CA1254892A - 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.
This divisional application discloses a process for preparing a compound having the formula IX

IX
wherein:
R5 is hydrogen, or C1-6 alkyl, and R6 is C1-12 alkyl or substituted C1-12 alkyl, C3-12 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 from the group consisting of halogen, C1-6 alkoxy, C1-6 alkanoyl, C6-14 aroyl, cyano, trihalomethyl, amino, C1-6 monoalkylamino, C2-12 dialkylamino, C6-12 aryl, C6-12 aryloxy, C1-6 alkanoyloxy, C7-14 aroyloxy, heterocyclic group selected from the group consisting of 5- or 6- membered heterocyclic aromatic or alicyclic ring having 1 or 2 hetero atoms selected from the 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 halogen, C1-6 alkoxy, C1-6 alkanoyl, cyano, trihalomothyl, amino, C1-6 alkyl amino, or C2-12 dialkyl-amino groups which comprises reacting a mitosane of formula X

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 adiazotizable aromatic amine.

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 7-0-substituted mitosane analogs thereof have similar utility.
., .
This is a division of Canadian application serial number 489,937 filed September 3, 1985.

Nomenclature - The systematic Chemi~al ~bs~racts name for mitomycin A ~ased on the xecent revision [Shirhata et al~, J.
Am. Chem. Soc., 105, 7199 (1983)l is:

llaS-(la~,8~,8a,8b~ 8-~ ((aminocarbonyl)oxy)me~hyl]-6,8a-dime~hoxy-l,la,2,8,8a,Bb-hexahydro-5-methyl-arizino~2',3',3,4,]pyrrolo[1,2-a]indole-4,7-dione ;

; ' ~

, ~h ~, according to which the az.iri~opyrroloindole ring systern is numbered as follows:

4 a Chemical Abstracts A trivial syst~m of nomenclature which has found wide use in the mitomycin literature identifies thP foregoing ring system including ~everal of the characteristic substituents of the mitomycins as mitosaneO

~0 O C~;20CONH

;~Hla Mitosane According to this system, mitomycin A is 7,9a-dimethoxymi~osane and mitomycin C is 7 amino-9a-methoxymitosane. As to the stereo-chemical configur~tion of the products of this invention, it is intended whe~ identifying them by the root name "mitosane" or by structural ormula to identify the stereochemieal configuration .: :

: ' thereof as the same as that of mitomycin A or C.

R ~ ~ ~ R Mitomycin A RrRl~OCH3 l l ~ Mitomycin C R-N~I R~-OCH3 c~3~ Y ~ N ~ ~
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 thexapy o~ 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 Yor]c 13~21, Physicians' Desk Reference 37~h :~:di~ion, 1983, pp. 747 and 748)o Mitomycin C and its production by fermentation is the subject o~ U.S. Patent No. 3,660,578 patented May 2, 1972 clai~ing priority from ear.lier applications including an application filed in Japan on Apri:L 6, 1957~

The structures of mitomycins A, B, C, and of porfiro-mycin were first published by J. S. Webb et al. o~ Iederle L~iboratories Division American Cyanam.id Company, J ~m. Chem S_, 84, 3185-3187 (1962~. One of the chemical transfo~na~ions used in this structure s tudy to relate mitomycin A and mi tomycin C was tha conversion o~ the former, 7,9a-dimethoxymitosane, by reaction with a3u~on~a to the latter, 7-amino-9a-methoxymitosaneO
Displac~ment 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 l, la, 8a and 8b have be~n shown to be as indieated above with respect to the Chemical Abstracts nomenclature [Shirhata et al., J. Am. Chem. Soc.~ 108, 7199-7200 (1983)~ The earlier literature refers tG the enanti-om~r . , ~5--The following articles and patents deal inter alla with the conversion of mitomycin A to a 7 substituted amino mi tomyc in 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 alO ~ J. ~ntibiotics, XXI, 189~198 (1968);

Konishita et al., J Med~ C~em., 14, 103-lG9 (1971);

Iyengar et al., J. Med. Chem., 24, 975-981 ~1981), Iyengar, Sami, Remers and Bradner, ~bstracts 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. 3,420,846t issued January 7, 1969;

; Mats~i et al., U.S. Patent No. 3,450,70S, issued 3une 17~ 1969;

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

Nakaslo et al., U.S. Patent No. 4,~31,936, issued November 4, 1980;

R~mers, U.S. Patent No. 4,268,676, issued May 19, 19810 The following patent applications deal with the prepa-ration of 7-substituted amino mitomycin C derivativ2s in which the substituent lncorporates a disulfide linkage.

~6--Kono et al~, European Patent Application No. 116,20 a (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 aI~, J.
Antibiotics, 23, 804-809 (1980).

Mitomycin C is the principal mitomycin produced by fermentation and is the commercially available form. Current technology for the conversion of mitomycin C to mitomycin A
suffers from a number of deficiencies. Hydrolysis of mitomycin C
to the corresponding 7-hydroxy-9a-methoxy mltosane, and then methylation of that substance requires diazomethane, a ~ery hazardou substance to handle on a manufacturing scale, and the 7-hydroxy.intermecliate is very unstable [Matsui et al., J.
Antibio~ics, XXI, 189-198 ~19S8)]. One attempt to avoid these difficulties involves the use of 7-acyloxymitosanes (Kyowa Hakko Xogyo KK Japanese Patent No. J5 6073-085, Farmdoc No. 56227 Dt31)~ Alcoholysis of mitomycln A as descxibed by Urakawa et al., J Antibiotics, 23, 804-809 (19801 is limited to the produc~
tion of only specific 7-alkoxy structural types by the availabil-ity and reactivity of the a~cohol starting materials.

SUM~ARY OF T~IE 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 co~.pounds may be represen~ed ~y ~he following general forrnula ; EIz 1~ ~
Alk2 ~ 3 ~3 ~ N ~ -R~

wherein R2 is an organlc group, vlz. the structural component of an organic thiol of the formula R2SH, and Alk2 and Rl have ~he meanings given below. These compounds are alternatively de scribed by Formulas II and III.

a c~ R
R3 Alkl-SS-Alk2~ ~ 3 II

CH3 ~ ~ _~1 o ~ ~2~C~
R--SS--Alk2--~ ~ III

C~3 N ~ _~1 ~herein:

Alkl is a st~aight or branched chain al~ylene group having 1 to 6 carbon atoms when R3 is jvined thereto through a carbon atom thexeof, and 2 to 6 carbon atGms when R3 is joined thereto through a ~ulur, oxygen or nitrogen atom thereof, and R3 .
, B ~
and -SS are in that i.nstance joi.ned to aifferent carbon atoms, Alk2 is a straight or branched chain alky.lene 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 nitxogen are attached to different carbon atoms of Alk2, wherein said A substituent is selected from the group consisting of one or two Cl 6 allcyl, Cl_6 alkanoyl, Cl_6 alkoxy, halogen, Cl 6 alkoxy-carbonylt cyano, C1_6 alkylamino, C1 6 dialkyl-amino, C~ 6 alka~oylamino and Cl 6 alkoxycarbonyl, Alk1 and Alk2 may contain a double bond, R1 is hydrogen, lower alkyl, lower alkanoyl, benzoyl or substituted benzoyl wherein ~aid substitu2nt is lower alkyl, lower alkoxy, halogen, amino or ~itro, R3 is selected from the group consisting of halogen, carboxy, alkanoyloxy having 1 to 7 carbon atoms, hydrc>xy wherein the oxygen atom is connected to Alkl having 3 to 6 car~on atoms, alkylamino or ~lialkylamino having 1 to 12 carbon atoms, N-alkoxy al~ylamino having 2-7 carbon atoms, alkanoylamino having 1-7 carbon atoms, henzoylamino or B~substi~utPd benzoylamino, naphthoylamino or B substituted naphthoylami~o, phenylamino or B-substitllted phenylamino, cycloalkyl or B-substituted cycloalkyl each having

3 to 8 ring members, cycloalkenyl or B-substi tu ted cycloaLkenyl each having 5 to 8 ring membexs, phenyl or B 5ubstituted phenyl; naphthyl or B~ubstitut:~d naphthyl, a heterocyclic group selected f.r.om the gro~p consisting of :
.

heteroaromat.i~ and heteroalicyclic group~ ha~in~
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 sul~ur, pyridylamino or thiazolylamino, alkoxy or alkylthio each having 1 to 6 carbon ato~s, alkoxycarbonyl or alkylaminocarbonyl each having 2 to 7 carbon atom~/ 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 (-NHCONHa:Lkyl) having 2 to 7 carbon a~oms, 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 car~on atoms and hydroxyalkylamino or ,N -dihydroxyalkyl ~nino each having 2 to 8 carbon atoms~ wherein said B substituent is selected fxom the group consisting of one or two lower alkyl, lower alkanoyl, lower alkoxy, halogenl a~lno, carboxy~ hydroxy and nitro groups, and R4 is selected rom the group consisting ~f alkyl having 1 to 12 carbon atoms, alkenyl or alkynyl ~ach having 3 to 12 carbon atoms, cycloalkyl or B-subs~ituted cycloalkyl having 3 to 8 ring ~mber~, cycloalkenyl or B-substituted cyclo-alkenyl each having 5 to 8 ring members, phenyl or B s~bstituted phenyl, naphthyl or B substituted naphthyl, a heterocyclic group selected from the group consisting of heteroaromati~ and heteroali-cyclic groups having from 1 to 2 rings, rorn 3 to 8 ring membexs in each ring, and from 1 to 2 h~teroatoms in each ring ~elected from oxygen, nitrogen a.rld sulfur, provided that the .
:' - :.~ , ` . ;
:: , .

iL~:D~

hete.rocyclic yroup is co~nected through a carbon atom which is attached to at least another carbon at~ (i , e ., the carbon atom attached to the -SS-may not itself be attached to two other heteroatoms), wherein said B substituent is seleoted 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 ad~aeent sulfur atom together constitute S-cysteinyl wherein said S-cystein~l group may be esterified, salified or joined within a non-toxic ~nd non-allergenic peptidel or a nontoxic pharmaceutically accepta~le 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 subst~nces. They are employed in a mannex similar to mutomycin. C. The dosages employed axe adjusted in proportion to their toxiciti~s relative.to the toxicity o~ mitomycin C. In cases where the~new compound is less toxic, a higher dose is employed.

In a further aspect of the present invention, there is ~: provided a new process or the production of mitosanes of Formulas II and III. This new process comprises reacting a mito-sane of Formula IV
O
~2~CNH~

H~ ~3 IV
3~--N ~-Rl ~' O
with a triazene of Formula V or Formula VI

Ar-NsN MH-Alk2-SS-Alk1 R3 Ar-N=N-NH-Alk2-SS-R4 VI
.

.~ ;

wherein R, R3, R4, Alkl and Alk2 are as defined above and Ar .is the organic residue o a diazotiæable aromatic amine.

In a va.riation o~ the present invention, there is provided an alternate process for the production of mitosaAes of Forn~ulas II and IIï~ This process comprises reacting a thiol of Formula VII or VIII
R3AlklSH R4SH

VII VIII

with a mitosane derivative of Formula Ib O ~ l ~ss-~lk2-~

The disul~ide mitosanes of Formula Ib are prepared by the triazene method described herein, More specifically, the mitosane of ~ormula Ib wherein Alk2 is ethylene and Rl is hydrogen is described in Example 20 a.nd in co-pending application : Serial No. 646,88a, filed September 4, 1984.

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

R6~ IX
CH3 ~ N~ R5 ~ O
: wher~in:
R5 is hydrogen, or Cl 6 al~cyl, and R6 is Cl_l2 alkyl or substituted Cl_l2 alkyl~ C3_12 .

.

-12~ 2~
cycloalkyl ox substituted C3 1~ cycloaLkyl wherein the caYbon atom thereof which is attached to the mitosane 7-oxygen atom bears from 1 to 2 h~drcgen atoms and said substituents are selected fro~n '~he group consisting of halogen, Cl_6 alkoxy, Cl~6 alkanoyl, C6_l~ aroyl, cyano, trihalomethyl, amino~ Cl 6 monoalkylamino, C~ 12 dialkylamino, 6-12 ry ~ ~6-12 ~ryloxy, C1~6 alkanoyloxy/ C
aroyloxy, heterocyclo having 1 or ~ rings and from 5 to 12 ring atoms includlng up to 4 heteroatoms selected from nitrogen, oxygen, and sulur, and wherein each of ~aid alkoxy, alkanoyl, aroyl r aryl, aryloxy, alkanoyloxy, aroyloxy, and he~ero-cyclo substituents optionally contains frorn 1 to 2 substituents selected from halogen, Cl 6 alkoxy, C1 6 alkanoyl, cyano, trihalomethyl, amino, Cl 6 alkyla~Lino, or C;~ 12 dialkylamino groups.

.Many of the compounds o ~ormula IX are known compounds having inhibitory activity against experimental animal tumors ln vivo. A number of novel compounds con~orming to Formula IX hav2 __ also been prepared ~y thi~ process, and are oonsidered part of the present invention. In particular the substances identified herlein as compounds of Example Nos. 14, lS, 16 and 19 are novel substances, and also have antitumor activity against experimental animal tu~rs, These compounds are part of the present invention. They are employed in a manner similar to mitomycin C.
The dosages employ~d are adjusked in proportion to the:ir toxicities relative to the toxicity o:E mitomycin CO In c~ses where the new compc3und i5 less toxic, a higher dose is employed.

The new procPss or production of compounds o~ FormNla ' 3~
--13~
IX comprises reacting a mitosane o~ Formula X

~10~ C 2 CH3 ~ R'5 with a triazene of Formu:La XI

~r N-N~NH~R6 XI

wherein ~5 and R6 are as defined a~ove and Ar is the organic residue of a diazGtizable aroma~ic amine.

The terms "lower alkyl", "lower alkoxyll and "lower alkanoyl" as used herein and in the claims (unless the con~ext inclicates otherwise) mean straight or branched chain alkyl, alkoxy or alkanoyl group~ containing :Erom 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t butyl, amyl, hexyl, etc. Preferably, these groups contain from 1 t:o 4 carbon atoms and, most preferably, they contain 1 or 2 carbon atoms. Unless otherwi~e speci~ied in the particular instance, the term "halogen" as used h~rein nd in the claims is intended to include chlorin~, fluorine, bromine and iodine. The tersn "nontoxic pharmaceutically acceptable sal~" 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~ camphorsul~onic~
levulinic and th~ like~ Such bases are well-known and .include, e.g. nontoxic metallic salts such as sodium, potassium, calcium and magnesium, the ammonium ~alt and salts with nontoxic amines, e.g. trialkylamines, procaine, diben~ylamine, pyridine, .

~ ~ .
~' .

N-methylmorpholine, N-methylpiper1dine and the likeO The salts are made by methods known in ~he art.

DESCRIPTION OF THE INVENTION
__ _ The present invention provides a new process for t~e preparation of cornpounds of Formula IX which comprises reac~ing a mitosane of Formula X with a triazene of Formula XI as shown in Scheme 1.

Scheme 1 o O I H;~OCNHz ~ El3 P.r~ N-NH-R6 ~>~ IX
C 3~--N .~ R~; XI
O
X

wherein R5 and R6 are as defined above and Ar is the organic residue of ~ diazotizable aromatic am.ine.

The 1-substituted-3-~ryltriazenes of Formula xI and more specifically l-alkyl-3-aryltriazenes make up a class of r~agents which are known to be useful for reacting with car~
boxylic acids to form th~ c~rresponding lower alkyl esters.
1-Methyl-3-(4-methylphenyl)triazen~ may be prepared according to the general procedures described ~y E. H. White et alO in ~y~, 48, 102-105 (1968) and as described herein in Procedure 1 ~uwever, this procedure works well only with water-soluble amines, and a second procedu.re which is described by E~ H. White et al., Tetrahedron Let~ers, No. 21~ 761 (1961) and also de scribed herein in Procedure 2 is more suitable for the prepara-tion of triazenes o~ water-1nsoluble amines.

~lS~
The reagent l-me th~l~ 3- (4-methylphenyl) triazelle 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. ~y~, 48, 102~105 (1968)]
and cephalosporan.ic acids which yields the desired ~3-compound without isomerization to the ~2-isomer [Mangia, Tetrahedron L~tters, No. S2, 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 _t 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 axylamlne having 6 to 12 carbon atoms which readily forms a diazonium salt may be used as the ~ource of the aryl portion of the 1,3 disub-stituted t.riazene. Some examples of triazenes produced in this fa~hion and used in ~he present invention are:

l-ln-butyl)-3-~4 methylphenyl)triazene;
l-~1-methylethyl)-3-(4-methylphe:nyl)triazene;
}-(4-methylphenyl~-3-[2-(4-morphollnyl)ethyl]txiazene;
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)triaz~ne;
1- ~4-chlorophenyl) -3~ (tetrahydrofuran-2-ylmethyl)triazene.

Other triazenes 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 ~. A. Da.niels et al., Can. J. Chem. r 55~ 3751~3754 (1977) are exemplary.

--16 ~

X ~N=N-NH CH;~-Y

a X - ~1, Y = CN
b X = N02 ~ Y = CN
c X = C02Me, Y = CN
d X ~ Ac, Y = CN
e X = N02 ~ Y = C02Et X = CO2Me, Y = CO2Et g X = CO ;2Me, 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-ven tion.

l-(n-butyl)-3-(a=napthyl) triazene l-(n-~e~y}~-3-phenyltriazene l-ethyl-3-(2,4-dimethylphenyl~triazene 1- ( 1-methylethyl~ -3- ( 4-methoxyphenyl ) triazene For the preparation of mito.mycin A, we prefer to use 3-methyl-1-(4-methylph~nyl)triaæene as methylating r~agent.
Preferably, at least two molecular proportions of the latter per molecular proportion of 7-hydroxy-9a methoxymitosane are employed and the reaction is preerab1y carried Ollt in a liquid organic solvent for the 7-hydroxy-9a-methoxymitosane starting material.
Preferred solvents are the lower alkanols, lower aIkanoic lower alkyl est~rs, the dilower alkyl ethers, the cyclic aliphatic ethers, and the lower polyhalogena~ed aliphatic hydrocarbons.
These solvents con~ain up ~o 6 carbon atoms, but those boiling at temperatures of less than 100C are preferred. Specific pre-ferred solvents axe methylene chloride, methanol, diethyl ether, ethyl acetate, and mixtures thereof. The reaction may be carried out at the reflux temperature o~ the reaction mixture or up to about 60 C. At temperatures in exce S5 of this the mitosane reactant is incli.ned to decompose with a resultant reduction ir , ~ "' `'` , -17~
yield. I~ is preferred to c~rry out the reaction at room temper-ature or below, for instance within the range of O to 25C.

A convenient way to determine when the reaction is complete is by thin layer chromatography. Mitomycin A is deep purple in color and can be readily distinguished from the start-ing material and from by~products. In the solvent system methylene chloride/methanol (90/10) mitomycin A exhibits R~ =
O.36. Chromatography on neutral alumina may be used for puri-fication of the product.

~ he foregoing xeaction conditions and precautions are generally applicable to the preparation of other 7-R60-mutosanes 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-po~ds 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 _..

O
22 Ar-N=N-NH-Alk2-SS Alkl-R3 ,OCH3 ~ _ _ ~ II or III
or Ar N=N-~H-Alk2-SS-R
3 ~ ~ R~ 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.

Aryltxiazenes of Formula V or ~I may be prepared in a similas fashion as described above for the preparation of .

. -18-aryltriazenes of Formula XI except that the alkyl amines utili~ed therein are replaced by ~minodisulfic1es of Formula XII

R2-SS-Alk2NH2 XI~
which are alternatively clescribed by Formulas XIII and XIV

R3-Alkl SS-Alk2NH2 and R4~SS~Alk2NH2 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 R3Alkl~H or R4SH with a Bunte salt of the formula or with a sulfenylthiocarbonate of the formula NH ~lk~SS~OC:H3 XVI

Rlayman et al., J. ~ Chem., 29, 3737-3738 (1964) __ _ have prepared the following by the Bu:nte salt method: -2-aminoethyl n~butyl disul~ide;
2~a~inoethyl n-hexyl disulfide;
2-aminoe~hyl nwoctyl disulfide;
2~am.inoethyl n-d~cyl disul~ide;
2-~minoethyl phenyl disulfide;
2-aminoethyl benzyl disulfide.
..
Meth~nol was found to be the preferred reaction solvent for the reaction of the Bunte salt with the thiol. Reaction te~peratures of 0 to -10C were ound to be preferred using ~his solvent. ~igher temperatures were necessary with other solventsv The chief drawba~k of this method is the formation of symmetrical disulfides as a by-product, presumably as a result of dispropor~
tionation of the desired mixed disulfide.

The mixed disul~ide startiny materials of Formulas XIXI
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., 92, 7629~7631 (1970). Typically, this preparative procedure involves adding the thiol to a methanol solution of the amino-alkylsulfenylthio~
carbonate of Formula ~VI and allowing the reaction to proceed at lO a temperature in the range of from 0 to 25C. Reaction times vary from virt~al:Ly instantaneous to several hours dependiny upon the particular thiol empl.oyed. The progress of the reaction can be followed by measuring the presence of unreacted t.hiol in the reac~ion 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 o aminodisulides of Formula XII with aryldiazonium salts in a s.imilar fashion as described hexein for the preparation of aryltriaæenes 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 1,3 disubstituted triazen~. Some examples of disulfide triazenes produced in this fashion and used in the presen~ invention are l-12-(2-acetamidoethyldithio)ethyl3~3-(4-methylphenyl)triazene;
1-[2-t3 nitro-2-pyridyldithio)~thyl]-3-(4~methylphenyl)txlazene.

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

: 1 12~- ~3-nitro-2-pyridyldithio) ethyl]-3-(4-chlorophenyl)triazene;
l-[2-~3-nitro-2-pyrilyldithio)propyl]-3-(4-methylphenyl)triazene;

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

l-t2-(phenyldithio)e~hyl] 3-(4-mekhylphenyl)triazene;

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

1-[2D(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-ch}oro-2-naphthyldithio)ethyl~-3-(4-methylphenyl)-triazene;

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

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

. ' In a prefexxed emhodimen~ of the present invention, there is provided an alternate process for the preparation of disulfide mitosanes having the Formula Ia C
CH O~NH
R2-SS-CH2CH20 ~ ~ OCH3 Ia C~3 ~ N ~ H

wherein R is an organic gxoup Vi2. the structural component of -an organic th ol of the formula R SH, which is alternatively described by R3Alk1 or R4 wherein R3, R4 and Alkl are as defined above.

. For the preparation of the clisul~ide mitosanes o~
Formula Ia, it is preferred to utilize the 9a-methoxy-7~ 13-nit:ro-2-pyridyldithio) ethoxy]mit:osane of Formula XVII in a ~hiol exchange process with an appropriate organic thiol of the formula R2SH as shown in xeaction Scheme 30 The driving force behind the formation of the disul~ides of Formulzl Ia is the stability o the by-product, namely 3-nitro-2 mercaptopyridine, which solely exists as the thione XVIII.

Scheme 3 -R

~ 2 ~ C~20~N~2 (~\)--ss-ca2cJ~ ~ t R25a >

XYI

O
RSS-C~2CH20~ ¢~2 C 3 ~E~ N ~a N

XVIII
Ia Alternatively, if it is d2sired to prepare mitosanes of Fonmula II or III wherein Alk2 is other than ethylenP, such as trimethylene or pr~pylene, then the appropriate triazene of Formula V or VI is utilized in the procedure depicted in Scheme 2 to produce disulfi.de mitosanes having the Formula Ib ~ ~ NH2 ~J \~ SS-Alk O ~ 2 OCH

CH3 ~ ~ -R

wherein Alk2 and Rl are as defined above.

There ~xe 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 ~or preparing either lipophilic or moderately soluble disulfides of Formula Ia, whereas yeneral procedure B is ~nployed for water-sol~ble disulfides of Formula Ia which are preferably isolated as sodium salts or as zwitterion c forms D Preferably, at least one equivalent of the mercaptan R SH per equivalent of mitosane of Formula XVII is elnployed, and ~he reaction may be carried ou~ in the presence of about one equivalent of base per equivalent of mercaptan R SH. Preferred bases are the tertiary amines, e.g.
triethylamine, N-methylmorpholine, N--methylp~peridine, pyridine, 2,6-lutidine and the inorganic bases, e.g. sodium ~icarbonate ~
potassium carbonate, potassium bicarbonate and the like. Suit able inert solven ts for the reaction of starting materials o~
Formula XVII and R SH are the lower alkanols, lower alkanoic lower alkyl esters, lower aliphatic ketone~, tbe cyclic aliphat.ic ethers, th~ lower polyhalogenated aliphatic hydrocarbons and water. The organic solvents contain up to 8 carbon atorns, bu~
those boilin~ at temperatllres of less than 100C are preEerred.
Sp~cific preferred solve.nts are methylene chloride, me thas~ol, acetone, water and mixtur45 th~reof~ The reaction may be carried c: ut at the reflux temper~ture of the reaction mixture or up to -2~-about 60C. It i5 preferred to carry out the reacti.on at room temper~ture or below, for instance within the range of 0 to 25C.

The foregoing reaction conditions and precautions axe 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 R3AlklSH or R4SH which may be converted via reaction with ~he 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 mixtur~ of products and the use of heteroaromatic thiols which may not react with compounds of the Formula Ia or Ib.

~ .

--~5--HSCH (CH3) 2 HS (CH2) 3CH3 HS CH2 CH (CH3 ) 2 fH3 HS~-C-CH3 HS<¦

H S-O

~S~

NS {3 - 26 -HS~ CH 2-CH=CH2 HS - C~I2 -CH=C ~ 3 3 2 HS CN C~ C CH

HS (CH2)nORl n = 2-4; Rl = H, CCH31 CH3 HS (CH2)~CXR n = 1-3; X = O, NH; NR; R/Rl = H, CH3 o HS IC~2)nNHR n = 2-4; Rl = CH3, CH2CH3~ CH2C~2CH3' CCH3 HS (C}I2) nNRlR2 n sr 2-4; Rl fR2 = CH3, CH2CH3 H S-CEI2CH2 SC~3 NS-CH2CN2~HC (CH3 ) 3 HS-CH2-8-CH2NHR1 ~1 ~ CH3 ~ ~CH3 ~H 3 NS-CN2CN2~NO

HS-CH2C~2-N \ ¦ n - 3 5 IC 2)n CH2NRlR2 HS-CH-CH2NRlR2 Rl = CH3; R2 = H, CH3 HS-CH2-fH-CH2NHRl Rl - CH3 NHR

HS-CH2-7H-NH2 ~ peptide 5 HS-CH2-1H-CH2-NH2 * p~ptides Hs~cH2-cH-cH2-co~H * peptides ~S-CH2CH2CH ~ peptides ~H2 ~ HCOCH2CH2CHC02H

CON~CH2C0 HS-CH=CH-NHCCH3 ~5-CH2- I H-cH2oH
0~1 ~3 HS-CH2CH-CH~OH

~P~

HS-CH21 H-CH2NR R2 Rl = CH3; R~ = H, CH3 H S - CH 2 CH2 -~ -CH 3 H S -CH 2CH2 NH~

,--_ HS-CH2CH2NH~
. N
N

H S-CH 2 CH2 -NH-</ 3 s ~H3 HS-CH2CH2-O- -~ H3 ~H3 HS~

HS ~ N (CH3 ) ~;2 HS~ . - . , H S~3 NHCCE13 .

' 3 ~
;!9--H S ~2 H

~S~

H S /[ 1~1 HS/~

HSJ~
N ~CH3 ~ 2 ~S-C~2~
E~S- C~ 2-CH=CH~3 H S-CH2 CH 2~3 N~ CEi3 HS- ~CH;~ ~ tl - 1, 2 H5 ~CH2)n~ n -- 1, 2 HS-(CH2)n~N n = 1, 2 /~
HS (C 2)n ~ n = 2, 4 HS~((H2)n~N~;~ n - 2 - 4; Rl - OCH2C~I3 Rl HS- (CH2~ ;3 n = 1, 2 HS- (CH2)n~3 n - 1, 2; X s O, S, NH

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

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

'A~
E~S (CH~)n~x~ ;2 n - 1, 2; X = O, S, NH

~~X
HS- ~C~2)n~ ,J n = 1, 2; X - O, S, NH

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

N (C~13 ) 2 H2~
N

HS-CH2~ Rl ~ H, CH3 . .

3~;~
--31~
HS - CH ~C\NR1 R 1 _ H, CH 3 CH2-CH2-N/--\k X -- O, NH, NC:H3, S

HS-CH2~ Rl = H, CH3 Rl , ElS - CH 2 {~ l = H, CH 3 Rl HS-CH2~ Rl = El, CH3 -C Rl = H, CH3 1 = H, C~ 3 R

~IS 1~\NR1 R1 = H ~ CH 3 n = 1-3; m = 1-3 ~ ~R2 E~S~CH2~< ~ R2 = }I, CH3; R -- H, C:H3 MS-CH2f NRl R L s H, CH3 oJ

. . ~ . . .

~32--HS~ ~NRl ~ = H, C~ 3 ~ I
t H S /~/~ ( 3 ) 2 HS~ --N~ I CH3 o HS~ --OH

HS ~ ~ 0~

Usefulness of compounds of Formulas I and IX in the antineoplastic therapeutic methods of the invention is de~lonstrated by the results of ln vlvo 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.

Canpo~ds according to the present invention are believed to possess anti bacterial activity against gram positive and gram-negativ~ microorganisms in a manner similar to that observed for the naturally occurring mitomycins and are thus potentially useful as therapeutic agents in treating bacterial in~ections in humans and animals.

Activitx A~ainst P-388 Murine Leukemia Table I contains the results of laboxatory tests with CDFl mice implanted intraperitoneally with a tumor 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 m1ce 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 b~ing 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 gra~s each were employed and a loss in weight of up to approximately 2 grams was not considered excessive. The ~e~ults 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 d~ys. The "maximum effect" in the following Table is ~xpressed as % T~C and the dose giving that effect is given. The values in parenthesis are the values obtained with mutomycin C as the positive control in the same experiment. Thus a measure ~f the relative activity of the present ubstance~ to mitomycin C can be estimated. A
minimum effect in terms of 9~ T/C was considered to be 125. The min~mum effective dose reported in the following Table is that dose giviny a ~ l'/C o approximately 1~5. The two values given in each instance in the "average weight change~ column are respectively the average weight change per mouse at the maximum effec~ive dose and at the minimum effective dose.

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~ alnst B16 Melanoma Table II contains results of antitumor tests using the B16 melanoma grown in mice. BDF1 mice were employed and inoculated subcutaneously with the t~or 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. Contxol animals inoculated in the same way as the test animals and treated with the injection vehicle and no drug exhibited a mean ~urvival time of 24 days. The survival ~ime relative to that of the cont.rols (% T/C) was used as a measure of effectiveness, and thP maximal effective dose and minimal effective dose fo.r each test compound was determined. The minim~l effective dose was defined as that dose exhibi~ing a % T/C value of 125. For each dosage level, the test animals were treated with the test compound on days 1, 5 ~nd 9 by the intravenous route .

TAB
B16 Melanoma , Compound of Maximum Effect Minimum Average Example No.~ T/C Dosel.Effective Dose 28 167(112)3 1.~(3)3 ~0 ~ -0.9; +1.~
>214(145) 2.4(3) ~1.6 -~4; 1.9 . .
110~ ) 3.2(3) 3.2 +0~5; +0.5 26 152~45) 1.~(3) <~ 0.6; ~0 1. mg/kg of body weight 2. average grams per day for each max~mal and minlmal effec~ive dose 3. values in parentheses are for mitomycin C tested in the s~me run ~ 5 In view of the antitumor acti~ity observed in experimental animal tumors, ~he invention includes use oE the substances of the present invention for inhibiting mammalian tumors. For this purpose, they are administered systematically to a mammal bearing a tumor in substantially nontoxic antitumor effective dose.

The comp~unds of the pre~ent 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 ox smaller doses may be employed depending upon the particular tumor sensitivity. They are readily distributed as dry pharmaceutical compositions containing diluents, buffers, stabilizers, solubilizers and ingredients contributing to pharmaceutica~
elegance. These compositions are then constituted with an inj ectable liquid medium extemporaneously just prior to use.
Suitable injectable liquids include water, isotonic saline and the like.

DE SC RI PT I ON ~F SPEC IF IC E~3 OD ~ENT S

In the following procedures and examples, all temperatures are gi~en in degrees Centigrade, and melting points ~re uncorrected. Proton nuclear magnetic resonance ( H NMR) spectra were ~ecorded on a Varian XL100, ~oel FX-90Q or ~ruker WM 360 spectrometer in either pyridine-d5 or D20 as indicated.
When pyridine-d5 is used as the solvent, the pyridine resonance a~ ~-8.57 is used as an internal reference, whereas with D~0 as solvent TSP i~ used as the internal reference. Chemical shifts are reported in ~ units ~nd coupling constants in ~ertz.
Splitting patterns are designated as follows: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad signal; dd, doublet of doublet; dt~ doublet ~f triplet. Infrared spectra were determined either on a Be~kma~ Model 4240 spectrometer or a NicQlet*5DX FT-IR spectrometer and are reported in reciprocal centimaters. Ultra~riolet ~W~ spec~ra were determined either on a Cary Mo~el 29Q spectrometer or a Hewlik~
Pac kard 84 50A spectrometer equipped with a multidiode array * trad~ mark detector. Thin layer chxomatography (TLC) was carried out on 0. 25mm Analtçch silica gel GF plates. Flash chromatography was run with either Woelm nPutral alumina (DCC grade) or Woelm silica gel (32-63ym) and the .indicated solvents. All evaporations of solvents were performed under reduced pressure and below 40~C.

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

Prooedure 1 Eo H. White et al., Or~ Syn., 48, 102-1~5 (1968).

l-Methyl-3-p~tolyltrlazene. ~-Toluidine l50.2 g, 0.47 mole) is added to a 2-1 flask equipped with a 200-ml dropping funnel a~d an efficient stirrer, and the flask is immersPd in an ice-salt bath at ca. -10. A solution of 46.8 g (0.55 mole3 cf potassium nitrite i~ 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 wi~h ~tirring. The potassium nitrite solution is slowly added wi~h continued stirring during 1-2 hours untii a positive starch-potassium iodide test ic obtained (Note 1~, ,~nd the mixtu-re is stirred for an additional hour to ensure the reaction of all the toluidine.

The solution o~ p-toluenediazonium chloride is then brought to pH 6. 8-7. 2 at 0 with cold, concentrated, aqueous ~odium ca~onate, whereupon the solution becomes red to orange in color and a small ,~mount of red material set~les out. The cold, neutral solution is transferred to a dropping funnel i~d added lowly to a vigorously stirred mixture of 150 g oE sodium carbo~ate, 300 ml o~ 30-35~ aqueous methylamine (Note 2), and 100 q of crushed ice in a 3-l ~lask. ~he reaction mixture is kept at ca. -lQ during the addition, which requires i~out ~5 minutes (Not~ 3). The solution is extracted with three 1-l portions o~ ether. The e~hereal extracts are dried with anhydrous sodium sulfate and e~aporated on a rotary evaporator at * trade mark.

a~

room 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, 6~%) 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.pO 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 of 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 lmpurity is 1,5-di-p-tolyl-3-methyl-1,4,-pentazadiene (m.p. 148). This can be removed by fractional crystallization, but it i5 easier to sublLme the triaz~ne from the reaction mix~ure.

. 5. The sublimate contains a trace of 1,3-di-p-tolyl-triazene, as show~ by thin-layer chromatography. Recrystal-lization yields the pure 1-methyl-3-p-tolyltriazene.

6. This proc~dure works well only with water-soluble amines. Procedu~e 2 given below i5 more suitable for the preparation of triazenes of water-insoluble amines.

~41-Procedure 2 E. ~. White et al., Tetrahederon Letters No. 21, _ _ _ _ _ _ ' p. 761 (1961).

A solution o~
~-chlorobenzenediazonium hexafluorophosphate (xec~stallized 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 dimethylformamide (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 mlxture 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 recrystalli~ed from pentane at low te~nperatures.

Procedure 3 .1 3h~ v 9~ = e Mitomycin C (2.2 g, 6.6 mmoles) was dissolved in 140 ml t).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 acetat~ (4 x 500 ml). The combined ethyl acetate extracts were dried (Na2SO4) and evaporated under r~duced 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, ~3%).

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).

~s~

Procedure 4 ,_ Mltom~cin A~ A 100 mg (0.30 mmole) quantity of

7-hydroxy9a-methoxymitosane and lO0 mg (0.67 mmole) quantity of 3-methyl-1-p-tolyltriazene was dissolved in 2 ml methylene chloride and lO ml diethyl etherO The solution, ater gently refluxing for 6 hours was stirred at xoom temperature for 18 hours. TLC [methylene chloride:methanol (90:10)] revealed the appearance of a deep purple spot at Rf=Oo36 wlth a trace amount of impurity at R~-0. 410 The reaction mixture was concentrated to 10 dryness and chromatosraphed on Woelm neutral alumina employing methylene chloride and methylene chloride:methanol (30:1) as eluting solvents . Fractions containing the component at R~=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 amoxphous purple powder (25 mg, 24%), mp 161.

Anal Calc'd for C16H19N36~ C~ 54-96; H~ ~-44; N~ 12~0 Found: C, 53.96; 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, ~H), 3.54(dd, lH), 3.96(dd, lH), 4.0~(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 solven~ 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 i.n Procedure 1) and ice, and the suspension was s~irred at -5C
(ice-salt bath). To this suspension was added dropwise, a cold susp~nsion of p-chlorobenzenediazonium hexafluorophosphate f~

~Aldrich Chemical Co,) in ice, water, Na~C03 (solution about pH 7). After the addition was complete, the reaction muxture was extracted with diethyl ~ther. The combined diethyl eth~r extract was backwashed with water, dried (Na2SO~) and concentrated. rhe yellowish solid residue was purified by column chromatography over Woelm alumina using hexane-methylene chloride (1:1) as eluting solvent (lH NMR recorded).

The triaze~es 1-7 of Table III which follows were prepared according to general Procedure l describe~ 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 prep~red according to general Procedure 5 described above.

r~ r~ Z
u ~ ~ z u~
O ~7 N ; N
N r1 M NUN ~N U
.,,~ ~ x h U U U U V
Z ~ Z Z Z Z Z Z
Z Z Z ~ Z Z Z
Z Z Z Z Z Z Z
o u r u c~ ~ u = ~: = 2 Z
Z ~"
F

~CO ~U

r-~j 3 ~i ~z~
~J ti~ ~j X ~ _N

r 5 r; ~U b~ ~

Z~ U U U ~O ~ ~ ~ U

;3 ,i ~i --4 5~

~ o~ o/ \o ~
~, E~ t~ ~ /
Z Z Z
1~ 11 11 :~ Z Z
~ U
~ U ~.
a. ~ ~

=~, = Z

-~, , ~1 ~
~¦ e .

La~
-~6 Example 11 -~ -3-(4-methyl~henyl) triazene .

A solution of 4-methylphenyldiazonium chloride is prepared as described in ~rocedure 1 from ~-toluidine and adjusted to pH 6.8-7.2 at 0C as described in that procedure.
solution containing 21.15 mmoles of the dia~onium 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-pyridyldithio) ethylamine, 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 o 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 drapwise during 2 one hour period from the dropping funnel. When addition had been completed the reaction mixture was allowed to warm to room tempexature and was then extracted with three 400 ml portions of ether. Drying and evaporation o~ the extracts yielded the desired product which was purified by chromatography using an alumina packed column, one inch in diameter and ten ~nches long, using hexane:methylene chloride (4:1);
hexane:methylene chloride ~3 2); hexane:methylene chloride (1:4);
and finally methylene chloride containing 1% methanol or development and elution of the column, The appropriate fractions (identified by TLC) were com~ined and evaporated to yield 2.5 g of the title compound.

Examples 12-19 General procedure ~ -b~ SY~bL
(12 A solutiQn of triazene (2 ~ 4 equivalents ) in CH2Cl~:methanol ~4:1) was added to a solution of 7-hydroxy-9a-methoxymitosane (prepared in Procedure 3) i51 CH2C12:methancl (4:1). The reaction mixture was stirred at room temperature and the progress of the reaction was monitored by thin layex 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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-51- ~5~
Example 20 y'di~,hio)ethoxy]mitosane (20) 7-Hydroxy-~a-methoxymitosane, 580 mg (1.73 mmoles) is placed in a round bottom flask and dissolved in 60 ml of methylene chloride. The tria~ene of Example 11, approxImately 2.5 g (5.7 ~moles) was added to the solution in the flask and the mixture was stirred at 5C ~or 14 hours and then at room temperature for 8 hours. The progress of the reaction was nitored by silica TLC using methylene chloride:methanol (9~
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 ~ombined and evaporated to yield the title compound, 470 mgO

Ar~al. Calc'd for C22H23N5O~S2 (corrected for 0.5 mole~ of CH2Cl2):

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(s, 3H) f 3.20(m, 2H), 3.39(d, lH), 3.83Idd, 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).

By adaptation of the procedures o:E 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.

N o CH~OCNH2 ~ S~(CH2)n~ ~ ~ R
n = 2-6 Rl = H, or C1 6 alkyl Exam ples 21-34 The 7-alkoxydithio-9a-methoxymitosane 21-34 vf 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 mitosan~ compounds 21-34 are repoxted m Table VI whioh also ~ollows.

Procedure ~

To a deoxygenated solution of mitosane of Example 20 (~001 mmole) in acetone (3-5 ml) is added with stirring, under argon, triethylamine (~1.1 equivalents) followed by dropwise or portionwise addition of a mercaptana (1 equivalent) in acetone ~ ml). In most o~ the reactionsb, ~he prcgress 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 t.he starting mitosane of Example 20 and the product have ~e$y close Rf~values on TLC, a high pressure li~uid chroma~.ography (HPLC) monitoring (~Bondapak-C18 column' is employed.
* trademark.

,. .

~2 (10% CH30H in CH2Cl~). The completion of reaction is signaled by the disappearance of spot corresponcling to the starting material and appearance of the product spot. At this point the reaction mixture is c~ncentrated under reduced pressure (at ~30C) and the residue chromatographed on a neutral Woelm alumina column (1/4" x 10") sluxry packed with 2-5% CH30H in CH2C12. 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 me~hoxy~
mitosane is characteriz~d.

Pr~cedure B

To a solution of mitosane of Example 20 (~0.1 mmole) in 2-5% acetonea in methanol (10 ml) is added saturated aqueous NaHC03 solutionb (~6 drops), followed ~y addition of mercaptan (1 equivalent) in metha~olC ~l ml). Th~ progress of the reaction is monitored by TLC (silica gel, 10% CH30~ in CH2Cl2). At ~he completion of reaction, the reaction mixture is diluted with water (15 ml) and concentrated to about lO ml under reduced presqure at about 30~C. The resulting solution is chromatographed on a reverse phase C-18 column with stepwise gradient elution tlOO% H20 to 80% CH30H in H20). The product, eluting as a major red band, i5 collected and concentrated to yield the 7-alkoxydithio-g2l-methoxymitosane as an amorphous solid. I~ ~urther purifica~ion is needed, the above chroma~ography qtep is repeated.

a) Methylene chloride can also be used, but acetone is preferred.
b) In the case where the m~rcaptan is Ll-cysteine, this base is not employed, c) Water is employed if the starting thiol is water soluble.
d) Elu~ion with water separates the yellow pyridyl thione by-product from the product, which is retained on the column~

TABLE V
7-Alko~thio-9a-methoxymitosanes A or RS~ CC 3 RSH + ~ ~ CH

Example Thiol No. (RSH) Procedure Prodllct 21 ethyl 2-mercapko- A R=-CH2CH20CCH3 acetate 2.2 .3-mercapto-1,2~ A R=-CH2CH~OH)CH20H
propanediol 23 3-mercaptopropionic B R=-CH2CH2COO Na acid 24 cysteine B R--CH2CH(NH3 )COO

thiophenol A R - ~

26 p-nitrobenzenethiol A R = ~ N02 27 p-me~ho*ybenzenethiol ~ R - ~ OCH3 28 p=aminobenzenetlliol A R ~ - ~ -NH~

~55--TABLE V (continued) Example Thiol No. (RSH) Procedure Product _ _ _ _____ _ _ _ _ _ COO Na 29 2-rnercaptobenzoic B R = ~3 acid 2-nitro-4-mercapto- B ~ = ~NO~
benzoic acid COO Na 31 4-mercaptopyridine A R = -~N

N ~, 3 2 ~-merc:aptomethyl-l~ A R = -f'E~
methylim:Ldazole .
c~3 NH
1 2 _ IHCOCH;2CH2CHCCo Na 33 glutathione B R--CH2CH
CONHCH;~COO Na~

3 4 dimethylamine B R--CH2CH2N (CH3 ) 2 ethanethiol ~5 o U~ U~
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Claims (12)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. The process for preparing a compound having the Formula IX

IX

wherein:
R5 is hydrogen, or C1 - 6 alkyl, and R6 is C1 - 12 alkyl or substituted C1-12 alkyl, C3-12 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 from the group consisting of halogen, C1-6 alkoxy, C1-6 alkanoyl, C6-14 aroyl, cyano, trihalomethyl, amino, C1-6 monoalkylamino, C2-12 dialkylamino, C6-12 aryl, C6-12 aryloxy, C1-6 alkanoyloxy, C7-14 aroyloxy, heterocyclic group selected from the group consisting of 5- or 6- membered heterocyclic aromatic or alicyclic ring having l or 2 hetero atoms selected from the 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 halogen, C1-6 alkoxy, C1-6 alkanoyl, cyano, trihalomethyl, amino, C1-6 alkylamino, or C2-12 dialkyl-amino groups which comprises reacting a mitosane of Formula X

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.

2. The process of claim 1 wherein said triazene of Formula XI is 3-methyl-1-(4-methylphenyl)triazene.

3. The process of claim 1 wherein at least two molecular proportions of said triazene relative to said mitosane of Formula X are employed.

4. The process of claim 1 wherein a reaction inert organic liquid solvent for said mitosane of Formula X is employed as reaction medium.

5. The process of claim 1 wherein a lower alkanol, a lower alkyl alkanoic ester, a dilower alkyl ether, a lower polyhalo-genated aliphatic hydrocarbon or a cyclic aliphatic ether having up to 8 carbon atoms is employed as reaction medium.

6. The process of claim 1 wherein methylene chloride, methanol, diethyl ether, ethyl acetate or a mixture of two or more thereof its employed as reaction medium.

7. The process of claim 1 wherein the reaction temperature is from 0' to 60 C.

8. The process of claim 1 wherein the reaction temperature is from 0° to 25°C.

9. The process of claim 1 wherein R6 is 2-(benzylthio)ethyl and R5 is hydrogen.

10. The process of claim 1 wherein R6 is 2-(2-pyridyl)ethyl and R5 is hydrogen.

11. The process of claim 1 wherein R6 is 2-(4-morpholinyl)ethyl and R5 is hydrogen.

12. The process of claim 1 wherein R6 is (1,3-dioxolan-2-yl)methyl and R5 is hydrogen.