CA1291293C - Substituted 7-oxomitosanes - Google Patents

Abstract

ABSTRACT

The present invention provides novel mitomycin analogs containing a disulfide group 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. Mitomycin A is an antibiotic having antitumor activity, and the 7-0-substituted mitosane analogs thereof have similar utility.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention provides novel mitomycin analogs containing a disulfide group 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 provides a method of producing mitomycin A and derivatives thereof. Mitomycin A is an antibiotic of established utility, and the 7-O-substituted mitosane analogs thereof have similar utility.

Nomenclature - The systematic Chemical Abstracts name for mitomycin A based on the recent revision [Shirhata et al., J.
Am. Chem. Soc., 105, 7199 (1983)] is:

[laS-(la~,8~,8aa,8b~)]-8-[((aminocarbonyl)oxy)methyl]-6,8a-dimethoxy-l,la,2,8,8a,8b-hexahydro-5-methyl-arizino[2',3',3,4,]pyrrolo[1,2-a]indole-4,7-dione ~g .
~ ,.

.: .

_3 ~ D~ "~
according to which the azirinopyrroloindole ring system is numbered as follows:

5~

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.

CH ~ Hla : 0 3 2 Mitosane .

According to this system, mitomycin A is 7,9a-dimethoxymitosane and mitomycin C i5 7-amino-9a-methoxymitosane. As to the stereo-chemical 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 configuration 4 ~ 3 thereof as the same as that of mitomycin A or C.

R~"~ R Mitomycin A R=Rl=OCH3 11 11 >~ Mitomycin C R=NH2, Rl=OCH3 CH3 1~--N ~NH
o 2. Disclosure Statement Mitomycin C is an antibiotic which is produced by fermentation and is presently on sale under Food and Drug Admin-istration approval in the therapy of disseminated adenocarcinoma o f 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 Reference 37th Edition, 1983, pp. 747 and 748). Mitomycin C and its production by fermentation is the subject 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.

The structures of mitomycins A, B, C, and of porfiro-mycin were first published by J. S. 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 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-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, la, 8a and 8b have been shown to be as indicated above with respect to the Chemical Abstracts nomenclature ~Shirhata et al., J. Am. Chem. Soc., 105, 7199-7200 (1983)]. The earlier literature refers to the enanti-omer.

'~' _5_ ~ 3 The following articles and patents deal inter alia with the conversion of mitomycin ~ to a 7-substituted amino mitom~cin C derivative having antitumor activity. The object of this re-search was to prepare derivatives which were more active, and particularly which were less toxic than mitomycin C:

Matsui et al., J. Antlbiotics, XXI, 189-198 (1968);

Konishita et al., J. Med. C~em., 14, 103-109 (1971);

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

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, issued July 25, 1967;

~` Matsui et al., U.S. Patent No. 3,420,846, issued January 7, 1969;

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

Matsui et al., U.S. Patent No. 3,514,452, issued ` May 26, 1970;

Nakano et al., U.S. Patent No. 4,231,936, issued November 4, 1980;

.
Remers, U.S. Patent No. 4,268,676, issued May 19, 1981.

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

Kono et al., European Patent Application No. 116,208 ~1984), Vyas et al., 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 et al., J.
Antibiotics, 23, 804-809 (1980).

Mitomycin C is the principal mitomycin produced by fexmentation and is the commercially available form. Current technology for the conversion of mitomycin C to mitomycin A
suEfers from a number of deficiencies. Hydrolysis of mitomycin C
to 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 the 7-hydroxy intermediate is very unstable [Matsui et al., J.
An~ibiotics, XXI, 189-198 (1968)]. One attempt to avoid these difficulties involves the use of 7-acyloxymitosanes (Xyowa Hakko Xogyo 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 produc-tion of only specific 7-alkoxy structural types by the availabil-ity and reactivity of the alcohol starting materials.

SUMMARY OF THE INVENTION

The present invention is concerned with a group of mitomycin A analogs having a dithio organic substituent - 7 ~ ~9~ 3 incorporated in the alkoxy group at the 7-position. These compounds may be represented by the following general formula C~ ~ ~
R2-SS-Alk2-0 ~ ~ ~ 1 I

CH3 N ~ -R

wherein R2 is an organic group, viz. the structural component of an organic thiol of the formula R2S~, and Alk2 and R1 have the meanings given below. These compounds are alternatively de-scribed by Formulas II and III.

O C~20CNH2 R -Alkl--SS-Alk2 ~ 3 II

C~3 ~ N ~ ~ -R4-55_~

wherein:

Alkl 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 ,2 ~ 3 and -SS- are in that instance joined to different carbon atoms, AIk2 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 connected thereto through oxygen, sulfur or nitrogen are attached to different carbon atoms of Alk2, wherein said A substituent is selected from the group consisting of one or two Cl 6 alkyl, Cl 6 alkanoyl, Cl 6 alkoxy, halogen, Cl 6 alkoxy-carbonyl, cyano, Cl 6 alkylamino, Cl~6 dialkyl-- amino, Cl 6 alkanoylamino and Cl 6 alkoxycarbonyl, Alkl and Alk2 may contain a double bond, Rl is hydrogen, lower alkyl, lower alkanoyl, benzoyl or substituted benzoyl wherein said substituent is lower alkyl, lower alkoxy, halogen, amino or nitro, R3 is sel~cted from the group consisting of halogen, carboxy, alkanoyloxy having 1 to 7 carbon atoms, alkylamino or dialkylamino having l 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 B ring members, cycloalkenyl or B-substituted cycloalkenyl each having 5 to 8 ring members, : phenyl or ~ substituted phenyl, naphthyl or :B-substituted naphthyl, a heterocyclic group selected from the group consisting of ~'1 : , .. , : .
. .
:: ;
,.

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 or thiazolylamino, alkoxy or alkylthio each having 1 to 6 carbon atoms, alkoxycarbonyl 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, alkoxycarbonylamino having 2 to 6 carbon atoms, ureido (-NHCONH2), N-alkylureylene (-NHCONHalkyl) having 2 to 7 carbon atoms, N3-haloalkylureylene having 3 to 7 carbon atoms, N3-haloalkyl-N3-nitrosoureylene having 3 to 7 carbon atoms, dialkylaminocarbonyl having 3 to 13 carbon atoms, dialkylaminoalkoxy having 4 to 13 carbon atoms, alkanoylaminoalkoxy having 3 to 7 carbon atoms and hydxoxyalkylamino 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 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 cyclo-alkenyl 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 a 5- or 6- membered cyclic aromatic or alicycllc ring having from 1 to 2 heretoatoms in each ring selected from oxygen, nitrogen and sulfur, wherein said B substituent is , .,~.,;.,. :
'-, :. ', :, ~ '' ,: :, - , ." ,, .. . ~ .
.. . .
; '.,' '` ' ' selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, carboxy, hydroxy or nitro groups, and R4 and the adjacent sulfur atom together constitute S-cysteinyl 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 are employed in a manner similar to mitomycin C. The dosages employed 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 `employed.

In a furthex aspect of the present invention, there is provided a new process for the production of mitosanes of Formulas II and III. This new process comprises reacting a mito-sane of Formula IV
O

C~3 ~ N ~ _Rl O \~
with a triazene of Formula V or Formula VI

Ar-N=N-NH-Alk 2- SS -Alk l-R3 Ar -N=N-NH-Alk 2 ~ SS -R4 V VI

L~

~' ' ' .

` ~ ,.
.

3~

wherein Rl, R3, R4, Alkl and Alk~ are as defined above and Ar is the organic residue of a diazotizable aromatic amine.

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

R3AlklSH R4SH
VII VIII

with a mitosane derivative of Formula Ib SS-Alk2-O ~ Rl 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 Rl is hydrogen is described in Example 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

CE~20CNH2 R O ~ IX

CH3 ~ N ~ -R5 O
wherein:
R5 is hydrogen, or Cl 6 alkyi, and R6 is Cl 12 alkyl or substituted Cl 12 alkyl, C3 12 . .

. . .

~ 73 cycloalkyl or substituted C3 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 substituents are selected ~rom the group consisting of halogen, Cl~6 alkoxy, Cl_6 alkanoyl, C6_14 aroyl, cyano, trihalomethyl, amino, Cl 6 monoalkylamino, C2 12 dialkylamino, C6 12 aryl, C6_12 aryloxY' C1-6 alk Y 7 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 alkoxy, alkanoyl, aroyl, aryl, aryloxy, alkanoyloxy, aroyloxy, and hetero-cyclo substituents optionally contains from 1 to 2 substituents selected from halogen, Cl 6 aLkoxy, Cl 6 alkanoyl, cyano, trihalomethyl, amino, Cl 6 alkylamino, or C2_12 dialkylamino groups.

Many of the compounds of Formula IX are ~nown compounds having inhibitory activity against experimental animal tumors ln 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 their toxicities relative to the toxicity of mitomycin C. In cases where the new compound i5 less toxic, a higher dose is employed.

The new process for production of compounds of Formula -13~ 3~"~
IX comprises reacting a mitosane of Formula X

Il 3 ~--R5 X

with a triazene of Formula XI
Ar-N=N-NH-R6 XI

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

The terms "lower alkyl", "lower alkoxy" and "lower alkanoyl" as used herein and in the claims (unless the context indicates otherwise) mean 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-butyl, amyl, hexyl, etc. Preferably, these groups contain from l 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 claims is intended to include chlorine, fluorine, bromine and iodine. The term "nontoxic pharmaceutically acceptable salt" is intended to include salts of the compounds of Formulas I and II with any nontoxic pharmaceutically acceptable acid or base. Such acids are well-known and include hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, maleic, ~umaric, succinic, oxalic, benzoic, methanesulfonic, tartaric, citric, camphorsulfonic, levulinic and the like. Such bases are well-known and include, e.g. nontoxic metallic salts such as sodium, potassium, calcium and magnesium, the ammonium salt and salts with nontoxic amines, e.g. trialkylamines, procaine, dibenzylamine, pyridine, .
" . . : .
.', ...

`. .. . . . . .
. .

~14-N-methylmorpholine, N-methylpiperidine and the like. The salts are made by methods known in the art.

DESCRIPTION OF TH E INVEN TI ON
_ The present invention provides a new process for the preparation of compounds of Formula IX which comprises reacting a mitosane of Formula X with a triazene of Formula XI as shown in Scheme l.

Scheme 1 HO~ ~ ~ ,OCH
l l ~ ~ Ar-N=N-NH-R6 ~ IX
CH3~ ~ ```N ~ -R5 XI
o X

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

The l-substituted-3-aryltriazenes of Formula XI and more specifically l-alkyl-3-aryltriazenes make up a class of reagents which are known to be useful for reacting with car-boxylic acids to form the corresponding low r alkyl esters.

1-Methyl-3-t4-methylphenyl)triazene may be prepared according to the general procedures described by E. H. White et al. in Or~.

Syn., 48, 102-105 (1968) and as described herein in Procedure 1.

However, this procedure works well only with water-soluble amines, and a second procedure which is described by E. H. White et al., Tetrahedron Letters, No. 21, 761 (1961) and also de--scribed herein in Procedure 2 is more suitable for the prepara-tion of triazenes of water-insoluble amines.

,, ,~ , , ~iX~3~33 The reagent l-methyl-3-(4-methylphenyl)triazene pre-pared in the above fashion has been previously employed to prepare methyl esters of carboxylic acids such as 2,4-dinitro-benzoic acid [E. H. White et al. Org. S~n., 48, 102-105 (1968)]
and cephalosporanic acids which yields the desired ~3-compound without isomerization to the ~2-isomer [Mangia, Te~ràhedron Letters, No. 52, pp. 5219-20 (1978)]. The reagent has 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. (Wiederkeher et al. U.S. Patent No. 4,069,324 issued January 17, 1978).

Other l-(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 arylamine having 6 to 12 carbon atoms which readily forms a diazonium salt nay be used as the source of the aryl portion of the 1,3-disub-stituted triazene. Some examples of triazenes produced in this fashion and used in the present inventioh are:

1- (n-butyl)-3- (4-methylphenyl)triazene;
l-(l-methylethyl) -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)triazeneO

Other triazenes have been described in the literature which are suitable reactants ~or use in the present process to provide 7-(substituted alkoxymitosane) of Formula IX. Those described by T. A. Daniels et al., Can. J. Chem., 55, 3751-3754 (1977) are exemplary.

~,.. ' ' ' '' ' ' .: ''"""' .

-16- ~; q~:g~ 3 X ~ N=N-NH-CH2-Y

a X = H, Y - CN
X 2' ~ CN
c X = COzMe, Y = CN
d X = Ac, Y = CN
e X = NO2, Y = CO2Et f X = CO2Me, Y = CO2Et g X = CO2Me, Y - COPh h X = NO2, Y - -CH(OCH3)2 The following further exemplify suitable triazene starting materials of Formula XI for use in the present in-vention.

l-(n-butyl)-3-(a-napthyl)triazene l-(n-hexyl~-3-phenyltriazene 1-ethyl-3-(2,4-dimethylphenyl)triazene l-(l-methylethyl)-3-(4-methoxyphenyl)triazene For the 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 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 100C are preferred. Specific pre-ferred solvents are methylene chloride, methanol, 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 60C. At temperatures in excess of this the mitosane reactant is inclined to decompose with a resultant reduction in - .

.. .

.
. : :

-17~ 3 yield. It is preferred to carry out the reaction at room temper-ature or below, for instance within the range of O to 25~C.

A convenient ~ay to determine when the reaction is complete is by thin layer chromatography. Mitomycin A is deep purple in color and can be readily distinguished ~rom the start ing 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 puri-fication of the product.

The ~oregoing reaction conditions and precautions are generally applicable to the preparation of other 7-R50-mitosanes of Formula IX according to the present process.

The new process of the present invention utilizing l-substituted-3-aryltriazenes may also be used to prepare com-pounds 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.
.
Scheme 2 C~I20CNH2 Ar-N=N-NH-Alk2-SS-Alkl-R3 H ~ ,OCH3 +> II or III
Il l ~ or Ar-N=N-NH-Alk2-SS-R
3 ~ ~ N ~ ~ -Rl VI
O
IV

wherein Rl, R3, R4, Alkl and Alk2 are as defined above, and Ar is the organic residue of a diazotizable aromatic amine.

Aryltriazenes of Formula V or VI may be prepared in a similar fashion as described above for the preparation of : . , . ' ~ '' ' ~ .

aryltriaxenes of Formula XI except that the alkyl amines utilized therein are replaced by aminodisulfides of Formula XII

R2-SS-Alk2NH2 XII
which are alternatively described by Formulas XIII and XIV

R3-Alkl-SS-Alk2NH2 and R4-SS-AlkzNH2 XIII XIV

Aminodisulfides of Formula XIII and Formula XIV are known compounds and may be prepared by various methods. For instance, they may be made by reaction of the appropriate thiol R3AlklSH or R4SH with a Bunte salt of the formula or with a sulfenylthiocarbonate of the formula NH2Alk2SS~CH3 XVI
.
Klayman et al., J. Org. Chem., 29, 3737-3738 (1964) have prepared the following by the Bunte salt method:

2-aminoethyl n-butyl disulfide;
2-aminoethyl n-hexyl disulfide;
2-aminoethyl n-octyl disulfide;
2-aminoethyI n-decy~ disulfide;
2-aminoethyl phenyl disulfide;
2-aminoethyl benzyl disulfide.
' Methanol was found to be the preferred reaction solvent for the reaction of the Bunte salt with the thiol. Reaction temperatures of 0 to -10C were found to be preferred using this solvent. Higher temperatures were necessary with other solvents.
The chief drawback of this method is the formation of symmetrical 2~

disulfides as a by-product, presumably as a result of dispropor-tionation of the desired mixed disulfide.

The mixed disulfide ~tarting 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 et al., J. Am. Chem. Soc., 921 7629-7631 (1970). Typically, this preparative procedure involves adding the thiol to a methanol solution of the amino-alkylsulfenylthio-carbonate of Formula XVI and allowing the reaction to proceed at lO a temperature in the xange of from 0 to 25C. 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 amount of triethylamine may be added as reaction accelerator.

The l-(substituted disulfide)-3-aryltriazenes of Formula V or VI are prepared by the reaction of aminodisulfides of Foxmula XII with aryldiazonium salts in a similar fashion as described herein for the preparation of aryltriazenes of Formula 20 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 l,3-disubstituted triazene. Some examples of disulfide triazenes produced in this fashion and used in the present invention are 1-[2-(2-acetamldoethyldithio)ethyl]-3-(4-methylphenyl)triazene;
l-[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.
l-[2-(3-nitro-2-pyridyldithio)ethyl] 3-(4-chlorophenyl)triazene;
l-[2-(3-nitro-2-pyridyldithio)propyl]-3~(4-methylphenyl)triazene;
? ~q '' .

. , .

-20~ 'r~
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-[2-(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-[2-(4-chloro-2-naphthyldithio)ethyl]-3-(4-methylphenyl)-triazene;

1-[2-(cyclopropylmethyldithio)ethyl]-3-(4-methylphenyl)triazene;

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

.. .

4~ 3 In a preferred embodiment of the present invention, ~here is provided an alternate process for the preparation of disulfide mitosanes having the Formula Ia CH20C~NHz R -SS-CH2CH20 ~ Ia CH3 ~ N ~ H

wherein R is an organic group viz. the structural component of an organic thiol of the formula R2SH, which is alternatively described by R3Alkl or R4 wherein R3, R4 and Alkl are as defined above.

For the preparation of the disulfide mitosanes of Formula Ia, it is preferred to utilize the 9a-methoxy-7-[2-(3-nitro-2-pyridyldithio)ethoxy]mitosane of Formula XVII 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 Ia is the stability of the by-product, namely 3-nitro-2-mercaptopyridine, which solely exists as the thione XVIII.

.

. ; :

.

~~` -22~ D~
Scheme 3 -C~2c~2-~ + R25~ >

C~I

XVII

R2-55-C~2C~20~ ~ ~ 2 ~3 ~ N

Ia XYIII

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 i5 utilized in the procedure depicted in Scheme 2 to produce disulfide mitosanes having the For~ula Ib ~_55-~lk2-~ }~

wherein Alk2 and Rl are as defined above.

There are two general synthetic procedures described herein for the preparation of lipophilic as well as hydrophylic mitosanes of the Formula Ia. General procedure A is employed for preparing either lipophilic or moderately soluble disulfides of Formula Ia, whereas general procedure B is employed for water-soluble disulfides of Formula Ia which are preferably isolated as sodium salts or as zwitterionic forms. Preferably, at least one equivalent of the mercaptan R2SH per equivalent of mitosane of Formula XVII is employed, and the reaction may be carried out in the presence of about one equivalent of base per equivalent of mercaptan R2SH. Preferred bases are the tertiary amines, e.g.
triethylamine, N-methylmorpholine, N-methylpiperidine, pyridine, 2,6-lutidine and the inorganic bases, e.g. sodium bicarb~nate, potassium carbonate, potassium bicarbonate and the like. Suit-able 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 aliphatic hydrocarbons and water~ The organic solvents contain up to 8 carbon atoms, but those boiling at temperatures of less than 100C are preferred.
Specific preferred solvents are methylene chloride, methanol, acetone, water and mixtures thereof. The reaction may be carried out at the reflux temperature of the reaction mixture or up to :- ., ,- :: ~

.

, -24~ 2~
about 60C. I~ is preferre~ to carry out the xeaction at room temperature or below, for instance within the range of 0 to 25C.

The foregoing reaction conditions and precautions are generally applicable to the preparation of other disulfide mitosanes of Formulas Ia and Ib according to the general proce-dure depicted in Scheme 3.

The following is an enumeration of representative thiols of the Formula R3Alk1SH or R4SH which may be converted via reaction with the Bunte salt XV or sulfenylthiocarbonate XVI to produce intermediates Formulas XIII and XIV, 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 of Formula Ia or Ib to produce products of the present invention. However, the only limitations to the 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 use of heteroaromatic thiols which may not react with compounds of the.Formula Ia or Ib.

-25- ~ g~2~;3 2(~ 3 HSCH (CH3) 2 HS (CH2) 3CH3 HS-CH-CH CH

HS CH2 CH (CH3 ) 2 l H3 HS<¦

,C
HS

o HS O

- ' , ' ~'`:
"' ' . ' ,''~' ,' ' ' HS{~

HS-CH 2-CH=CH

HS-CH2-CH=C(CH3)2 HS-CH -C-CH

HS CH C~C CH

HS (CH2)nORl n = 2-4; Rl = H, CCH3, CH3 ~S (C~12) nCX~ n = 1-3; X = O, NH, NRl; R~'Rl = H, C~3 HS (CH2)nNHR1 4 Rl = CH3t CH2CH3~ CH2CH2 3 3 HS (CH2)nNRlR2 n = 2-4; Rl/R2 = CH3, CH2CH3 H s-CH2FH2 SCH3 HS-CH2CH2NHC (CH3 ) 3 HS-CH2- 1 -CH NHRl Rl = CH3, CCH3 l~H 3 H S-C~l2c 32N~O

. .

.

~C~I2 HS-CH2CH2-N\ ¦ n = 3-5 (CH2 ) n CH2NRl R2 HS--CH--CH2NRlR Rl = CH3; R2 = H, CH3 HS--CH --CH--CH NHRl Rl = CH3 NHR

HS - CH -7H-NH + peptides HS-CH2-1CH-CH2-NH2 + peptides HS-CH ~-CH-CH 2-CO2H + peptides ICH 2CO;2H
HS-CH2CH2CH + peptides lH2 H S -CH= CH -NHCCH 3 OH

l H3 -28- ~ ç,;
HS-CH21CH-CH2NRlR2 Rl = CH3; R2 = H, C~3 ~CH 3 H S - CH 2 CH2 -~ -CH 3 HS--CH2CH2NH~ ' H S-CH 2CH2 NH~

H S-CH2CH2 -NH~

HS--CH2CH2--O ~, C 3 HS~

HS ~3 N (CH3 ) 2 HS ~ 3 HS~ ~ . . .

H S ~ NHeCH 3 -:
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HS--CH2CH2~ NHCCH3 HS- (CH2) n4~ n = 1, 2 N

HS (CH2)n~ n = 1, 2 N

HS- (CH2) n~N n = 1, 2 /~N
S t C 2 ) n ~N n = 2, 4 HS (CH2)n ~ n = 2 - 4 Rl = OCH2CEI3 HS- (CH2) n~ n = 1, 2 HS-(CH2)n~ n = 1, 2; X = O, S, NH

HS- (CH2)n~ n = 1, 2, X = O, S, NH

HS (CH2)n~ n = 1, 2; X - O, S, NH

2)n~ NH2 n = 1, 2; X = O, S, NH

HS- (CR2)n~ n = 1, 2; X = O, S, NH

HS-(CH2)n~N~NH2 n = 1, 2; X = O, S, NH

~=<N (CH3 ) 2 HS-CH2~

HS-CH2~ Rl = H, CH3 . - ' -31- ~ 3 HS - CH ~NRl R ~ - H, CH 3 HS-CH2-CH2-N~X X = O, NH, NCH3, S

HS-CH2~ Rl = H, CH3 N

Rl HS-CH2{> Rl = H, CH3 Rl HS-CH2-CNRl Rl = H, CH3 HS-CNRl Rl = H, CH3 HS~C~ . Rl = H, CH3 .

HS~\Rl Rl = H, CH3 HS- (CH2) ~ ~ 2)m n = 1-3; m = 1-3 HS CH2~iR Rl R2 = H, CH3; Rl = H, CH3 HS-CH2f NRl Rl = H, CH3 o , ~32~ 3 /\ 1 1 HS--CH 2~C NR R = H, CEI 3 HS /~~ ~N ( CH3 ) ;~

HS~ --NHCCH3 H S~NH\~\ OH

OH
HS~ ~/\ OH

Usefulness of compounds of Formulas I and IX in the antineoplastic therapeutic methods of the invention is demonstrat'ed by the results of ln 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 gram-positive and gram-negative microorganisms in a manner similar to that observed for the naturally occurring mitomycins and are thus potentially useful as therapeutic agents in treating ~acterial infections in humans and animalsO

-33- ~ 3~ ,s~,~3 Activity Against P-388 Murine_Leukemia Table I contains the results of laboratory tests with CDFl mice implanted intraperitoneally with a tumor inoculum of 106 ascites cells of P-3~8 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 of ten saline treated control mice was included in each series of experiments.
The mitomycin C treated groups were included as a positive control. A 30 day protocol 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 results were determined in terms of % T/C which is the ratio of the mean survival time of the treated group to 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 mitomycin C can be estimated. A
minimum effect in terms of % T/C was considered to be 125. The minimum 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.

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'' ,.' , , - _ 3 7 . . ~ ~ ~ 3~ t3 Activity Against B16 Melanoma Table II contains results of antitumor tests using the B16 melanoma grown in mice. BDFl 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 determined. Control animals inoculated in the same way as the test animals and treated with the injection 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 effectiveness, 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 on days 1, 5 and 9 by the intravenous route.

TABLE II
B16 Melanoma Compound of Maximum Effect Minimum Average Example_No.~ T/C Dosel Effective Dosel Wt. Change2 -28 167tll2) 1.6(3) ~0.4 _o,g; +1.4 >214~145) 2.4(3) <1.6 -2.4; -1.9 110(112) 3.2(3) 3.2 +0.5; +0.5 26 152(145) 1.6(3) <1.6 -0.6; -0.6 1. mg/kg of body weight 2. average grams per day for each maximal and minimal effective dose 3. values in parentheses are for mitomycin C tested in the same run -38- ~ 3 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, ~hey are administered systematically to a mammal bearing a tumor in substantially non~oxic antitumor effective dose.

The compounds of the present invention are intended primarily for U52 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 particular tumor sensitivity, They are readily distributed as dry pharmaceutical compositions containing diluents, buffers, stabilizers, solubili~ers and ingredi~nts contributing to pharmaceutical elegance. These compositions are then constituted with an injectable li~uid medium extemporaneously just prior to use.
Suitable injectable liquids include water, isotonic saline and the like.

DESCRIPTION OF_SPECIFIC EMBODIMENTS

In the following procedures and examples, all temperatures are given in degrees Centigrade, and melting points are uncorrected. Proton nuclear magnetic resonance (lH NMR) spectra were recorded on a Varian XL100, Joel FX-90Q or Bruker WM 360 spectrometer in either pyridine-d5 or D20 as indicated.
When pyridine-d5 is used as the solvent, the pyridine resonance at ~=8.57 is used as an internal reference, whereas with D2O as solvent TSP is used as the internal reference. Chemical shifts are reported in ~ units and coupling constants in Hertz.
Splitting patterns are designated as follows: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad signal; dd, d~ublet of doublet; dt, doublet of triplet. Infrared spectra were determined either on a Beckma~ 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 ~ ~ * trade mark.

:.

detector. Thin layer chromatography (TLC) was carried out on 0.25mm Analtech silica gel GF plates. Flash chromatography was run with either Woelm neutral alumina (~CC grade) or Woelm*silica gel (32-63~m) and the indicated solvents. All evaporations of solvents were performed under reduced pressure and below 40C.

The l-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. l-Methyl-3-(4-methylphenyl)triazene may be prepared as follows:

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

l-Methyl-3-P-tolyltriazene. ~-Toluidine (50.2 g, 0.47 mole) is added to a 2-1 flask equipped with a 200-ml dropping funnel and an efficient stirrer, and the flask is immersed in an ice-salt bath at _ . -10. A solution of 46.8 g (0.55 mole) of potassium nitrite in 150 ml of water is placed in the dropping funnel, and 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 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, aqueous sodium carbonate, whereupon the solution becomes red to orange in color and a small amount of 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 car~onate, 300 ml of 30-35% aqueous methylamine (Note 2), and 100 g of crushed ice in a 3-1 flask. The reaction mixture is kept at ca. -10C during the addition, which requires about 45 minutes (Note 3). The solution is extracted with three 1-1 portions of ether. The ethereal extracts are dried with anhydrous sodium sulfate and evaporated on a rotary evaporator at * trade mark.

, .

~ 3~2~1~
room temperature to give 65 ~ 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 minimum amount of ether, and the solution is diluted with 2 volumes of hexane and cooled to 0 to give flat plates with a slightly yellow cast; m.p. 79-81. The yield o~
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 of potassium nitrite has been stopped.

2. 40% aqueous methylamine may be substituted.

3. The reaction is over when a drop of solution no longer gives a red color with a solution of ~-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 fractional crystalli~ation, but it is easier to sublime 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. Recrystal-lization yields the pure l-methyl-3-p-tolyltriazene.

6. This procedure works well only with water-soluble amines. Procedure 2 given below ls more suitable for the preparation of triazenes of water-insoluble amines, . .
:.' .
'' ,, .. ` ':; ' .

~ 3~ 3 Procedure 2 E. H. White et al., Tetrahederon Letters No. 21, p. 761 (1961).

l-n-8utyl-3-p-chlorophenyltriazene. ~ solution of ~-chlorobenzenediazonium hexafluorophosphate (recrystallized from acetone-methanol) (2.87 g, 10.1 mmoles) in dimethylformamide (dimethylamine-free) was added slowly to a stirred mixture of n-butylamine (0.73 g, 10.0 mmoles), powdered sodium carbonate (15 g), and dimeth~lformamide (30 ml) stirred and maintained at -5. The diazonium salt solution may be used at 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-naphthol (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 triazene may be isolated at this point and recr~stallized from pentane at low temperatures.

.
Procedure 3 ....

7-Hydroxy-9a-methoxymitosane. Mitomycin C (2.2 g, 6.6 mmoles) was dissolved in 140 ml 0.lN methanolic NaOH (50%) and the reaction mixture was stirred at room temperature for 30 hours. The solution was then adjusted to ca. pH 4.0 with lN HCl and extracted with ethyl acetate (4 x 500 ml). The combined ethyl acetate extracts were dried (Na2SO4) and evaporated under reduced pressure at about 30 to 35C 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%).

lH NMR (pyridine-d5, ~): 2.05(s, 3H), 2.14(bs, lH), 2.74(bs, lH), 3.13(d, lH), 3.24(s, 3H), 3.56(d, lH), 4.00(dd, lH), 4.37(d, lH), 5.05(t, lH), 5.40(dd, lH), 5.90(bs, 2H).

, . . .

~ '3~ ~3 Procedure ~

Mitomycin A. A 100 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 ~or 6 hours was stirred at room temperature for 18 hours. TLC [methylene chloride:me~hanol ~90:10)] revealed the appearance of a deep purple spot at Rf=0.36 with a trace amount o~ impurity at Rf=0.41. The reaction mixture was concentrated to lO dryness and chromatographed on Woelm neutral alumina employing methylene chloride and methylene chloride:methanol 130:1) as eluting solvents. Fractions containing the component at Rf=0.36 were pooled and concentrated to dryness. Precipitation of the dry residue from methylene chloride and hexane afforded the title compound as a fine amorphous purple powder (25 mg, 24%), mp 161.

Anal. Calc'd for C H N3O6: C, 54.96; H, 5.44; N, 12.02 Found: C, 53.~6; H, 5.37; N, 11.99 IR(KBr),Vmax, cm 1 3400, 3300, 2950, 1700, 1630, 1575, 1200, 1060.

20 lH NMR (pyridine-d5, ~): 1.82(s, 3H), 2.74(dd, lH), 3.12(d, lH), 3.24(s, 3H), 3.54(dd, lH), 3.96(dd, lH), 4.02(s, 3H), 4.22(d, lH), 4.84(bs, 2H), 5.02(t, lH), 5.38(dd, lH).

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

Procedure S
-In a 250 ml one neck round bottom flask was placed solid Na2CO3, 35% aqueous solution of amine (amount as in Procedure 1) and ice, and the suspension was stirred at -5C
(ice-salt bath). To this suspension was added dropwise, a cold suspension of p-chlorobenzenediazonium hexafluorophosphate r ~

.
.

(Aldrich Chemical Co.) in ice, water, Na2C03 (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 (Na2S04) and concentrated. The yellowish solid residue was purified by column chromatography over Woelm alumina using hexane-methylene chloride (1:1) as eluting solvent (lH NMR recorded).

Examples 1-10 The triazenes 1-7 of Table III which follows wer~
prepared according to general Procedure 1 described above wherein the triazene of Example 1 is exemplified. The triazenes were purified by column chromatography on Woelm alumina.

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

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Example 11 1-[2-(3-Nitro-2-pyridyldithio)ethyl]-3-(4-methylphenyl) triazene A solution of 4-methylphenyldiazonium chloride is prepared as described in Procedure 1 from ~-toluidine and adjusted to pH 6.8-7.2 at 0C 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 a 250 ml 3 neck, round bottom flask contain-ing 5.34 g (20.0 mmoles) of 2-(3-nitro-2-pyrldyldithio) ethylamine, 7 g of sodium carbonate, and 150 ml of dioxane which had been added to the flask in that sequence. Satura~ed 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 reaction mixture was allowed to warm to room temperature and was then extracted with three 400 ml 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 hexane:methylene chloride (4:1);
hexane:methylene chloride (3:2); hexane:methylene chloride (1:4);
and finally methylene chloride containing 1% methanol for development and elution of the column. The appropriate fractions ~identified by TLC) were combined and evaporated to yield 2.5 g of the title compound.

Examples 12-19 General procedure for preparing 7-alkoxy-9a-methox~mitosanes (12-19) A solution of triazene (2.4 equivalents) in CH2C12:methanol (4:1) was added to a solution of 7-hydroxy-9a-methoxymitosane (prepared in Procedure 3) in CH2Cl2: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 CH2C12). 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 amorphous solid. The products produced are ide~tified as Example Nos.
12-19 in Table IV.

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5~ ,'2~3 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 mmoles) was added to the solution in the flask and the mixture was stirred at 5C for 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~ 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 C H N O S : C, 45.65; H, 4.09; N, 11.82 (corrected for 0.5 mole% of CH2C12):

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

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

H NMR (pyridine-d5, ~): 1.81(s, 3H), 2.00(bs, lH), 2.61(bs, lH), 2.98(bs, lH), 3.08 (5, 3H), 3.20(m, 2H), 3.39(d, lH), 3.83(dd, lH), 4.07(d, lH), 4.59-4.89(m, 3H), 5.21(dd, lH), 7.16(dd, lH), 8.31(dd, lH), 8.71(dd, lH).

.3~2~3 By adaptation of the procedures of Examples 11 and 20 to other ~-(3 nitro-2-pyridyldithio)alkyl amines having 2 to 6 carbon atoms in the alkyl group, mitosane derivatives of the following formula may be prepared.

NO CH2 ~ NH2 S~-(C~2)n~ ~ "OCH3 ~ 2 CH3 n = 2-6 Rl = H, or Cl 6 alkyl Examples 21-34 The 7-alkoxydithio-9a-methoxymitosanes 21-34 of Table V
which follows were prepared according to general Procedure A or B
described below and indicate~ in Table V. The physical data for the mitosane compounds 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 (~l.l equivalentsl followed by dropwise or portionwise addition of a mercaptana (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 high pressure liquid chromatography (HPLC) monitoring (~Bondapak-C18 col D ) is employed.
* trademark. , ., '`~ ,.
., : , , .
.

(10% CH30H in CH2Cl2~. The completion of reaction is signaled by the disappearance of spot correspondlng to the starting material and appearance of the product spot. At this point the reaction mixture is concentrated under reduced pressure (at ~30C) and the residue chromatographed on a neutral Woelm alumina column (l/4" x 10") slurry packed with 2-5~ CH30H in CHzCl2. 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% CH30H in CH2C12 is further carefully purified by flash silica gel chromatography using 5-7% CH30H in CH2Cl2 as the eluting solvent. The major band corresponding to product is isolated and the amorphous 7-alkoxydithio-9a-methoxy-mitosane is characterized.

Procedure B

To a solution of mitosane of Example 20 i~0.1 mmole) in 2-5% acetonea in methanol (10 ml) is added saturated aqueous NaHC03 solutionb (~6 drops), followed by addition of mercaptan (1 equivalent) in methanolC (1 ml). The progress of the reaction is monitored by TLC (silica gel, 10~ CH30H in CH2Cl2). At the completion of reaction, the reaction mixture is diluted wlth water (15 ml) and concentrated to about 10 ml under reduced pressure at about 30C. The resulting solution is chromatographed on a reverse phase C-18 column with stepwise gradient elution (100% H20 to 80% CH30H 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.

_ a) Methylene chloride can also be used, but acetone is preferred.
b) In the case where the mercaptan is L-cysteine, this base i5 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 the column.

TABLE V
?-Alkoxy~dithio-9a-methoxymitosanes A or RSS~ ~OCH3 RSH + 20 B ~ CH

-~xample Thiol No. (RSH) Procedure Product .

21 ethyl 2-mercapto- A R=-CH2CH2OCCH3 acetate 22 ~3-mercapto-1,2- A R=-CH2CH(OH)CH2OH
propanediol 23 3-mercaptopropionic B R=-CH2CH2COO Na acid 24 cysteine B R=-cH2cH(NH3 )COO

thiophenol A R = ~

26 p-ni~robenzenethiol A R = ~ NO2 27 p-mPthoxybenzenethiol A R = ~ OCH3 28 p-aminobenzenethiol A R - ~ NH2 ;2~t3 TABLE V (continued) , Example Thiol No. (RSH)Procedure Product .

COO Na 29 2-mercaptobenzoic B R -acid r~
2-nitro-4-mercapto~ 2 benzoic acid '~~~ +
COO Na 31 4-mercaptopyridine A R = ~ N

32 2-mercap~omethyl-l- A R = -CH2 ~/ 3 methylimidazole N

1 2 +
NIHCOCH2CH2CHCOO Na 33 glutathione B R=-CH2CH
CONHCH2COO Na+

34 dimethylamine ~ R=-CH2CH2W(CH3)2 ethanethiol .

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Claims (25)

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

II

III

wherein:

Alk1 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 R 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 connected thereto through oxygen, sulfur or nitrogen are attached to different carbon atoms of Alk2, wherein said A substituent is selected from the group consisting of one or two C1-6 alkyl, C1-6 alkanoyl, C1-6 alkoxy, halogen, C1-6 alkoxy-carbonyl, cyano, C1-6 alkylamino, C1-6 dialkyl-amino, C1-6 alkanoylamino and C1-6 alkoxycarbonyl, Alk1 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 alkylamino or 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 carbon atoms, cycloalkenyl or B-substituted cycloalkenyl each having 5 to 8 carbon atoms, phenyl or B-substituted phenyl, naphthyl or B-substituted naphthyl, a heterocyclic group selected from the group consisting of a 5- or 6- membered cyclic aromatic or alicyclic ring having 1 to 2 heteroatoms selected from oxygen, nitrogen and sulfur, pyridylamino or thiazolylamino, alkoxy or alkylthio each having 1 to 6 carbon atoms, alkoxycarbonyl 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, alkoxycarbonylamino having 2 to 6 carbon atoms, ureido (-NHCONH2), N-alkylureylene (-NHCONHalkyl) having 2 to 7 carbon atoms, N3-halo-alkylureylene having 3 to 7 carbon atoms, N3-halo-alkyl-N3-nitrosoureylene having 3 to 7 carbon atoms, dialkylaminocarbonyl having 3 to 13 carbon atoms, dialkylaminoalkoxy having 4 to 13 carbon atoms, alkanoylaminoalkoxy 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 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 cyclo-alkenyl 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 a 5- or 6- membered cyclic aromatic or alicyclic ring having from 1 to 2 heteroatoms selected from oxygen, nitrogen and sulfur, wherein each B substituent is selected from the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogen, amino, carboxy, hydroxy or nitro groups, and R4 and the adjacent sulfur atom together constitute S-cysteinyl wherein said S-cysteinyl group may be esterified, salified or a nontoxic pharmaceutically acceptable salt thereof.

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

3. The compound of Claim 2 having Formula II wherein Alk1 is ethylene and R3 is acetylamino.

4. The compound of Claim 2 having Formula II wherein Alk1 is ethylene and R3 is acetyloxy.

5. The compound of Claim 2 having Formula II wherein Alk1 is methylene and R3 is 1,2-dihydroxyethyl.

6. The compound of Claim 2 having Formula II wherein Alk1 is ethylene and R3 is carboxy or a nontoxic pharmaceutically acceptable salt thereof.

7. The compound of Claim 2 having Formula II wherein Alk1 is methylene and R3 is 1-carboxy aminomethyl or a nontoxic pharmaceutically acceptable salt thereof.

8. The compound of Claim 2 having Formula II wherein Alk1 is methylene and R3 is 1-methylimidazol-2-yl.

9. The compound of Claim 2 having Formula II wherein Alk1 is ethylene and R3 is dimethylamino.

10. The compound of Claim 2 having Formula III wherein R4 is phenyl.

11. The compound of Claim 2 having Formula III wherein R4 is 4-nitrophenyl.

12. The compound of Claim 2 having Formula III wherein R4 is 4-methoxyphenyl.

13. The compound of Claim 2 having Formula III wherein R4 is 4-aminophenyl.

14. The compound of Claim 2 having Formula III wherein R4 is 2-carboxyphenyl or a nontoxic pharmaceutically acceptable salt thereof.

15. The compound of Claim 2 having Formula III wherein R4 is 4-nitro-3-carboxyphenyl or a nontoxic pharmaceutically accept-able salt thereof.

16. The compound of Claim 2 having Formula III wherein R4 is 4-pyridyl.

17. The compound of Claim 2 having the formula or a nontoxic pharmaceutically acceptable salt thereof.

18. A compound of Claim 1 having Formula Ib Ib wherein R1 and Alk2 are as defined in Claim 1.

19. The compound having the formula XVII

20. A pharmaceutical composition comprising a compound of Claim 1 or 19 in association with a pharmaceutically acceptable solvent, diluent, adjuvant or carrier.

21. The process for the 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-Alk1-R3 Ar-N=N-NH-Alk2-SS-R4 V VI
with one equivalent of a mitosane of Formula IV

IV
wherein R1, R3, R4 Alk1 and Alk2 are the same as defined in Claim l and Ar is phenyl or C-substituted phenyl, naphthyl or C-substitutednaphthyl having 6 to 12 carbon atoms, wherein said C substituent is selected from the group consisting of l or 2 lower alkyl, lower alkoxy, halogen, acetoxy, carboalkoxy and nitro 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;
provided that when a mitosane compound of Formula Ib is obtained Ib one equivalent of compound Ib may optionally be reacted with at least one equivalent of thiol of Formula R3Alk1SH or Formula R4SH wherein R1, R3, R4, Alk1 and Alk2 are the same as defined in claim l 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 appriecable amount of the product of Formula II or Formula III is obtained.

22. The process of claim 21 wherein said triazene of Formula VI is 1-[2-(3-nitro-2-pyridyldithio)ethyl]-3-(4-methylphenyl)triazine.

23. The process of claim 21 wherein Alk2 is ethylene and R1 is hydrogen.

24. The process of claims 21 or 23 wherein one equivalent of base is employed in the reaction.

25. The process of claim 21, wherein a lower alkanoyl, 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 during the conversion of a compound of structural Formula Ib to product of Formula II or III,