Note: Descriptions are shown in the official language in which they were submitted.
.".............................. ' 129Z9~0
HOECHST AKTIENGESELLSCHAFT HOE 85/F 042 Dr.Ka/Fr
Heterocycl;c disulfides and their use as immunomodulators
It is known that the defense mechanisms of the liv;ng body,
which are br;efly termed the hùmoral immun;ty and cellular
;mmun;ty, cooperate to neutralize and eliminate foreign
bodies which may give rise to pathogenetic changes and be
injurious, principally microorganisms or neoplastic cells.
Immunological investigations have shown that there are
connections between a decrease in immunological activity
provoked by internal or external factors and an increase
in the infect;ous or tumorous diseases. In addit;on,
other d;seases arise owing to changes in the functioning
of the ;mmune system. These include, for example, auto-
immune diseases or d;sorders caused by immune complexes.
~hus, there has for a long time been a search for immuno-
stimulants, that ;s to say substances wh;ch are able tomodify the immunological act;vity of the recipient, pre-
ferably to increase it, and which allow, by reason of
their high efficacy and good tolerability, ~ide use to
support the body's defenses. Examples which have been
tested for their stimulation of immunity are BCG and C.
parvum, as well as extracts of M. tuberculosis and of the
~rucellae.
However, at the concentrations at which these substances
are used they produce marked side effects such as, for
example, local granulomas to varying extents. The lack
of knowledge of the exact nature of these substances makes
it difficult to carry out a systematic investigation with
reasonable reproduc;b;lity of the clin;cal results. ~hus,
in this connection there is a requirement for new immuno-
stimulants which are chemically defined substances andhave low toxic;ty, such as, for example, bestat;n which is
an immunostimulant of low molecular we;ght, is undergo;ng
intensive investigation at present and is, in general, a
7~
129Z990
scientific reference substance.
It has no~ been found, surprisingly, that the compounds
according to the invention have a potent immunostimulating
and immunorestorative action, as is expressed, for example,
in the DTH react;on on sheep erythrocytes, in the activa-
t;on of mononuclear phagocytes and in a pronounced CSF
activity. These immunostimuLating effects can also be
observed, for example, in an increase in the po~er of
resistance to infections. Furthermore, the compounds
according to the invention have, surprisingly, cytostatic
activity, for example against ~16 melanoma of the mouse.
The present invention thus describes a class of substances
~hich have immunopharmacological and cytostatic activity,
are chemically defined, have low toxicity and are, as such
or combined with other active compounds, valuable medica-
ments. The compounds actord;ng to the invention have a
LDso value above 1,000 mg/kg on intravenous injection in
mice. The effective immunomodulatory and cytostatic amount
for vertebrates, preferably warm-blooded mammals is in the range
from about 0,5 to about 100 mg/kg of body weight for each parenteral or
oral dose, and this sho~s no toxic side effects and is thus
very ~ell suited for the treatment of diseases of the immune
system.
Thus the present invention relates to the use of compounds
of the general formula I or I'
Het-S-S-Het Het -S-S-Het'
(1) (1')
for immunostimulation, immunorestoration and cytostatic
treatment and to the use of these compounds for th~ pre-
paration of a medicament to be used for this purpose.
The invention also relates to pharmaceutical agents for
immunostimulation, immunorestoration and cytostatic treat-
ment, ~hich contain a compound of the general formula I or I'
1292990
-- 3 --
and to the use of a pharmaceutical agent of th;s type for
the medical indication mentioned.
In the compounds of the general formula I, Het represents
an opt;onally substituted S- or 6-membered heterocycle.
The heterocycle can contain, for example, 1 - 4 hetero-
atoms, in particular N, where appropriate in combination
with S or 0.
The follo~ing fundamental ring systems may be mentioned
as examples of Het: thienyl, furyl, imidazolyl, pyrazolyl,
thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl,
thiadiazolyl, oxadiazolyl, tetrazolyl, pyridyl, pyrazinyl,
pyrimidinyl, pyridazinyl, triazinyl and benzo-fused deri-
vatives, such as benzoxazolyl, benzothiazolyl and benzi-
midazolyl, ;t also be;ng possible for the ring systems
to be entirely or partially hydrogenated, such as, for
example, dihydrotriazinyl.
The preferred ring systems have 5 members and one suLfur
or oxygen atom and 1 or 2 nitrogen atoms, such as thia-
20lyl, in particular 2-thiazolyl and 5-thiazolyl, 1,3,4-
thiadiazolyl, in particùlar 1,3,4-thiadiazol-5-yl, oxadia-
zolyl, such as 1,3,4-oxadiazol-5-yl. Other preferred ring
systems have 5 members and 1 to 4, in particular 2 to 4,
nitrogen atoms such as, for example, imidazolyl, prefer-
ably 2-imidazolyl, triazolyl, preferably 1,3,4-triazol-5-
yl and 1,2,4-triazol-5-yl. aenzo-fused derivatives are
also preferred, in particular benzoxazol-2-yl, benzo-
thiazol-2-yl and benzimidazol-2-yl.
Other suitable and preferred r;ng systems have 6 members
and 1 to 3, preferably 1 or 2, nitrogen atoms, such as,
for example, pyridyl, preferably 2-pyridyl, 3-pyridyl and
4-pyridyl, pyrimidyl, preferably 2-pyrimidyl and 4-pyrimi-
dyl, triazinyl, preferably 1,2,4-triazin-3-yl, and 2,5-
and 4,5-dihydro-1,2,4-triazin-3-yl.
125~Z99O
The Het radical can be substituted, examPLes of suitable
substituents being the following:
Straight-chain and branched alkyl groups having 1 - 6,
preferably 1 - 3 carbon atoms, such as, for example,
methyl, ethyl, n- or i-propyl, n- or tert.-butyl, prefer-
ably methyL, which can, ~here appropriate, in turn be
substituted by
halogen, such as, for example, chlorine or bromine,
hydroxyl, alkoxy having 1 - 4 carbon atoms, such as,
for example, methoxy or ethoxy, amino, alkylamino or
dialkylamino having 1 - 4 carbon atoms in each alkyl
radical, such as, for example, methyLamino, ethylamino,
dimethylamino and diethylamino,
mercapto,
alkylthio having 1 - 4 carbon atoms, such as, for
example, methylthio and ethylthio,
alkoxycarbonyl having 1 - 4 carbon atoms ;n the alkyl
moiety, such as, for example, methoxycarbonyl and
ethoxycarbonyl,
aminocarbonyl, N-alkylaminocarbonyl or N,N-dialkylamino-
carbonyl having 1 - 4 carbon atoms per alkyl moiety,
such as, for example, N-methylaminocarbonyl and N-ethyl-
aminocarbonyl or N,N-dimethylaminocarbonyl and N,N-
diethylaminocarbonyl,
carboxyl, sulfo, phospho, 1H-tetrazol-5-yl,
aryl, such as, for example, phenyl,
halogen, such as, for example, chlorine or brom;ne,
hydroxyl, oxo, oxido,
alkoxy having 1 - 4 carbon atoms, such as, for example,
methoxy or ethoxy,
amino, alkylam;no or dialkylamino having 1 - 4 carbon
atoms per alkyl moiety, such as, for example, methyl-
amino, ethylamino, dimethylamino or diethylamino,
or acylamino, ~here acyl may represent the radical of an
aliphatic mono- or dicarboxylic acid having 2 - 5 carbon
atoms, such as, for example, acetyl, propionyl, 3 - car-
boxypropionyl, 4 - carboxybutyryl preferably acetyl,
.
lZ92990
aLkylthio, alkenylthio and alkynylthio having 1 - 4 carbon
atoms in the alkyl moiety and 2 - 4 carbon atoms in the
alkenyl and alkynyl moiety, such as, for example~
methylthio, ethylthio, vinylthio, allylthio, ethynyl-
thio or propynylthio, which can optionally be substi-
tuted by carboxyl, sulfo, phospho, 1H-tetrazol-5-yl or
aminocarbonyl,
alkenyl having 2 - 4 carbon atoms, such as, for example,
vinyl or allyl, which can be optionally substituted by
halogen such as, for example, chlorine or bromine,
hydroxyl,
alkoxy having 1 - 4 carbon atoms, such as, for example,
methoxy or ethoxy,
alkoxycarbonyl having 1 - 4 carbon atoms in the alkyl
moiety, such as, for example, methsxycarbonyl or
ethoxycarbonyl,
aminocarbonyl, N-alkylaminocarbonyl,
N,N-dialkylaminocarbonyl having 1 - 4 carbon atoms per
alkyl moiety, such as, for example, N-methylamino-
carbonyl and N-ethylaminocarbonyl or N,N-dimethylamino-
carbonyl and N,N-diethylaminocarbonyl, carboxyl, sulfo,
phospho, 1H-tetrazol-5-yl,
carboxyl or
alkoxycarbonyl having 1 - 4 carbon atoms in the alkyl
moiety, such as, for example, methoxycarbonyL or
ethoxycarbonyl.
~ithin the general formula I, the use according to the
invention also covers new compounds.
Accordingly, the present invention also relates to new low-
molecular weight heterocyclic disulfides of the general
formula I'
Het'-S-S-Het' lI')
in which Het' represents a 5-membered ring system having one
sulfur and 1 or 2 nitrogen atoms or having 1 - 3 nitrogen
lZ9Z~91~I
atoms, such as, for example, thiazolyL`, in particular 2-
thiazolyL and 5-thiazolyl, 1,3,4-thiadiazolyl, in parti-
cuLar 1,3,4-thiadiazol-5-yl, imidazolyl, ;n particular
;2-imidazolyl, and triazolyl, in particular 1,2,4-triazol-
5-yl. Benzo-fused derivatives are also preferred, in
particular benzothiazol-2-yl and benzimidazol-2-yl.
Het' can also represent a 6-membered ring system having
1 to 3 nitrogen atoms, such as, for example, pyridyl, pre-
ferably 2-pyridyl, pyrimidyl, preferably 2-pyrimidyl, and
triazinyl, preferably 1,2,4-triazin-3-yl, it also being
possible for the ring systems to be entirely or partially
hydrogenated, such as, for example, dihydrotriazinyl.
The radical Het' can be substituted, examples of suitable
substituents being the following:
Straight-chain and branched alkyl groups having 1 - 6,
preferably 1 - 3 carbon atoms, such as, for example,
methyl, ethyl, n- or ;-propyl, n- or tert.-butyl, prefer-
ably methyl, which can, where appropriate, in turn be
substituted by
halogen, such as, for example, chlorine or bromine,
hydroxyl,
alkoxy having 1 - 4 carbon atoms, such as, for example,
methoxy or ethoxy,
amino, alkylamino or dialkylamino having 1 - 4 carbon
atoms in each alkyl radical, such as, for example,
methylamino, ethylamino, dimethylamino and diethylamino,
mercapto,
alkylthio having 1 - 4 carbon atoms, such as, for
example, methylthio and ethylthio,
alkoxycarbonyl having 1 - 4 carbon atoms in the alkyl
moiety, such as, for example, methoxycarbonyl and
ethoxycarbonyl,
aminocarbonyl, N-alkylaminocarbonyl or N,N-dialkylamino-
carbonyl having 1 - 4 carbon atoms per alkyl moiety,
such as~ for example, N-methylaminocarbonyl and N-ethyl-
aminocarbonyl or N,N-dimethylaminocarbonyl and N,N-
lZ92990
-- 7 --diethylaminocarbonyl,
carboxyl, suLfo, phospho, 1H-tetrazol-5-yl,
aryl, such as, for example phenyl,
halogen, such as, for example, chlorine or bromine,
hlydroxyl, oxo, oxido,
alkoxy having 1 - 4 carbon atoms, such as, for example,
methoxy or ethoxy,
amino, alkylamino or dialkylamino having 1 - 4 carbon
atoms per alkyl moiety, such as, for example, methyl-
amino, ethylamino, dimethylamino or diethylamino,or acylamino, where acyl may represent the radical of an
aliphatic mono- or dicarboxylic acid having 2 - 5 carbon
atoms, such as, for example, acetyl, propionyl, 3 - car-
boxypropionyl, 4 - carboxybutyryl, preferably acetyl,
alkylthio, alkenylthio and alkynylthio having 1 - 4 carbon
atoms in the alkyl moiety and 2 - 4 carbon atoms in
the alkenyl and alkynyl moiety, such as, for example,
methylthio, ethylthio, vinylthio, allylthio, ethynyl-
thio or propynylth;o, ~h;ch can optionally be substi-
tuted by carboxyl, sulfo, phospho, 1H-tetrazol-5-yl or
aminocarbonyl,
alkenyl having 2 - 4 carbon atoms, such as, for example,
vinyl or allyl, which can be optionally substituted by
halogen such as, for example, chlorine or bromine,
hydroxyl,
alkoxy having 1 - 4 carbon atoms, such as, for example,
methoxy or ethoxy, alkoxycarbonyl having 1 - 4 carbon
atoms in the alkyl moiety, such as, for example,
methoxycarbonyl or ethoxycarbonyl,
aminocarbonyl, N-alkylaminocarbonyl, N,N-dialkylamino-
carbonyl having 1 - 4 carbon atoms per alkyl moiety,
such as, for example, N-methylaminocarbonyl and N-ethyl-
aminocarbonyl or N,N-dimethylaminocarbonyl and N,N-di-
ethylaminocarbonyl,'
carboxyl, sulfo, phospho, 1H-tetrazol-5-yl,
carboxyL or
alkoxycarbonyl having 1 - 4 carbon atoms in the alkyl moiety,
such as, for example, methoxycarbonyl or ethoxycarbonyl~
. . lZ~Z990
The heterocycles Het and Het' can be subst;tuted once or
several times, for example once to three times, in the
manner described above. Ho~ever, preference is given to
heterocycles Het and Het' ~hich carry one or 2 sùbstitu-
ents in the heterocyclic ring or in the carbocyclic ring~hich is fused on. Those ~hich are particularly preferred
are ones in ~hich at least one of these substituents car-
ries an acid group, in particular a carboxyl group such
as, for example carboxyalkyl or carboxyalkylthio.
Preference is also given to heterocycles Het and Het' ~h;ch
carry in the heterocyclic moiety or in the fused-on carbo-
cyclic ring one or t~o substituents, at least one of ~hich
is a directly bonded ac;d group such as, for example, car-
boxyl or hydroxyl.
If the substituents on the heterocycle Het and Het', such
as, for example, alkyl, alkenyl, alkylthio, alkenylthio
or alkynylthio, are further substituted in the manner des-
cribed above, then they can also carry more than one sub-
stituent, for example 1 - 3 other substituents. However,
single substitution is preferred.
~here the compounds of the formulae $ and I' carry acid
groups they can also be in the form of their physiologic-
ally tolerated salts, for example as alkali metal and
alkaline earth metal salts such as, preferably, the Na, K,
Ca or Mg salts or, for example, ammonium salts or substi-
tuted ammonium salts such as, for example, NH4~, ethanol-
ammonium, diethanolammonium, trialkylammonium, such as,
for example, triethylammonium, tetraalkylammonium, or
salts with basic amino acids such as, for example, lysine
or arginine.
lz~2sso
- 8A -
In accordance with one embodiment of the invention, a compound
of the formula I' is provided
Het'-S-S-Het' (I)
wherein Het' represents a heterocyclic ring system selected from
the group consisting of thiazolyl, l,3,4-thiadiazolyl, triazolyl,
triazinyl, and partially hydrogenated triazinyl, and wherein said
~et- is substituted by C1-C6-alkyl, which may be unsubstituted or
substituted by halogen, hydroxyl, C1-C4-alkoxy, mercapto, C1-C4-
alkylthio, C~-C4-alkoxy-carbonyl, or carboxyl; phenyl; halogen,
hydroxy; oxo; C~-C4-alkoxy; C2-C5-acylamino wherein acyl is the
radical of an aliphatic mono- or dicarboxylic acid; C1-C4-
alkylthio, C2-C4-alkenylthio or C2-C4-alkynylthio, wherein said C1-
C4-alkylthio, C2-C4-alkenylthio or C2-C4-alkynylthlo may be
un~ubstituted or ~ub~tituted by carboxyl; Cz-C4-alkenyl which may
be unsubstituted or substituted by halogen, hydroxyl, C1 -C4-
alkoxy, C~-C4-alkoxy-carbonyl, or carboxyl; or C~-C4-alkoxy-
carbonyl, wherein at least one of the above-defined substituents
of Het' is or carries carboxyl or hydroxyl.
The invention also relates to a proce~s for the preparation of
the compounds, according to the invention, of the formula I'.
. 1292990
The process according to the invention comprises conver-
sion of a compound of the formula II
Het'-SH ~II)
in ~hich Het' has the meaning detailed above, by reaction
with an oxidizing agent, into the compounds according to
the invention and, where appropriate, conversion of the
abovementioned substituents, according to the invention,
of Het' ;nto others of the abovementioned substituents,
according to the invention, of Het'.
Examples of suitable oxidizing agents which may be men-
tioned are oxygen, hydrogen peroxide, where appropriate
with addition of iron salts such as, for example, Mohr's
salt, or with the addition of bases such as, for example,
sodium hydroxide, potassium hydroxide, sodium bicarbonate
or potassium b;carbonate, organic peracids such as, for
example, peracetic acid, perbenzoic acid or m-chloroper-
benzoic acid, elementary iodine or bromine, where approp-
riate with the addition of bases such as, for examPle~
sodium hydroxide or potassium hydroxide, FeCl3 or
K3~Fe~CN)6)
The oxidizing agents which may be mentioned as preferred
are: oxygen, hydrogen peroxide, organic peracids such as,
for example, peracetic acid, perbenzoic acid and m-chloro-
perbenzoic acid, and elementary iodine. The ox;dation is
preferably carried out in water or an organic solvent such
as, for example, methanol, ethanol, isopropanol, ethyl
acetate and halogenated hydrocarbons such as, for example,
dichloromethane and chloroform. It is also possible to
use a mixture of these solvents. ~hen work;ng vith oxygen,
hydrogen peroxide and peracids, the use of solvents which
are known to be able to form explosive peroxides, such as,
for example, ether, tetrahydrofuran, dioxane and acetone,
methyl ethyl ketone, isopropanol, etc. should be avoided.
` 1292990
-- 10 --
~he compounds according to the invention can also be pre-
pared by reaction of a compound of the formula III
Het'-X (III)
in which Het' has the meaning detaiLed above, and X rep-
resents a reactive leaving group such as halogen, prefer-
ably chlorine or bromine, such as -OS02R, R preferably
meaning methyl, trifluoromethyl, phenyl, tolyl or naphthyl,
or such as -OPO~OR'2), R' preferably meaning phenyl, with
Me2S2, Me representing an alkali metal, preferably
sodium, and, where appropriate, conversion of the above-
mentioned substituents, according to the invention, of
Het' into others of the abovementioned substituents,
according to the invention, of Het'.
Furthermore, the compounds according to the invention can
be prepared by reaction of a compound of the formula IV
Het'SS02Me (IV)
;n wh;ch Het' and Me have the meanings detailed above,
w;th iodine in an aqueous medium and, where appropriate,
conversion of the abovementioned substituents, according
to the invention, of Het' into others of the abovementioned
substituents, according to the invention, of Het'.
The reaction can be carried out in a temperature range
between about -40C and the boiling point of the solvent
or ~ixture of solvents, preferably between about -10C
and about +40C.
It is quite generally possible to convert by processes
known from the literature the substituents contained in
the heterocycle Het' in the disulfides of the formula I'
into other substituents according to the invention Thus,
for example, an alkoxycarbonyl or aminocarbonyl group can
be converted by hydrolysis, or in the case of the amino-
lZ92990
carbonyl group also by nitrosation, into a free carboxylgroup.
The active compound can be administered alone or combined
w;th one or more, preferably one other medicament having
favorable effects on infections, caused, for example, by
bacteria, fungi, or viruses, and on tumorous diseases~
According to the invention, the active compounds can be
administered both parenterally and orally. Suitable for
parenteral administration are solutions or suspensions of
the active compound in a pharmaceutically tolerated
vector, preferably a vegetable oil such as, for example,
peanut oil or sesame oil, and alcoholic solutions of the
active compound, for example, in ethanol, propanediol or
glycerol or in mixtures of the abovementioned solvents.
To prepare aqueous solutions, the active compound is
preferably used in the form of phys;olog;cally tolerated
salts wh;ch are soluble in water. lt is possible for the
formulat;ons to contain the customary auxiliaries and
excip;ents. Examples of these are f;llers, emuls;f;ers,
lubricants and buffers and flavor-modifying agents.
In the text which follo~s, the action of the compounds on
the immune response of the mouse and their immunostimulant
activities in various in vivo standard methods are illus-
trated by way of example. The various test methods employed
are known to be particularly ~ell suited for the assessment
of immunostimulants and their quality of action.
Experiment 1
Effect on the cellular immunological reaction, of the
delayed type, to sheep erythrocytes (delayed type hyper-
sens;t;v;ty, DTH)
Either 106 or 109 red blood cells from sheep were adm;ni-
stered intravenously to each animal in groups of 5 female
NMRI mice we;ghing 18 - Z0 9. Sheep erythrocytes are
lZ9Z99O
regarded in immunology as a standard test substance (anti-
gen) for the induction of ceLluLar and humoraL immune re-
actions. In particuLar, this test provides information
about the functioning of the T-cell-dependent component (T-
helper cells) of the immune system. The test substanceobtained as in exemplary embodiment 8 (bis(5-carboxymethyl-
4-methyl-2-thiazolyl) disulfide) was adm;nistered intraper;-
toneally (2x/day) in the concentrations 20 mg/kg, 30 mg/kg and
40 mg/kg in physiological saline solution on days -3, -2, -1
and 0. After 5 days, each animal received 2 x 108 sheep
erythrocytes injected into the underside of the foot and,
24 hours later, the swelling of the foot was measured.
The foot s~elling is induced by a skin reaction of the
delayed type (delayed type hypersensitivity, DTH) and is,
as is kno~n to the expert, a measure of the cellular
immune response (Collins, F.M. and Mackaness, G.B., J.
Immunol. 101, 830-845, 1968). The results compiled in
Table 1 illustrate that there is an increase in the cellu-
lar immune response o~;ng to the adm;n;stration of the sub-
~; stance obtained accord;ng to the invention, for exampleafter immunization with 106 sheep erythrocytes. A maximum
st;mulation can be observed ;n th;s experimental design
on administration of 30 mg/kg test substance.
Table 1
Immunization of mice ~ith sheep erythrocytres-action on
the cellular immune response (DTH reaction)
2x/day i.p. administration % foot s~elling with
on days -3, -2 -1 and 0 of 106 erythrocytes
30PBS* 24.3 + 1.3
20 mg/kg 28.3 ; 6.7
Test substance 30 mg/kg 36.5 ~ 6.5
40 mg/kg 31.4 ~ 8.7
* PBS = phosphate-buffered saline (NaCl:8,000 mg/l,
KCl: 200 mg/l, Na2HP04.2H20: 1,440 mg/l,
KH2P04:200 mg/l
lZ9Z99O
Experiment 2
Effect on the stimulation of non-specific immunity
activat;on of mononuclear phagocytes
This entailed investigation of the effect of the test sub-
stance obtained as in exemplary embodiment 8 on the stimu-
lation of peritoneal macrophages in 6-8 week-old NMRI mice.
Female mice received the test substance in parenteral or
oral doses of 25 mg/kg, Sû mg/kg, 100 mg/kg and 200 mg/kg.
Buffered saline solution was administered to a control
group. The mice were sacrificed three days after the in-
jections, and the peritoneal macrophages of the animals
were examined for their state of activation. The secret-
ion of the lysosomal enzymes (g-galactosidase, g-glucuroni-
dase, and N-acetyl-g-D-glucosaminidase) was determined as
one measure of macrophage activation. In addition, it was
possible to examine the pinocytosis in comparable macro-
phage cultures by the uptake of colloidal gold ~198Au) - as
is known to the expert. The level of oxidative metabolism
in macrophages is taken as another measure of their state of
act;vation. This activity is measured with the assistance
of a biolumate by determination of the chemiluminescence.
For this purpose, either 3 x 106 macrophages were cul-
tured with 1 ml of TC 199 culture medium in Petri dishes
of diameter 30 mm or 106 macrophages were cultured with
100 ~ul of medium in round-bottomed polyethylene tubes
(for determination of the chemiluminescence), with 5% C02
and at 37C.
After incubation for one hour, the cultures were washed
to remove flDating cells. The chemiluminescence (tube
culture) was then determined directly, whereas the Petri
dishes were incubated for a further 24 hours at 37C and
then the enzyme and pinDcytosis activities in the cultures
were determined. The following results were obtained.
1~92990
- 14 -
Table 2
Action on the ox;dat;ve metaboLism ;n mouse per;toneal
macrophages (chem;lum;nescence ;n RLU*/15 m;nutes).
1 x adm;n;strat;on intraperitoneal oral
of
,
PBS2.97 + 0.28 x 10' 3.65 + 0.81 x 10'
Test 25 mg/kg 7.87 + 0.28 x 105 6.41 + 0.42 x 105
substance 50 mg/kg 9.72 + 0.82 x 105 8.99 4 0.3~ x 105
100 mg/kg 12.85 + 2.37 x 105 11.85 + 0.92 x 105
200 mg/kg 24.40 + 3.39 x 105 15.60 + 1.98 x 105
*RLU = relative light units
Both parenteral and oral treatment of NMRI m;ce ~ith the
test substance prepared as in Example 8 stimulate macro-
phage activity and thus have an immunity-stimulating
action. Thus, the oxidative metabolism of macrophages,
~ith the generation of oxygen radicals and the measurable
light associated ~ith this, ;s markedly ra;sed. At dos-
ages of 25 mg/kg up~ards, there is a dose-dependent in-
crease in the macrophage activity with both parenteral
and oral administrat;on.
It can be seen from Table 3 that macrophages from control
mice release only small amounts of lysosomal enzymes (B-
glucuronidase, B-galactosidase and N-acetyl-B-D-glucos-
aminidase) ;nto the culture supernatant. Mononuclear
phagocytes from mice which have been treated parenterally
or orally with the test substance for 72 hours secrete
markedly more of the abovementioned acid hydrolases
(B-Glu, B-Gal and N-Ac-Glu), and thus sho~ a dose-effect
curve ~hich demonstrates a super;or;ty over the controls
for all the enzymes measured. It is evident that the test
substance has a stimulant action on macrophage act;v;ty
and contributes to an ;ncrease in enzyme release~
129;~9~0
- 15 -
Table 3
Effect of the test substance on the release of the
lysosomal hydrolase enzymes from mouse peritoneal macro-
phages.
5 1 x i.p./p.o. ~-Glu B-Ga~ N-Ac-Glu
Administration mU/ml mU/ml mU/ml
PBS 755/ 484 1306/ 1702 1238/1168
25 mg/kg 1001/ 897 2584/ 4917 2786/1947
Test 50 mg/kg1370/1133 11058/ 9179 3315/28620 substance 100 mg/kg 1791/1593 17596/13195 4305/3676
200 mg/kg 2136/1886 22351/17357 6548/5621
The quant;tative determ;nat;on of the p;nocytosis act;v;ty
of mononuclear phagocytes was carr;ed out by the method
of Dav;es et al. ~Davies, P., Allison, A.C. and Has~ell,
A.D.; Piochem. Biophys. Res. Com. 52, 627, 1973~. Radio-
active collo;dal gold ~198Au) w;th a part;cle size of
20 nm and a spec;fic act;vity of 4-12 mCi/mg of Au was
used for this. The results in Table 4 illustrate the
effect of the test substance obtained as in Example 8 on
th~ efficiency of endocytosis. The pinocytosis of colloi-
dal gold (198Au) by mouse peritoneal macrophages from
animals treated ~ith the compound according to the inven-
tion is significantly and dose-dependently higher than by
macrophages from untreated an;mals.
Table 4
The effect of the test substance on the eff;ciency of
pinocytos;s of mouse macrophages.
1 x administration of ;ntraperitoneal oral
P~S 0.286 x 103 cpm 0.198 x 103 cpm
25 mg/kg 0.341 " " 0.272 " "
Test 50 mg/kg 0.396 " " 0.358 " "
substance 100 mg/kg 0.462 " " 0.416 " "
200 mg/kg 0.587 " " 0.506 " "
lZ~2990
- 16 -
Experiment 3
Increase in the res;stance of Balb/c mice to infection
with Candida albicans
a) Prophylactic treatment:
Balb/c mice were treated intraperitoneally with the test
substance (compound of exemplary embodiment 8) at a dose
of 2 x 60 mg/kg/day for 4 days. 24 hours after the last
administration of the test substance, these animals and
the control animals, which had received administrations
of physiological saline solution in the same volumes and
time intervals, are infected intravenously with Candida
albicans (5 x 105 CFU/mouse). The relevant mean survival
times can be calculated from the death rate after the
infection. 50% of the animals in the control group die
after 9.7 days, while the group treated with the test
substance showed a mean surv;val time of 16.1 days. With
the selected administration regimen and with these doses
tprophylactic administration), the test substance induces
a significant increase in the resistance of the Balb/c
mouse to Candida albicans.
Table 5
Mean survival times after C. albicans infection (5 x 105 CFU)
~ubstance Mean Confidence limits
2 x 60 mg/kg/ survival
25 day i.p. time (days) 95% 99Z
PBS 9.7 8.4-1û.6 7.8-10.9
Test substance 16.1 14.8-17.4 14.4-17.8
b) Therapeutic treatment:
.
For the therapeutic treatment of a chronic Candida albicans
infection, female Balb/c mice (15/group) were infected
intravenously on day 0 with Candida albicans ~1 x 105
~ Z92990
- 17 -
CFU/mouse). After the infection had taken place, the
animals were treated intraperitoneally on each of 8 con-
secutive days (days 3 - 10) with 60 mg/kg of the test
substance (compound of exemplary embodiment 8). The con-
trol animals received injections of physiological salinesolution. Urine was collected from the animals on days 8,
14 and 21, and the organism count was determined. On day
30, the animals were sacrificed and the organism count and
the formation of necroses in the kidneys were determined.
Table_6 shows that the test substance on therapeutic admin-
istration markedly reduces all the parameters of chronic
Candida albicans infection (organism count in the urine
and kidneys and formation of necroses) and thus exerts a
therapeutic effect on the disease. ~hereas organisms are
detectable in the urine of a high percentage of the con-
trol animals, there is a significant reduction in the
organism count in the treated animals.
In addit;on, the treatment reduces the colonization of the
kidneys by the organism from 63X to 27X with, at the same
t;me, a reduction ;n the formation of necroses from 87X
to 10X.
TabLe 6
Therapeutic treatment of a chronic Candida albicans
infection ~1 x 105 CFU)
Substance Animals with Kidneys with Necrotic
60 mg/kg positive findings positive kidneys
i.P. organism
findings
Day 3-10 Day 8 Day 14 Day 21 Day 30 Day 30
PBS 4115 8/15 10/15 19/30 26/30
(27%) (53%) (67%) (63%) ~87X)
Test 1/15 1/15 4/15 8/30 3/30
substance ( 7X) ( 7X) (27%) (27X) (10%)
129Z990
- 18 -
Exper;ment 4
Stimulation of the DTH reaction by the compounds according
-
to the invention
NMRI mice were treated with the substances accord;ng to
the invention as described in experiment 1.
The DTH reaction was checked as a test to determine the
immunostimulation.
Table 7 shows the relative activity of the test substance,
related to the compound from Example 8 whose maximal acti-
vation corresponds to 100X (difference between controland stimulation). It can be seen from the table that the
DTH reaction in the animals pretreated with the test sub-
stances is more pronounced than in the corresponding
control animals.
Table 7
Compound from Dose Admin;stration DTH reaction
Example No. (mg/kg) ~SRBC)
2 20 1 x i.p. day 0 88Z
200 " 61%
6 100 " 100%
8 100 " 100%
" 112X
12 100 " 96%
13 200 " 147X
25 14 100 " 118%
Experiment 5
Stimulation of macrophage activity by the compounds
according to the invention
NMRI mice were treated with the compounds according to the
invention as descr;bed in experiment 2.
1292990
.
- 19 -
The macrophage function (chem;lum;nescence and enzyme
act;v;ty) was checked as a test to determ;ne the ;mmuno-
stimulation.
_able 8 shows the relat;ve act;vity of the test substances,
related to the compound from Example 8 whose maximal
act;vation (d;fference between control and stimuLation)
corresponds to 100%. It can be seen from the table that
in comparison with macrophages from untreated an;mals, the
chem;lum;nescence reaction of these cells was also greatly
stimulated by all the test substances, and the;r content
of lysosomal enzymes was markedly raised.
Table 8
Compound fromMacrophage activity
Example No.Chemiluminescence Exocytos;s
15 (100 mg/kg i.p.)
1 30X 48X
Z 72% 53X
37Z 45%
6 26X 33X
8 100% 100X
66Z 21X
12 39X 54X
13 81X 77%
14 97X 93Z
Exper;ment 6
St;mulation of the de~ense against infection by Candida
alb;cans by prophylact;c administration of the compounds
accord;ng to the invention
Balb/c m;ce were treated w;th the compounds accord;ng to
the ;nvention as descr;bed in experiment 3a.
~ As Table 9 sho~s, the mean survival time of the mice atter
129Z99o
- 20 -
C.albicans infection increases signif;cantlY compared with
an untreated control group.
Table 9
-
Example Dose Relative prolongation of the
5 No. (mg/kg) mean survival times *
1 x i.p. day after infection
. _ . .
1 1 144
2 1 123
142
6 10 145
8 1 130
135
12 10 205
14 10 107
*Mean surv;val time of the control group = 100
Experiment 7
Effect on the stimulation of defense against the B16
melanoma tumor
Primary tumor growth was induced w;th 2 x 105 live B16
melanoma cells in female C57B1/6 mice (10 animals/group)
weighing 18 - Z0 9. After development of a particular
tumor size (0.65 cm in diameter), the primary tumor was
removed. The untreated animals then died of metastases in
the lung. After the tumor induction, the animals were
treated intraperitoneally on days 3, 5, 7, 9, 11 and 13
after amputation of the primary tumor with 50, 100 and
200 mg/kg of the test substance obtained as ;n Example 8.
The relevant mean survival times can be calculated from
the death rate after amputation of the primary tumor owing
to the development of pulmonary metastases. According to
this, 50% of the animals in the control group die after 26
days. The groups treated with the test substance showed
129Z990
- 21 -
(cf. Table 10) a significant increase in the mean survival
time to 43, 40 and 35 days with the appropriate dosages.
Table 10
Therapeutic treatment of 816 melanoma tumor
. . _ .
Administration 6 x i.p. (mg/kg) Mean survival time
day 3 5, 7, 9 11, 13 (days)
,
PBS 26
43
Test substance 100 40
10200 35
Experiment 8
Effect on formation of bone marrow colonies
-
This examined the effect of the test substance obtained as
;n Example 8 on the st;mulat;on of bone marrow colonies
in 6 - 8 week-old B2D2F1 mice. Female mice received the
test substance ;ntraperitoneally in the doses 2.5 and 5
mg/kg. The animals were sacrificed one day later, and the
bone marrow cells were isolated and cultured by generally
known methods (Metcalf, Immunology 21, 427, 1971 and
Stanley et aL., J. Exp. Med. 143, 631, 1979). Ac is cus-
tomary, L-cell supernatant (15~) was used as CSF source
(colony stimulating factor) for the development of the
bone marrow colonies. The colonies were counted 8 days
after the start of culturing. As can be seen in Table 11,
a single dose of 2.5 or 5 mg of the test substance results
in a marked increase in the formation of bone marrow ceLl
colonies both with and without addition of CSF ~colony
stimulating factor) in vitro.
:~ZS~Z990
- 22 -
Table 11
In vivo-effect on the formation of bone marrow colonies
.
Test substance Number of bone marro~ colonies (day 8)
1 x i.p. (mg/kg) with CSF (15Z) without CSF (15%)
S PBS 41 + 6 0
Test 2.5 94 + 11 24 1 2
substance 5.0 163 ~ 8 44 I S
The examples which follow illustrate the invention but
do not restrict it.
Example 1
9is(3-hydroxy-1-phenyl-1,2,4-triazol-5-yl) disulfide
2.9 9 (15 mmol) of 3-hydroxy-1-phenyl-5-mercapto-1,2,4-
triazole are initially introduced into
100 ml of methanol and, at room temperature,
15 3.2 ml ~33 mmol) of 35% strength hydrogen peroxide in
methanol are added dropwise. The solution initi-
ally turns yellow and, after 20 min, the new com-
pound slowly prec;pitates out. After 4 hours, the
precip;tate is filtered off with suction, washed
- 20 with methanol and dried.
Yield 2.1 9 = 72% of theory.
Melting point: 232C decomposition
NMR (d6-DMSO) ~ = 11.58 ppm, s, ZH, OH
7.38 ppm, m, 10H, aromat. H
129Z99O
- 23 -
Example 2:
Bis~5-carboxybenzimidazol-2-yl) disulfide
.
Preparation in analogy to Example 1 from 2-mercaptobenz;mi-
dazole-5-carboxylic acid
Yield 70% of theory
Melting point 235 - 8C
NMR (d6-DMS0) ~ = 9.4 ppm, d, NH
7.4 - 8.4 ppm, m, aromat. H
Example 3:
Bis(3-carboxy-2-pyridyl) disulfide
Preparat;on in analogy to Example 1 from 2-mercaptopyri-
dine-3-carboxylic ac;d
Yield: 56Z of theory
Melting point: 206C
NMR (d6-DMS0) ~ = 8.55 ppm, q, ZH
8.22 ppm, q, 2H
7.53 ppm, q, 2H
Example 4:
Bis(5-aminocarbonylmethyl-4-methyl-1,3-thiazol-2-yl)
disulfide
Preparation in analogy to Example 1 from 5-aminocarbonyl-
methyl-2-mercapto-4-methyl-1,3-thiazole
Yield 92.1%
lZ92990
- 24 -
Melting po;nt 200 - 203C
NMR (d6-DMSO) 6 = 7.33, br d, 4H NH2
3.62 S, 4H, CH2
2.26 s, 6H, CH3
S Example_5:
Bis[4-(2-chloroallyl)-6-hydroxy-5-oxo-4,5-dihydro-1,2,4-
triazin-3-yl] disulfide
2.2 9 of 4-(2-chloroallyl)-6-hydroxy-3-mercapto-5-oxo-
4,5-dihydro-1,2,4-triazine were dissolved ;n
25 ml of methanol and, at room temperature,
0.86 ml of 35X H202 was added dropwise, and the mixture
was then stirred for 1 hour during which the pro-
duct crystallized out. It was filtered off with
suction, washed with methanol and dried in air.
Yield 1.2 9
Melting po;nt: 204 - 5C (decomposition)
NMR (d6-DMSO) 6 = 12.7 ppm ~br. s., OH)
5.5 ppm (dd, = CH2)
4.8 ppm (s, CH2N)
Example 6:
~is(6-hydroxy-2-methyl-5-oxo-2,5-dihydro-1,2,4-triazin-
3-yl) disulfide
1.59 9 of 6-hydroxy-3-mercapto-2-methyl-5-oxo-2,5-dihydro-
1,2,4-triazine were dissolved in
30 ml of methanol and, at room temperature,
0.43 ml of 35X H22 was added dropwise, and the mixture
was then stirred for 30 minutes. The solution was
clarified with charcoal, evaporated to dryness in
, .
- 129Z990
- 25 -
a rotary evaporator, and the residue ~as tritura-
ted ~ith ice-~ater. The p;ecipitate was fiLtered
off, ~ashed with ice-water, dried in air, suspen-
ded in methanol, filtered off, washed uith a
little methanol, and dried ;n vacuo~
Yield 0.8 9
Melting point: 220C (decomposition)
NMR (d6-DMSO) ~ = 3.87 ppm (s, CH3)
Example 7:
Bis(6-hydroxy-4-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-3-
-
yl) disulfide
3.7 9 of 6-hydroxy-3-mercapto-4-methyl-5-oxo-4,5-d;hydro-
1,2,4-triazine ~ere dissolved in
400 ml of methanol and
38 ml of H20. At room temperature,
2.5 ml of 35X H22 were added dropwise. After stirr-
ing the mixture for 30 minutes, it was concentra-
ted to 100 ml, and the precipitate was filtered
off with suction and dried.
Yield 1.5 9
Melting point: 237C
NMR (CF3C02D): ~ = 3.67 ppm s, 2H, OH
3.8 ppm, s, 6H, CH3
Example 8:
Bis(5-carboxymethyl-4-methyl-2-thiazolyl) disulfide
23.62 9 of 5-carboxymethyl-4-methyl-2-mercaptothiazole
~ere dissolved in
129Z99O
- 26 -
250 ml of methanol, and the mixture was filtered off and,
~hile cooling in a water bath,
12.5 ml of 35Z H22 were added s(owly. The mixture was
then stirred in the ~ater bath for 30 minutes and
at room temperature for 2 hours. After filtration
and washing with methanol, 22.8 9 of the title
compound ~ere isolated.
Melting point: 162C
NMR (Cf3C02D): ~= 2.6 ppm, s, 6H, CH3
4.2 ppm, s, 4H, CH2
Example 9:
B;s(5-carboxymethyl-1,3-thiazol-2-yl) disulfide
0.88 9 of 2-mercapto-1,3-thiazol-5-ylacetic acid is dis-
solved in about 10 ml of methanol and, ~hile stir-
ring at room temperature,
0.5 ml of 33Z H22 solution is added. After 0.5 h, the
suspension of crystals ~as cooled, filtered, and
the residue from filtration ~as dried. Yield 0.6 9
of decomposition point 150C. Comparison by
thin-layer chromatography on Merck silica gel
plates shows reaction complete ~Rf: 0.3; mobile
phase: ethyl acetate: 65, ethanol: 25, ~ater: 10,
formic acid: 1)
Example 10:
Bis(4-carboxymethyl-1,3-thiazol-2-yl) disulfide
~hen
1.75 9 of 4-carboxymethyl-2-mercaptothiazole is reacted
in analogy to Example 8, then 0.95 9 of the title
compound is obtained.
129Z~90
, - 27 -
Melting point: 163 - 5C
NMR (DMS0-d6): ~ = 3.7 ppm, s, 4H, CH2
7.6 ppm, s, 2H, CH
9.4 ppm, s, 2H, C02H
Example 11
Bis(2-carboxymethylthio-1,3,4-thiadiazol-5-yl) disulfide
1.4 9 of 2-mercapto-1,3,4-thiadiazol-5-ylthioacetic acid
are dissolved in 10 ml of methanol and, while
cooling in ice,0 0.65 ml of 35X H22 is added dropwise. The mixture is
then stirred at room temperature for 1 hour and
filtered. The crystals are washed with a little
methanol and are dried in vacuo.
Yield: 1.3 g
Melt;ng point: 179t (decomposition)
NMR (DMS0-d6): 6 = 13 ppm, broad, C02
4.2 ppm, s, CH2
Example 12:
Bis(5-carboxy-1,3-thiazol-2-yl) disulfide
21 9 of 2-mercaptothiazole-5-carboxylic acid are dis-
solved in
100 ml of methanol by warming gently. ~hile cooling in
ice,
10.7 ml of 35% H22 are added slowly, and the mixture
is then stirred at room temperature for 45 minutes.
The solid is filtered off with suction, washed
with methanol and dried in vacuo.
- lZ9Z99O
- 28 -
Yield 21.5 9
Melting point: 280C (decomposition)
NMR ~DMS0-d6): ~ = 8.48 ppm, s, thiazole-H
Example 13:
Bis(5-carboxy-4-methyl-2-thiazolyl) d;sulf;de
1.75 9 t10 mmol) of 4-methyl-2-mercaptothiazole-5-
carboxylic ac;d are suspended ;n
50 ml of methanol and, while cooling in ice,
0.86 ml of 35Z H22 is added. The solut;on is then
stirred at 0C for 1/2 hour and at room tempera-
ture for 1 hour. The crystals which have formed
are filtered off ~ith suction, ~ashed ~ith a
little methanol and dried in vacuo.
Y;eld 1.52 g, melting point: 240 - 1t (Jecomposition)
NMR ~DMS0-d6): ~ z 2.6 ppm, s, CH3
Example 14:
Bis(4-carboxymethy~-5-methyl-2-thiazolyl) disulf;de
St ge 1
Methyl 4-bromopropionylacetate
22 9 of methyl propionylacetate are dissolved in
60 9 of CH2Cl2, and
8.7 ml of Br2 in
30 ml of CH2Cl2 are added drop~ise. The mi~ture is
then stirred at room temperature for 1 1/4 h, a
further
0.66 ml of Br2 in
lZ9Z99O
- 29 -
5 ml of CH2Cl2 is added, and the mixture is stirred
for a further 1 1/4 h.
It is washed ~ith
3 x 50 ml of H20 each time,
1 x 20 ml of saturated NaHC03 and
2 x 50 ml of H20 each time, dried over Na2S04 ~nd
evaporated. 38.6 9 of oil are obtained.
Stage 2
Methyl 2-mercapto-5-methyl-4-thiazolylacetate
30.1 9 of the oil from stage 1, dissolved in
80 ml of ethanol, are added drop~ise to a solution of
24 9 of freshly prepared ammonium dithiocarbamate in
200 ml of ethanol and
200 ml of H20 at room temperature. The mixture is then
stirred for 2 1/2 h and
7.5 ml of trifluoroacetic acid are added. After hav;ng
been stirred for 1 hour, the mixture is evaporated
to dryness and
200 ml of CHCl3/H20 t1:1) are added. After two further
washings with Hz0, the solution is dried over
Na2S04 and evaporated. ~he solid residue is
suspended in ether and filtered off.
Yield 8.9 9
Melting point: 149C (decomposition)
NMR td6-DMSO); 6= 11 ppm, broad, SH
3.7 ppm, s, OCH3
3.5 ppm, s, CH2
2.1 ppm, s, CH3
1292990
- 30 -
Stage 3
2-Mercapto-5-methyl-4-th;azolylacetic acid
1~98 9 of stage 2 were d;ssolved, w;th warming in
2U ml of methanol. ~he solution was then stirred at
room temperature for 40 min,
50 ml of H20 were added, and the mixture was acidified
to pH 1.0 with concentrated HCl. After stirring
for 1 h while cooling in ice, the solid was fil-
tered off with suction and washed with ice-water
until-free of chloride. Drying in vacuo over
P2s produced 1.67 9
NMR (d6-DMS0): 6 = 13.9 ppm, broad, C02H
3.5 ppm, s, CH2
2.08 ppm, s, CH3
Stage 4
0;s~4-carboxymethyl-5-methyl-2-thiazolyl) d;sulf;de
430 mg of stàge 3 were suspended in
20 ml of methanol and d;ssolved by heating. At room
temperature0 0.25 ml of 35% H22 was added dropwise, and the m;xture
was then st;rred for 1 h. After hav;ng been con-
centrated to about 5 ml, the m;xture was filtered,
and the solid was washed with a little ice-cold
methanol.
Yield: 280 mg
NMR td6-DMS0): 6 = 3.65 ppm, s, CH2
2.37 ppm, s, CH3
1~:929~0
- 31 -
ExampLe 15:
_is(5-carboxymethyl-1,3-thiazol-2-yl) d;sulfide
0~8 9 of 2-mercapto-5-carboxymethyl-1,3-thiazole were
dissolved ;n 10 ml of methanol and, ~hile stirring,
S 0.5 ml of 33% strength H20z solution was added
dropwise. The mixture was allowed to stir over-
night, and the product which had crystallized out
was filtered off.
Yield 0.6 9 of melting point 150C (decomposition),
homogeneous by thin-layer chromatography (Rf =
0.3; silica gel; mobile phase: ethyl acetate/
ethanol/water/formic acid (60:25:15:1)
IR (KBr disk): ~ - 1710 cm (COOH).
1H-NMR (d6-DMSO): ~ ~ppm): 3.8 ~5, CH2-th;azole, 2H)
7.6 (s, thiazole-4H, 1H).
Example 16:
Bis(5-methoxycarbonylmethyl-1,3-thiazol-2-yl) disulfide
0.5 g of 2-mercapto-5-methoxycarbonylmethyl-1,3-thiazole
was dissolved in 5 ml of methanol and, while
stirring, 0.3 ml of 33X strength H22 solution
was added dropwise. The mixture was allowed to
stir overnight, and the product which had crystal-
lized out was filtered off. 0.3 g of meLting
point 72C, homogeneous by thin-layer chromato-
graphy on silica gel using ethyl acetate as the
mobile phase (Rf = 0.8).
IR ~KBr disk): ~ = 1735 cm 1 (COOCH3).
1H-NMR (d6-DMSO): ~ (ppm): 3.6 (s, CH2-thiazole, 2H),
9~99o
-
- 32 -
3.7 (s, COOCH3, 3H),
7.0 ts, thiazoLe-4H, 1H).
Example 17:
Bis(4-carboxy-1,3-thiazol-5-yl) disuLfide
5 9 of bis(4-methoxycarbonyl-1,3-thiazol-5-yl) disul-
fide ~ere suspended in 100 ml of methanol. To
this was added a solution of 1.5 9 of NaOH in
20 ml of H20, and the mixture ~as heated under
reflux for 1 h. After the solution had been
cooled, methanol uas removed in a rotary evapora-
tor, the aqueous phase ~as extracted once with a
little ethyl acetate and acidified ~ith 2 NHCl
solution. The product ~as filtered off, dried and
recrystallized from ethyl acetate. 2 9 of melting
point 154C were obtained.
IR ~KBr disk): ~ = 1680 c~ 1 (COOH)
H-HMR ~d6-DMSO): 6 (ppm), 8.6 ~s, thiazole-2H).
Examples 18 to 22 ~ere synthesized as described in
Example 1.
Example 18:
Bis(5-acetylamino-1,3-thiazol-2-yl) disulfide
1H-NMR (d6 - DMSO):
= 2.16 ppm (s, 6H, -CH3), 7.56 ppm (s, 2H, thiazole-H),
11.6 ppm ~br s, 2H, -NH-)
Example 19:
Bis(5-acetylamino-1,3,4-thiadiazol-2-yl) disulfide
1H-NMR (d6 - DMSO):
~ = 2.20 ppr (s, 6H, -CH3), 12.84 ppm (br s, 2H, -NH-)
lZ9Z99~
- 33 -
Example 20:
_is(5-glutaromonoamido-1,3-thiazol-2-yl) disulfide
1H-NMR (d6 - DMS0):
~i = 1.6 - 2.6 ppm (m, 12H, six CH2 groups), 7.50 ppm
(s, 2H, thiazole-H), 12.63 ppm (br s, 2H, -NH-), 13.05 ppm
(br s, 2H, -C02H)
Example 21:
Bis(5-succinomonoamido-1,3-thiazol-2-yl) disulfid_
1H-NMR (d6 - DMS0):
6 = 2.4 - 2.7 ppm (m, 8H, -CH2), 7.50 ppm (s, 2H,
thiazole-H), 12.66 ppm (br s, 2H, -NH-), 13.15 ppm (br s,
2H, -C02H)
ExampLe 22:
~ist5-(2-carboxyethyl)-4-methyl-1,3-thiazol-2-yl~
_isulfide
1H-NMR ~d6 ~ DMS0):
6 = 2.27 ppm ~s, 6H, -CH3), 2.55 ppm ~d, 4H, thiazole-
CH2), 2.96 ppm (t, 4H, CH2-C02-)
Example 23:
; Bis~(5-carboxymethyl-6-hydroxy-4-methyl)-2-pyrimidinyl~
disulfide
4 9 of 5-carboxymethyl-6-hydroxy-2-mercapto-4-methylpyri-
midine were dissolved in 200 ml of 1 molar sodium bi-
carbonate solution and, while cooling in ice, 10 ml of 35%
H22 were added dropwise. The mixture was stirred at
room temperature for 4 h and, while cooling in ice, was
acidified to pH 2.0 with 1 N HCl. The precipitate was
filtered off with suction and provided, after drying,
1.5 9 of the title compound.
Melting point 331C (decomposition), IR 1630, 1700 cm 1
~ = 2.5 ppm (s, 6H, -CH3), 3.26 ppm, (s, 4H, -CH2)
