CY1585A - Pyrroloindole derivatives - Google Patents

Description

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GB 2 164 038 A

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SPECIFICATION

Pyrroloindole derivatives

5 Background of the invention

1. Field of the invention ■ .

The present invention provides novel mitomycin analogs containing a disulfide group {Class 260, Subclass 326.24) and processes for the preparation thereof. These compounds are mitomycin A analogs in which the 7-alkoxy group bears an organic substituent incorporating a disulfide group. The present invention also 10 provides a method of producing mitomycin A and derivatives thereof (Class 260, Subclass 326.24).

Mitomycin A is an antibiotic of established utility, and the 7-O-substituted mitosane Einalogs thereof have similar utility.

Nomenclature - The systematic Chemical Abstracts name for mitomycin A based on the recent revision [Shirhata eta/., J. Am. Chem. Soc., 7(75,7199(1983)] is:

15 [1aS-(1ap,8p,8aa, 8bp)]-8-[((aminocarbonyl)oxy)methylI-6,8a-dimethoxy-1,1a,2,8,8a,8b-hexahydro-5-methylarizino[2',3',3,4,]pyrrolo[1,2-a]indoIe-4,7-dione according to which theazirinopyrroloindole ring system is numbered as follows:

7

6

20 . 8b

N-1

25

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Chemical Abstracts

A trivial system of nomenclature which has found wide use in the mitomycin literature identifies the foregoing ring system including several of the characteristic substituents of the mitomycins as mitosane.

30

35

CH2OCONH2

•i0

35

40

Mitosane

40

According to this system, mitomycin A is 7,9a-dimethoxymitosane and mitomycin C is 7-amino-9a-methoxymitosane. As to the stereochemical configuration of the products of this invention, it is intended when identifying them by the root name "mitosane" or by structural formula to identify the stereochemical 45 configuration thereof as the same as that of mitomycin A or C.

45

50

CH2OCONH2

Mitomycin A R=R1=OCH3 Mitomycin C R=NH2, R^OCH^

50

55 2. Disclosure statement 55

Mitomycin Cis an antibiotic which is produced by fermentation and is presently on sale under Food and Drug Administration approval in the therapy of disseminated adenocarcinoma of the stomach or pancreas in proven combinations with other approved chemotherapeutic agents and as palliative treatment when other modalities have failed (Mutamycin® Bristol Laboratories, Syracuse, New York 13221, Physicians' Desk 60 Reference 37th Edition, 1983, pp. 747 and 748). Mitomycin C and its production by fermentation is the subject 60 of U.S. Patent No. 3,660,578 patented May 2,1972 claiming priority from earlier applications including an application filed in Japan on April 6,1957.

BNSDOCID: <GB 2164038A_I_>

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GB 2 164 038 A

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The structures of mitomycins A, B, C, and of porfiromycin were first published by J. S. Webb eta/, of Lederle Laboratories Division American Cyanamid Company, J. Am. Chem. Soc., 84,3185-3187 (1962). One of the chemical transformations used in this structure study to relate mitomycin A and mitomycin C was the conversion of the former, 7,9a-dimethoxymitosane, by reaction with ammonia to the latter, 7-amino-9a-5 methoxymitosane. Displacement of the 7-methoxy group of mitomycin A has proven to be a reaction of considerable interest in the preparation of antitumor active derivatives of mitomycin C. Recently the stereochemical configurations of positions 1,1a, 8a and 8ab have been shown to be as indicated above with respect to the Chemical Abstracts nomenclature [Shirhata eta/., J Am. Chem. Soc., 105,7199-7200 (1983)]. The earlier literature refers to the enantiomer.

10 The following articles and patents deal inter alia with the conversion of mitomycin A to a 7-substituted amino mitomycin C derivative having antitumor activity. The object of this research was to prepare derivatives which were more active, and particularly which were less toxic than mitomycin C:

Matsui et at., J. Antibiotics, XXI, 189-198 (1968);

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

15 Iyengar eta/., J. Med. Chem.,24,975-981 (1981);

Iyengar, Sami, Remers and Bradner, Abstractsof Papers, 183rd Annual Meeting of the American Chemical Society, Las Vegas, Nevada, March 1982, Abstract No. MEDI72;

Cosulich eta!., U.S. Patent No. 3,332,944, issued July 25,1967;

Matsui era/., U.S. Patent No. 3,420,846, issued January 7,1969;

20 Matsui et a/., U.S. Patent No. 3,450,705, issued June 17,1969;

Matsui etal., U.S. Patent No. 3,514,452, issued May 26th, 1970;

Nakano etal., U.S. Patent No. 4,231,936, issued November4,1980;

Remers, U.S. PatentNo. 4,268,676, issued May 19,1981.

The following patent applications deal with the preparation of 7-substituted amino mitomycin C 25 derivatives in which the substituent incorporates a disulfide linkage.

Kono etal., European Patent Application No. 116,208 (1984);

Vyas eta!., U.K. Patent Application No. 2,140,799 (1984).

7-Alkoxy substituted mitosanes related structurally to mitomycin A are described as useful antibiotics having activity in experimental animal tumors in an article by Urakawa etal., J. Antibiotics, 23,804-809 30 (1980).

Mitomycin C is the principal mitomycin produced by fermentation and is the commercially available form. Current technology for the conversion of mitomycin Cto mitomycin A suffers from a number of deficiencies. Hydrolysis of mitomycin Cto the corresponding 7-hydroxy-9a-methoxy-mitosane, and then methylation of that substance requires diazomethane, a very hazardous substance to handle on a manufacturing scale, and 35 the7-hydroxy intermediate is very unstable [Matsui et al., J. Antibiotics, XXI, 189-198 (1968)]. One attempt to avoid these difficulties involves the use of 7-acyloxymitosanes (Kyowa Hakko Kogyo KK Japanese Patent No. 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 production of only specific 7-alkoxy structural types by the availability and reactivity of the alcohol starting materials.

40

Summary of the invention

The present invention is concerned with a group of mitomycin A analogs having a dithio organic substituent incorporated in the alkoxy group at the 7-position. These compounds may be represented by the following general formula

R -SS-A13c,-0

OCH.

CH.

wherein:

R2is an organic group, viz. the structural component of an organic thiol of the formula R2SH, and AlK2and 55 R1 have the meanings given below. These compounds are alternatively described by Formulas II and III.

R3-Alk.-SS-Alk.

■OCH.

r-R-

CH

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GB 2 164 038 A

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0

5

R4-SS-Alk.

t-R"

,1

III

5

wherein:

10 Alk-i is a straight or branched chain alkylerie group having 1 to 6 carbon atoms wheren (disjoined thereto 10 through a carbon atom thereof, and 2 to 6 carbon atoms when R3 is joined thereto through a sulfur, oxygen or nitrogen atom thereof, and R3 and -SS— are in that instance joined to different carbon atoms,

Alk2 is a straight or branched chain alkylene group having 2 to 6 carbon atoms optionally bearing an A substituent wherein the sulfur and oxygen atoms connected thereto and any optional A substituent 15 connected thereto through oxygen, sulfur or nitrogen are attached to different carbon atoms of Alkz, wherein 15 Said A substituent is selected from the group consisting of one ortwo C-,.6 alkyl, C^s alkanoyl, C-|.6 alkoxy, halogen, alkoxycarbonyi, cyano, C-|.6 alkylamino, CV6 dialkylamino, C-|_6 alkanoylamino and C^e alkoxycarbony],

Alk-i and Alk2 may contain a double bond,

20 R1 is hydrogen, lower alkyl, lower alkanoyl, benzoyl or substituted benzoyl wherein said substituent is 20 lower alkyl, lower alkoxy, halogen, amino or nitro,

R3 is selected from the group consisting of halogen, carboxy, alkanoyloxy having 1 to 7 carbon atoms,

hydroxy wherein the oxygen atom is connected to Al^ having 3 to 6 carbon atoms, alkylamino or dialkylamino having 1 to 12 carbon atoms, N-alkoxy-alkylamino having 2-7 carbon atoms, alkanoylamino 25 having 1-7 carbon atoms, benzoylamino or B-substituted benzoylamino, naphthoylamino or B-substituted 25 naphthoylamino, phenylamino or B-substituted phenylamino, cycloalkyl or B-substituted cycloalkyl each having 3 to 8 ring members, cycloalkenyl or B-substituted cycloalkenyl each having 5to 8 ring members,

phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and heteroalicyclic groups having from 1 to 2 rings, from 3 to 8 ring 30 members in each ring and from 1 to 2 heteroatoms in each ring selected from oxygen, nitrogen and sulfur, 30 pyridylamino orthiazolylamino, alkoxy or alkylthio each having 1 to 6 carbon atoms, alkoxycarbonyi or alkylaminocarbonyl each having 2 to 7 carbon atoms, aminocarbonyl, phenoxycarbonyl or B-substituted phenoxycarbonyl, phenoxy or B-substituted phenoxy, naphthoxy or B-substituted naphthoxy, alkoxycar-bonylamino having 2 to 6 carbon atoms, ureido (—NHCONH2), N-alkylureylene (—NHCONHalkyI) having 2 to 35 7 carbon atoms, N3-haloaIkylureylene having 3 to 7 carbon atoms, N3-haloalkyl-N3-nitrosoureylene having 3 35 to 7 carbon atoms, dialkylaminocarbonyl having 3 to 13 carbon atoms, diaikylaminoalkoxy having 4to 13 carbon atoms, alkanoylamirioalkoxy having 3 to 7 carbon atoms and hydroxyalkylamino or N,N-dihydroxyalkyl amino each having 2 to 8 carbon atoms, wherein said B substituent is selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, carboxy, hydroxy 40 and nitro groups, and 40

R4 is selected from the group consisting of alkyl having 1 to 12 carbon atoms, alkenyl oralkynyl each having 3 to 12 carbon atoms, cycloalkyl or B-substituted cycloalkyl having 3 to 8 ring members, cycloalkenyl or B-substituted cycloalkenyl each having 5 to 8 ring members, phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and 45 heteroalicyclic groups having from 1 to 2 rings, from 3 to 8 ring members in each ring, and from 1 to 2 45

heteroatoms in each ring selected from oxygen, nitrogen and sulfur, provided that the heterocyclic group is connected through a carbon atom which is attached to at least another carbon atom (i.e., the carbon atom attached to the -SS- may not itself be attached to two other heteroatoms), wherein said B substituent is selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, 50 carboxy, hydroxy or nitro groups, and R4 and the adjacent sulfur atom together constitute S-cysteinyl 50

wherein said S-cysteinyl group may be esterified, salified or joined within a non-toxic and non-allergenic peptide, or a nontoxic pharmaceutically acceptable salt thereof.

The compounds of the present invention are inhibitors of experimental tumors in animals. In particular, the substances identified herein as compounds of Example Nos. 17,20 and 21-34 are novel substances. They 55 are employed in a manner similarto mitomycin C. The dosages employed are adjusted in proportion to their 5E> toxicities relative to the toxicity of mitomycin C. In cases where the new compound is less toxic, a higher dose is employed. .

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GB 2164038 A

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In a further aspect of the present invention, there is provided a new process for the production of mitosanes of Formulas II and ill. This new process comprises reacting a mitosane of Formula IV

10 3

with a triazene of Formula V or Formula VI Ar-N=N—NH—Alk2—SS—Alk-|—R3 Ar-N=N-NH—Alkz—SS—R4

iv

15

V

VI

wherein R-1, R3, R4, Alk, and Alk2areas defined above and Ar is the organic residue of a diazotizable aromatic amine.

20 In a variation of the present invention, there is provided an alternate process for the production of mitosanes of Formulas II and III. This process comprises reacting a thiol of Formula VII or VIII

R^I^SH 25 R4SH

with a mitosane derivative of Formula lb no

30

SS-Alk2-0.

35

VII

VIII

lb

:-R

10

15

20

25

30

35

The disulfide mitosanes of Formula lb are prepared by the triazene method described herein. More specifically, the mitosane of Formula lb wherein Alk2 is ethylene and R1 is hydrogen is described in Example 40 20 and in co-pending application Serial No. 646,888, filed September 4,1984.

In another aspect of the present invention, there is provided an improved method for preparing compounds having Formula IX

o II

45

ch2ocnh2

ix

50 0

wherein:

Rs is hydrogen, or alkyl, and

R6 is C-|_i2 alkyl or substituted Ci_-|2 alkyl, C3-12 cycloalkyl or substituted C37.12 cycloalkyl wherein the carbon atom thereof which is attached to the mitosane 7-oxygen atom bears from 1 to 2 hydrogen atoms and said 55 substituents are selected from the group consisting of halogen, C,.e alkoxy, C,.s alkanoyl, C6.14 aroyl, cyano, trihaiomethyl, amino, C-|.6 monoalkylammo, C2.12 dialkylamino, C^ary!, Ce-i2 aryloxy, C-,.e alkanoyloxy, C_14 aroyloxy, heterocyclo having 1 or 2 rings and from 5 to 12 ring atoms including up to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, and wherein each of said aikoxy, alkanoyl, aroyl, aryl, aryloxy, alkanoyloxy, aroyloxy, and heterocyclo substituents optionally contains from 1 to 2 substituents selected 60 from halogen, alkoxy, C,.6 alkanoyl, cyano,trihaiomethyl, amino, C1.6 alkylamino, or C2.i2 dialkylamino groups.

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BNSDOCID: <GB 2164038A_I_>

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GB 2 164 038 A

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Many of the compounds of Formula IX are known compounds having inhibitory activity against experimental animal tumors in vivo. A number of novel compounds conforming to Formula IX have also been prepared by this process, and are considered part of the present invention. In particular the substances identified herein as compounds of Example Nos. 14,15,16 and 19 are novel substances, and also have antitumor activity against experimental animal tumors. These compounds are part of the present invention. They are employed in a manner similar to mitomycin C. The dosages employed are adjusted in proportion to theirtoxicities relative to the toxicity of mitomycin C. In cases where the new compound is less toxic, a higher dose is employed.

The new process for production of compounds of Formula IX comprises reacting a mitosane of Formula X

10

15

10

15

with a triazene of Formula XI

20

Ar—N = N-NH-R

XI

20

wherein:

R5 and R6 are as defined above and Ar is the organic residue of a diazotizable aromatic amine.

25 The terms "lower alkyl", "lower aikoxy" and "lower alkanoyl" as used herein and in the claims (unless the 25 context indicates otherwise) means straight or branched chain alkyl, alkoxy or alkanoyl groups containing from 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyi, amyl, hexyl, etc. Preferably, these groups contain from 1 to 4 carbon atoms and, most preferably, they contain 1 or 2 carbon atoms. Unless otherwise specified in the particular instance, the term "halogen" as used herein and in the 30 claims is intended to include chlorine, fluorine, bromine and iodine. The term "nontoxic pharmaceuticaily 30 acceptable salt" is intended to include salts of the compounds of Formulas I and II with any nontoxic pharmaceuticaily acceptable acid or base. Such acids are well-known and include hydrochloric, hydrobro-mic, sulfuric, sulfamic, phosphoric, nitric, maieic, fumaric, succinic, oxalic, benzoic, methanesulfonic,

tartaric, citric, camphorsulfonic, levulinic and the like. Such bases are well-known and include, e.g. nontoxic 35 metallic salts such as sodium, potassium, calcium and magnesium, the ammonium salt and salts with 35

nontoxic amines, e.g. trialkylamines, procaine, dibenzylamine, pyridine, N-methylmorpholine, N-methylpiperidine and the like. The salts are made by methods known in the art.

Description of the invention

40 - The present invention provides a new process forthe preparation of compounds of Formula IX which 40

comprises reacting a mitosane of Formula X with a triazene of Formula XI as shown in Scheme 1.

Scheme 1

45

50

x

'wherein:

55 Rsand R6 are as defined above and Arts the organic residue of a diazotizable aromatic amine. 55

The 1-substituted-3-aryltriazenes of Formula XI and more specifically 1-alkyl-3-aryltriazenes make up a class of reagents which are known to be useful for reacting with carboxylic acids to form the corresponding lower alkyl esters. 1 -M ethyl-3-(4-methyiphenyl)triazene maybe prepared according to the general procedures described by E. H. White etal. in Org. Syn., 48,102-105 (1968) and as described herein in 60 Procedure 1. However, this procedure works well only with water-soluble amines, and a second procedure 60 which is described by E. H.White eta!., Tetrahedron Letters, No. 21,761 (1961) and also describes herein in Procedure 2 is more suitable forthe preparation of triazenes of water-insoluble amines.

BNSDOCID: <GB -216403BA_L>

6 GB 2 164 038 A

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The reagent 1 -methyl-3-(4-methylphenyl)triazene prepared in the above fashion has been previously employed to prepare methyl esters of carboxylic acids such as 2,4-dinitrobenzoic acid [E. H. White ef a/. Org. Syr>., 48.102-105 (1968)] and cephalosporanic acids which yields the desired A3-compound without isomerization to the A2-isomer [Mangia, Tetrahedron Letters, No. 52, pp. 5219-20 (1978)]. The reagent has ' 5 also been employed to produce a 3-methoxy-cephalosporin derivative by reaction with the corresponding 3-hydroxy-3-cephem-4-carboxylate in benzene solution at the reflux temperature. (Wiederkeherefa/. U.S. Patent No. 4,069,324 issued January 17,1978).

Other 1-(lower alkyl)-3-aryltriazenes of Formula XI may be prepared similarly by reaction of other lower alkyl amines with aryldiazonium salts in similar fashion. Any aryiamine having 6 to 12 carbon atoms which 10 readily forms a diazonium salt may be used as the source of the aryl portion of the 1,3-disubstituted triazene. Some examples of triazenes produced in this fashion and used in the present invention are: 1-(n-butyl)-3-(4-methylphenyl}triazene;

1-(1-methylethyl)-3-(4-methylpheny!)triazene;

1-(4-methy!phenyl)-3-[2-(4-morpholinyl)ethyl]triazene; 15 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-chl orophenyl )-3-(tetrahydrofu ran-2-ylmethyl )triazene.

20 Othertriazenes have been described in the literature which are suitable reactants for use in the present process to provide 7-(substituted alkoxymitosane) of Formula IX. Those described by T. A. Daniels eta!., Can. J. Chem., 55,3751-3754 (1977) are exemplary.

•25 jvn=n-nh-ch2-y a X = H, Y = CN b X = N02, Y = CN 30 c X = C02Me, Y = CN '

d X = Ac, Y = CN eX = N02,Y = C02Et .

fX = C02Me,Y = C02Et gX = C02Me, Y = COPh 35 h X = N02, Y = -CH(OCH3)2

The following further exemplify suitable triazene starting materials of Formula XI for use in the present invention.

1-(n-butyl)-3-(a-napthyI)triazene 40 1-(n-hexyl)-3-phenyltriazene

1-ethyl-3-(2,4-dimethylphenyl)triazene

1-(1-methylethyl)-3-(4-methoxyphenyl)triazene

Forthe preparation of mitomycin A, we prefer to use 3-methyl-1-(4-methylphenyl)triazene as methylating reagent. Preferably, at least two molecular proportions of the latter per molecular proportion of 45 7-hydroxy-9a-methoxymitosane are employed 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 alkanoic lower alkyl esters, the dilower alkyl ethers, the cyclic aliphatic ethers, and the lower polyhalogenated aliphatic hydrocarbons. These solvents contain up to 6 carbon atoms, but those boiling at temperatures of less than 100°C are preferred. Specific preferred solvents are methylene chloride, methanol, 50 diethyl ether, ethyl acetate, and mixtures thereof. The reaction may be carried out at the reflux temperature of the reaction mixture or up to about 60°C. At temperatures in excess of this the mitosane reactant is inclined to decompose with a resultant reduction in yield. It is preferred to carry out the reaction at room temperature or below, for instance within the range of 0 to 25°C.

A convenient way to determine when the reaction is complete is by thin layer chromatography. Mitomycin 55 A is deep purple in color and can be readily distinguished from the starting material and from by-products. In the solvent system methylene chloride/methanol (90/10) mitomycin A exhibits Rf = 0.36. Chromatography on neutral alumina may be used for purification of the product.

The foregoing reaction conditions and precautions are generally applicable to the preparation of other 7-R60-mitosanes of Formula IX according to the present process.

• 60 The new process of the present invention utilizing 1-substituted-3-aryltriazenesmayalsobeusedto prepare compounds of the Formula II or III which comprises reacting a mitosane of Formula IV with a triazene of Formula V or VI as shown in Scheme 2.

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GB 2 164 038 A

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Scheme 2

Ar-N=N-NIi-Alk2-SS-Alk1-R

or Ar-N=N^NH-Alk2-SS-R

VI ■

II or III

10

IV

wherein: •

R\ R3, R4, Aik, and Alk2 are as defined above, arid Aris the organic residue of a diazotizable aromatic amine. . ..

15 Aryltriazenesof Formula V or VI maybe prepared in.a similar fashion as described above for the preparation of aryltriazenes of Formula XI except that the alkyl amines utilized therein are replaced by aminodisulfides of Formula XII - •. —

20

R2—SS—Alk2NH2 ■

which are alternatively described by Formulas XIH and XIV R3-Alk1-SS-AlkzNH2

XII

XIII

10

15

20

25 and 25

R4—SS—Alk2NH2 / XIV

Aminodisulfides of Formula XIII and Formula XIV are known compounds and may be prepared by various 30 methods. For instance, they may be made by reaction of the appropriate thiol R^I^SH or R4SH with a Bunte 30 salt of the formula

NH2Alk2SS03Na • . . XV

35 or with a sulfenylthiocarbonate of the formula 35

0 •

NH2Alk2SSCOCH3 XVI

40 40

Klayman etal., J. Org. Chem., 29,3737-3738 (1964) have prepared the following by the Bunte salt method: 2-aminoethyi n-butyl disulfide;

2-aminoethyl n-hexyl disulfide;

2-aminoethyl n-octyl disulfide;

45 2-aminoethyl n-decyl disulfide; 45

2-aminoethyl phenyl disulfide; _ ■

2-aminoethyl benzyl disulfide .

Methanol was found to be the preferred reaction solvent forthe reaction of the Bunte salt with the thiol. 50 Reaction temperatures of 0° to -10°C were found to be preferred using this solvent. Higher temperatures 50 were necessary with other solvents. The chief drawback of this method is the formation of symmetrical disulfides as a by-product, presumably as a result of disproportionate of the desired mixed disulfide.

The mixed disulfide starting materials of Formulas XIII and XIV are preferably prepared via reaction of the appropriate thiol, with a sulfenylthiocarbonate of Formula XVI. This is the method of S. J. Brois etal., J.Am. 55 Chem. Soc., 92,7629-7631 (1970). Typically, this preparative procedure involves adding the thiol to a 55

methanol solution of the amino-alkylsulfenylthiocarbonate of Formula XVI and allowing the reaction to proceed at a temperature in the range of from 0°to 25°C> Reaction times vary from virtually instantaneous to several hours depending upon the particular thiol employed. The progress of the reaction can be followed by measuring the presence of unreacted thiol in the reaction vessel. If the reaction is sluggish, a catalytic 60 amount of triethylamine may be added as reaction accelerator. 60

BNSDOCID: <GB 2164038A_J_>

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GB 2 164 038 A

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The 1-{substituted disulfide)-3-aryltriazenes of Formula V or VI are prepared by the reaction of aminodisulfides of Formula XII with aryldiazonium salts in a similar fashion as described herein forthe preparation of aryltriazenes of Formula XI. Any arylamine having 6 to 12 carbon atoms which readily forms a diazonium salt may be used as the source of the aryl portion of the 1,3-disubstituted triazene. Some 5 examples of disulfide triazenes produced in this fashion and used in the present invention are 5

1 -[2-(2-acetamidoethyldithiol)ethyl]-3-|4->methylphenyl)triazene; 1-[2-(3-nitro-2-pyridyldithio)ethyl]-3-(4-methylphenyl>triazene.

The following further exemplify suitable triazene starting materials of Formula V or VI for use in the present invention.

10 1-[2-(3-nitro-2-pyridy!dithio)ethyl]-3-{4-chlorophenyl)triazene; 10

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;

l-[2-(butyldithio)ethyl]-3-(4-methy!phenyl)triazene;

15 1-[2-(4-methoxyphenyldithia)ethylJ-3-{4-methylphenyl)triazene; 15

1-[2-(4-nitropheny(dithio)ethyll-3-(4-methylphenyl)triazene; 1-{2-[(2-benzoylaminoethyl)dithio]ethyil}-3-(4-methylphenyl)triazene; 1-[2-(4-chloro-2-naphthyldithio)ethyl]-3-(4-methylphenyl)-triazene; 1-[2-(cyclopropylmethyldithio)ethyt]-3-(4-methylphenyl)triazene;

20 1-{2-[(2-phenoxyethyl)dithio]ethyl}-3-(4-methylphenyl)triazene. 20

In a preferred embodiment of the present invention, there is provided an alternate process for the preparation of disulfide mitosanes having the Formula la

25

30

R -SS-CH2CH20.

la

25

30

35

40

wherein:

R2is an organic group viz. the structural component of an organic thiol of the formula R2SH, which is alternatively described by RlAlk, or R4 wherein R3, R4 and Alki are as defined above.

Forthe preparation of the disulfide mitosanes of Formula la, it is preferred to utilize the 9a-methoxy-7-[2-(3-nitro-2-pyridyldithio)ethoxy]mitosane of Formula XVil in a thiol exchange process with an appropriate organic thiol of the Formula R2SH as shown in reaction Scheme 3. The driving force behind the formation of the disulfides of Formula la is the stability pf the by-product, namely 3-nitro-2-mercaptopyridine, which solely exists as the thione XVIII.

Scheme 3 '

35

40

SS-C^CHj-O.

+ R SH

xvii r -ss-ch2ch2q,

r^f°2 H

XVIII

la

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GB 2 164 038 A

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Alternatively, if it is desired to prepare mitosanes of Formula II or III wherein Alk2 is other than ethylene, such as trimethylene or propylene, then the appropriate triazene of Formula V or VI is utilized in the procedure depicted in Scheme 2 to produce disulfide mitosanes having the Formula lb

5

lb

5

10

!-R'

,1

10

wherein Alk2 and R1 are as defined above.

There are two general synthetic procedures described herein forthe preparation of lipophilic as well as 15 hydrophylic mitosanes of the Formula la. General prpcedyre A is employed for preparing either lipophilic or 15 moderately soluble disulfides of Formula la, whereas general procedure B is employed for water-soluble disulfides of Formula la which are preferably isolated as sodium salts or as zwitterionic forms. Preferably; at least one equivalent of the mercaptan RZSH per equivalent of mitosane of Formula XVII is employed, and the reaction ma^ be carried out in the presence of about one equivalent of base per equivalent of mercaptan 20 R2SH. Preferred bases are the tertiary amines, e.g.triethylamine, N-methylmorpholine, N-methylpiperidine, 20 pyridine, 2,6-lutidine and the inorganic bases, e.g. sodium bicarbonate, potassium carbonate, potassium bicarbonate and the like. Suitable inert solvents for the reaction of starting materials of Formula XVII and R2SH are the lower alkanols, lower alkanoic lower alkyl esters, lower aliphatic ketones, the cyclic aliphatic ethers, the lower polyhalogenated aliphatip hydrocarbons and water. The organic solvents contain up to 8 25 carbon atoms, but those boiling at temperatures of less than 100°C are preferred. Specific preferred solvents 25 are methylene chloride, methanol, acetone, water and mixtures thereof. The reaction may be carried out at the reflux temperature of the reaction mixture or yp to about 60°C. It is preferred to carry out the reaction at room temperature or below, for instance within the range of 0 to 25°C.

The foregoing reaction conditions and precautions are generally applicable to the preparation of other 30 disulfide mitosanes of Formulas la and lb according to the general procedure depicted in Scheme 3. 30

The following is an enumeration of representative thiols of the Formula R^I^SH or R4SH which may be converted via reaction with the Bunte salt XV or sulfenylthiocarbonate XVI to produce intermediates Formulas XIII and XIVr which in turn are converted to products of the present invention as described. In the case of the preferred embodiment, the representative thiols may be employed in a reaction with mitosanes 35 of Formula la or lb to produce products of the present invention. However, the only limitations to the 35

methodology of the present invention is the use of thiols containing terminal primary alkyl amines which may lead to a mixture of products and the qse of heteroaromatic thiols which may not react with compounds of the Formula la or lb.

BNSDOCID: <GB 2164038A_I_>

10

GB 2 164038 A

10

hsch3 hsch2ch3 HSCH2chzch3 • hsch(ch3}2 hs(ch2)3ch3

hs-ch-ch2ch3

I

ch3

HSCH2ch(ch3)z ch3

I ' -hs-c-ch3

•I <

ch3

hs-ch2-ch=ch2

hs-ch2-ch=c(ch3)2

hs-ch2-c=ch hs-ch2-o=c-ch3

HS'

HS

BNSDOCID: <GB 2164038A_L>

11

gb 2 164 038 a 11

0

II

hs(ch2)nor1 n = 2-4; R1 = H, CCH3, CH3 0

II

hs(ch2)ncxr n = 1-3; x = O, NH, NR1; r/R1 = H, CH3

0

HSfCHzJnNHR1 n = 2-4; R1 = CH3, CH?CH3f CH2CH2CH3, CCH3

hs(CH2)nnr1 r2 n = 2-4; R1/R2 = CH3, CH2CH3

hs-ch2ch2sch3

hs-ch2ch2nhc{ch3)3

ch3 o

I II

hs-ch2-c-ch2nhr1 r1 = ch3, cch3

I

ch3

ch2

HS-CH2CH2-N ! n = 3-5 (CH2}n ch2nr1r2

hs-ch-ch2nr1r2 r1 = ch3; r2 = h, ch3

hs-ch2-ch-ch2nhr1 r1 - ch3

I

nhr1

HS-CH2-CH-NH2 + peptidps C02H

HS-CH2-CH-CH2-NH2 + peptides

I

co2h

HS-CH2-CH-CH2-C02H + peptides

I

nh2

BNSDOCID: <GB 2164038A_J_>

12 GB 2 164 038 A

12

ch2co2h

HS—CH2CH2CH + peptides

I

nh2

nh2

I

nhcoch2ch2chco2h

I

hs-ch2ch I

C0NHCHzC02H 0

hs-ch=ch-nhcch3

hs-ch2-ch-ch2oh

I

oh ch3

I

hs-ch2ch-ch2oh hs-ch2ch-ch2nr1r2 r1 = chs; r2 = h, ch3

I

0ch3 hs-ch2ch2-n-gh3

och3

hs-ch2ch2-o-c-ch3

BNSDOCID: <GB 2164038A_l_>

13

G8 2 164 038 A 13

■o hs-

(/ v»

(cb3i2

■o-

och.

hs

nhch.

NHQCH j

hs-

\\ (t hs n hs o hs n(ch3)2

hs-ch,

// w hs-ch 2-ch,h^3

h s -ch2 ch 2 -{/ 7~ nhc:ch3

BNSDOCID: <GB 2164038A_1_>

14 GB 2 164 038 A

14

HS-(CH2)n

HS (CH2)n

7 \\

N~

N

HS-(CH2) n-</ NN

/^-N

HS— (CH_) -N I

2 n \^N

HS-(CH2)„-N

. vjj

HS-(CH2>

-O

n = 1, 2

n = 1, 2

. n = 1, 2

n = 2, 4

n = 2-4j R = OCH2CH3

n = 1, 2

bs"'ch2i«-^^

»„o

HS- (CH

HS-(CH

rr n

HS— (CH,) -sf V-NH, A n X "

»<^x

HS- (CH2)

hs— (ch,)

2 n N ^

HS-CH.

. /N<CH3>2

•o n = 1, 2i X = O, S, NH

n = 1, 2; X = 0, S, NH

n = 1, 2; X = O, S, NH

n = 1, 2; X = O, S, NH

n = 1, 2? X = O, S, NH

n = 1, 2; X = 0, S, NH

HS-CH.

N.

R = H, CH,

BNSDOCID: <GB 2164036A l_>

15

GB 2 164 038 A

15

HS-CH,-C , NK'

r1 = h, ch3

hs-ch--ch--n x

2 2 \ /

x = o, nh, nch3, s r = h, ch.

r = h, ch.

r = h, ch.

HS-<^ \R]

r = h, ch.

-Q

r = h, ch.

i hs-c nr r = h, ch,

hs- (ch.

y Wr

n = 1-3; re = 1-3

hs-ch

2 X -ci-p1

r2 = h, ch3; r1 = h, ch3

HS-CH2-r ^

r = h, ch-

hs-chj-^ nr1

r1 = h, ch3

n(ch3)2

.nh-

HS-

'oh

BNSDOCID: <GB 2164038A_I_>

16

GB 2 164 038 A

16

5 5

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

Compounds according to the present invention are believed to possess anti-bacterial activity against 10 gram-positive and gram-negative microorganisms in a manner similar to that observed forthe naturally 10 occurring mitomycins and are thus potentially useful as therapeutic agents in treating bacterial infections in humans and animals.

A ctivity against P-388 murine leukemia 15 Table I contains the results of laboratory tests with CDFi mice implanted intraperitioneally with a tumor 15 inoculum of 106 ascites cells of P-388 murine leukemia and treated with various doses of either a test compound of Formula I or II, or with mitomycin C. The compounds were administered by intraperitoneal injection. Groups of six mice were used for each dosage amount and they were treated with a single dose of the compound on the day after inoculation. A group often saline treated control mice was included in each 20 series of experiments. The mitomycin C treated groups were included as a positive control. A 30 day protocol 20 was employed with the mean survival time in days being determined for each group of mice and the number of survivors at the end of the 30 day period being noted. The mice were weighed before treatment and again on day six. The change in weight was taken as a measure of drug toxicity. Mice weighing 20 grams each were employed and a loss in weight of up to approximately 2 grams was not considered excessive. The 25 results were determined in terms of % TIC which is the ratio ofthe mean survival time of the treated group to 25 the mean survival time of the saline treated control group times 100. The saline treated control animals usually died within nine days. The "maximum effect" in.the following Table is expressed as % T/C and the dose giving that effect is given. The values in parenthesis are the values obtained with mitomycin C as the positive control in the same experiment. Thus a measure of the relative activity of the present substances to 30 mitomycin C can be estimated. A minimum effect in terms of % T/C was considered to be 125. The minimum 30 effective dose reported in the following Table is that dose giving a %T/C of approximately 125. The two values given in each instance in the "average weight change" column are respectively the average weight change per mouse at the maximum effective dose and at the minimum effective dose.

BNSDOCID: <G8 2164038A_J_>

TABLE I

Inhibition of P-388 Murine Leukemia

Compound of Example No.

Maximum Effect Minimum Effect Average

% T/C

Dose

Dose Wt. Change'

12'

n-Bu

167 (306) 1.6(3.2)

0.2 +0.1; -0.4

13'

i-Pr

167(306) 1. 6(3.2)

0.05 -0.7; +0.1

14

-ch,ch,-n 0.

2 2 Y__y

222(306) 6.4(3.2)

<0.05

-1.7; -0.5

15

-ch2ch2

/ \\

233(306) 3.2(3.2)

<0.05 -2.4; +0.2

16

-ch2ch2sch2

7 V

156(306) 6.4(3.2)

0.2 -0.7; +0.3

TABLE I (continued)

Compound of Example No.

17 -ch2ch2ssch2

ch3conhch2

183 -CH2CH2OCH3

/°

19 -ch,ch t \

O

Np2

20 -CH2CH2SS-^/

26 -ch2ch2ss-

Maximum Effect % T/C Dose1

Min imum Effect

Average

2

Wt. Change

183(306) 6.4(3.2)

211(172) 0.4(3.2)

267 (172.) 0.4 (3.2)

206 (>333) 6.4 (4. 8)

.178 (>333) 12.8 (4. 8)

0.1 -1.2; -0.3

<0.05 -0,6; +1.3

<0.05 +0.9; +1.7

•0.2 -1.0; +1.5

0.2 +0.4; +2.1

TABLE I (continued)

Compound of Maximum Effect Minimum Effect Average

Example No. R % T/C Dose1 Dose1 Wt. Change2

28

-ch2ch2ss

■€>

194(>333)

3.2(4.8)

<0.1

-3.1; +1.8

1. mg/kg of body weight

2. average grams per day for each maximal and minimal effective doses

3. Urakawa et al., J. Antibiotics, 23, 804-809 (1980)

4. values in parentheses are for mitomycin C tested in the same run

20 GB 2 164038 A

20

Activity against B16 melanoma

Table II contains results ofantitumor tests using the B16 melanoma grown in mice. BDF-, mice were employed and inoculated subcutaneously with the tumor implant. A 60-day protocol was used. Groups of ten mice were used for each dosage amount tested, and the mean survival time for each group was 5 determined. Control animals inoculated in the same way as the test animals and treated with the injection 5 vehicle and no drug exhibited a mean survival time of 24 days. The survival time relative to that of the controls (% T/C) was used as a measure of effectivenuss, and the maximal effective dose and minimal effective dose for each test compound was determined. The minimal effective dose was defined as that dose exhibiting a % T/C value of 125. For each dosage level, the test animals were treated with the test compound 10 on days 1,5 and 9 by the intravenous route. 10

TABLE II BW Melanoma

15 15

Compound of Maximum Effect Minimum Average

Example No. %T/C Dose1 Effective Dose1 Wt. Change2

20 28 167(112)3 1.6(3)3 >0.4 -0.9;+1.4 20

>214(145) 2.4(3) <1.6 -2.4;-1.9

20 110(112) 3.2(3) 3.2 +0.5;+0.5

25 26 152(145) 1.6(3) >1.6 -0.6;-0.6 25

1. mg/kg of body weight

30 2. average grams per day for each maximal and minimal effective dose 30

3. values in parentheses are for mitomycin C tested in the same run.

In view of the antitumor activity observed in experimental animal tumors, the invention includes use of the substances of the present invention for inhibiting mammalian tumors. For this purpose, they are 35 administered systematically to a mammal bearing a tumor in substantially nontoxic antitumor effective 35

dose.

The compounds of the present invention are intended primarily for use by injection in much the same way and for some of the same purposes as mitomycin C. Somewhat larger or smaller doses may be employed depending upon the particulartumor sensitivity. They are readily distributed as dry pharmaceutical 40 compositions containing diluents, buffers, stabilizers, solubilizers and ingredients contributing to phar- 40

maceutical elegance. These compositions are then constituted with an injectable liquid medium extemporaneously just priorto use. Suitable injectable liquids include water, isotonic saline and the like.

Description of specific embodiments 45 In the following procedures and examples, all temperatures are given in degrees Centigrade, and melting 45 points are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian XL100, Joel FX-90Q orBrukerWM 360 spectrometer in either pyridine-^ or D20 as indicated. When pyridine-c/E is used as the solvent, the pyridine resonance at 8=8.57 is used as the internal reference,

whereas with DaO as solvent TSP is used as the internal reference. Chemical shifts are reported in S units and 50 coupling constants in Hertz. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, 50 quartet; m, multiplet; bs, broad signal; dd, doublet of doublet; dt, doublet of triplet Infrared spectra were determined either on a Beckman Model 4240 spectrometer or a Nicolet 5DX FT-IR spectrometer and are reported in reciprocal centimeters. Ultraviolet (UV) spectra were determined either on a Cary Model 290 spectrometer or a Hewlitt Packard 8450A spectrometer equipped with a multidiode array detector. Thin layer 55 chromatography (TLC) was carried out on 0.25mm Analtech silica gel GF plates. Flash chromatography was 55 run with either Woelm neutral alumina (DCC grade) or Woelm silica gel (32-63(xm) and the indicated solvents. All evaporations of solvents were performed under reduced pressure and below 40°C.

The 1-alkyl-3-aryitriazenes make up a class of reagents which are known to be usefuifor reacting with carboxylic acids to form the corresponding lower alkyl esters. 1-Methyl-3-(4-methylphenyl)triazene may be 60 prepared as follows: 60

BNSDOCID: <GB 2164038A_I_>

21

GB 2 164 038 A

21

Procedure 1E. H. White et ai., Org. Syn.,48,102-195 (1968).

l-Methyl-3-p-tolyltriazene. p-Toluidine (50.2 g, 0.47 mole) is added to a 2-1 flask eq .tipped with a 200-ml dropping funnel and an efficient stirrer, and theftask is immersed in an ice-salt bath < t ca. -10°. A solution of 46.8 g (0.55 mole) of potassium nitrite in 150 ml of water is placed in the dropping fur nel, and a mixture of 5 250 g of crushed ice and 140 ml of concentrated hydrochloric acid is added to the p-toluidine with stirring. 5 The potassium nitrite solution is slowly added with continued stirring during 1-2 hours until a positive starch-potassium iodide test is obtained (Note 1), and the mixture is stirred for an additional hour to ensure the reaction of all the toluidine.

The solution of p-toluenediazonium chloride is then brought to pH 6.8-7.2 at 0°with cold, concentrated, 10 aqueous sodium carbonate, whereupon the solution becomes red to orange in color and a small amount of 10 red material settles out. The cold, neutral solution is transferred to a dropping funnel and added slowly 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 3-1 flask. The reagtion mixture is kept at ca. -10° during the addition, which requires about 45 minutes (Note 3). The solution is extracted with three 1-1 portions of ether. The ethereal 15 extracts are dried with anhydrous sodium sulfate and evaporated on a rotary evaporator at room 15

temperature to give 65 g of crude 1-methyl-3-p-tolyltriazene (Note 4). This is placed in a water-cooled sublimer, and the triazene is sublimed at 50° (1 mm.); 43.3 g (0.29 mole, 62%) of a yellow, crystalline sublimate, m.p. 77-80°, is obtained (Note 5). The sublimate can be recrystallized from hexane to give the triazene as white needles, m.p. 80.5-81.5°. More conveniently, it is dissolved in the minimun amount of ether, 20 and the solution is diluted with 2 volumes of hexane and cooled to 0° to give flat plates with a slightly yellow 20 cast; m.p. 79-81°. The yield pf pure triazene is 33-37 g (47-53%) (Note 6).

Notes

1. The individual tests with starch-potassium iodide paper should be made 1-2 minutes after the addition

25 of potassium nitrite has been stopped. 25

2. 40% aqueous methylamine may be substituted.

3. The reaction is over when a drop of sqlution no longer gives a red color with a solution of |J-naphthol in aqueous sodium carbonate.

4. The chief impurity is 1,5-di-p-tolyl-3-methyl-1,4-pentazadiene (m.p, 148°). This can be removed by

30 fractional crystallization, but it is easierto sublime the triazene from the reaction mixture. 30

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

6. This procedure works well only with water-soluble amines. Procedure 2 given below is more suitable forthe preparation oftriazenes of water-insoluble amines.

35 35

Procedure 2 E. H. White eta!., Tetrahederon Letters No. 21, p. 761 {1961).

1-n-Butyi-3-p-chiorophenyitriazene. A solution of p-chlorobenzenediazonium hexafluorophosphate (re-crystallized from acetone-methanol) (2.87 g, 10.1 mmoles) in dimethylformamide (dimethylamine-free) was added slowiy to a stirred mixture of n-butylamine (0.73 g, 10.0 mmoles), powdered sodium carbonate (15 g), 40 and dimethylformamide (30 ml) stirred and maintained at -5°. The diazonium salt solution may be used at 40 room temperature; however, a purer product is usually obtained if the diazonium salt solution is prepared in and delivered from a cooled separatory funnel maintained at ca. -50°. The mixture was warmed to 0° and stirred until a negative test was obtained with 2-naphthoi (only a few minutes are usually required). Ether was added, the mixture was filtered, and the filtrate was washed thoroughly with water, then dried. (The 45 triazene may be isolated at this point and recrystallized from pentane at low temperatures. 45

Procedure 3

7-Hydroxy-9a-methoxymitosane. Mitomycin C (2.2 g, 6.6 mmoles) was dissolved in 140 ml 0.1N methanolic NaOH (50%) and the reaction mixture was stirred at room temperature for 30 hours. The solution 50 was then adjusted to ca. pH 4.0 with 1N HCI and extracted with ethyl acetate (4 x 500 ml). The combined E<0 ethyl acetate extracts were dried (Na2S04) and evaporated under reduced pressure at about 30 to 35°C to obtain a solid residue, which upon dissolving in ether and treating with excess hexane afforded a purple precipitate. The precipitate was collected and air dried to afford the title compound as a fine purple powder (1.4 g, 63%).

55 1H NMR (pyridine-rfs, 8): 2.05(s, 3H), 2.14(bs, 1H), 55

2.74(bs, 1H),3.13(d, 1H). 3.24 (S, 3H),3.56 (d, 1H),

4.00 (dd, 1H), 4.37 (d, 1H), 5.05 (t, 1H), 5.0 (dd, 1H),

5.90 (bs, 2H).

BNSDOCID: <GB 2164038A_I_>

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GB 2 164 038 A

22

Procedure 4

Mitomycin A. A100 mg (0.30 mmole) quantity of 7-hydroxy9a-methoxymitosane and 100 mg (0.67 mmole) quantity of 3-methyl-1-p-tolyltriazene was dissolved in 2 ml methylene chloride and 10 ml diethyl ether. The solution, after gently refluxing for 6 hours was stirred at room temperature for 18 hours. TLC [methylene 5 chloride: methanol (90:10)3 revealedthe appearance ofa deep purple spotatRf=0.36 with a trace amount of 5 impurity at Rf=0.41. The reaction mixture was concentrated to dryness and chromatographed on Woelm neutral alumina employing methylene chloride and methylene chloride: methanol (30:1) as eluting solvents. Fractions containing the component at Rf=0.36 were pooled and concentrated to dryness. Preparations of the dry residue from methylene chloride and hexane afforded the title compound as a fihe amorphous 10 purple powder (25 mg, 24%),'mp 161°. 10

Anat. Calc'd for C16H19N306: 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. ■

NMR (pyridine-^, 8): 1.82(s,3H),2.74(dd,1H),3.12(d, 1H),

15 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). 15

The yield in procedure 4 is raised to 63% by employing methylene chloride as reaction solvent and room temperature for a 24 hour period.

Procedures

20 In a 250 ml one neck round bottom flask was placed solid Na2CC>3,35% aqueous solution of amine (amount 20 as in Procedure 1) and ice, 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, Na2C03 (solution about H 7). Afterthe addition was complete, the reaction mixture was extracted with diethyl ether. The combined diethyl ether extract was backwashed with water, dried (Na2S04) 25 and concentrated. The yellowish solid residue was purified by column chromatography over Woelm alumina 25 using hexane-methylene chloride (1:1) as eluting solvent CH NMR recorded).

Examples 1-10

The triazenes 1 -7 of Table lit which follows were prepared according to general Procedure 1 described 30 above wherein the triazene of Example 1 is exemplified. The triazenes were purified by column 30

chromatography on Woelm alumina.

The triazenes 8-10 of Table III were prepared according to general Procedure 5 described above.

BNSDOCID: <GB 2164038A_I_>

23

GB 2 164 038 A

23

24

GB 2 164 038 A

24

10

15

20

25

30

35

r-\

9

O

A

o o o u

0

10

15

20

25

30

35

40 Example 11 40

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

A solution of 4-methylphenyldiazonium chloride is prepared as described in Procedure 1 fromp-toluidine and adjusted to pH 6.8-7.2 at 0°C as described in that procedure. A solution containing 21.15 mmoles of the diazonium salt in 45 ml of solution was prepared in this fashion and placed in a dropping funnel connected to 45 a 250 ml 3 neck, round bottom flask containing 5.34 g (20.0 mmoles) of 2-{3-nitro-2-pyridyldithio) ethylamine, 45 7 g of sodium carbonate, and 150 ml of dioxane which had been added to the flask in that sequence.

Saturated aqueous sodium carbonate solution, 6 ml, and 10 g of ice were added to the flask. The flask was chilled in an ice bath and the contents stirred mechanically. The diazonium salt solution was then added dropwise during a one hour period from the dropping funnel. When addition had been completed the 50 reaction mixture was allowed to warm to room temperature and was then extracted with three 400 ml £>0

portions of ether. Drying and evaporation of the extracts yielded the desired product which was purified by chromatography using an alumina packed column, one inch in diameter and ten inches long, using hexaneimethylene chloride (4:1); hexanermethylene chloride (3:2); hexane:methylene chloride (1:4); and finally methylene chloride containing 1 % methanol for development and elution of the column. The 55 appropriate fractions (identified by TLC) were combined and evaporated to yield 2.5 g of the title compound. 55

Examples 12-19

General procedure for preparing 7-alkoxy-9a-methoxymitosanes (12-19)

A solution of triazene (2.4 equivalents) in CH2CI2:methanol (4:1) was added to a solution of 60 7-hydroxy-9a-methoxymitosane (prepared in Procedure 3) in CH2Ci2:methanol (4:1). The reaction mixture was stirred at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC) (10% MeOH in CH2CI2). The 7-alkoxy-9a-methoxymitosane product appears as a dark purple spot on the TLC. The reaction mixture is chromatographed on Woelm alumina when the reaction is judged to be complete on the basis of the TLC, and the 7-alkoxy-9a-methoxymitosane is obtained as an 65 amorphous solid. The products produced are identified as Example Nos. 12-19 in Table IV.

60

65

BNSDOCID: <GB 2164038A l_

25

GB 2 164 038 A

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BNSDOCID: <GB 2164038A_I_>

TABLE XV (continued)

Exomple No,

Formula

NMR (Pyridine-dj, 5) ppm

IR (cm-1)

Elemental Analysis

14

c,d

IS

16

/ \ N O

\^_/

■o

1.96(s, 311), 2.40(m, 411), 2.56(t, 2H) , 2.76 (bs, 111), 3.16(d, 1H) , 3.24 (s, 311), 3.54 (d, 1H) , 3.6B(m, 4H) , 4.00 (dd, 111), 4.26(d, 1H) , 4.60(m, 211), 5.04 (t, 1H) , 5.44 (dd, 111).

1.68(3, 311), 1.96(bs, 1H), 2.72(bs, 111), 3. 08 (bs. III), 3. 20 (t, 2H) , 3.20(1;, 3H) , 3.S0(d, 1H) , 3.92 (dd, 1H) , 4.16(d, 1H) , 4.92 (m), 5.36 (dd, 111).

r»-c»2cii2sch2

a

1.92(s, 311), 2.12 (bs, 1H), 2.76(bs, 111), 2.84 (t, 2H) , 3.16 (bs, 111), 3.26(s, 311), 3.56 (d, 1H) , 3.86 (s, 211), 4.04(dd, III), 4.28(d, 111), 4.601m, 211) , 5.12 (t, 1H) , 5. 48 (dd. 111), 7.46 (m) ,' 7. 76 (bs) .

3480, 3260,

2940, 1725,

1620, 1210 1055

3430, 3300,

2930, 1715,

1625, 1210, 1060

3420, 3280,

2930, 1895, 1620, 1210, 1065

Ti C, 52.60,' II, 5.95) N, 11.41 F: C, 52.75J H, 6.00; N, 11.44 (Corrected for 0.5 mole \ of CH„C1,I.

T: C, 59.99, H, 5.49) H, 12.72 F: C, 59.94; II, S.66; N, 12.63

T: C, 59.37; H, S.60; N, 8.6S

S, 6.60

Fs C, 59.26; H, S.66; N, 8.63,

S, 6.66

17 R=-CH2CH2SS-CH2CH2HHC0CH;) 1.081s,' ill) , .2.04 (s, 3H), 2.*4(bs, 1H) , 3300, 2920,

2.76 (bs, 1H), 3.08 (m, 5H)', 3.24 (s, 3H) , 1710, 1625,

3.52(d, 1H), 3.76 (t, 211), 3.98 (dd, 1H) , 1210, 1065 4.24 (d, 1H) , 4.72 (t, 211), 5.04(t, 1H) ,

5. 40 (dd, 111).

TABLE IV (continued)

Example ^11 NMR (Pyridine-d^, 8) IR (cm '| Elemental Analysis3

No. Formula ppm

18b R=-CI!2CH2OCH3 l.B8(s, 311), 2.00(bs, in), 2.72(bs, 1H) , 3440, 3280, T: C, 54.96; H, 5.89; N, 10.68

3.08 (d, II!), 3.20 (s, 311), 3.24(s, 3H) , 2950, 1700, F: C, 54. 45; H, 5.85; N, 10.42

3.52 (m, 3H) , 3.96(dd, 1H), 4.20(d, 1H) , 1630, 1200,

4.56 (m, 211), 5.04 (t. 111), 5.40(dd, 1H) . 1065

19 R=-CH2-ai^ J 1.92 (s, 311), 2.72 (m, 111), 3.08(d, 1M , 3460, 3360, T: C, 54.15; H, 5.50; N, 9.97

O 3.20(s, 311), 3.52 (dd, 111), 3.84 In, 4H) , 3200, 2960, F: C, 53 .44; H, 5.46; N, 9.74

3.94 (dd, 111) , 4.20(d, 111), 4.60(m, 211), 1720, 1625,

4.9611, III), 5.28(m, III), 5.36(dd, 111), 1210, 1060 7.52(bs, 211).

a) T = theory, F = found b) Urakawa et al., J. Antibiotics, 23, 804-809 (1980)

c) Molecular ion observed in (+) and (-) CIMS

d) Also preparable by alcoholysls of mitomycin A, Urakawa et al., loc, cit.

28 GB 2 164 038 A

Example 20

9a-Methoxy-7-[2-(3-nitro-2-pyridyldithio)ethoxy]mitosane (20)

7-Hydroxy-9a-methoxymitosane,580 mg (1.73 mmoles) is placed in a round bottom flask and dissolved in 60 ml of methylene chloride. The triazene of Example 11, approximately 2.5 g (5.7 mnoles) was added to the 5 solution in the flask and the mixture was stirred at 5°Cfor 14 hours and then at room temperature for 8 hours. The progress of the reaction was monitored by silica TLC using methylene chloride:methanol (9:1). The reaction was kept at room temperature for an additional 26 hours and then worked up by column chromatography on a column 1/3 of an inch wide by 12 inches long packed with alumina. The solvents employed in sequence for development and elution were 200 ml portions each of methylene chloride; 0.5% 10 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, 470 mg.

Anal. Calc'd for C22H23N508S2: C, 45.65; H, 4.09; N, 11.82 (corrected for 0.5 mole% of CH2CI2):

Found: C, 45.74; H, 4.14; N, 11.61.

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

1H NMR (pyridine-ds, 8); 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 adaptation of the procedures of Examples 11 and 20 to other a>-(3-nitro-2-pyridyldithio)alkyl amines having 2 to 6 carbon atoms In the alkyl group, mitosane derivatives of the following formula may be 20 prepared.

<Ct-SS- (CH,) -O

.och,

ch n = 2-6

30 R1 = H, or C^g alkyl Examples 21-34

The 7-alkoxydithio-9a-methoxymitosanes 21-34Table V which follows were prepared according to general Procedure A or B described below and indicated in Table V. The physical data forthe mitosane compounds 35 21-34 are reported in Table VI which also follows.

Procedure A

To a deoxygenated solution of mitosane of Example 20 (—0.1 mmole) in acetone (3-5 ml) is added with stirring, under argon, triethylamine (—1.1 equivalents) followed by dropwise or portionwise addition of a 40 mercaptan® (1 equivalent) in acetone (1-2 ml), in most of the reactionsb, the progress of the reaction is monitored by silica gel thin layer chromatography a) In cases where starting mercaptan is impure >1 equivalent of thiol is required.

b) in cases where the starting mitosane of Example 20 and the product have very close Rf values on TLC, a 45 high pressure liquid chromatography (HPLC) monitoring (|j,Bondapak-C18 column) is employed.

(10%CH3OH in CH2CI2). The completion of reaction is signaled by the disappearance of spot corresponding to the starting material and appearance of the product spot. At this point the reaction mixture is concentrated under reduced pressure (at ~ 30°C) and the residue chromatographed on a neutral Woelm alumina column 50 (1/4" x 10") slurry packed with 2-5% CH3OH in CH2CI2. This procedure separates the desired mitosane product from the pyridyl thione by-product, which is retained on the column. The product thus eluted using 2-5% CH3OH in CH2CI2 is further carefully purified by flash silica gel chromatography using 5-7% CH3OH in CH2CI as the eluting solvent. The major band corresponding to product is isolated and the amorphous 7-alkoxydithio-9a-methoxymitosane is characterized.

28

5

10

15

20

25

30

35

40

45

50

BNSDOCID: <GB_

S 2164038A l_>

29

GB 2 164 038 A

29

Procedure B

To a solution of mitosane of Example 20 (—0.1 mmole) in 2-5% acetone3 in methanol {10 ml) is added saturated aqueous NaHC03 solutionb (~6 drops), followed by addition of mercaptan (1 equivalent) in methanol® (1 ml). The progress of the reaction is monitored by TLC (silica gel, 10% CH3OH in CH2CI2). At the 5 completion of reaction, the reaction mixture is diluted with water (15 ml) and concentrated to about 10 ml 5 under reduced pressure at about 30°C. The resulting solution is chromatographed on a reverse phase C-18 column with stepwise gradient elution (100%d H20 to 80% CH3OH in H20). The product, eluting as a major red band, is collected and concentrated to yield the 7-alkoxydithio-9a-methoxymitosane as an amorphous solid. If further purification is needed, the above chromatography step is repeated. 10 10

a) Methylene chloride can also be used, but acetone is preferred.

b) In the case where the mercaptan is L-cysteine,this base is not employed.

c) Water is employed if the starting thiol is water soluble.

d) Elution with water separates the yellow pyridyl thione by-product from the product, which is retained on

15 the column. 15

TABLE V

7-Alkoxydi th io-9a-methoxymi to s ane s RSS^^0

8 ch cnh2

A or

RSH + 20 >

Example No.

Thiol (RSH)

Procedure

Product

21 ethyl 2-mercapto-

acetate

O II

A R=-CH2CH2OCCH3

2 2 . 3-mercapto-l,2-

propanediol a r=-ch2ch(oh)ch2oh

2 3 3-mercaptopropxonic acid

B R=-CH2CH2COO~Na+

24

cysteine

B R=~CH2CH(NH3 )COO

25

thiophenol

26 p-nitrobenzeriethiol

A R =

A R

2 7 p-methoxybenzenethiol a R

28 p-aminobenzenethiol

•r = ^j^nh2

BNSDOCID: <GB 2164038A_I_>

30

GB 2 164 038 A

TABLE V (continued)

Example No.

Thiol (RSH)

Procedure

Product

29 2-mercaptobenzoic acid

30 2-nitro-4-mercapto-benzoic acid

COO Na+

B" R =

R =

COO~Na+

31

4-me rcapt opyridine

R =

3 2 2-mercaptomethyl-l-

methylimidazole

R =

-CH^ f

N -1

CH,

33 glutathione

NH,

I ^ _ 4 NHCOCH CH,CHC00 Na

R=—CH-CH

2I - +

CONHCH2COO Na

3 4 dimethylamine ethanethiol

B R=-CH2CH2N(CH3)2

BNSDOCID: <GB 2164038A_L>

TABLE VI

7-Alkoxydithlo-9a-methoxymitosanes

Example No.

Formula

1H NMR Data (Pyridine-d,.)a (6, ppm)

UV (CH3OH)

IK

nm)

IR (KBr) (Vmax' 01,-1 >

21 R=-CH2CH2OCCH3

J

„occ

1.76(s,3H),1.84(s,3H),1.97(t,lH, 7.9Hz),2.59(bs,1H),2.87(t,2H, 6.7Hz),2.96(t,2H,6.2Hz), 2.99(bs,1H),3.07(s,3H),3.37 (d,1H,12.1Hz),3.84(dd.lH,11.2,

4.3Hz),4.05(d,lH,12.1Hz),4.27 *

(t,2H,6.7Hz),4.56(m,2H),4.92(t, 1H,10.8Hz),5.25(dd,lH,11.2,4.3Hz)

520,324, 216

3370,3310,2930, 1740,1720,1640, 1560,1520,1335, 1220, 1200, 1065, 1030.

TABLE VI (continued)

Example No.

Formula

NMR Data (Pyridine-d,.)' (6, ppm)

UV (CH^OH)

(X , run) * max'

IR (KBr)

(v , cm max'

?H

22 R=-CH2CHCH2OH

1.77(s,3H),1.99(bs,1H),2.60(bs,1H), 215,32 4,

3.03(t,2H,6.3Hz),3.00(m,lHj, 3.07(s,3H),3.15(m,lH),3.28 (dd, 1H,4.5,13.2Hz),3.37(d,1H,12.4Hz), 3.84(dd,1H,4.5,11.2Hz),3.94 (bs, 2H),4.06(d,1H,12.4Hz),4.28(bS,lH), 4.57(m,lH),4.64(m,1H),4.92(lH,t, 10.6Hz),5.25(dd,1H),6.29(bs,lH), 6.69(bs,1H),7.5{m,2H).

520

3440,29 65,1720, 1655,1635,15 80, 1455,1410,1340, 1305,1215,1070, 1020.

23 R=-CH2CH2COO~Na+

1. 77 (s, 3H) , 2. 60 (bs, 111) ,2.78(t,2H, 7.4Hz),2.94(m,3H),3.00(s,3H), 3.17(t,2H,7.4Hz),3.38(d,1H,12.4Hz), 3.83(dd,lH,4.0,11.0Hz),4.06(d,lH, 12.5Hz),4.58(m,2H),4.84(m,lH), 5. 20 (dd, 1H, 4.1,10. 6Hz), 7. 64 (m, 2H) .

215,323, 510

3430,2920,1715, 1650,1620,1575, 1450,1405,1340, 1300,1210, 1110, 1065.

TABLE VI (continued)

Example *H NMR Data (Pyridine-dg)a UV (CH3OH) IR (KBr)

No. Formula (6, ppm) cm"1)

illcUv illdA

24

25

f2

R=CH2CHCOOH

R=

7 V

1.92(s,3H),2.95-3.15(m,5H),3.29 216,324,

(s,3H),3.36(dd,lH,4.0,15.0Hz),4.02 381,52 6 (d,lH,13Hz),4.10 (dd,lH,4.0,8.7Hz), 4.30(t,lH,10.5Hz),4.48(m,2H),4.66

(dd,1H,4.8,10.8Hz).

1.75(s,3H),1.99 (bs,lH),2.61 (bs,lH), 20 6,324,

2. 98 (m,3H) ,3.08(s,3H) ,3.39(d, 111) , 524

3.82(dd,lH,4.3,11.0Hz),4.06 (d,lH,

12.4Hz),4.49(m,1H),4.57(m,lH),4.92

(m,1H),5.23(dd,lH,4.3,11.0Hz),7.11

(d,lH,7.2Hz),7.19 (dd,2H,7.2,7.2Hz),

7.50(d,2H,7.2Hz).

3430,29 20,1715, 1635,1575,1495, 1450,1340,1300, 1210,1060.

3440,29 20,1715, 1650, 1630,1575", 1450,1335,1300, 1210,1105,1055.

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TABLE VI (continued)

Example 1H NMR Data (Pyridine-d^a UV (CH3OH) IR (KBr)

No. Formula (6, ppm) *\nax' nm' *Vmax' cm

30 R=

NO-

COO Na'

31

- O

1.75(s,3H),2.58(bs,lH),2.93(t,2H, 215,321, 6.3Hz) ,2.90 (m,lH) ,3. 38 (d',lH,520 520 12.8Hz),3.82(dd,lH,4.3,10.9Hz), 4.06(d,lH,12.1Hz),4.53(m,2H),4.88 (t,lH,10.5Hz) ,5.21 (dd, 111,4. 2, 10.7Hz),7.45(d,lH,8.5Hz),7.65 (d,lH,8.3Hz),8.14(bs,lH). -

4

1.76(3H,s),1.95(bs,IH),2.60(bs, 215,241,

IH),3.01(t,2H,6.1Hz),3.00(m,lH), 32 3,52 2 3.08(s,3H),3.39(d,lH,11.5Hz),

3.83 (dd, IH, 4. 2,11. 0 Hz) , 4 . 07 (d, IH, 12.4Hz),4.52(m,2H),4.90(bs,lH), 5.25(dd,lH,4.3,10.5Hz),7.39 (dd, 2H,1.5,4.5Hz),7.6(m,2H), 8.48(dd,2H,1.5,4.5Hz) .

3440,29 30,1720, 1620,1580,1520, 1450,1400,1340, 1215,1070

3430,2920,1720, 1650,1630,15 75, 1450,1405,1335, 1300,1210,10 65.

TABLE VI (continued)

Example No.

Formula

*H NMR Data (Pyridine-d^)a (6, ppm)

UV (CH3OH)

(X

max'

nm)

IR (KBr)

(v , cm *) max'

32

R=-CH

><3

N'

I

ca.

1.74(s,3H),2.59(bs,lH),2.73(t,2H, 6.4Hz),2.98(d,IH,4.2Hz),3.08(s, 3H),3.40(m,IH),3.43(s,3H), 3.84 (dd ,1H,4.4,11.0Hz) , 4. 06 (d, IH,12.5Hz),4.11(s,2H),4.46(m,2H), 4.89(m,IH),5.25(dd,lH,4.4,10.5Hz), 6.95(q,2H).

218,323, 5 20

3430,2960,1720, 1650,1630,1580, 1490, 1450, 1335, 1300,1210,1105, 1065.

TH2

ch.chcoo Na rC°CH2-2

33 R=-CH_CH

I — +

CONHCH2COO Na

1.91 (s,3H) ,2.16(dd,2H,7.4,14.2Hz) 2.55(dt,2H,2.3,8.2Hz),2.95-3.10 (m,5H),3.28(s,3H),3.28(dd,lH, 4.6,14.1Hz),3.61(d,lH,13.1Hz), 3.70(dd,lH,4.4,10.5Hz),3.70-3.90 (m,4H),4.02(d,2H,13.1Hz),4.30 (t,IH,10.5Hz),4.46(m,2H),4.65(dd, IH,4.5,10.7Hz).

326,366, 524

3380, 32 90,29 20 , 1715, 1650, 1635_, 1580, 14 55, 1405, 1340,1300,1210, 1105,1065,1030.

34

r=-ch2ch2n(ch3)2

1.77(s,3H),1.93(bs,lH),2.14(s, 6H) ,2.61 (m,3H) , 2. 80-3 .05 (in, 5H) ,3.08(s,3H),3.40(m,lH), 3.84(dd,lH,4.3,10.3Hz),4.06 (d,lH,12.5Hz),4.57(m,2H), 5.26(dd,IH,4.3,10.3Hz)

a) D^O was used for mitosanes of Examples 24 and 33.

38 GB 2 164 038 A

38

Claims (39)

1. A compound selected from the group having Formula II or Formula III

R -Alk, -SS-Alk,

t-R'

R -SS-Alk,-i t-R'

CH.

25 wherein:

Alki is a straight or branched chain alkylene group having 1 to 6 carbon atoms when R3 is joined thereto through a carbon atom thereof, and 2 to 6 carbon atoms when R3 is joined thereto through a sulfur, oxygen • or nitrogen atom thereof, and R3 and —SS- are in that instance joined to different carbon atoms,

Alk2 is a straight or branched chain alkylene group having 2 to 6 carbon atoms optionally bearing an A 30 substituent wherein the sulfur and oxygen atoms connected thereto and any optional A substituent connected thereto through oxygen, sulfur or nitrogen, are attached to different carbon atoms of AIk2, wherein said A substituent is selected from the group consisting of one or two alkyl, C-,-6 alkanoyl, Ci_6 alkoxy, halogen, alkoxycarbonyi, cyano, 0^6 alkylamino, Cv6 dialkylamino, 0^6 alkanoylamino and Ci_6 alkoxycarbonyi,

35 Alkt and Alk2 may contain a double bond,

R1 is hydrogen, lower alkyl, lower alkanoyl, benzoyl or substituted benzoyl wherein said substituent is lower alkyl, lower alkoxy, halogen, amino or nitro,

R3 is selected from the group consisting of halogen, carboxy, alkanoyloxy having 1 to 7 carbon atoms, hydroxy wherein the oxygen atom is connected to Alki having 3 to 6 carbon atoms, alkylamino or 40 dialkylamino having 1 to 12 carbon atoms, N-alkoxy-alkylamino having 2-7 carbon atoms, alkanoylamino having 1-7 carbon atoms, benzoylamino or B-substituted benzoylamino, naphthoylamino or B-substituted naphthoylamino, phenylamino or B-substituted phenylamino, cycloalkyl or B-substituted cycloalkyl each having 3 to 8 ring members, cycloalkenyl or B-substituted cycloalkenyl each having 5 to 8 ring members, phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the 45 group consisting of heteroaromatic and heteroalicyclic groups having from 1 to 2 rings, from 3 to 8 ring members in each ring and from 1 to 2 heteroatoms in each ring selected from oxygen, nitrogen and sulfur, pyridylamino orthiazolylamino, alkoxy oralkylthio each having 1 to 6 carbon atoms, alkoxycarbonyi or alkylaminocarbonyl each having 2 to 7carbon atoms, aminocarbonyl, phenoxycarbonyl or B-substituted phenoxycarbonyl, phenoxy or B-substituted phenoxy, naphthoxy or B-substituted naphthoxy, alkoxycar-50 bonyiamino having 2 to 6 carbon atoms, ureido (—NHCONH2), N-alkylureylene (-NHCONHalkyl) having 2 to 7 carbon atoms, N3-ha(oaikyfureylene having 3 to 7 carbon atoms, N3-haloalkyl-N3-nitrosoureylene having 3 to 7 carbon atoms, dialkylaminocarbonyi having 3 to 13 carbon atoms, dialkylaminoalkoxy having 4to 13 carbon atoms, alkanoylaminoalkoxy having 3 to 7 carbon atoms and hydroxyalkylamino or N,N-dihydroxyalkylamino each having 2 to 8 carbon atoms, wherein said B substituent is selected from the group 55 consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, carboxy, hydroxy and nitro groups, and

R4 is selected from the group consisting of alkyl having 1 to 12 carbon atoms, alkenyl or alkynyl each having 3 to 12 carbon atoms, cycloalkyl or B-substituted cycloalkyl having 3 to 8 ring members, cycloalkenyl or B-substituted cycloalkenyl each having 5 to 8 ring members, phenyl or B-substituted phenyl, naphthyl or 60 B-substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromatic and heteroalicyclic groups having from 1 to 2 rings, from 3 to 8 ring members in each ring, and from 1 to 2 heteroatoms in each ring selected from oxygen, nitrogen and sulfur, provided that the heterocyclic group is connected through a carbon atom which is attached to at least another carbon atom, wherein said B substituent is selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, 65 halogen, amino, carboxy, hydroxy or nitro groups, and R4 and the adjacent sulfur atom together constitute

BNSDOCID.- <GB 2164038A (_>

5

10

15

20

25

30

35

40

45

50

55

60

65

39

GB 2 164 038 A

39

S-cysteinyl wherein said S-cysteinyl group may be esterified, salified or joined within a non-toxic and non-allergenic peptide, or a nontoxic pharmaceuticaily acceptable salt thereof.

2. A compound of Claim 1 wherein Alk2 is ethylene and R1 is hydrogen.

The compound of Claim 1 or 2 of Formula II wherein Alk-i is ethylene and R3 is acetylamino.

The compound of Claim 1 or 2 of Formula II wherein Alk! is ethylene and R3 is acetyioxy.

The compound of Claim 1 or 2 of Formula II wherein Alkn is methylene and R3 is 1,2-dihydroxyethyl.

The compound of Claim 1 or 2 of Formula II wherein Alk, is ethylene and R3 is carboxy or a nontoxic pharmaceuticaily acceptable salt thereof.

7. The compound of Claim 1 or 2 of Formula II wherein Alki is methylene and R3 is 1-carboxy 10 aminomethyl or a nontoxic pharmaceuticaily acceptable salt thereof.

8. The compound of Claim 1 or 2 of Formula II wherein Alk-! is methylene and R3is 1-methylimidazol-2-yl.

The compound of Claim 1 or 2 of Formula II wherein Al^ is ethylene and R3 isdimethylamino.

The compound of Claim 1 or 2 of Formula III wherein R4 is phenyl.

The compound of Claim 1 or 2 of Formula III wherein R4 is 4-nitrophenyl.

The compound of Claim 1 or 2 of Formula III wherein R4 is 4-methoxyphenyl.

The compound of Claim 1 or 2 of Formula III wherein R4 is 4-aminophenyl.

The compound of Claim 1 or 2 of Formula III wherein R4 is 2-carboxyphenyl or a nontoxic pharmaceuticaily acceptable saltthereof.

15. The compound of Claim 1 or 2 of Formula 111 wherein R4 is 4-nitro-3-carboxyphenyl or a nontoxic 20 pharmaceuticaily acceptable saltthereof.

16. The compound of Claim 1 or 2 of Formula 111 wherein R4 is4-pyridyl.

17; The compound of Claim 2 having the formula

15

3.

4.

5.

6.

9.

10.

11.

12.

13.

14.

10

15

20

25

30

NH_

I ^

H02CCHCH2CH2C0NH

CHCH-SSCH_CH,0 I 2 2 2 H02CCH2NHC0

25

30

or a nontoxic pharmaceuticaily acceptable salt thereof.

18. A compound of Claim 1 having Formula lb

35

40

lb

35

40

45 wherein R1 and Alk2 are as defined in Claim 1.

19. The compound of Claim 2 having the formula

45

50

55

XVII

50

55

20. The method of inhibiting growth of a mammalian tumor which comprises systemic administration to a mammal bearing a tumor a substantially nontoxic antitumor effective dose of a compound of any of Claims 1-19.

60

21. A pharmaceutical composition comprising a compound of any of claims 1-19 and a pharmaceuticaily 60 acceptable solvent, diluent, adjuvant or carrier.

: <GB 2164038A l_>

40

GB 2 164 038 A

40

22. The process forthe preparation of a compound of Claim 1 which comprises reacting at least one equivalent of a triazene of Formula V or Formula VI

Ar-N=N—NH-Alk2—SS-Alk!—Ft3 Ar-N=N—NH—Alkz-SS—R4 with one equivalent of a mitosane of Formula IV

V Vi

10

15

IV

wherein:

R1, R3, R4, Alkn and Alk2 are the same as defined in Claim 1 and Ar is the organic residue of a diazotizable 20 aromatic amine under reaction conditions in an inert organic solvent at a temperature from about 0° to 60°C until an appreciable amount of the product of Formula II or Formula III is obtained.

23. The process of Claim 22 wherein said triazene of Formula VI is 1-{2-{3-nitro-2-pyridyldithio)ethyl]-3-{4-methylphenyl) triazene.

24. The process for the preparation of a compound of Claim 1 which comprises reacting at least one 25 equivalent of thiol of Formula F^AI^SH or Formula R4SHwith one equivalent of a mitosane of Formula lb

10

15

20

25

30

lb

35 wherein:

R1, R3, R4, Alki and Alk2 are the same 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 from about 0° to 60°C until an appreciable amount of the product of Formula II or Formula III is obtained.

25. The process of Claim 24 wherein Alk2 is ethylene and R1 is hydrogen.

40 26. The process of Claim 24 or 25 wherein one equivalent of base is employed in the reaction.

27. The process of Claim 24,25 or 26 wherein a lower alkanol, a lower alkanoic lower alkyl ester, a lower aliphatic ketone, a cyclic aliphatic ether or a lower polyhalogenated aliphatic hydrocarbon having up to 8 carbon atoms or water is employed as reaction medium.

28. The process for preparing a compound having Formula IX

45

50

IX

wherein:

55 R5 is hydrogen, or C!.6 alkyl, and

R6 is Cn_12 alkyl or substituted Ci_i2 alkyl, C3.12 cycloalkyl or substituted C^ cycloalkyl wherein the carbon atom thereof which is attached to the mitosane 7-oxygen atom bears from 1 to 2 hydrogen atoms and said substituents are selected from the group consisting of halogen, Cn.6 alkoxy, Ci.6 alkanoyl, C6.14 aroyl, cyano, trihaiomethyl, amino, C-,_6 monoalkylamino, C2.12 dialkylamino, C6.12 aryl, C6-i2 aryloxy, C|.6 alkanoyloxy, 60 C704 aroyloxy, heterocyclo having 1 or 2 rings and from 5 to 12 ring atoms including up to 4 heteroatoms seleci .id from nitrogen, oxygen, and sulfur, and wherein each of said alkoxy, alkanoyl, aroyl, aryl, aryloxy, alkanoyloxy, aroyloxy, and heterocyclo substituents optionally contains from 1 to 2 substituents selected from \elogen, C^ alkoxy, Ci_6 alkanoyl, cyano,trihaiomethyl, amino, Ci.6 alkylamino, or C2.12 dialkylamino groups which comprises reacting a mitosane of Formula X

30

35

40

45

50

55

60

_2164038A_I_>

41

GB 2 164 038 A

41

O

Hi

I!

o ch2ocnh2 ^OCH3

5

X

5

CH

■R'

,5

with a triazene of Formula XI

10

10

Ar-N=N-NH-R6

XI

wherein:

RB and R6 are as defined above and Ar is the organic residue of a diazotizable aromatic amine.

15

29. The process of Claim 28 wherein said triazene of Formula XI is 3-methyl-1-(4-methylphenyl)triazene. 15

30. The process of Claim 28 or 29 wherein at least two molecular proportions of said triazene relative to said mitosane of Formula X are employed.

31. The process of Claim 28,29 or 30 wherein a reaction inert organic liquid solvent for said mitosane of Formula X is employed as reaction medium.

20 32. The process of Claim 28,29 or 30 wherein a lower alkanol, a lower alkyl alkanoic ester, a dilower alkyl 20 ether, a lower polyhalogenated aliphatic hydrocarbon ora cyclic aliphatic ether having up to 8 carbon atoms is employed as reaction medium.

33. The process of Claim 28,29 or 30 wherein methylene chloride, methanol, diethyl ether, ethyl acetate ora mixture of two or more thereof is employed as reaction medium.

25 34. The process of any of claims 28-33 wherein the reaction temperature is from 0° to 60°C. 25

35. The process of Claim 34 wherein the reaction temperature is from 0° to 25°C.

36. The compound IX of Claim 28 wherein R6 is 2-(benzylthio)ethyl and Rs is hydrogen.

37. The compound IX of Claim 28 wherein R6 is 2-(2-pyridyl)ethyl and R5 is hydrogen.

38. The compound IX of Claim 28 wherein R8 is 2-{4-morpholinyl)ethyl and Rs is hydrogen.

30 39. The compound IX of Claim 28 wherein R6 is (1,3-dioxolan-2-yl)methyl and R5 is hydrogen. 30

40. A process as claimed in claim 22 or 24, substantially as described in respect of any of the foregoing Examples.

41. A compound as claimed in claim 1, prepared by a process as claimed in any of claims 22-27, or claim 40.

35 42. A pharmaceutical composition comprising a compound asclaimed in any of claims 2-19, orclaim 41, 35 and a pharmaceuticaily acceptable solvent, diluent, adjuvant or carrier.

43. A process as claimed in claim 28, substantially as described in respect of any of the foregoing Examples.

44. A compound of the formula IX as defined in claim 28, prepared by a process as claimed in any of

40 claims 28-35,orclaim43. 40

45. A pharmaceutical composition comprising a compound as claimed in any of claims 36-39, or claim 44, and a pharmaceuticaily acceptable solvent, diluent, adjuvant or carrier.

Amendments to the claims have been filed, and have the following effect:-

45 (a) Claims 36to 39 above have been deleted or textually amended. 45

(b) New or textually amended claims have been filed as follows:-

36. The process of Claim 28 wherein R6is2-(benzylthio)ethyl and R5 is hydrogen.

37. The process of Claim 28 wherein R6 is 2-(2-pyridyl)ethyl and R5is hydrogen.

50 38. The process of Claim 28 wherein R6 is 2-(4-morpholinyl)ethyl and R5 is hydrogen. 50

39. The process of Claim 28 wherein R6is (1,3-dioxolan-2-yl)methyl and Rs is hyd-ogen.

Printed in the UK for HMSO. D8818935, 1/8B. 7102.

Published by The Patent Office, 25 Southampton Buildings, London, WC2A 1AY, from which copies may be obtained.

: <GB 2164038A l_>