is methyl or hydroxymethyl, as well as pharmaceutically acceptable salts thereof. The antitumor agent according to claim 2, wherein R 1 is - (CH ₂ ) _n - (X) - (Y), n = 0 to 4, X is oxy, thio, -N (R _a ) - or is absent and Y means an amino acid residue provided with one or a wood protecting group. The antitumor agent of claim 2, wherein R 1 is -CH ₂ (a sulfur-bound N-acylcysteine residue), (S) -2 - [(R) -alpha-methylbenzylaminocarbonyl] -2- (acylamino) ethylthiomethyl, or (R) -2 - [(R) -alpha-methylbenzylaminocarbonyl] -2- (9) · - (acylamino) ethylthiomethyl. A dimeric compound consisting of the two anti-tumor agents of claim 1 linked by a linker. 29th antitumor compounds of formula I wherein Ri is - (CH2) _n - (X) - (Y) n represents an integer of 1-4, preferably 1, X is oxy, thio, or -N _(Ra) - preferably a thio group, Y represents a 2 to 15-membered branched or unbranched carbon chain, respectively. containing 1, 2 or 3 non-peroxide oxy, thio or -N (R _a ) - groups, the chain is substituted on a carbon atom of 1, 2 or 3, substituents from oxo, carboxy, mercapto, -N (Rb) (R _c ) , C 3 -C 6 cycloalkyl, aryl, heteroaryl, a saccharide, amino or peptide residue and is saturated or unsaturated, preferably Y is 2 to 6, 6 membered branched or unbranched uhiíkový chain substituted on a carbon atom by 1, 2 or 3 substituents selected from oxo, heteroaryl, amino acid residue or peptide residue, R2 is hydrogen or C1C6alkyl, R ₃ is hydrogen, C1C6alkyl, C1 C 6 alkoxy, C 1 -C 6 alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents hydrogen or C 1 -C 6 alkyl and R 5 represents hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R 5 together represent ethylene R6 is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC1-C6alkyl, -C (= O) NR f R _g, a saccharide residue or a peptide or amino C1-C6 alkyl optionally substituted with 1 or 2 substituents selected from hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate residues or -C (= O) NR _f R _g , R _a represents a hydrogen atom, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl and R b, R _c , R f and R g independently represent C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R b and R _c or R f and R g together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group Y or R ₃ is optionally substituted with 1, 2 or 3 substituents from the group C 1 -C 6 alkyl (, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, halogenC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. The antitumor agent of claim 29, which is or a pharmaceutically acceptable salt thereof. • ······· · · · · · ··· ··· ·· ·· 31. An antitumor compound of formula II _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R ₅ is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together are ethylenedioxy, R 6 is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 substituents from hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R g, R 2 represents a carboxy group, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _d R _e , a saccharide, amino acid or peptide residue or C 1 -C 6 alkyl, optionally substituted with 1 or 2 substituents from hydroxy, C 1 -C 6 C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate residue, or -C (= O) NR d R _e , · · · · · · · · · · · · · · · · · · · · · · · · · ·· R _d , R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or a morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 , halogenC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the atom nitrogen to which a pyrrolidine, piperidine or morpholine ring is bound, as well as pharmaceutically acceptable salts thereof. The antitumor agent of claim 32, wherein R 7 is C 1 -C 6 alkyl, substituted with 1 or 2 substituents selected from hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _d R _e . The antitumor agent of claim 32, wherein R 7 is hydroxymethyl. The antitumor agent of claim 32, which is or a pharmaceutically acceptable salt thereof. φ φ An antitumor agent comprising a compound according to claim 1, 2, 3, 29 or 32 and a reagent capable of binding to a tumor-associated antigen. 36. The antitumor agent of the general formula wherein A is - (CH _{2) n} - (X) - (Y) n = 1-4, X is oxy, thio, or -N (R _a ) -, Y is a peptide residue or C 1 -C 6 alkyl substituted with a peptide residue, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ is hydrogen or C 1 -C 6 alkyl; R 5 is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R ₆ represents hydrogen, carboxyl, C 1 -C 6 alkanoyl, ♦ 9 9 · «· 9 9 99 99 • ♦ ♦ 9 9 9 9 9 • 9 9 9 9 C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue or C 1 -C 6 alkyl, optionally substituted with 1 or 2 substituents from hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid , peptides or carbohydrate or the group -C (= O) NR _f R _g , Rd, R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form Rd and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 alkyl, halogenC 1 , hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj are independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached are bound by a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. 37. Antitumor agents of formula I wherein Ri means - (ΟΗ ₂ ) _Π - (Χ) - (Υ) n = 1 to 4, ·· 00 • 0 ·· ·· • · 0 0 0 0 0 · · 0 0 · 00 00 X is oxy, thio, or -N (R _a ) -, Y is C 2 -C 6 alkenyl, optionally substituted with 1 or 2 substituents from oxo, hydroxy, carboxy, halogen, mercapto, nitro, -N (R _b ) (R _c ), C 3 -C 6 cycloalkyl, aryl, heteroaryl, saccharide residue, amino acids or a peptide, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R 5 is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, haloC 1 -C 6 alkyl, -C (= O) NR _f R _g , a saccharide, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 substituents from the group hydroxy, C1-C6alkoxy, C1-C6alkanoyloxyskupina, carboxy, amino acid residue, peptide or carbohydrate or -C (= O) NR _f R _g, R _d , R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or a morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 , halogenC 1 -C 6 alkyl, hydroxy, halogen, carboxy, mercapto, nitro or. 4 · · · ·· ·· -N (Rh) (R j), wherein R _h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. 38. The antitumor agent according to claim 1, wherein: R 1 is -CH 2 - (a sulfur-bound cysteine residue) -R _x , where R _x is an amino acid or peptide residue of 2 to 24 amino acids, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R ₅ is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, haloC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy substituents, C 1 -C 6 alkoxy, C 1 -C 6 alkanoy (oxy, carboxy, amino acid, peptide or carbohydrate residue, or -C (= O) NR _f R _g , R _d , R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, 00 00 0 0 00 00 • · · 0 0 0 · 0 0 0 Φ • 0 0 0 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 C 1 -C 6 alkanoyl, phenyl or benzyl or forms R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted 1, 2 or 3 substituents from the group C1-C6alkyl, C1-C6alkoxy, C1-C6alkanoyl, C1-6alkanoyloxy, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, halogenC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (-) R h) (R j) wherein R h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof substances. A compound according to claim 1, wherein R 1 is -CH ₂ - (a sulfur-bound N-acylcysteine residue) R _x , wherein R _x is an amino acid or peptide residue of 2 to 24 amino acids, R ₂ represents C 1 -C 6 alkyl, R ₃ represents hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6aikylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R ₅ is hydroxy, C 1 -C 6 alkoxy or 9 • 9 " 9 9 9,999 9,9 9 9 9 9 9 9 9 9 9 C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue, or C 1 -C 6 alkyl, optionally substituted with 1 or 2 substituents from hydroxy, C1-C6alkoxy, C1-C6alkanoyloxy, carboxy, *. an amino acid residue, a peptide or a carbohydrate, or a group -C (= O) NR _f R _g , Rd, R e, R f and R g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or when R <I R _e or R f and R _g together with the nitrogen atom to which they are attached form a pyrrolidine, piperidine or morpholine ring wherein any aryl, heteroaryl, aryloxy or heteroaryloxy R ₃ is optionally substituted 1, 2 or 3 C 1 -C 6 alkyl, C 1 -C 6 alkoxy substituents, C1-C6alkanoyl, C1-6alkanoyloxy, C1-C6alkoxycarbonyl, hydroxyC1-C6alkyl, halogenC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj independently represent hydrogen, C1 -C6alkyl, C1-C6alkanoyl, phenyl or benzyl, or together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. Anti-cancer agents according to claim 1, of the general formula I (I) 9 9 9 9 9 9 99 99 where 9 R 1 is -CH 2 - (a glutathione residue bonded via a sulfur atom), R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R 5 is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy substituents, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate residues, or -C (= O) NR _f R _g , R _d , R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or a morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted with 1,2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 , halogenC1-C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the atom nitrogen to which a pyrrolidine, piperidine or morpholine ring is bound, as well as pharmaceutically acceptable salts thereof. 9 · 99 ti • 9 · 9 9 99 9 9 9 9 99 ·· 99 99 9 9 9 99 99 41. The antitumor agent according to claim 1, wherein: EMI15.0 [0100] Rt is 2 - [(R) -alpha-methylbenzylaminocarbonyl] -2 (acylamino) ethylthiomethyl, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R ₅ is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together are ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue, or C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy substituents, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R _g , Rd, R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or morpholine ring , φ · · · φ φ φ φ φ φ φ φ φ φ · · φ φ φ φ · · · · · φ φ φ φ φ φ φ φ φ φ lová lová lová lová group, aryloxy or heteroaryloxy R ₃ is optionally substituted with 1, 2 or 3 substituents selected from C1-C6alkyl, C1-C6alkoxy, C1 6 alkanoyl, C1-6alkanoyloxyskupina, C 1 -C 6 alkoxycarbonyl, hydroxyC1-C6alkyl, halogenC1-C6 alkyl, hydroxy, halogen , carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or a morpholine ring; pharmaceutically acceptable salts thereof. 42. The antitumor agent of claim 1, wherein R 1 is - (CH ₂ ) _n - (X) - (Y), n = 1 to 4, X is oxy, thio, or -N (R _a ) -, Y represents a branched or unbranched carbon chain of 2 to 15 carbon atoms, substituted on the carbon atom by 1 or 2 substituents from the group carboxy, C 3 -C 6 cycloalkyl, heteroaryl, a sugar, amino acid or peptide residue, said chain being saturated or unsaturated, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R 5 is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, haloC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy substituents, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R _g , Rd, R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or morpholine a ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R ₃ is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 alkyl, halogenC 1 -C 6 alkyl -C6alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj), wherein Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the nitrogen atom, to which are bound a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof. The antitumor agents according to claim 1, of the general formula (I), wherein the flfl flfl flfl flfl. R 1 is - (CH 2) n - (X) - (O. n = 1, X is thio, Y represents a 2 to 6-membered branched or unbranched carbon chain substituted on the carbon atom by 1, 2 or 3 substituents selected from oxo, heteroaryl or the remainder of the peptide, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R 5 is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R ₅ together form ethylenedioxy, R 6 is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, halogenC 1 -C 6 alkyl, -C (= O) NR _f R _g , a carbohydrate, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 substituents from hydroxy, C 1 -C6alkoxy, C1-C6alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R _g , R _d , R _e , R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or a morpholine ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy group R 3 is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 haloC 1 -C 6 alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (Rj) wherein Rh and Rj independently represent hydrogen, C1-C6alkyl, C1-C6alkanoyl, phenyl or benzyl or form together with the nitrogen atom to which they are attached a pyrrolidine, piperidine or morpholine ring, as well as a pharmaceutically acceptable acceptable salts thereof. 44. The antitumor agent according to claim 1, wherein the compound of formula I is a compound of formula (I) R 1 is hydroxy, carboxy, aryl, heteroaryl or a saccharide, amino acid or peptide residue, _R2 is C1-C6alkyl, R ₃ is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C C6alkylthioskupinu, aryl, heteroaryl, aryloxy or heteroaryloxy, R ₄ represents a hydrogen atom or C 1 -C 6 alkyl and R ₅ is hydroxy, C 1 -C 6 alkoxy or C 1 -C 6 alkanoyloxy or R ₄ and R 5 together are ethylenedioxy, R ₆ is hydrogen, carboxy, C 1 -C 6 alkanoyl, C 1 -C 6 alkoxycarbonyl, haloC 1 -C 6 alkyl, -C (= O) NR _f R _g , a saccharide, amino acid or peptide residue or C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy substituents, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyloxy, carboxy, amino acid, peptide or carbohydrate, or -C (= O) NR _f R _g , Rd, R e, R f and R _g independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form R _d and R _e or R f and R _g together with the nitrogen atom to which they are attached pyrrolidine, piperidine or morpholine a ring, wherein any aryl, heteroaryl, aryloxy or heteroaryloxy R ₃ is optionally substituted with 1, 2 or 3 substituents from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkanoyl, C 1-6 alkanoyloxy, C 1 -C 6 alkoxycarbonyl, hydroxyC 1 -C 6 alkyl, C 6 alkyl, hydroxy, halogen, carboxy, mercapto, nitro or -N (Rh) (R j), wherein R _h and R j independently represent hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkanoyl, phenyl or benzyl or form together with the nitrogen atom, to which a pyrrolidine, piperidine or morpholine ring, as well as pharmaceutically acceptable salts thereof, are bonded. 45. A pharmaceutical composition comprising as an active ingredient an antitumor agent according to any one of claims 1, 2, 3, 29, 30, 31, 34, 36, 37, 38, 39, 40; 41, 42, 43 or 44 in combination with a pharmaceutically acceptable diluent or carrier. The antitumor agents according to claims 1, 2, 3, 29, 30, 31, 34, 36, 37, 38, 39, 40, 41, 42, 43 or 44 for use in medical purposes. The antitumor agent according to claim 46 for use in the treatment of cancer. Use of the antitumor agents according to claims 1, 2, 3, 29, 30, 31 34, 36, 37, 38, 39, 40, 41, 42, 43 or 44 for the manufacture of a pharmaceutical composition for the treatment of cancer. The use of claim 48, wherein the cancer is a solid tumor. • 9 50. A method of inhibiting tumor cells, comprising contacting the cells in vitro or in vivo with an effective amount of the antitumor agents of any one of claims 1, 2, 3, 29, 30, 50, 50, 50, 31, 34, 36, 37, 38, 39, 40, 41.42, 43 or 44. 51. A method of treating cancer comprising administering an effective amount of an antitumor agent according to any one of claims 1, 2, 3, 29, 30, 31, 34, 36, 37, 38, 39, 40, 41. 42, 43 or 44.