Note: Descriptions are shown in the official language in which they were submitted.
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.
O CH20CONH2
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.
O CH2 OCONH2
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
: ,
.. , : .
. .
:: ;
,.
- 9 -
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
, .,~.,;.,. :
'-, :. ',
:, ~ '' ,: :,
- , ." ,, .. . ~ .
.. . .
; '.,' '` ' '
-- 10 --
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 H2 OCNH2
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~
--11--
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
-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 ~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
CHZOCNM2
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
CX2 OCNH2
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
--15--
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
: . ,
. ' ~ '' ' ~ .
-18-
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
2 2S 03 XV
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~
--19--
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
-21-
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
-23-
Formula V or VI i5 utilized in the procedure depicted in Scheme 2
to produce disulfide mitosanes having the For~ula Ib
~_55-~lk2-~ }~
CH3 -R
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
HSCH3
2(~ 3
HSCH2CH2CH3
HSCH (CH3) 2
HS (CH2) 3CH3
HS-CH-CH CH
CH3
HS CH2 CH (CH3 ) 2
l H3
HS-C-CH3
1H3
HS<¦
,C
HS
o
HS O
- ' , ' ~'`:
"' ' . ' ,''~' ,' ' '
--26--
HS{~
HS-CH20
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
. .
.
-27-
~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
C02H
HS-CH2-1CH-CH2-NH2 + peptides
C02H
HS-CH ~-CH-CH 2-CO2H + peptides
NH2
ICH 2CO;2H
HS-CH2CH2CH + peptides
NH2
lH2
NHCOCH2CH2CHC02H
HS-CH2CH
CONHCH2C02H
H S -CH= CH -NHCCH 3
HS-CH2-CH-CH20H
OH
l H3
HS-CH2CH-CH20H
-28- ~ ç,;
HS-CH21CH-CH2NRlR2 Rl = CH3; R2 = H, C~3
~CH 3
H S - CH 2 CH2 -~ -CH 3
CH3
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~ ~ . . .
NHCH3
H S ~ NHeCH 3
-:
'
..
, . .
29-
HS~C02H
NH2
HS~ ~>
HS~
HS O
~q
HS~N
N ( CH3 ~ 2
HS-CH2~3
HS - CH 2-CH=CH~
HS--CH2CH2~ NHCCH3
HS- (CH2) n4~ n = 1, 2
N
HS (CH2)n~ n = 1, 2
N
- 3 0 -
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
7L
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.
-34-
a) ~
o o o o o
I + I + +
C~ .
o o ,~ ~ o
3 +
In In U~
IY ~ ~ O o o
~ 0 o o o o o
.
~ _~ ~ _ _ _ _
a) ~ ~ ~ ~ ~3
o
au al a _ _ _ _ _
'~ ~ ~
H :~ ~ . _ _ _ ~ _
1-l co ~ ~.)
11~ ~ o=Z ~ /~ - d~ O O O ~0
~1 ~) ~ / \
o ~ .
,R 0=~=0
~s~
a~
.
O Z
~ O .
:~ ~1 r~
0~ ~ ~ ~ Ln ,
O X
' .
,',. '
- -35~
~ ~ ~ t~ I
a
o ~ ,~
I + ~ +
U .. ~
a~ o e:
,1 o o ,~ o
3 I I + I +
~J
O
U~
o O N
U~
o VO
q
~,~
.~
_~
U~ .
O ~ ~ ~ ~r er
a _ . _ _ _ _
~D O O ~ ~
_,
X
,~ _
C~
o r_
E-~ ~ _I ~1 A A
_ - _ _ _
dP I`') ~1 1` ~ CO
co ~ u~ o r~
~ ~Z ~3
U~ X C~
~ O ~ O~ ~0 ~ ~
~ :q ~ ~ ~ ~ .
~ U ~ U U U U
a)
.,1
O ~ .
O O
Z
H ~ ~1
C~ ~ t` ~ ' ~ o U~
~1 1~
m r~
~ o X
E~ ~
.
:
-36~ "~
.
,
~ ~ +
s~ ~
~ U ~`
V
,i
E a v
U~
a ~ .
~ ~ V
~ ~ 4
X U . ~
E~ _
dP ~ ~ C~
a~ C a~ O
_, ~ I ~
E ~ .
~ 3`~
U~ ~ ~ U~
U~
m~
u .,~
~ ~ a
5: 3 P~
u . a.
l ~ ~a ~ s~
:~ ~ (:~ '
.,,
a
~ ,
o ~ . o a~
V O O O' 3 u~
Z ~ ~ ~ o
~ X
H ~ aJ
~ _l tJ~
~ .~ ~0
'; ' '
:"
:, . ~ . , .: ,
:.. ". ' ' "
'' ,.'
,
,
- _ 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
-
3 3
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.
:.
-39-
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
-41-
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
-42-
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
16 19
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 ~
.
.
-43-
(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.
-44-
~ ~ N ~z~ ~ W
C U U W W W W y
Z lZI lZI Z Z Z Z
U U U U U U U
w m~ U :~ U :: ~ u
~ . ~ . .
$ .~ t
H~
~5 ~`
~3¢ : ~
'~ _ U
; W~ 3' ~7r
:
z
~ _~ ~ ~ ~ ~ ~D 1`
:~
.
. ' , ~ : ' !' . ~ . ' .
,` ' , : . . . ..
'' . ~ ' ' '
-45- ~¢~ 3
C O O~ O
.,~ ~ ~ ~
E~ ~ U Y
Z~ Z Z
z æ
11 11 11
Z Z Z
U O U
U U C~
V
o ..
, .
~ o . .
1~ _~ Q Cl~ O
~ 8 ~
... ... .. .
.
.
:
-46-
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-
-47-
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.
.
.
--48--
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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
- 22 23 5 8 2
(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
--52--
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. ,
., '`~ ,.
., : , , .
.
-53-
(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.
-54
TABLE V
?-Alkoxy~dithio-9a-methoxymitosanes
CH2 OCNH2
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
CH3
1 2 +
NIHCOCH2CH2CHCOO Na
33 glutathione B R=-CH2CH
CONHCH2COO Na+
34 dimethylamine ~ R=-CH2CH2W(CH3)2
ethanethiol
.
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