NL8502383A - SUBSTITUTED 7-OXOMITOSANS. - Google Patents

Description

VO 7335

Substituted 7-oxomitosanes.

The present invention provides new mitomycin analogs containing a disulfide group, as well as methods for preparing them. These compounds are mitomycin A analogues in which the 7-alkoxy group carries an organic substituent containing a disulfide group.

Mitomycin A is an antibiotic that has been proven useful, and its 7-0-substituted mitosan analogs have similar utility.

The present invention provides new mitomycin analogs containing a disulfide group (Class 260, Subclass 326.24), as well as methods for preparing it. These compounds are mitomycin A analogues in which the 7-alkoxy group carries an organic substituent which is a disulfide group. contains. The present invention also provides a method of preparing mitomycin A and derivatives thereof (Class 260, Subclass 326.24). Mitomycin A is an antibiotic whose utility has been proven, and its 7-O-substituted mitosan analogs have similar utility.

Nomenclature - The Systematic Chemical Abstract Name for Mitomycin A Based on a Recent Revision [Shirhata et al., J. _Am. Chem. Soc., 105, 7199 (1983)] is: 20 (1S- (lae, 8β, 8aa, 8bB) -8 - [((aminocarbonyl) oxy) methyl] - 6,8a-dimethoxy-1,1a, 2, 8,8a, 8b-hexahydro-5-methyl-arizino [21,31,3,4] pyrrolo [1,2-a] indole-4,7-dione by which the azirinopyrroloindole ring system is numbered according to formula 1 of the formula sheet (Chemical Abstract Numbering).

A trivial nomenclature system that has been widely used in mitomycin literature identifies the aforementioned ring system including several of the characteristic substituents of the mitomycins as mitosane of formula 2.

According to this system, mitomycin A is 7,9a-dimethoxymitosane and mitomycin C is 7-amino-9a-methoxymitosane. Regarding the stereochemical configuration of the products of the invention, when designated by the strain name "mitosane" or a structural formula, it is intended to indicate the stereochemical configuration as in mitomycin A or C.

15 0 2 38 3: ï Λ «-2-

Mitomycin A corresponds to formula 3 wherein R ^ R ^ OCH ^.

Mitomycin C corresponds to formula 3 wherein R = NH 2 and R 4 OCH 4.

Mitomycin C is an antibiotic prepared by fermentation and currently marketed with the Food and Drug Administration's approval in the therapy of metastatic adenocarcinoma of the stomach or pancreas in proven combinations with other approved chemotherapeutic agents and as a palliative treatment when other have failed (Mutamycin - Bristol Laboratories, Syracuse, New York 13221, Physicians' Desk Reference 37th 10 Edition, 1983, pp. 747 and 748). Mitomycin C and its preparation by fermentation is the subject of U.S. Patent 3,660,578 of May 2, 1972, which takes precedence over previous filings, including one filed in Japan on April 6, 1957.

The structures of mitomycins A, B, C, and of porfiromycin were first published by JS Webb et al. Of Lederle Laboratories Division American Cyanamid Company, J. Am. Chem. Soc., 84, 3185-3187 (1962). One of the chemical transformations used in this structural study to relate mitomycin A and mitomycin C was the conversion of the former compound, 7,9a dimethoxymitosane, by reaction with ammonia in the latter compound, 7-amino-9a-methoxymitosane. Displacement of the 7-methoxy group of mitomycin A was found to be a reaction of great importance for the preparation of anti-tumor active derivatives of mitomycin 25C. Recently, the stereochemical configurations of positions 1, 1a, 8a and 8b have been shown as the above has been indicated in connection with the Chemical Abstract nomenclature [Shirhata et al., J. Am. Chem. Soc., 105, 7199-7200 (1983)]. The previous literature refers to the enantiomer.

The following articles and patents deal, inter alia, with the conversion of mitomycin A to a 7-substituted amino mitomycin C derivative with anti-tumor activity. The aim of this study was to prepare derivatives that are more active and in particular toxic water than mitomycin C: 35 Matsui et al., J. Antibiotics, XXI, 189-198 (1968);

Konishita et al., J. Med. Chem. 14, 103-109 (1971);

Iyengar et al., J. Med. Chem., 24, 975-981 (1981); 8502383

4K

-3-

Iyengar, Sami, Remers and Bradner, Abstracts of Papers, 183rd Annual Meeting of the American Chemical Society, Las Vegas, Nevada, March 1982, Abstract No. MEDI 72;

Cosulich et al., U.S. patent no. 3,332,944, July 25, 1967; 5 Matsui et al., U.S. patent Nb. 3,420,846, January 7, 1969;

Matsui et al., U.S. patent no. 3,450,705, June 17, 1969;

Matsui et al., U.S. patent no. 3,514,452, May 26, 1970;

Nakano et al., U.S. patent Nb. 4,231,936, November 4, 1980;

Remers, U.S. patent no. 4,268,676, May 19, 1981.

The following patent applications relate to the preparation of 7-substituted amino-mitomycin-C derivatives in which the substituent comprises a disulfide bridge.

Kono et al., European Patent Application Nb. 116,208 (1984);

Vyas et al., British Patent Application No. 2,140,799, (1984).

In an article by Urakawa et al., J. Antibiotics, 23, 804-809 (1980), 7-alkoxy-substituted mitosans, which are structurally related to mitomycin A, are described as useful antibiotics with activity in experimental durables.

Mitomycin C is the major mitomycin produced by fermentation and is the commercially available form. Current technology for the conversion of mitomycin C to mitomycin A has a number of shortcomings. Hydrolysis of mitomycin C into the corresponding 7-hydroxy-9a-methoxy-mitosane and subsequent methylation of that substance requires diazomethane, a substance which is very dangerous for production scale handling, and the 7-hydroxy intermediate is very unstable [Matsui et al. , J. Antibiotics, XXI, 189-198 (1968)].

An attempt to avoid these difficulties includes the use of 7-acyloxymitosamen (Kyowa Hakko Kogyo KK Japanese Patent Nb. J5 6073-085, Farmdoc NO. 56227 D / 31). Alcoholysis of mitomycin A as described by Urakawa et al., J. Antibiotics, 23, 804-809 (1980) is limited to the preparation of only specific 7-alkoxy structures due to the availability and reactivity of the starting materials used alcohol.

The present invention relates to a group of mito-35 mycine A analogs with a dithio-organic substituent incorporated in 8502333 - - κ -4 - the 7-position alkoxy group. The compounds can be represented by the general formula I, wherein R is an organic group, namely the structural component of an organic thiol of the formula II R SH, and Alk 2 and R have the meanings set forth below.

These compounds are also described by formulas II and III, in which:. Alk. a straight or branched chain alkylene group having 1-6 carbon atoms of 13 when R is connected thereto via a carbon atom, and 2-6 carbon atoms when R ^ is connected thereto via a sulfur, oxygen or nitrogen atom thereof, and R ^ and -SS- in that case, is attached to different carbon atoms,

Alk2 represents a straight or branched chain alkylene group of 2-6 carbon atoms optionally bearing an A substituent, wherein the sulfur and oxygen atoms associated therewith and the optional A substituent attached thereto via oxygen, sulfur or nitrogen different carbon atoms of Alk 2 are bonded, wherein the .alpha.-substituent is selected from the group consisting of one or two C 1 -alkyl, C 1. alkanoyl, C-alkoxy, halogen, 1-10-1-b g alkoxycarbonyl, cyano, g alkylamino, C 1-8 dialkylamino, C.. alkanoylamino and C, c alkoxycarbonyl / 1-b 1-b

Alk 2 and Alk 2 may contain a double bond, R * represents hydrogen, lower alkyl, lower alkanoyl, benzoyl, or substituted benzoyl, wherein the substituent is lower alkyl, lower alkoxy, halogen, amino or nitro, R 4 is selected from the group consisting of halogen, carboxy, alkanoyloxy of 1-7 carbon atoms, hydroxy in which the oxygen atom is connected with Alk ^ of 3-6 carbon-30 atoms, alkylamino or dialkylamino of 1-12 carbon atoms, N-alkoxy-alkylamino with 2-7 carbon atoms, alkanoylamino of 1-7 carbon atoms, benzoylamino or B-substituted benzoylamino, naphthoylamino or B-substituted 850 2 3 8 3 * * -5-substituted naphthoylamino, phenylamino or B-substituted phenylamino, cycloalkyl or B- substituted cycloalkyl each having 3-8 members in the ring, cycloalkenyl or B-substituted cycloalkenyl each having 5-8 members in the ring, phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and heteroa 1-2-membered licyclic groups, having 3-8 members in each ring and having 1-2 hetero atoms in each ring, selected from oxygen, nitrogen and sulfur, pyridylamino or thiazolylamino, alkoxy or alkylthio, each having 1- 6 carbon atoms, alkoxycarbonyl or alkyl aminocarbonyl each containing 2-7 carbon atoms, aminocarbonyl, phenoxycarbonyl or B-substituted phenoxycarbonyl, phenoxy or B-substituted phenoxy, naphthoxy or B-substituted naphthoxy, alkoxycarbonylamino with 2-6 carbon atoms, -NHC0NH N-alkyl urylene (-NHCONHalkyl) with 2-7 carbon atoms, N-haloalkyl urylene with 3-7 carbon atoms, 3-N, haloalkyl-N-nitrosoureylene with 3-7 atoms, dialkylaminocarbonyl with 3 -13 carbon atoms, 20 dialkylaminoalkoxy of 4-13 carbon atoms, alkanoylaminoalkoxy of 3-7 carbon atoms and hydroxyalkylamino or Ν, Ν-dihydroxyalkylamino each of 2-8 carbon atoms, wherein the B substituent is selected from the group consisting of one or two low alkyl, low alkanoyl, low alk oxy, halogen, amino, carboxy, hydroxy and nitro groups, and 4 R is selected from the group consisting of alkyl of 1-12 carbon atoms, alkenyl or alkynyl of 3-12 carbon atoms each, cycloalkyl or B-substituted cycloalkyl of 3 -8 membered ring, cycloalkenyl or B-substituted cycloalkenyl 30 each having 5-8 membered ring, phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and heteroalicyclic groups with 1-2 rings, 3-8 members in each ring and 1-2 heteroatoms in each ring, selected from oxygen, nitrogen and sulfur, provided that the heterocyclic group is attached via an 8502333 '* 6 carbon atom bonded to at least one other carbon atom (ie the -SS- bonded carbon atom cannot itself be attached to two other heteroatoms), the B substituent being selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, carboxy, hydroxy 4 or nitro groups, and R and the adjacent sulfur atom together form S-cysteinyl wherein the S-cysteinyl group may be esterified, salted or incorporated into a non-toxic and non-allergenic peptide, or a non-toxic pharmaceutically acceptable salt thereof.

The compounds of the present invention are inhibitors of experimental tumors in animals. In particular, the substances identified herein as the compounds of Examples XVII, XX and XXI-XXXIV are novel substances. They are used in a similar manner to mitomycin C. The dosages used are adjusted in proportion to their toxicities relative to the toxicity of mitomycin C. In cases where the new compound is less toxic, a higher dose is used.

In another aspect of the present invention, a new method for preparing mitosans of formulas II and III is provided. This new process involves reacting a mitosane of formula IV with a triazene of formula V or formula VI wherein 13 4 R, R, R, Alk ^ and Alk ^ as defined above Ar is the organic residue of a diazotizable aromatic amine .

In a variant of the present invention, another method is provided for the preparation of mitosans of formulas II and III. This method involves reacting a thiol of formula VII or formula VIII with a mitosane derivative of formula Ib.

The disulfide mitosanes of formula Ib are prepared by the triazene method described herein. More specifically, the mitosane of formula Ib wherein Alk2 is ethylene and R1 represents hydrogen is described in Example XX.

In another aspect of the present invention there is provided an improved process for preparing compounds of IX wherein: 8502383 * 1-7- represents hydrogen or alkyl of 1-6 carbon atoms, and g R represents 12 alkyl or substituted alkyl, C3-12 cyclalkyl or substituted cycloalkyl wherein the carbon atom thereof connected to the 7-oxygen atom of mitosane carries 1-2 hydrogen atoms and the substituents are selected from the group consisting of halogen, alkoxy, alkanoyl, C- ,. aroyl, cyano, trihalomethyl, amino, C, mono-6-14 lo alkylamino, C 2-12 dialkylamino C 6-12 ary 3 C 6-12 ar 10 oxy, C-alkanoyloxy, C aroyloxy, heterocyclo having 1 to 14 1 or 2 rings and 5 to 12 ring atoms, including up to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and wherein each of the alkoxy, alkanoyl, aroyl, aryl, arvloxy, alkanoyloxy, aroyloxy, and heterocyclo substituents optionally contains 1-2 substituents selected from halogen, alkoxy, alkanoyl, cyano, trihalomethyl, amino, C. alkylamino, or C-. dialkyl-1-b 2-12 amino groups.

Many of the compounds of formula IX are known compounds with inhibitory activity against experimental animal tumors in vivo. A number of new compounds corresponding to formula IX have also been prepared by this method, and are believed to be part of the present invention. In particular, the substances designated herein as compounds of Examples XIV, XV, XVI, and XIX are novel substances, and also have anti-tumor activity against experimental animal tumors. These compounds are part of the present invention. They are used in a similar manner to mitomycin C. The dosages used are adjusted in proportion to their toxicities relative to the toxicity of mitomycin C. In cases where the new compound is less toxic, a higher dose is used.

The new method of preparing compounds of formula IX includes a reaction of a mitosane of formula X with a triazene of formula XI wherein R and R are as defined above and Ar is the organic residue of a diazotizable aromatic amine.

Q Λ ^ t ·: o 7 J *, v <ς yj «Y o * *% -8-

The terms "lower alkyl," "lower alkoxy," and "lower alkanoyl" used herein mean (unless the context dictates otherwise) straight or branched chain alkyl, alkoxy, or alkanoyl groups of 1-6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl , isobutyl, tert-butyl, 5 amyl, hexyl, etc. Preferably, these groups contain 1-4 carbon atoms, and most preferably contain 1 or 2 carbon atoms, unless otherwise indicated, the term "halogen" intended to include chlorine, fluorine, bromine and iodine. The term "non-toxic pharmaceutically acceptable salt" is intended to include salts of the compounds of formulas I and II with any non-toxic pharmaceutically acceptable acid or base. Such acids are well known and include hydrogen chloride, hydrobromide, sulfuric, sulfamic, phosphoric, nitric, maleic, fumaric, succinic, oxalic, benzoic, methanesulfonic, tartaric, citric, campulphonic, levulinic and the like. Such bases are also well known and include, for example, non-toxic metal salts such as sodium, potassium, calcium and magnesium salts, the ammonium salt, and salts with non-toxic amines, for example trialkylamines, procaine, dibenzylamine, pyridine, N-methylmorpholine, N methylpiperidinine and the like. The salts are prepared by methods known per se.

The present invention provides a new process for the preparation of compounds of formula IK which comprises a reaction of a mitosane of formula X with a triazene of formula XI as depicted in Scheme 1, wherein R and R are as defined above and Ar is the organic residue of a diazotizable aromatic amine. ~

The 1-substituted-3-aryltrizenes of formula XI and more particularly 1-alkyl-3-aryltrizenes form a class of reagents known to be useful for reaction with carboxylic acids to form the corresponding lower alkyl ester. The compound 1-methyl-3- (4-methylphenyl) triazene can be prepared according to the general procedures described by E.H. White et al. In Org. Syn., 48, 102-105 (1968) and as described in this description under Procedure 1 35. However, this procedure only works well with water-soluble amines, and a second procedure described by it K i-} \; * «<ij -w '4 # £ = V.' / V / 0 * - - 9- EH White et al., Tetrahedron Letters, No. 21, 761 (1961) and also described herein under Procedure 2, is more suitable for the preparation of trizenes from water-insoluble amines.

The reagent 1-methyl-5- 3- (4-methylphenyl) triazene prepared in the manner described above has previously been used for the preparation of methyl esters of carboxylic acids such as 2,4-dinitrobenzoic acid [E.H. White et al., Org. Syn. 48, 102-105 (1968)] and cephalosporanic acids yielding the desired Δ-compound without isomerization to the Δ-isomer [Mangia, Tetrahedron Letters, Nb. 52, p. 5219-20 (1978)]. Reagent 10 has also been used to prepare a 3-methoxy-cephalosporin derivative by reaction with the corresponding 3-hydroxy-3-cephem-4-carboxylate in benzene solution at the reflex temperature. (Wiederkeher et al., U.S. Patent No. 4,069,324, January 17, 1978).

Likewise, other 1- (lower alkyl) -3-aryltrizenes of formula XI may be prepared by reacting other low allylamines with aryl diazonium salts in a similar manner. Any arylamine of 6-12 carbon atoms that readily forms a diazonium salt can be used as the source of the aryl portion of the 1,3-diisubstituted triazene. Some examples of trizenes prepared in this manner and used in the present invention are as follows: 1- (n-butyl) -3- (4-methylphenyl) triazene; 1- (1-methyl-ethyl) -3- (4-methylphenyl) triazene; 1- (4-methylphenyl) -3- [2- (4-morpholinyl) ethyl] triazene; 1- (4-methylphenyl) -3- [2- (2-pyridyl) ethyl] triazene; 1- (2-benzylthiolethyl) -3- (4-methylphenyl) triazene; 1- (4-chlorophenyl) -3- (2-methoxyethyl) triazene; 1- (4-chlorophenyl) -3- (1,3-dioxol-2-ylmethyl) triazene; 1- (4-chlorophenyl) -3- (tetrahydrofuran-2-ylmethyl) triazene.

Other tririazenes have been described in the literature which are suitable reagents for use in the present process to prepare 7- (substituted alkoxymitosane) of formula IX. Examples thereof are those described by T.A. Daniels et al.,

Can. J. Chem., 55, 3751-3754 (1977). These are compounds of formula IV in which the symbols have the following meanings: fr ^ r \ r * λ ÖO'U Δ 0 b - -10-

a X = Η, Y = CN b X = NC> 2, Y = CN C X = C02Me, Y = CN d X = Ac, Y = CN

5 e X = N02, Y = C02Et f X = C02Me, Y = C02Et g X = C02Me, Y = COPh h X = no2, Y = -CH (OCH3) 2

Further examples of suitable triazene starting materials of formula XI for use in the present invention are as follows: 1- (n-butyl) -3- (a-naphthyl) triazene; 1- (n-hexyl) -3-phenyltriazene; 1-ethyl-3- (2,4-dimethylphenyl) triazene; 1- (1-methylethyl) -3- (4-methoxyphenyl) triazene.

For the preparation of mitomycin A, 3-methyl-1- (4-methylphenyl) triazene is preferably used as the methylation reagent. Preferably, at least two molecular amounts of the latter compound are used per molecular amount of 7-hydroxy-9a-methoxy-mitosane and the reaction is preferably carried out in a liquid organic solvent for the 7-hydroxy-9a-methoxymitosane starting material. Preferred solvents are the lower alkanols, lower alkyl esters of lower alkanoic acids, the di-lower alkyl ethers, the cyclic aliphatic ethers, and the low polyhalogenated aliphas

hydrocarbons. These solvents have up to 6 carbon atoms, but solvents at temperatures below 100 ° C

cooking is preferred. Particularly preferred solvents are methylene chloride, methanol, diethyl ether, ethyl acetate, and mixtures thereof. The reaction can be carried out at the reflux temperature of the reaction mixture or up to about 60 ° C. At temperatures above 30, the mitosane reaction component tends to decompose leading to a decrease in yield. It is preferred that the reaction is carried out at room temperature or below, for example in the range of 0 to 25 ° C.

-9502 38 3 -11-

A convenient way to determine when the reaction is complete is by thin layer chromatography. Mitomycin A has a deep purple color and can be easily distinguished from the starting material and by-products. In the solvent system raethylene chloride / methanol (90/10), mitomycin A exhibits an R 1 value of 0.36. Neutral alumina chromatography can be used to purify the product.

The above reaction conditions and precautions are broadly applicable to the preparation of other 7-R O mitosans of formula IX according to the present process.

The novel process of the present invention using 1-substituted-3-aryltrizenes can also be used to prepare compounds of formula II or III in which a mitosane of formula IV is reacted with a triazene of formula V or formula VI as deduced in scheme 2, wherein R, R, 4 R, Alk2 and Alk2 are as defined above and Ar is the organic residue of a diazotizable aromatic amine.

Aryl triazene of formula V or VI may be prepared in a similar manner as described above for the preparation of aryl trizenes of formula XI, except that the alkyl amines used therein are replaced by amino disulfides of formula XII which are also described with the formulas XIII and XIV.

Amino disulfides of formula XIII and formula XIV are known compounds and can be prepared by various methods.

For example, they can be prepared by reacting a suitable thiol 3 4 R Alk.SH or R SH with a Bunte salt of the formula XV or with "a sulfenyl thiocarbonate of the formula XVI.

By Klayman et al., J. Org. Chem., 29. 3737-3738 (1964), the following compounds have been prepared by the Bunte salt method: 2-aminoethyl n-butyl disulfide? 2-aminoethyl n-hexyl disulfide; 2-aminoethyl n-octyl disulfide; 2-aminoethyl n-decyl disulfide; 2-aminoethyl phenyl disulfide; 2-aminoethyl benzyl disulfide.

8502383 * λ * -12-

Methanol was found to be the preferred reaction solvent for the reaction of the Bunte salt with the thiol. Reaction temperatures from 0 ° C to -10 ° C have been found to be preferable using this solvent. Higher temperatures were required when using other solvents. The main drawback of this method is the formation of symmetrical disulfides as a by-product, probably due to a disproportionation of the desired mixed disulfide.

The mixed disulfide starting materials of formula XIII and XIV are preferably prepared via a reaction of the appropriate thiol with a sulfenyl thiocarbonate of formula XVI. This is the method of S.J. Brois et al., J. Am. Chem. Soc., 92, 7629-7631 (1970). Typical of this preparation procedure is the addition of the thiol to a methanol solution of the amino alkyl sulfenyl thio carbonate of formula XVI and continuing the reaction at a temperature in the range of 0-25 ° C. The reaction times vary from almost instantaneous to several hours depending on the thiol used. The progress of the reaction can be monitored by measuring the presence of unreacted thiol in the reaction vessel. When the reaction proceeds slowly, a catalytic amount of triethylamine 20 can be added as a reaction accelerator.

The 1- (substituted disulfide) -3-aryltrizenes of formula V or VI are prepared by reacting amino disulfides of formula XII with aryl diazonium salts or a similar method as described herein for the preparation of aryl trizenes of formula XI. Any aryl-25 amine of 6-12 carbon atoms that readily forms a diazonium salt can be used as a source for the aryl portion of the 1,3-two-substituted triazene. Some examples of disulfide trizenes prepared in this manner and used in the present invention are as follows: 1- [2- (2-acetamidoethyldithiol) ethyl] -3- (4-methylphenyl) triazene; 1- [2- (3-nitro-2-pyridyldithio) ethyl] -3- (4-methylphenyl) triazene.

Further examples of suitable triazene starting materials of formula V or VI for use in the present invention are as follows: 8502383 Λ i -13- 1- [2- (3-nitro-2-pyridyldithio) ethyl] -3- (4- chlorophenyl) triazene; 1- [2- (3-nitro-2-pyridyldithio) propyl] -3- (4-methylphenyl) triazene; 1- [2- (2-pyridyldithio) ethyl] -3- (4-methylphenyl) triazene; 1- [2- (phenyldithio) ethyl] -3- (4-methylphenyl) triazene; 1- (2- (butyldithio) ethyl] -3- (4-methylphenyl) triazene; 1-f2- (4-methoxyphenyldithio) ethyl] -3- (4-methylphenyl) triazene; 1- [2- (4 - nitrophenyldithio) ethyl] -3- (4-methylphenyl) triazene; 1- {2 - [(2-benzoylaminoethyl) dithio] ethyl} -3- (4-methylphenyl) triazene; 1-f2- (4-chloro-2 -naphthyldithio) ethyl] -3- (4-methylphenyl) triazene; 10 1-f2- (cyclopropylmethyldithio) ethyl] -3- (4-methylphenyl) triazene; 1- {2- [2-phenoxyethyl) dithio] ethyl} - 3- (4-methylphenyl) triazene.

In a preferred embodiment of the present invention, there is provided another process for the preparation of disulfide mitosans of formula la, wherein R is an organic group, viz. The structural component of an organic thiol of formula R SH, which is 3 3 4 is also described with R Alk ^ or R wherein R, R and Alk ^ are as defined above.

For the preparation of the disulfide mitosanes of formula la it is preferred to use the 9a-methoxy-7- [2— (3-nitro-2-pyridyldithio) ethoxy] mitosane of formula XVII in a thiol-2 exchange process with a suitable organic thiol of formula R SH as depicted in reaction scheme 3. The driving force behind the formation of the disulfides of formula Ia is the stability of the by-product, namely 3-nitro-2-mercaptopyridine, which only exists in the form of the thion of formula XVIII.

When it is desired to prepare mitosans of formula II or III in which Alk 2 does not represent ethylene, such as trimethylene or propylene, the appropriate triazene of formula V or VI is used in the procedure depicted in scheme 2 for the preparation ve 30 disulfide mitosanes of formula Ib wherein Alk2 and R1 are as defined above.

Two general synthetic procedures have been described here for the preparation of both lipophilic and hydrophilic mitosans of formula Ia. General Procedure A is used for the preparation of lipophilic or moderately soluble disulfides of Formula Ia, while General Procedure 8 is used for water-soluble disulfides with formula Ia, which are preferably isolated in the form of sodium salts or in the form of zwitterions. Preferably, at least 2 one equivalent of the mercaptan R SH per equivalent of mitosane of formula XVII is used, and the reaction may be conducted in the presence of about one equivalent base per equivalent of mercaptan of 2 of the formula R SH. Preferred bases are the tertiary amines, for example triethylamine, N-methylmorpholine, N-methylpiperidine, pyridine, 2,6-lutidine and the inorganic bases, for example sodium-10 bicarbonate, potassium carbonate, potassium bicarbonate and the like. Suitable inert solvents for the reaction of starting materials of formula XVII and R SH are the low alkanols, low alkyl esters of low alkanoic acids, low aliphatic ketones, the cyclic aliphatic ethers, the low polyhalogenated aliphatic hydrocarbons and water. The organic solvents have up to 8 carbon atoms, but the solvents boiling at temperatures below 100 ° C are preferred. Specifically preferred solvents are methylene chloride, methanol, acetone, water and mixtures thereof. The reaction can be carried out at the reflux temperature of the reaction mixture or up to about 60 ° C. It is preferred that the reaction be carried out at room temperature or below, for example in the range of 0-25 ° C.

The above reaction conditions and precautions are generally applicable to the preparation of other disulfide mitosanes of formulas Ia and Ib according to the general procedure completed in scheme 3.

Following is a list of representative thiols of the formula R 4 Alk ^ SH or R SH which can be reacted via a reaction with the Bunte salt of formula XV or sulfenyl thiocarbonate of formula XVI to form intermediates of formulas XIII and XIV, 30 which in turn are converted into products of the present invention as described. In the case of the preferred embodiment, the representative thiols can be used in a reaction with mitosans of formula Ia or Ib to form products of the present invention. However, the only limitations to the method of the present invention is the lack of thiols containing terminal primary alkylamines which can lead to a mixture of 8502 38 3 -15 products and refuse to use heteroaromatic thiols. react with compounds of formula la or ib.

The thiols are indicated on the formula sheet with formulas 5-90.

Usefulness of compounds of formulas I and IX in antineoplastic therapeutic methods of the invention is demonstrated by results of in vivo selection procedures in which the compounds are administered in varying dosage amounts to mice in which a P-388 leukemic or B16 melanomic condition has been induced.

Compounds of the present invention are believed to have anti-bacterial activity against gram-positive and gram-negative microorganisms in the same manner as has been observed for the naturally occurring mitomycins and are therefore, in principle, useful as therapeutic agents for the treatment of bacterial infections in humans and animals.

Activity against P-388 murine leukemia

Table A contains the results of laboratory tests with CDF-mice in which a tumor graft was implanted intraperitoneally of 6 20 10 -ascites cells of P-388 mouse leukemia and treated with different doses of a test compound of formula I or II, or with mito-mycine C. The compounds were administered by intraperitoneal injection. Groups of six mice were used for each dose amount and they were treated with a single dose of compound 25 on the day after inoculation. A group of ten saline-treated control mice was included in each series of experiments. The mito-mycine C treated groups were included as a positive control.

A 30-day protocol was used in which the average survival time in days for each group of mice was determined and the number of survivors at the end of the 30-day period was recorded. The mice were weighed before treatment and again on the sixth day.

The weight change was taken as a measure of drug toxicity. Mice weighing 20 grams each were used and a weight loss of up to about 2 grams was not considered excessive. The results were determined in terms of% T / C which is the ratio of the mean survival time of the treated group to the average survival time of the saline treated control group x 100. Control animals treated with saline usually died within 9 days. The maximum effect in the following 5 table is expressed as% T / C and the dose giving that effect is indicated. The values in brackets are the values obtained with mitomycin C as a positive control in the same experiment. Thus, a measure of the relative activity of the subject substances relative to mitomycin C can be estimated. A minimum effect in 10 terms of% T / C was considered 125. The minimum effective dose listed in the following table is the dose giving a% T / C of approximately 125. The two values given in each case in the mean weight change column are the mean weight change per mouse at the maximum effective dose and at the minimum effective dose, respectively.

8502333 -17-

CM

CP

g • Η u φ Ό cl nj ^ • ρΜΐηοΊΓ'Ιί'ΐί'ΙΓ'Ο'τΗΟΟ μ% »·. ··» »*** '· 4) φ OOOOOO-i — i — iCNlT-i Ό> I + I + + I + + + + + rt οι

P -P

Ό Λ ....

Η -Η τ-ιΓ'Γ ^ '^ Ρ'ίΜνΟσ'Ο'Τ'-ι g *> D »0« | + llllll + l + l OT ° 0 Φ

Ό C

U Φ U

Ή -2 O

• w Yy R

4) II Φ O — l sii s * oi in ο ο 0 in Φ6

I §H <NOOON-.OO CM CS ^ £ + J

£ .HO OOOOOOOOOOO Φ «

Oil GO VV vv V <u a 1! % 01 2 !! -5 01 3 ^ ojoicMCMCMCNioacMoorara τ * ® & II + Jr ^ noc ^ ononr ^ · 5J'¾ί, ^ I, yy j Si, oo ΰ1ί ^ Ν ^ <ί ^ Ί <Ι (130Ι ® «I 7 Ü ÏÏ Q ^ -Γ VO m 0 “0 0 o“ VO c7 0 a ο g

? A '! (U ^ <0 <31 M

é · '- g - a §11 2' d ^ φ ^ π -π - - - - - - - 000 321 ° (U v · Γ) OOOOOOCNCNnrOO β 0 ® 8 * §ooooot-i- ^ oo ® ^ 0 ! i dP ^ ¢ - ^ 00 ^ 1 ^^ 00321. ^ J | .μ vOvOCNrnLfiCO'HvDOr ^ O M CO Π3 £ * II HHCSlfVlHHCVlCNICSrlH 0

S !! sV

t-tcNO ^ roinot ^ o O '^ σισισισιίπαισισισι -amo φφφφοφφφ ^, o>

Ol Η -I Η -I cvjrH ^ Mrj £ {7 <

^ (v * dd: 33! B3333 Φ + JC

OMSgSgCJgSgS ^ Q] £?

CflCUfiUMM CMMMUS IIOOOOSBOOO Q mjN

[ΙΙτίΙΙΗΐΗΉΉϋΉΉΉΉ i + JIfl S 1 -u I g φ.

* λ φ 1¾ -η

<SM

- Φ * Λ 3 θ ’« 3 -

Π. 01 + JG

"* S C -4 Φ S 2 § ns φ οι 2 c ιβ ίο οι ^ ο Ό Φ 3 και ηη >> ηηηΧΧηη -j rj

** J β a a * ^ s η 3 * £ s ^ 3 | S

Ss? * * £ * & 5333 2 i I * 2 3 ^ g> a ¢ 3 ï H O G ....

g ïi 5 hi ^ -i 5502333 * κ -18-

Activity against Bl6 Melanoma

Table B contains the results of anti-tumor tests using B16 melanoma grown in mice. BDP1 mice were used and inoculated subcutaneously with the tumor implant. A 60-day protocol was used. Groups of ten mice were used for each dose quantity tested, and the mean survival time was determined for each group. Control animals inoculated in the same manner as the test animals and treated with the drug-free injection vehicle showed an average survival time of 24 da-10 gene. Mean survival to that of controls (% T / C) was used as a measure of effectiveness, and the maximum effective dose and minimum effective dose for each test compound was determined. The minimum effective dose was defined as the dose showing a% T / C value of 125. For each dose level, the test animals were treated with the test compound on days 1, 5 and 9 intravenously.

TABLE B

Compound of Maximum Effect Minium Mean Example No.% T / C Dose * * Effective Dose * Weight Change 20 XXVIII 167 (112) 3 1.6 (3) 3 <0.4 -0.9; +1.4> 214 (145) 2.4 (3) <1.6. -2.4; -1.9 XX 110 (112) 3.2 (3) 3.2 +0.5; +0.5 XXVI 152 (145) 1.6 (3) <1.6 -0.6; -0.6 1. See footnote 1 to Table I.

25 2. See footnote 2 to Table I.

3. See footnote 4 to Table I.

ύ ΰ ϋ I 0 & - »4 -19-

In connection with the anti-tumor activity observed in experimental animal tumors, the invention includes the use of the substances of the present invention to effect inhibition of mammalian tumors. For this purpose, they are systematically administered to a mammal having a tumor at a substantially non-toxic anti-tumor effective dose.

The compounds of the present invention are primarily intended for use by injection in much the same manner and for some of the same purposes as mitomycin C. Slightly larger or smaller doses may be used depending on the tumor sensitivity involved. They are easily distributed in the form of dry pharmaceutical preparations containing diluents, buffers, stabilizers, solubilizers and ingredients that contribute to the pharmaceutical elegance of the preparation. These compositions are then improvised into an appropriate form for administration just prior to use using an injectable liquid medium. Suitable injectable liquids include water, isotonic saline solutions and the like.

Description of Specific Embodiments In the following procedures and examples, all temperatures are given in degrees Celsius, and the melting points are not corrected. Proton nuclear magnetic resonance (* H NMR) spectra were recorded on a Varian XL100, Joel FX-90Q or Bruker WM 360 spectrometer in pyridine-dj. or D ^ O as indicated. When pyridine-d ^ is used as a solvent, the pyridine resonance at 5 = 8.57 is used as internal reference, while in the case of D20 as solvent, TSP is used as internal reference. Chemical shifts are indicated in δ units and coupling constants in Hertz. Kbps splitting patterns are denoted as follows: s, singlet; d, dou-30 beet; t, triplet; q, quartet; m, multiplet; bs, wide signal; dd, doublet of doublet; dt, doublet of triplet. Infrared spectra were determined on a Beekman Model 4240 spectrometer or on a Nicolet 5DX FT-IR spectrometer and are indicated in reciprocal centimeters.

Ultraviolet (UV) spectra were determined on a Cary Model 290 spectro-35 meter or on a Hewlitt Packard 8450A spectrometer equipped with a multidiode array detector. Thin layer chromatography (TLC) 8502383 * κ -20- was performed on 0.25 mm Analtech silica gel GF plates. Flash chromatography was performed with Woelm neutral alumina (DCC grade) or Woelm silica gel (32-63 µm) and the indicated solvents. All solvent evaporations were performed under reduced pressure and below 40 ° C.

The 1-alkyl-3-aryltrizenes form a class of reagents known to be useful in reacting them with carboxylic acids to form the corresponding lower alkyl esters.

1-Methyl-3- (4-methylphenyl) triazene can be prepared as follows: Procedure 1 E.H. White et al., Org. Syn., 48, 102-195 (1968).

1-Methyl-3-p-tolyltriazene. p-Toluldine (50.2 g, 0.47 mol is added to a 2-liter flask equipped with a 200-ml dropping funnel and an efficient stirrer, and the flask is immersed in an ice-salt bath at about -10 ° C A solution of 46.8 g (0.55 mol) of potassium nitrite in 150 ml of water is placed in the dropping funnel, a mixture of 250 g of crushed ice and 140 ml of concentrated hydrochloric acid is added to the p-toluidine with stirring The potassium nitrite solution is added slowly, with further stirring for 1-2 hours, until a positive starch potassium iodide test is obtained (Note 1), and the mixture is stirred for an additional 1 hour to complete the reaction of all of the toluldine. to ensure.

The solution of p-toluenediazonium chloride is then brought to a pH of 6.8-7.2 with cold, concentrated aqueous sodium carbonate at 0 ° C, after which the solution turns red to orange and a small amount of red colored material deposits. . The cold, neutral solution is transferred to a dropping funnel and slowly added to a vigorously stirred mixture of 150 g of sodium carbonate, 300 ml of 30-35% aqueous methylamine (Note 2), and 100 g of crushed ice in a 3-liter flask. The reaction mixture is kept at about -10 ° C during the addition, which takes about 45 minutes (Note 3). The solution is extracted with three 1 liter portions of ether. The ether extracts are dried with anhydrous sodium sulfate and evaporated on a rotary evaporator at room temperature to obtain 65 g of crude 1-methyl-3-p-tolyltriazene (Note 4). This is placed in a water-cooled sublimator, and the triazene is sublimed at 50 ° C (1 mm); 8502383 -21- is obtained 43.3 g (0.29 mol, 62%) of a yellow-colored, crystalline sublimate having a melting point of 77-80 ° C (Note 5). The sublimate can be recrystallized from hexane to give the triazene as white needles, m.p. 80.5-81.5 ° C.

It is better to dissolve this in the minimal amount of ether, and dilute the solution with 2 volumes of hexane and cool to 0 ° C to obtain flat platelets with a light yellow tint; mp 79-81 ° C. The yield of pure triazene is 33-37 g (47-53%) (Note 6).

Notes 1. The individual tests with starch potassium iodide paper are preferably conducted 1-2 minutes after the addition of potassium nitrite is complete.

2. 40% aqueous methylamine can be used instead.

3. The reaction is over when a drop of solution no longer turns red with a solution of β-naphthol in aqueous sodium carbonate.

4. The major impurity is 1,5-di-p-tolyl-3-methyl-1,4-pentazadiene (melting point 148 ° C). This can be removed by fractional crystallization, but it is easier to sublimate the triazene from the reaction mixture.

5. The sublimate contains a trace of 1,3-di-p-tolyl-triazene as shown by thin layer chromatography. Recrystallization gives the pure 1-methyl-3-p-tolyltriazene.

6. This procedure only works well with water-soluble amines. Procedure 2 shown below is more suitable for the preparation of trizenes from water-insoluble amines.

Procedure 2 E.H. White et al., Tetrahedron Letters No. 21, 30 p. 761 (1961).

1-n-Butyl-3-p-chlorophenyl triazene. A solution of p-chloro-benzenediazonium hexafluorophosphate (recrystallized from acetone-methanol; 2.87 g, 10.1 mmol) in dimethylformamide (dimethylamine-free) was slowly added to a stirred mixture of n-butylamine (0.73 g, 10.0 mmol), powdered sodium carbonate (15 g) and dimethyl formamide (30 ml), were stirred and kept at -5 ° C 8002383 -22%. The diazonium salt solution can be used at room temperature; however, a purer product is usually obtained when the diazonium salt solution is prepared in and dispensed from a cooled separatory funnel held at a temperature of about -50 ° C. The mixture was warmed to 0 ° C and stirred until a negative test was obtained with 2-naphthol (usually only a few minutes were needed). Ether was added, the mixture was filtered, and the filtrate was washed thoroughly with water and then dried. (The triazene can currently be isolated and recrystallized from pentane at low temperatures). Procedure 3 7-Hydroxy-9a-methoxymitosane. Mitomycin C (2.2 g, 6.6 mmol) was dissolved in 140 ml of 0.1N methanolic NaOH (50% ig) and the reaction mixture was stirred at room temperature for 30 hours. The solution was then adjusted to a pH of about 4.0 with 1N HCl and extracted with 4 x 500 ml ethyl acetate. The combined ethyl acetate extracts were dried (Na 2 SO 4) and evaporated under reduced pressure at about 30-35 ° C to give a solid residue which, after dissolving in ether and treatment with excess hexane, gave a purple-20 color precipitate. The precipitate was collected and air dried to yield the title compound as a fine purple powder (1.4 g, 63%).

^ H NMR (pyridine-d ^, 6): 2.05 (s, 3H), 2.14 (bs, 1H), 2.74 (bs, 1H), 3.13 (d, 1H), 3, 24 (s, 3H), 3.56 (d, 1H), 25 4.00 (dd, 1H), 4.37 (d, 1H), 5.05 (t, 1H), 5.40 (dd, 1H), 5.90 (bs, 2H).

Procedure 4

Mitomycin A. An amount of 7-hydroxy-9a-methoxymitosane of 100 mg (0.30 mmol) and an amount of 3-methyl-1-tolyltriazene of 100 mg (0.67 mmol) were dissolved in 2 ml of methylene chloride and 10 ml diethyl ether. The solution was stirred at reflux for 6 hours at room temperature after 6 hours of gentle reflux. Thin layer chromatography [methylene chloride: methanol (90; 10)] revealed the appearance of a deep purple spot at Rf = 0.36 with a 8502333-23 trace amount of impurity at R ^ = 0.41. The reaction mixture was concentrated to dryness and chromatographed on Woelm neutral alumina using methylene chloride and methylene chloride: methanol (30: 1) as eluent solvents. Fractions containing the component at Rd = 0.36 were brought together and concentrated to dryness. Precipitation of the dry residue from methylene chloride and hexane gave the title compound as a fine amorphous purple powder (25 mg, 24%), mp 161 ° C.

Anal, calculated for C: 54.96; H: 5.44; N: 12.02 found: C: 53.96; H: 5.37; N: 11.99 IR (KBr), max, cm -1: 3400, 3300, 2950, 1700, 1630, 1575, 1200, 1060.

1 H NMR (pyridine-dj., 5): 1.82 (s, 3H), 2.74 (dd, 1H), 3.12 (d, 1H), 3.24 (s, 3H), 3.54 (dd, 1H), 3.96 (dd, 1H), 4.02 (s, 3H), 4.22 (d, 1H), 4.84 (bs, 2H), 5.02 (t, 1H) 5.38 (dd, 1H).

The yield in procedure 4 is increased to 63% by using methylene chloride as the reaction solvent and from room temperature for a period of 24 hours.

Procedure 5

Into a 250 ml one-necked round bottom flask, solid Na 2 CO 2, 35% aqueous solution of amine (amount as in procedure 1) and ice was charged, and the suspension was stirred at -5 ° C (ice-salt bath). To this suspension was added dropwise a cold suspension of p-chlorobenzenediazonium hexafluorophosphate (Aldrich Chemical Co.) in ice, water, Na 2 CO 2 (solution about pH 7). After the addition was complete, the reaction mixture was extracted with diethyl ether. The combined diethyl ether extract. was backwashed with water, dried (Na2SQ4) and concentrated. The yellowish solid residue was purified by column chromatography on Woelm alumina using hexane-methylene chloride (1: 1) as the eluent solvent (1 H NMR incorporated).

Examples I-X

Tririazenes 1-7 of Table C below were prepared according to the general procedure 1 described above, 8502333-24, in which the triazene of Example I is exemplified. The tririazenes were saturated by column chromatography on Woelm alumina.

Triazenes 8-10 of Table G were prepared according to general procedure 5 described above.

8502383 y * -25- ro 2 o o o 33 2

CM

33

O

CM

33 O co ro cm $ n 2 aa - w o

Cj ro cm O

co S3 33 cm

^ U O X.

G η N "_ <N O

0) 33 33 33 33 CM

(UO CJ CJ O 33 n 2 „~ X £ £ y rtS 2 Β · 2 2? Δ M 2 2 ^ I _ | {η [| ii ^ ^ i? 2 2 2220002JI00 rf ^ ^ rt rt H 2 tH Tt yyy yyy yyy 33 * 3 «u Ii 10 onion onion 1) vQ 2 0) Φ

U U U <-H · Ή H O O H

ro ro ro 3 3 3 co O 3 g

222SSS®TjSS

cjooMMuoony

β, Q, ft IH S l8 ft ft 5 IM

3 2 o o ^ «j n σ> ^ § $ o). «2. .

Ïj c 3 2 ü οι o S g N U «O) (3 o o

h3 —I Ή * H

Q N Q

33 - <

Eh C

<31 a

<D

N ™ <e s

• H S

ij CN1 61 o

CM

2

CJ

m oj w

e CM CM

-5 2 x s CMCMO — tCMO2 <'0O'

rtj 532000 CM CM OO

22 - <- <- "K®" - * "" - 1 co 2 OU

CJ Φ <D <D 2 cm (U <D CM2 CMrHr-tH2®'jl ^ j 2 0 ^ 3 3 3 0 0 3 3

2 w ROSSOOSS

co co 2 μ ö yco coM U

33200002200 CJU —'ΉΉΗΗΟΟΟΟμμΉ 8

2 hhm >> hhmXX

Λ) WHH> HHH

Q) Η> H

Λ>

YOU

O

O

> * 8502383 -26-

Example XI

1- [2- (3-Nitro-2-pyridyldithio) ethyl] -3- (4-methylphenyl) triazene

A solution of 4-methylphenyldiazonium chloride is prepared from p-toluidine as described in procedure 1 and adjusted to pH 6.8-7.2 at 0 ° C as described in that procedure.

A solution containing 21.15 mmol of the diazonium salt in 45 ml solution was prepared in this way and placed in a dropping funnel connected to a 250 ml 3-neck round bottom flask containing 5.34 g (20.0 mmol) 2- (3-nitro-2-pyridyldithio) ethylamine, 7 g of sodium carbonate, and 150 ml of dioxane added to the flask in this order. Saturated aqueous sodium carbonate solution (6 ml), and 10 g of ice were added to the flask. The flask was cooled in an ice bath and the contents were moved with a mechanical stirrer. The diazonium salt solution was then added dropwise from the dropping funnel over a period of 15 hours. When the addition was complete, the reaction mixture was allowed to warm to room temperature and extracted with three 400 ml portions of ether. Drying and evaporation of the extracts gave the desired product, which was purified by chromatography using an alumina packed column,

20 with a diameter of 2.5 cm and a length of 25 cm, using hexane: methylene chloride (4: 1); hexane: methylene chloride (3: 2); hexane: methylene chloride (1: 4); and finally methylene chloride containing 1% methanol, for column development and elution. Correct fractions (determined by thin layer chromatography) were combined and evaporated to give 2.5 g of the title compound. Examples XII-XIX

General procedure for the preparation of 7-alkoxy-9a-methoxymitosanes (XII-XIX).

A solution of triazene (2.4 equivalents) in C5 Cl3: mole a 30 nol (4: 1) was added to a Solution of 7-hydroxy-9a-methoxy-mitosane (prepared in procedure 3) in CH ^ Cl2: methanol (4: 1). The reaction mixture was stirred at room temperature and the progress of the reaction was monitored by thin layer chromatography (10% MeOH in CH 2 Cl 2).

The 7-alkoxy-9a-methoxymitosane product appeared in the form of a dark purple spot on the thin layer chromatogram. The reaction mixture was chromatographed on Woelm alumina when the reaction was judged complete based on the thin layer chromatogram, and the 7-alkoxy-9a-methoxymitosane was obtained as an amorphous solid. The products obtained are shown in Table D as Example XII-XIX.

8502383 -28- • O 'Ο r-t r »οο o O ^ * - | · »H τ-Ι <-l 1-) .. ... · ·· i

Id a 2 2 2 <u m ω. » - · '· «-> i o * tn m o o H o * cn σ \ o nj * - - * c U3 l £> UI VO 0 id 0 ύ · ·> ·· ··? η a n i η «_ id? +) ... .v · ~ - S-) ö O "d · om <u Φ m co <o gj g * * * * Ql · Φ co 00 CN CN ή -> H in m in in Μ cn W % ri .. .. · «·· 0 o OU OOU cn (U a ·· ·· ·· ·· Cn o / —s 33 if - ** *

LD ^ o LO O

Q £ <-i lö fM H

I CN r * CN

C3 g co * "t * - ·

Li V— I I · * - ** ïö o o o tn

<rt M CO ^ <N LD

5 H OS ^ 0 O

CO CN t— * ^%

X

H

%

(D% C

h cr '' a “* - d ** ffi ^ * - * *> ^ -» -—V% s. * A ^ a a - a a r? a ® cr ^ oo as - ^ a co ** a 0 co * co cn cn ·> * · <in ui *. ·. a ““ * ^ ^ - Λ Ό "ö * 0 Ό" cn T3 'q ui -', d'T3 cn co * α "0 4J * - · Ό +> d co ^ ~ ^ w • d’ ^ ^

Jm 0 * 000 * CO “OO * VO CN O O 'MC so co - ο o' co co oo 'in - oo cQiö -co ·· - -“ “-η" ** · 3 οι »-η o * in.-π “O 'm cn o * in E-) omo - + j> o | -a * · * -a-' * rj - s οι a aa a aa 5 * -1 * ®

6 r * O ’" · * - * »- * η * Η (N O * 0 * CN

K -Η tn tn n π-t S '“a“ “-Λ- · *“ cö ““

a · η ft S '-', aa ό w ό s S '-' SS

yy j_i (¾ - cn - '- · - - ca - wi ^ ww

m>, Ο * Γ- CN O 'CO f' ND O Ot-iCOO

g q, o * *> in o cn »m o o * *. co co 1 ^ CN * · CN "CO" "US« HCOin ^ CO LD CN CO O '0 a "·" "* 1 2 ^ ·» - * * »%> -. ^ -— * »·. ·· · * s- 33 4K-> 33 y — v 53 «" "• k Γ5 53 g1 z as ^ aacN a ^ aa a -haa ^ 0 aa to coco co m th co yA ^ ^%%%% ^ co% <4 ^ ΛΜβΛ τ5 Λ CO Ό 1 ^ - | .- <^ WW ^ www C “Ο 00 O CO O '000 * 0 * CO vO O' Ο O 'CO O CN CN CO CNCNCN.CN (Tir ^ inCNO * k. k, »t 1.« 1 · * ** * · ** O CN (OO 'r ~ tH CN CO - O' i-iCNCOO'in co au £ a cn », ~ m Λ P = 1 aa a 3 Ö os cn '-fyaao

0 U> W

1 I

Ό rrj ·· 3 Λ λ Ο 0) Η Μ ^ ω · Η Η Η ΰ Ο X Η κ Μ 2 χ ο!> 8502383 -29-

CM PO

r ». kQ LD Π

* * CO CO

PM CM '' - - 00 00 · * ·· ·· ** z z z z m co O $

Tf cO cO cO

«» *. «· *» Mm mm .. .. M ··

Z Z Z Z I

σΓ -q · r ~ co a \ <? i po o cm co

«, *« * Co ► cO

cn (Ti σ »σ m in m co m co ü ü ü ra ü ffl K ·« ·· · ^ fa & * fa ^ ^ ^ ^%% »o tn o o m o o <n in

0-- ω σι - 2J fj? ) S

CM0 CM CM 00 CM <Ti CO O

PO - - CO - - CM - - * * *. ~ ~ »'' 'Ooino o o o m o o o ro po cm co cm ro cm co 2Γ * Γ! n · o co o ^ σ> co o poc ^ cm PI (\ | η h PO CM - - PO - - - »* · '^»> · * - _ BB λ <- »·· -»' '- - κζ - ζζ— 5ζ®ζ öl Ρ0— Γ0 - Z - z - z

Yy »- ΡΟΗ cfl» »oi - ~" * »1 _Q yy Ό Λ CO Ό" · CO "· 2 r 5 ü 3 Ow ^ + j. ΛοίΌ + ι 03 CM O Ό CO CO - w · - 2ΐΓ> t '· CM - r> CM ^ CM 2 Z 1_ C. - - O - Λ CM PM σ o CM PO -¾1 CM PO - - -' '' 'q N mi m ίο cMPOPom · »·» «» ^ ^ Γ '"* - ^ ^ ^ ^ ^ ^ ^ H ZZZ ZZ— -Γ- gg £ £ CQ HWH rJf-iffi® 5 5 55 jji · cn cn - mm cn cn * iig § ii»' s | «I £ £ 8 8. S 2 ï S 8 38 ??

* PO - Φ »- - - f» * ‘‘ J

- PO "- CM PO PO CMPOPOM * - co - - ·. Λ * po * ·« · '· »- ·" -C C? *, λ ^^ λΖ λ z - - z B - Sentence ZZZZ— 5! ^ ÜÜ Ξ ^ po - PO cm - - σ * • 01 ·· - »'» ~ ~ Ό ^ Ïin-Γ—

3 3 S -S 5 3 S S S 3 3 S S S

CD 00 O PM CM If Ό 00 co 00 CO CM ^ o co o in σ σι cd m cm ^ go r »m cm ^%% ^ ^ ^ ^ ^ ^ * - ro ro - cm po ^ m - cm po m CM P0 3! σι σ σ 3 * 1 s g g Μ y «

SS tS

> H M

M B S

Öa U 2 3 3 3 -30-

00 CM

li <f Γ ~ ^ - σι r- 2 2 σΓ οΓ a s a a * «., ·«. «*. I *.

<n m co co in mt «, * k *» ** uo lo in lo ·· * «* · ·· a a a a co in in -¾1 σι rr cc cf ^ n in in in · Μ 1« »·

O U U U

• a aa aa aa

Eh fc Eh 1¾ Ή a. N a aa "a '0 ooooomo CO Ο O co CO cm 10 cm r ·' cm m σι co ou maHaH ΓΠ 04 aH a — I ^ * '« aaaaa ooom oooo -' d'inmco coocNa-i σ io o ^ cm γ · »cm h Γ0 CM aH al rOrOa-la-l (β

+ J

Φ a-T IÖ

21 aa «a« a * a "a S

Λ aH "a- a— Λ» a- »a- ^ a-a id

tT> ^ B B - B «a X

H '® a-4 B Cf CM rt CO

0 M cn a-ι U

> X) - - '' D

u ~ ·> Ό Ό * S S Ό CU Μ Μ ^ Ό Ό w v Ό '> c ^ -' O'- '<

'-a "* 3 * CM O CO CO COCO

(M (M * M1 O '- CO Φ

Q a.aa * -a -00 ^ --G

• am in m in ή (J a-> ·· '' O 0 Μ M ·· Λ aa ·· a ^ aa * CO> 1 Μ a — I a — a S3 '-' '- Π3 aC' - ' σι £ 3 E η Β Β Η H a 'I · 0 EH · Γ0 a-4 al aH ^ W 4-1 CO "a ^' S · Η _Q» ιφ »'Ό Ό * σ» H g

- in Ό 4-> g Ό ^ g OU

Q aa V_a WwQa ^ 03 fj Ο O CO O 'CM M CM CO 1 -—a ro CN σι OO' in ·· CM '3II> cm' '' o - mm in cm to in co φ '' · G ω, a '' '' 'a — a aa, —a' Φ

33 a ^ a-a a-a a-aa-affJ ^ SJ Ol rH

m SB SB 33 E W <h W cm cm | -. g —'aHOCM mo a-H + Λ * ► n, ^ w 0

- T3 - (/] W M

in φ 0 S in in φ 4-> Λ uch --- aa aa - 'aa - · aa · γ · γ4 id CO CO CM CO CM O * 3 * CO CM' 4-> 00 to cn cm σι σι in »o G ii

a a a a a a, aa »a a, a, fj 'fl φ Q

aHOOTf 'aHOO' ^ r 'φ Λ S 0 Φ -H 0 Φ G + J Sa 0 g SL Ό m p λ! o> ή * 10 a y <u ο σ> O '·, <o m O • nl 2 Φ CM Φ II S Λ S3 rH'

u 0 fe · G <B

cm S <3 9 Ü ® Μ «Φ -W -p

0 0 0) M

1 4h -ri + j sh o U Φ -H ^ 0 <d

Φ td Η M

£ S 0 0 Λ + J td Ο o Η X X Φ

Η Η II (d H G

H X. £ 2 $

> Eh d a X

X .. ^ Λ U Λ * 0 <0 8502383 '-31-

Example XX

9a-Methoxy-7- [2- (nitro-2-pyridyldithio) ethoxy] mitosane (XX)

580 mg (1.73 mmol) of 7-hydroxy-9a-methoxymitosane was placed in a round bottom flask and dissolved in 60 ml of methylene chloride. About 2.5 g (5.7 mmol) of the triazene of Example XI was added to the solution in the flask and the mixture was stirred at 5 ° C for 14 hours and then at room temperature for 8 hours. The reaction progress was monitored by silica thin layer chromatography using methylene chloride: methanol (9: 1). The reaction liquid was kept at room temperature for an additional 26 hours and then worked up by column chromatography on a column which was 0.8 cm wide, 30.5 cm long and packed with alumina. The solvents used successively for development and elution were 200 ml portions of methylene chloride each; 0.5% methanol in methylene chloride; 1.0% methanol in methylene chloride; 1.5% methanol in methylene chloride; 2% methanol in methylene chloride; and 4% methanol in methylene chloride. The appropriate fractions were combined and evaporated to yield the title compound in an amount of 470 mg.

20 final, calculated for C 10 H 15 N 5 S: C: 45.65; H: 4.09; N: 11.82

- ZZ ZJ D o Z

(corrected for 0.5 mol.% CE ^ C ^): found: C: 45.74; H: 4.14; N: 11.61.

IR (KBr), Vmax, cm -1: 3440-3200, 3060, 2930, 1720, 1570, 1510, 1395, 1335, 1210, 1055.

NMR (pyridine-dj., Δ): 1.81 (s, 3H), 2.00 (bs, 1H), 2.61 (bs, 1H), 2.98 (bs, 1H), 3.08 (s, 3H), 3.20 (m, 2H), 3.39 (d, 1H), 3.83 (dd, 1H), 4.07 (d, 1H), 4.59-4.89 ( m, 3H), 5.21 (dd, 1H), 7.16 (dd, 1H), 8.31 (dd, 1H), 8.71 (dd, 1H).

By adapting the procedures of Examples XI and XX to other ω- (3-nitro-2-pyridyldithio) alkylamines with 2-6 carbon atoms in the alkyl group, mitosane derivatives of formula III can be prepared. In this formula, n has a value of 2-6 and R represents hydrogen or an alkyl group of 1-6 carbon atoms.

8502383

«V

-32-

Examples XXI-XXXIV

The 7-alkoxydithio-9a-methoxymitosans XXI-XXXIV of the following Table E were prepared according to the general procedure A or B described below and shown in Table E. The physical data for the mitosan compounds XXI-XXXIV are listed in Table F which also follows hereafter.

Procedure A

To a deoxygenated solution of mitosane of Example XX (1 MD, 1 mmol) in 3-5 ml of acetone is added with stirring under argon tri-thylamine (Μ, 1 equivalents), followed by dropwise or portionwise addition of one equivalent of a mercaptan in 1-2 ml of acetone (in cases where the starting mercaptan is impure, more than 1 equivalent of thiol is required). In most reactions (in cases where the starting mitosane of Example XX and the product have very close values on TLC, use is made of high pressure liquid chromatography (HPLC) tracking technique (^ Bondapak-C ^ g column). reaction progress followed by silica gel thin layer chromatography (10% CH 2 OH in CH 2 Cl 2) The completion of the reaction is indicated by the disappearance of the spot corresponding to the starting material and the appearance of the product spot. at this point, the reaction mixture is concentrated under reduced pressure at about 30 ° C and the residue is chromatographed on a neutral Woelm alumina column (0.6 cm by 25 cm) packed with a suspension of 2-5% 25 CHgOH in CH This procedure results in a separation of the desired mitosan product from the pyridylthion by-product, which is retained on the column, and the resulting product is eluted with 2-5% CH 2 OH in eluted product. Precisely purified by flash silica gel chromatography with 5-7% by volume in eluting solvent. The main band corresponding to the product is isolated and the amorphous 7-alkoxydithio-9a-methoxymitosane is characterized.

Procedure B

About 6 drops of 8502383 -33-saturated aqueous solution are added to a solution of mitosane of Example XX (^0.1 mmol) in 2-5% acetone (methylene chloride may also be used, but acetone is preferred) in 10 ml of methanol. NaHCOg solution (in case the mercaptan is L-cysteine, this base is not used), followed by addition of 1 equivalent of mercaptan in 1 ml of methanol (when the starting thiol is soluble in water, water is used). The progress of the reaction is monitored by TLC (silica gel, 10% CH 2 OH in CH 2 Cl 2). After the completion of the reaction, the reaction mixture is diluted with 15 ml of water and concentrated to about 10 ml under a reduced pressure at about 30 ° C. The resulting solution is chromatographed into a reverse phase C-18 column with 10 step gradient elution (100% H 2 O (elution with water separates the yellow pyridylthion byproduct from the product held on the column) to 80 % CH ^ OH in ^ O). The product eluted as a predominant red band is collected and concentrated to give the 7-alkoxydithio-9a-methoxymitosane as an amorphous solid. When further purification is needed, the chromatography step described above is repeated.

t 20 8502 333 -34-

TABLE E

7-Alkoxydithio-9a-methoxymitosanes (Scheme 5)

Pre- Thiol Procedure Product image (RSH) R =

No.

Ö XXI ethyl 2-mercaptoacetate A -CH ^ CH ^ OCCH ^

XXII 3-mercapto-1,2-propanedio 1 A -CH2CH (OH) CH2OH

XXIII 3-mercaptopropionic acid B -CH CH9C00 Na

XXIV cysteine B -CH2CH (NH3) COO

XXV thiophenol A phenyl XXVI p-nitrobenzenethiol A p-nitrophenyl XXVII p-methoxybenzenethiol A p-methoxyphenyl XXVIII p-aminobenzenethiol A p-aminophenyl XXIX 2-mercaptobenzoic acid B formula 112 XXX 2-nitro-4-mercapto _ _. r B formula 113 benzoic acid XXXI 4-mercaptopyridine A formula 114 XXXII 2-mercaptomethyl-1--, _, .. .. i A formula 115 methylimidazole XXXIII glutathione B formula 116 B -CH2CH2H (CH3) 2 8502383 - 35- * - - οοιηιη o-oo ro m O <no r- σ> vo σ »or" co σι o M r4 r4 Η «H in Ί-Ι I" ^ us * · »*» ** ^

SO o O O O lil * O KI

^ hcncno iSi £ 3rir! '-' 'm r' in cm θ 'η n1 n ^ pf) * —i * - * —I CM ΙΟ «Ί« i a «„ -4 H S ** »** · **« »'» o o o o o o m m m o

p »'fl4 VO CM CO rf Λ lil O M

(Ί ["· in CM O 10 ^ 4 σΐ O

s § ^ "y o 2 <n cm m σι σι

K

αχ * _ ie o vo m o g cm> —I --1 cm> r <in cm cm m D w fö

H

Id a ^ ^

3 * * * 2 * n n * cm jB

u ^ aaaos '-4 --4 η μ οι h · σι' '- * m iw a Ό - σι in a Λ' '- σι „~' yy Ό m ^ '» • sT' - ^ o - p. . w «-» o B ·· σι - σ r '^ * ft ιο' cm cm a Ό 'm 1 (1 c

c. »Σι n '--1' w σι 'cm B

l3 nj a - - '-ic: £ ^ ^, o οι - * η n fn * S η * X cn - n in - * - * w co * --V - jj rrt J * * · * N VO - A ^ SC% w (V, SI 1) ^ 3J if) BW - XX ^ ^ p (0 ^ NE CO *> Ή O cn CVJ * -1 £ 3 m j5i c t- * E m * * · vo w_ ^ - * * ax -η O 'p- ^ ^' oToa ^ m ^ · CÜO Ό * ** * -Ό CN ow ^ - <£ -Η H 10 111 'Ό' CM Λ O '2¾ yy —cmN' - '* σι' 'w --1 · ~ ·

^ m> v <-> · ·· 'p *' B in ci m co B

ÏÏ S 'g B o - <cm a' -4 'cm' --1 L aconvom a '^ 4 * n \ cn' '»^ nin''2

I r * «. «·. _yy »· K - W ** E - E A

0 01 * 0 U1 w 3 5, ^ Η ί w m

.¾ s ^ '-'P'B CMN ^ s NwoJ

- m ^ a-Η 'a σι ~' 'a cm vo 5 O' a '^ Ό' ω σν eg «^ 4 ον« · H tt) 'CM m Ό -P' '° S rn ΰ rrj O' <-IO »-i 'CM ΜΓ y. «. O 'ρ », -ι m«.-Τ'-! "v qc". Λ cn co cm '2 ® -ons λ ao' '' '^' ίο - 'c 33 0 g Β ^' σι · ^ 4® Β ^^ σιΒΒ-π 1j m CM »-ιηΝΒ-- | - * < a> '' acM 'I' cn - '' σι ^ ÏÏ rL tn Λ - NN -U 111 λ 'nw SI 5 ίο σι a --4 ^ cm Γ' 'ί1 ^ "J οι

m cm '' σι t ^ '- ^' OvocM

'' 'CM CM' 'a a CM' ''

rl (l | IS H rl H CM -I rl ^ 1 ^ 1 VO

a o

σ 'CM

s δ 8 a

° CM I

s s

CM CM

a a V v r-i,.

β H Ü

n a a I

m z κ o g 8502383 -36- »· * *. »*.» · * - - · in in o o m m o inmom ^ ^ τη (j \ o <-4 r * - o in [s η η r- · ^ co r · * in co o

* -! * · «* * -4 fH« «4 iH

s ** s «* ^ ^ ^ · * · ** · ** · oomo o m ο o ο o m m

N N Ο η N Is IJ CM m m O

σι vo mi * cm φ ui η o cn vo m ft CM fl fl f * CM fI f * f1 CM fl ft fl %% <- «» · ---- Ο Ο Ο O m O m Ο O oooo m in mo vo mmm, -ι mt mm * h vo ^ mo 'd'vo'fl'CN fl1 ia' ϊ «

PO t — I »H t — 4 T — i fO * -l« -4 * H rn ^ Hf — lrH

% Ro rt <m ^

CM CM CM CM

co co in ro ** * »» * mo vo vo

-1 fi ft CO O CM

cm m cm co cm m * "Γ0 * * * * rQ · * ^ ^ ^ OS * ^ * NNB · - N cm m NB« -i * NB CM CTl '' mt β o - B m co B p- * ^ jt * «* ® m '* · - fi p ~», -hg co ^ hO' m 41 -

C ^ fi - · h · ^ - B

* 41 - »» λ IB »* Ό cm ^ B '' 00 Β 01 B 'N * -if * -f- Ό O' cm Β O 'cm * i Β B BN'" 'ii cm' 4 * σι fi co * -I sn tl * Q '•' C 'CM' '01 M * CN Ό> 4J *' S 'Ό' O Λ '' »-— yy - + j -.-— - (<)" * jd '-OCMmo φ CO' — BB' ^ i CM— 'f <w ft —1> r — r- ^ cMVO-' OO 'vo oi ** - - - (_ * - - m oo' fO -C-.

CM'ie'C— B ^ '' CM'BB

h mog m 4 · 4 · m fi f · - · ^ 'w »" e »*% ^ ^. * - s /« “·.

/, J λ 'm 00 - »* -» »' - * * 'N N

M B - COinN g Ν - Β Br ^ OS® *

03 τ-ι Β '' Β ~ ΒΝ-1 rtB '-'- CMCM

<mm ^ vo moB m vo cm r-P 'g3 »*>, - i ~ t' * '' ο σ> t— -in * * d 1 Ü] ^ ^ η ΓΟ H CO t3 £ M ffl -

- - Ν Ν I - - - ft B

ooBB 'm' 'vo σι co' 'cm vo o 4 <m f1 σι ο o vo σι o —- —-' ^ H ^ ^ ^ ^ ^ - - Mffl * 0 ^ CM CO CM CM ^ CM ^^^ T r-4 CO Ê tH ^ T3 ^ «-4 O ^ 4 w« * * «* · * ·» · * · »· * - - - - ΤΗ o **« * · », - - ® * - «* 33 EC * <» * ^ -s ^ 4 S - in BBBB — IB —I Β BW * - · w P- 'mm fi «-» m cm mm t *' p · * * · * 'm' '---- ^' t3 ^ '-' m * - '016¾¾¾ cn ¾ ¾ a oi a' 4> n *% - »s_s w *» w * ww "*« - 'w »» w * w> «-J4 pr * N]

p- 4 · co vo o cm vo o co moo 'OB

ρ- σι m o cm σι m fi 4 · ρ »σι - - 'cm v - - - - ----« - Β B f * fi cm m 41 m -MmO *'? - cm fi * - »ft P '

B

+ s (0 o

2 O

K ^

O CM

Ο B

U §

CM

Β B

oh CM CM> 1

C B C

U O, 0) I I <w H>>

Η Η X

XX

X X

X

8502383 -37- ^%% v ^ ^ s%

o o in m in o O O m O o in o O

§ ° rrSS s i; o ^ o g {£ Ξ ~ en i o «· cm o Oi in ^ PI H cn in 'T cm M 2 ^ η Ξ N ^ ™ ^ ~ ~

^ K H *,, »^ <* ·« I

(—5 in in in in O li) O O O o O O 2 'O

Reversal O PI Ί θ 'Γ' (N Is «“ 3 2 η om o ë o 2 η o <n - omooo £ 2222 m _ ^ ^, η - ™ - -ίο in oooomoom ° ° 2 £! Ï! 2 2 2 2 Ξ o * - <O co - in in cm σ> oo ^! 2 S 2 2 2 2 2 ^ 2 2 2 £ Sj cm '2

SS S

%

** 33 ** · * · * 'O

S s ™ £ S3 N N

o5 * <n - 'SC' 'O - in cm' Ό ό '-, .. 2. -3> * ..Ε '-' νο - <+) tn Ό ^ J · £ ^ 22- ^ '- • in * · »^ e m o ^, _. as sc cm oo r ^ sb 33 co - eb o Ώ 'f - s j m * co 33 Ό t ^ g 3: i: S S -2 - «S S« ΐ * i.

? i s - 3 ° -; 5 5 - I- ° is-3 “l η o - a ιί - B - s -g Π“. a fr. cm 'OS · cm οι t-i in - «. co <ï iro cm »* 2-5. ^ - - 'cm o - “· j» * -. σο co cm n - <- - 33 'cm> - oo

ffl SC CM - - If CO 33CM - * - 'N- 5. ^ 5 E SJ

§ -. "*. o? ^ -" § s ω H s. N § 5 g 'ü ^ ^ 2 - s ~ ~ -io 33' + Γ co '2 ss' -SO Μ M 33 - * CM PM - * CM 't' S ° 5 2 «0 -0— - O - TO m CM Γ- 2.m - - ë - - O - M 33 «O - 33 Ό JT 'n H CM N - Ό CM CM 33 ~ ° + Μ ~ <i U · - _i _i ow ^ th o 5: ^ - - CM CM - - --C0 'c' 2. ffi - 31 - - - o - sc - «i -u - ς s ^ * 33 2 33 3S m - K-SS-'T ^ O 5 Ξ B 7Ï co ^ ^ ω co »ho M 2 *, 2 s ee * * ίΛ · *» CM ** * - *. · ^ »Λ ^ Ό ΓΊ morrtfrtN ^ 03.0 * 0 · * * 3 * N <- · 01 PO N CNJ Ό * SC 2 3 a N ci Ü w ^ EN - IE ^ - co m in CM O 33 OO £ -033 5! S “1 * 3 s

Os GO CO O - ω Γ- O CO - - - Γ- ^ 2. om EG - 33 ^^ •. *. «- 1 ** * ·«. * »E E *“ * * ** CNW η- · <h η η Μ η o thcocot-icM'i-’OO rH μ t h in pi

iH

& h i-i c 'i>, <u g>

G Ή C

O)>? ^ 4-1 £ ^ o 9 g

Sn λ Jj +) + 1 e 25 s § t l *

§ g H

κ I ^

X

8Ö02 33 3 -38- s V «·» »» '' * 1 ooo η n ooo oinÏÏH2 (I) co Ö1 rt N CM CM 'CT Μ Γ »n co σ n co cm o r-- n co r ~ n co o CM - - - - - - - - - - -, «.«. ss **** ·· ******** o η η nooooooono 'o να nor · * co co oi "- cm co o - η Λ' inod tn O1 oo vo -31 cm CO - - - - CM - - - CM - - - * »·» · »*» · »« ····· ·> »* · * ··%.

o o n o o o o o Ui o o o o σ cm n -> ï «m h co η n o co r * n co - ^ vo mp cm m1 co π CO - - - - CO - - - CO - - - ^ ^ ^

10-1 cm

CM CM -O "CM

CM n CO CM UI

»*»% *% O m o n co - cm - cm - cm CM Γ0 CM n CM co% 33

H

· »“ · *, —N <-N · * ** ** * ^ ^ N 33 - T3 - 33 * Z N 03 O 33 - IS) -. N -33 o 33 - * - cn o 33 n üjcM-tn - omn - - - - ^ CM (O 00 Λ * CO * * * 33 CM - - E ~ '»' N fl * * - CO - n 33 - - γ * ω uocoko ^ * - - λ * * co r * c: * ~ ^

s co *% ****** th m ffiro ^ osD

33 * CM * 33 - 33 - cm CM MO - cm O cm * - Η ra * ^ ι η B cm N - * * '' - η

Oi - o * cm 33 nr * * m • m 'M' * *, -M - n * - -CO - m - * 0 - 0 mo * - + J -P '33 - ~ Λ CO * * Ό> X3 "* 33 33" M cm - - - - Ο 03 - - * u ^ co - cm co Λ co - nn o * - 33 σ 33

0) σι ο— σ - 33 33 VOCO CMCOCM

*> n * * * * η * ο Ό η n * * * - - Λ irj gr * * CM Cl ^ 'CM - t3 * Γ * * cm 33 Ό -' r * · * vo O CO —'ÖS Ό 1¾ - - n * - * η o - * 33 - * * Ό - on— "N - - - - - ro cm - - '

* - * 00 * N - 33 33 * '* 33 33 COnNO

rj 33 m * 33 N r * - EB 33- - - - 33 'O' ¢ 0 - Λ co -M · 33 * - * - g co ^ n *

jg] * - - - * jt p- - Λ * * * * "* CO

Eh * η * - o · m * N * Ό - mo - · * O

g σι - N - r * * Λ Ό 33 +3 - 33 Λ O - - - * · w * jg ö ® * - «* - * σ - * n - - * nn - n CM 33 M * * - - CO ω * ω M1 n co 03 33 '33 <n co * cm co n co o co - * σι n' a * co cm - - cm * * * - - - * r * m * * * * * - - CM - "d * r * Ό cm co ^ X] - - - cm · Μ * *

M - - * * - N - - S

-33 - - r * - * co * - ^ P * 33 -— '

cm * * 33 33 ^ P -—. - * - M - · - * - - - 33 VO

UJ - 33 33 - - »33 33 ^ 33 33 * m * 33 33 - * co VO - - CO CO— CM Γ 'CO vO - - Γ * S% ^ * * * - - - - ig <g - * * 33 * 0 * 33 - * * φ * mSB'd'öOO— ms - s' -i mBS'd'd'd -

- CM · - W - 'N —W W N ^ CM —— “' - 'N

vo co n co co 33 no * co * 33 vo or * n33 r- * co o - cm - c ^ oOncMco r * * coocMn ** 173 *** h ^ n ^ ui r * ¢ 0 h cn w ^ ^ co th cn m

CM CO

- - - - - - φ Φ Φ

Η H

3 3 §

g B S

B u a a δ ΰ 3 S3 3 κ 3 8502383 -39- ΟΟΙΛΙΛ ο LH LH Q Ο

Ol CO Π O $ 2 2 £ ïï

r- in co i-i fli O Μ O

^, - 1, - (, - 1 W η rt <H rt ^ ^ k - - - - - o o o o o o m o m i ίΰn in η ei in in o co

φ ifl <f M CM cQ 'H' CO O

04 r- (1-1 -rH fd), _ (, -4, -4, -1 s ^ V W 1 »1 1» O O O O in O in O O m

(I) UI 31 O U) (D H O O

if tfl n1 n O co r- m co i- <n 1-1 1-t 1-1 il {1 ^ 1-11-11-1 ^ 1 m -ig cm ^ co (*> | - * * oo o co's *

^ CM CM CM

cm in co Lfi. N -

Oi - S3 ~ CT

- co K Kin ε 33 ---

^ 1 -CM 1 - 1 O — 1 N

w co θ 'in oo σιθ -i * KDSO - m cm - - - <co-m 1311-1--01 --To - ε -

- 131 -. - CM CO - I - cm ^ CM

lO - Kin 1-10 K o 11 g * · Ol * * VO Γ ** KO ”n - co" ^ K co co - £ 33 co KN · -1 CM rr KH Kin -P 33 ^ ΰ σι -o ^ - cm - 'r ^ 2

-j »rn * * * * -« * ^ Ö * ^ C JT LJ

η +1, -1 13 · N ι3 · ΗΝΝΠΟ "1SS2 Sd

5 O · - 1-1 - K i-i w K 33 - 1-1 ^ η ^ Η X

u ro ~ <j 5 o 1-1 m 13 * - ^ fiO X

> - rn -31 - O 131 - O - in - 01 5 O CM - - - CO ii - * - CM '<ü - 13 · KC ^ I iJ "31 -S- ^ -> ü * -01 cm- in '' * ° '2 - 13 - - 31 CO 3 * h - 5 j K CO K - - K - - - - KK m N CB * j

3 ^ 0 ^ 0) 131 (N N 3 3 31 h -1 5 X

S - -1 'C'l -, -rf »^ r <\ te —4 te te te te ^ ^' wp $ αΐ ^ Ό ^ Κ Ό - 33K-T3 Μ 33 O £ 0) ^ Λ- 1-1-ι-ί Φ n ii 1-1 K Ό jqmi-i | 0 ^ wffi cs s- * ww «" * ki ^ vocs ^^ ιη ro Φ C) lo k co * * 0 ε m cs o)

te es J ^ JT'S

cm - m N w CM r · rr ^ 1 - 1 g ^ m £ mt ® in - co o cm o o

- -incM - co cm r— o '' 'P

^ ^ ^ ^ t, k. ^ N «n-te *» · * n S r KKKcMin κ cm m co o · K «'H * c co ^ co * -4 ^ m - 04 ^ 8- ° 4 § te te te **** * “** ** [2 ^ ^ w Ό wso: ω cs X n E ε 3 04 S -5 c

«Ω m" ~: - "2" CO - S «- I

r- σι o1 - - σ> -P - - - ^ c - pootn ü - »- Ό B -OoimS- * ® '' 5 -4 cm co> - 'S ^ w - τη - 13 ^ ^ coco ^ f ω

P

i- (6 cm y o co 0 in '• O g> 1-4 ^ o yy

^ ^ Z X

0) 0} aT1 3 si CM Λ ES s: gi 00 u n »lp Ή Ό

P

0) s

ïï Η H

5 H X O

X g 5 _

* - s M

8502383

Claims (39)

A compound selected from the group of formula IX or formula III, wherein: Alk. a straight or branched chain alkylene group having 1-6 carbon-13 atoms when R is connected thereto via a carbon atom thereof, and 2-6 carbon atoms when R ^ is connected thereto via a sulfur oxygen or nitrogen atom thereof, and R ^ and -SS- in that case, represents different carbon atoms, Alk2 represents a straight or branched chain alkylene group containing from 2 to 6 carbon atoms optionally bearing an A substituent, wherein the sulfur and oxygen atoms associated therewith and any optional A substituent connected thereto via oxygen, sulfur or nitrogen have different 15 # - carbon atoms of Alk 2 are bonded, wherein the A substituent is selected from the group consisting of one or two C 1 alkyl, C 1 alkanoyl, C ... alkoxy, halogen, 1, 0 1-b 1-b C 2 alkg alkoxycarbonyl, cyano, C ^g alkylamino, C ^g dialkyllamino, C.c alka'noylamino and C.r alkoxycarbonyl, 1-bl-o Alk2 and Alk2 may contain a double bond, R represents hydrogen, lower alkyl, lower alkanoyl, benzoyl or substituted benzoyl, the substituent being lower alkyl, lower alkoxy, halogen, amino or nitro, R is selected from the group consisting of from halogen, carboxy, alkanoyloxy of 1-7 carbon atoms, hydroxy in which the oxygen atom is bonded to Alkj of 3-6 carbon atoms, alkylamino or dialkylamino of 1-12 carbon atoms, N-alkoxyalkylamino of 2-7 carbon atoms, alkanoyl-amino with 1-7 carbon atoms, benzoylamino or B-substituted benzoylamino, naphthoylamino or B-substituted 8302383 -41-naphthoylamino, phenylamino or B-substituted phenylamino, cycloalkyl or B-substituted cycloalkyl, each having 3-8 members in the ring, cycloalkenyl or B-substituted cycloalkenyl each having 5-8 members in the ring, phenyl or B-5 substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and heteroalic cyclic groups with 1-2 rings, 3-8 members in each ring and 1-2 heteroatoms in each ring, selected from oxygen, nitrogen and sulfur, 10 pyridylamino or thiazolylamino, alkoxy or alkylthio each containing 1-6 carbon atoms, alkoxycarbonyl or alkylamino -carbonyl each containing 2-7 carbon atoms, aminocarbonyl, phenoxycarbonyl or B-substituted phenoxycarbonyl, phenoxy or B-substituted phenoxy, naphthoxy or B-substituted naphthoxy, alkoxycarbonylamino with 2-6 carbon atoms, ureldo (-NHC0NEL), N-alkyl urylene (-NHCONHalkyl) 1 3 with 2-7 carbon atoms, N-haloalkyl urylene with 3 3 3-7 carbon atoms, N-haloalkyl-N-nitrosourylene with 3-7 carbon atoms, dialkylamino carbonyl with 3-13 20 carbon atoms, dialkylaminoalkoxy with 4 -13 carbon atoms, alkanoylaminoalkoxy of 3-7 carbon atoms and hydroxyalkylamino or Ν, Ν-dihydroxyalkylamino each having 2-8 carbon atoms, the B substituent being selected from the group consisting of one or two lower alkyl, lower alkanoyl, low alkoxy, halogen, amino, carboxy, hydroxy and nitro groups, and 4 R is selected from the group consisting of alkyl of 1-12 carbon atoms, alkenyl or alkynyl of 3-12 carbon atoms each, cycloalkyl or B-substituted cycloalkyl of 3-8 members ring, cycloalkenyl or B-substituted cycloalkenyl, each having 5-8 members in the ring, phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and heteroalicyclic groups having 1- 2 rings, 35 3-8 members in each ring and 1-2 heteroatoms in each ring, 2O9t383 -42- selected from oxygen, nitrogen and sulfur, except that the heterocyclic group is attached via a carbon atom attached to at least another carbon atom is bonded, the B substituent being selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, carboxy, hydroxy or nitro-4 groups, and R and the adjacent one sulfur atom s amen to form S-cystelnyl, wherein the S-cysteinyl group may be esterified, salted or bound within a non-toxic and non-allergenic peptide, or a non-toxic pharmaceutically acceptable salt thereof. The compound of claim 1 wherein Alk is ethylene and 1 ^ R is hydrogen. A compound according to claim 2 of formula II wherein Alk. 3 1 15 is ethylene and R is acetylammo. A compound according to claim 2 of formula II wherein Alk. 3 is 1 ethylene and R is acetyloxy. A compound according to claim 2 of formula II wherein Alk. 3 is 1 methylene and R is 1,2-dihydroxyethyl. A compound according to claim 2 of formula II wherein Alk. 3 is 1 ethylene and R is carboxy or a non-toxic pharmaceutically acceptable salt thereof. A compound according to claim 2 of formula II wherein Alk. 3 is 1 methylene and R 1 is carboxyaminomethyl or a non-toxic pharmaceutically acceptable salt thereof. A compound according to claim 2 of formula II wherein Alk. 3 is 1 methylene and R is 1-methylimidazol-2-yl. A compound according to claim 2 of formula II wherein Alk. 3 is 1 ethylene and R is dimethylamino. 4 A compound according to claim 2 of formula III wherein R is phenyl. 4 A compound according to claim 2 of formula III wherein R is 4-nitrophenyl. 4 A compound according to claim 2 of formula III wherein R 35 is 4-methoxyphenyl. 02381 4 -43-13 * A compound according to claim 2 of formula III wherein R is 4-aminophenyl. 4 A compound according to claim 2 of formula III wherein R is 2-carboxyphenyl or a non-toxic pharmaceutically acceptable salt thereof. 4 A compound according to claim 2 of formula III wherein R is 4-nitro-3-carboxyphenyl or a non-toxic pharmaceutically acceptable salt thereof. 4 A compound according to claim 2 of formula III wherein R 10 is 4-pyridyl. A compound according to claim 2 of formula 117, or a non-toxic pharmaceutically acceptable salt thereof. A compound according to claim 1 of formula Ib wherein R * and Alk2 are as defined in claim 1. A compound according to claim 2 of formula XVII. A method of inhibiting the growth of a mammalian tumor, comprising a systemic administration to a mammal carrying a tumor of a substantially non-toxic anti-tumor effective dose of a compound according to claim 1. A pharmaceutical composition comprising a compound according to claim 1 together with a pharmaceutically acceptable solvent, diluent, adjuvant or carrier. 22. A process for the preparation of a compound according to claim 1, comprising a reaction of at least one equivalent of a triazene of formula V or formula VI with one equivalent of a mitosulfan of formula IV, wherein R, R, R, Alk 2 and Alk 2 are as defined in claim 1 and Ar is the organic residue of a diazotizable aromatic amine under reaction conditions in an inert organic solvent at a temperature of 0-60 ° C until a decent amount of the product of formula II or formula III is obtained. The method of claim 22, wherein the triazene of formula VI is the compound 1- [2- (3-nitro-2-pyridyldithio) ethyl] -3- (4-methylphenyl) triazene. O 0 ü £ 0 ti 3 -44- The method of preparing a compound according to claim 1 comprising a reaction of at least one equivalent of 3 1 4 a thiol of the formula R Alk SH or the formula R SH with one equivalent of a mitosane with the formula Ib wherein R, R, R, Alk 2 and 5 z * -in as defined in claim 1 and optionally in the presence of at least one equivalent of a base in an inert solvent at a temperature of about 0-60 ° C, until a fair amount of the product of formula II or formula III is obtained. The method of claim 24, wherein Alk is ethylene and R * is hydrogen. The method of claim 24 or 25, wherein one equivalent base is used in the reaction. 27. A process according to claim 24 or 25, wherein a low alkanol, a lower alkyl ester of a low alkanoic acid, a low aliphatic ketone, a cyclic aliphatic ether or a low polyhalogenated aliphatic hydrocarbon with up to 8 carbon atoms or water as the reaction medium is applied. 28. A process for preparing a compound of formula IX, wherein: 5 R represents hydrogen or alkyl of 1-6 carbon atoms, and g R represents alkyl or substituted alkyl, C3-12 cycloalkyl or substituted cycloalkyl, wherein the carbon atom thereof, which is attached to the 7-oxygen-25 atom of mitosane, carries 1-2 hydrogen atoms, and the substituents are selected from the group consisting of halogen, C 1-8 alkoxy, g alkanoyl, C 9 aroyl, cyano, trihalomethyl, amino, C 1 -8 monoalkylamino, C 2-12 dialkylamino, cg-12 ary, -cg-12, oxy, g alkanoyloxy, aroyloxy, heterocyclo with 1 or 2 rings and 5-12 ring atoms in which up to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and wherein each of the alkoxy, alkanoyl, aroyl, aryl, aryloxy, alkanoyloxy, aroyloxy, and heterocyclic substituents optionally contains 1 to 2 substituents selected from halogen, C 4 -g alkoxy, C 4 -g alkanoyl, cyano, trihalo-8502 38 7 -45- no methyl, amino, alkylamino, or ^ -1 2-ylamino groups, comprising a reaction of a mitosane of formula X with a triazene of formula XI wherein R and R are as defined above and Ar is the organic moiety of a diazotizable aromatic amine. The method of claim 28, wherein the triazene of formula XI is the compound 3-methyl-1- (4-methylphenyl) triazene. 30. The method of claim 27 or 28, wherein at least two molecular amounts of the triazene are used in proportion to the mitosane of formula X. A process according to claim 27 or 28, wherein a reaction inert organic liquid solvent for the mitosane of formula X is used as the reaction medium. 32. A process according to claim 27 or 28, wherein a lower alkanol, a lower alkyl alkanoic acid ester, a dilage alkyl ether, a low polyhalogenated aliphatic hydrocarbon or a cyclic aliphatic ether with up to 8 carbon atoms is used as the reaction medium. 33. A process according to claim 27 or 28, wherein methylene chloride, methanol, diethyl ether, ethyl acetate or a mixture of two or more thereof is used as the reaction medium. The method of claim 27 or 28, wherein the reaction temperature is 0-60 ° C. The method of claim 27 or 28, wherein the reaction temperature is 0-25 ° C. g The compound of claim 28, wherein R 2 represents 2- (benzylthio) -ethyl and R represents hydrogen. A compound according to claim 28, wherein R 1 2- (2-pyridyl) -ethyl and R 2 represent hydrogen. A compound according to claim 28, wherein 2- (4-morpholinyl) ethyl and R 1 represent hydrogen. 6 The compound of claim 28, wherein R represents (1,3-dioxolan-2-yl) methyl and R 1 represents hydrogen. 850 2 3 8 3 United Patent Offices o.a. 8502383 S-GRAVENHAGE (HOLLAND) Belongs to letter dated October 18, 1985 Ln / AH:: 71P. * * i * W * f · (c \ r; ('. n £ I' ut \ ji rJO I Improvement of errata in the description associated with patent application No. 8502383 proposed by Applicant under date October 18, 1985 On page 12, line 30, "dithiol" is changed to "dithio"; on page 23, line 11, "max" is changed to "Vmax"; on page 45, lines 26, 28, 30, and 32, "Connection" is changed to "Method 8502383 545