The invention relates to novel 1,2-disubstituted-4-nitroimidazole compounds that are useful as potent antiprotozoal agents and simultaneously possess non-mutagenicity as compared to known 4- or 5-nitroimidazole compounds. Also, compositions and methods for antiprotozoal uses of said novel 1,2-disubstituted-4-nitroimidazole compounds are disclosed herein.

BACKGROUND OF THE INVENTION 
The invention relates to novel 1,2-disubstituted-4-nitroimidazole compounds 
being useful as potent antiprotozoal agents which possess non-mutagenic 
activity. The novel compounds disclosed herein are useful in formulating 
compositions comprising an effective amount of said novel compounds and 
methods of administering said compositions. 
Nitroimidazoles are a class of known compounds and have the reputation as 
mutagens. Metronidazole (flagyl), a 5-nitro-imidazole is mutagenic and 
consequently raises concern over safety when administered to patients. 
Also, the compound, 1-methyl-2-methylsulfonyl-4-nitroimidazole is a known 
compound and is disclosed in the Indian Journal of Chemistry, Section B, 
21B (11), pp. 1006-21. The utility of this compound was disclosed as being 
active as radio and chemosensitizers and no mention was made of any 
biological activities or relative mutagenicity. The novel compounds 
disclosed in the invention herein are not mutagenic in Ames strain TA100, 
and are highly potent against protozoal diseases and, therefore, offer a 
greater safety advantage to patients. Said novel compounds are of 
particular use against Trichomonas spp. such as T. foetus and T. 
vaginalis. 
SUMMARY OF THE INVENTION 
The invention relates to novel non-mutagenic 
1,2-disubstituted-4-nitroimidazole compounds that are useful as potent 
antiprotozoal agents. 
Accordingly, it is an object of the invention to provide a class of novel 
1,2-disubstituted-4-nitroimidazole compounds. 
A further object of the invention is to describe processes for the 
preparation of said novel nitroimidazole compounds. 
Another object of the invention is to provide pharmaceutical compositions 
for administering an effective amount of said novel compounds to patients 
(warm blooded animals). 
These and other objects and advantages of the invention will become 
apparent from the following description. 
DESCRIPTION OF THE INVENTION 
The invention relates to novel non-mutagenic 
1,2-disubstituted-4-nitroimidazole compounds that are potent antiprotozoal 
agents. Said novel 4-nitroimidazole compounds of the invention have the 
following general structure: 
##STR1## 
wherein: R.sub.1 is alkyl(C.sub.1 -C.sub.6) such as methyl, ethyl, 
isopropyl, pentyl and the like; or arylalkyl(C.sub.7 -C.sub.9), preferably 
phenylalkyl(C.sub.1 -C.sub.3) such as benzyl, phenylethyl and the like; 
R.sub.2 is alkyl(C.sub.1 -C.sub.12) such as methyl, ethyl, isopropyl, 
heptyl, decyl and the like; or phenyl; and 
n is 0, 1 or 2; 
with the proviso that when n is 2, R.sub.1 or R.sub.2 is other than methyl. 
The preferred compounds of the invention are: 
1-methyl-2-ethylsulfonyl-4-nitroimidazole; 
1-methyl-2-propylsulfonyl-4-nitroimidazole; 
1-methyl-2-isopropylsulfonyl-4-nitroimidazole; 
1-methyl-2-phenylsulfonyl-4-nitroimidazole; 
1-methyl-2-methylsulfinyl-4-nitroimidazole; 
1-methyl-2-methylthio-4-nitroimidazole; 
1-methyl-2-ethylthio-4-nitroimidazole; 
1-methyl-2-propylthio-4-nitroimidazole; 
1-methyl-2-isopropylthio-4-nitroimidazole; 
1-methyl-2-ethylsulfinyl-4-nitromidazole. 
The novel compounds disclosed herein are prepared by processes known in the 
art. Generally, said compounds are prepared by treating 1-alkyl or 
arylalkyl-2-mercaptoimidazole with a base in the presence of an alcohol 
solvent and an alkylhalide at room temperature and then with nitric acid 
and water. The reaction is heated to 100.degree. C. for about 1.0 to 1.5 
hours to obtain the corresponding 1-alkyl or 
arylalkyl-2-alkylthio-5-nitroimidazole. This product is then dissolved in 
an acid (weak), treated with hydrogen peroxide and heated to 100.degree. 
C. for approximately an hour. The resulting 1-alkyl or 
1-arylalkyl-2-alkylsulfonyl-5-nitroimidazole is dissolved in an inert 
solvent and potassium iodide added therein and heated gently to reflux to 
obtain the corresponding 1-alkyl or 
arylalkyl-2-alkylsulfonyl-4-nitroimidazole. 
Treating 1-alkyl or arylalkyl-2-alkyl or arylalkylthio-4-nitroimidazole 
with an organic peracid (m-chloroperbenzoic acid) in the presence of an 
inert solvent at room temperature for approximately 1.5 to 3.0 hours gives 
the corresponding 1-alkyl or arylalkyl-2-arylalkylsulfinyl or 
alkylsulfinyl-4-nitroimidazole. 
Refluxing 1-alkyl-2-methylsulfonyl-4-nitroimidazole with alkyl or 
arylalkylmercaptans in strongly basic solvent for approximately 2 hours 
gives the corresponding 1-alkyl or arylalkyl-2-alkylthio or 
arylalkylthio-4-nitroimidazole. 
Nitroimidazoles are known generally to be mutagenic compounds and are 
usable only in those instances where the disease being treated is of such 
a level of seriousness that the negative effects of the mutagenicity of 
the compound are balanced against the conditions resulting from the 
disease. Thus, the discovery of a non-mutagenic drug which could be used 
to treat protozoal diseases has been long sought. 
One very well accepted measure of the mutagenicity of chemicals, which has 
generally also been closely correlated with the carcinogenicity of such 
compounds, is the Ames Mutagenicity Test. This test involves the addition 
to a fermentation medium in which is growing a particular organism 
identified as Ames Salmonella TA100, and measuring the number of mutant 
organisms formed. Greater numbers of mutants over the background number of 
spontaneous mutants is an indication of greater mutagenicity of the 
compound. Generally a series of varying concentrations of the test 
compound is employed to determine threshold levels if possible. 
In one such Ames mutagenicity test 1-methyl-2-ethylthio-4-nitroimidazole 
was compared to two commercially available nitroimidazoles, 
ronidazole(1-methyl-2-[(carbamoyloxy)methyl]-5-nitroimidazole) and 
metronidazole(1-(2-hydroxyethyl-2-methyl-5-nitroimidazole). At 3 .mu.g per 
plate ronidazole had 358 mutants per plate while metronidazole and the 
instant compound were indistinguishable from background. At 30 .mu.g per 
plate, ronidazole had 2682 mutants per plate and metronidazole had 142 
mutants per plate while the instant compound was still indistinguishable 
from background. At 100 and 300 .mu.g per plate metronidazole had 443 and 
1374 mutants per plate respectively, while the instant compound was still 
at barely a threshold level of 30 and 65 mutants per plate respectively. 
The instant compound continued to show no significant levels at 30, 100, 
300 and 1000 .mu.g per plate by recording 0, 54 and 0 mutants per plate 
respectively. Such levels of mutagenicity are not statistically 
significantly different from background and as such, the instant compound 
would be considered non-mutagenic. 
Thus, considering the rapidly increasing mutagenic activity of ronidazole 
and metronidazole and the continuing statistically insignificant levels of 
mutagenic activity with the instant compound, it is apparent that the 
instant compound represents a considerable breakthrough in treating 
protozoal diseases with a new level of safety, unachieved and unachievable 
with prior therapies. 
The instant compounds have antiprotozoal activity, and is particularly 
active against the causative organisms of the protozoal parasitic diseases 
trichomoniasis and enterohepatitis. It is also effective against 
amoebiasis and trypanosomiasis, as well as against the PPLO organisms and 
schistosomes. 
Trichomoniasis is a protozoan disease caused by parasites of the genus 
Trichomonas. The compound of the invention is effective against the 
particularly troublesome form of trichomoniasis known as T. vaginalis 
vaginitis, caused by infestation of the vagina with T. vaginalis. In 
treating this disease, the compound may be administered either orally or 
topically. For oral administration unit dosage, forms such as tablets or 
capsules are normally employed which may contain from about 25 to about 
1000 mg of active ingredient. These are prepared by techniques known in 
the art, and contain the usual diluents, granulating agents, extenders 
and/or lubricating agents known to be satisfactory for the compounding of 
tablets and capsules. It is preferred to administer the compound of the 
invention orally at a dose level of from about 50-500 mg/day, in either 
single or divided doses with divided doses being used more frequently than 
a single dose. An example of a suitable compressed tablet contains the 
ingredients below: 
______________________________________ 
Mg per tablet 
______________________________________ 
1-methyl-2-ethylthio-4-nitro- 
250 
imidazole 
Dicalcium phosphate 
100 
Lactose 75 
Starch 50 
Guar gum 12 
Magnesium stearate 2-3 
______________________________________ 
If desired, tablets may be sugar coated or enteric coated by standard 
techniques. Alternatively, the antitrichomonal agent may be formulated in 
capsule form using fillers such as lactose, starch or kaolin. A typical 
capsule would contain 250 mg of, for instance, 
1-methyl-2-ethylthio-4-nitroimidazole, 2-3 mg of magnesium stearate and 
about 75 mg of lactose in a No. 0 size capsule. Tablets and capsules 
containing smaller quantities of active ingredient may be made by reducing 
proportionately the amounts of excipients and diluents illustrated above. 
Alternatively, the compound may be administered orally in liquid 
pharmaceutical vehicles such as solutions, emulsions, syrups or 
suspensions containing the diluents, flavoring agents and preservatives 
customarily employed in the pharmaceutical art. 
For topical application such as creams or ointments and suppositories these 
formulations may be prepared via conventional methods containing the 
active ingredient. To illustrate, a cream or ointment or suppository is 
prepared by mixing sufficient quantities of hydrophilic material and 
water, containing from about 5-10% by weight of the compound, in 
sufficient quantities to produce a cream or ointment or suppository having 
the desired consistency. 
Enterohepatitis is a disease occurring primarily in turkeys and is caused 
by the protozoan parasite Histomonas meleagridis. It is also known as 
turkey blackhead disease. The compound of this invention is useful in the 
prevention and treatment of this disease and for this purpose is 
administered to turkeys mixed with an element of turkey sustenance, i.e. 
in the feed or drinking water. Although the optimum dose level will depend 
on the severity of the infection, good control of enterohepatitis is 
obtained by orally administering to the turkeys a feed containing from 
about 0.003% to about 0.1% by weight of the instant compound. When the 
material is administered via the drinking water, somewhat higher levels 
may be employed, especially for therapeutic use. For instance, the 
drinking water may contain up to about 0.2% by weight of the active 
ingredient with good results. 
As previously stated, the compound described herein may also be employed 
against trypanosomiasis, amoebiasis and the pleuro-pneumonia like 
organisms which have come to be known as PPLO. 
The compound is effective against PPLO when the compound is administered to 
fowl or swine in feed containing from about 0.003% to about 1.0% by weight 
of carbamate. The preferred dosage level, however, is between from about 
0.003% to 0.08% by weight. 
It will be understood, however, that the specific dose level for any 
particular patient will depend upon a variety of factors including the 
activity of the specific compound employed, the age, body weight, general 
health, sex, diet, time of administration, route of administration, rate 
of excretion, drug combination and the severity of the particular disease 
undergoing therapy. 
Some studies which further exemplify the invention are listed below: 
Mice were infected through intraperitoneal (I.P.) injection of 
1.4.times.10.sup.7 T. foetus. Each compound disclosed in Table I below was 
dissolved in an aqueous vehicle, dosed to groups of mice (consisting of 
five) by gavage. The mice were dosed once per day for four consecutive 
days with doses ranging from 1-50 mg/kg. The effective dose of the 
compound, ED.sub.50, was calculated from the number of animals surviving a 
two week period. 
TABLE I 
______________________________________ 
Trichomonal Activity of 
1-(R.sub.1)-2-(R.sub.2)-4-Nitroimidazoles and Flagyl 
ED.sub.50 (T. foetus) 
Com- in vitro 
in vivo 
pound R.sub.1 R.sub.2 (.mu.g/ml) 
(mg/kg) 
______________________________________ 
1. --CH.sub.2 CH.sub.3 
--SO.sub.2 CH.sub.3 
25 10-50 
2. --CH.sub.3 
--SO.sub.2 CH.sub.2 CH.sub.3 
18.5 2-5 
3. --CH.sub.3 
--SO.sub.2 CH.sub.2 CH.sub.2 CH.sub.3 
27 5-10 
4. --CH.sub.3 
--SO.sub.2 --iPr 
21 5-10 
5. --CH.sub.3 
--SO.sub.2 Ph 17 10-25 
6. --CH.sub.3 
--SOCH.sub.3 14 2-5 
7. --CH.sub.3 
--SCH.sub.3 2.1 5-10 
8. --CH.sub.3 
--SCH.sub.2 CH.sub.3 
2.3 2 
9. --CH.sub.3 
--SCH.sub.2 CH.sub.2 CH.sub.3 
4.0 5 
10. --CH.sub.3 
--S--iPr 4.0 10-50 
11. FLAGYL 0.4 25-50 
______________________________________ 
As indicated in Table I above, the novel compounds of the invention as 
compared to Flagyl (a commonly used nitroimidazole) are from 1.5 to 25 
times more active. 
TABLE II 
______________________________________ 
Mutagenicity and Trichomonal Activities of 
4-Nitroimidazoles Relative to 5-Nitroimidazoles 
T. foetus In Vivo* 
Mutagenicity 
(ED.sub.50) 
Safety 
(percent In vivo Index (rel 
Compound Ronidazole) 
(mg/kg) to flagyl) 
______________________________________ 
1-methyl-2-ethylthio- 
&lt;0.1** 2 &gt;1750 
4-nitroimidazole 
1-methyl-2-methanol 
100 2-5 0.7 
carbamate (ester)- 
5-nitroimidazole 
(Ronidazole) 
1-(2-hydroxyethyl)-2- 
7 50 1.0 
methyl-5-nitro- 
imidazole (Flagyl) 
______________________________________ 
*In vivo Safety Index is the relative increase in safety (1/mutagenicity) 
observed at the ED.sub.50 dose. All values are expressed relative to 
flagyl. The larger the number the greater the relative safety compared to 
flagyl. Therefore, the representative compounds of this invention are 
greater than 1750 times safer than flagyl at the therapeutic dose. 
**Mutagenicity was undetectable. (Less than 0.1% of the mutagenicity of 
ronidazole). 
TABLE III 
______________________________________ 
Mutagenic Activity of 4-Nitroimidazoles in Ames 
TA100 Strain 
Mutagenicity* 
T. foetus 
(percent (ED.sub.50) In 
R1 R2 ronidazole) vivo (mg/kg) 
______________________________________ 
--CH.sub.3 
--SO.sub.2 CH.sub.2 CH.sub.3 
0.1 2-5 
--CH.sub.3 
--SO.sub.2 CH.sub.2 CH.sub.2 CH.sub.3 
0.1 5-10 
--CH.sub.3 
--SO.sub.2 --i-Pr 
0.1 5-10 
--CH.sub.3 
--SO.sub.2 Ph 0.1 10-25 
Flagyl 7.0 25-50 
Ronidazole 100.0 5-10 
______________________________________ 
*Mutagenicity values of all novel compounds disclosed herein are 
undetectable (&lt;0.1% relative to the mutagenicity of ronidazole). 
TABLE IV 
______________________________________ 
Single Strand DNA Breaks and 
Cytotoxicity In Rat Hepatocytes 
Relative* Relative** 
Treatment Concentration 
Elution Slope 
Viability (%) 
______________________________________ 
Control -- 1.00 100 
Flagyl 3.0 mM 5.33 107 
10.0 mM 7.1 101 
1-methyl-2- 
0.5 mM 1.33 107 
ethylthio-4- 
1.5 mM 1.23 105 
nitroimidazole 
5.0 mM 1.60 102 
______________________________________ 
*Measure of the extent of DNA strand cleavage. Only relative elution 
slopes .gtoreq.3.0 are considered significant. Therefore, 
1methyl-2-ethylthio-4-nitroimidazole did not cause significant DNA strand 
breakage up to 5.0 mM while Flagyl caused breaks at both 3.0 and 10.0 mM. 
**Relative viability is the number of hepatocytes surviving exposure to 
the nitroimidazoles relative to the control cells.

The following examples illustrate preparation of various novel 
1,2-disubstituted-4-nitroimidazole compounds of the invention. Said 
examples should be construed as illustrations rather than limitations 
thereof. 
EXAMPLE 1 
1-Methyl-2-ethylthio-4-nitroimidazole 
Ethyl mercaptan (5.14 g, 0.083 mole) was added to dimethylformamide (140 
ml) containing potassium hydroxide (4.6 g, 0.08 mole). This was stirred 
for 90 minutes until all the potassium hydroxide had dissolved. To this 
solution was added 1-methyl-2-methylsulfonyl-4-nitroimidazole (8.5 g, 0.04 
mole) and the reaction stirred for 2 hours at room temperature. The 
reaction mixture was then added to water (500 ml) and stirred in an ice 
bath for an additional hour. The crystalline product was filtered off and 
dried to give 5.6 g of crude product. This was recrystallized from 
ethanol/water to give 4.3 g of product. 
EXAMPLE 2 
1-Methyl-2-ethylsulfonyl-4-nitroimidazole 
1-Methyl-2-mercaptoimidazole (5 g, 0.044 mole) was dissolved in methanol 
(150 ml) and 50% sodium hydroxide solution was added (3.5 ml, 0.044 mole). 
Ethyl iodide (7.0 ml, 0.087 mole) was then added and the reaction stirred 
at room temperature for 90 minutes. The reaction mixture was concentrated 
to 50 ml on a rotovap and then diluted with water (130 ml). This was 
extracted into three volumes of ether which was dried over sodium sulfate 
and then evaporated to give a clear oil. The oil was treated with a 
solution of concentrated nitric acid and water (20 ml of 70% nitric acid 
and 8 ml of water) and heated at 100.degree. C. for 75 minutes. After 
cooling to room temperature, the reaction mixture was added to 200 ml of 
water and made basic with concentrated ammonium hydroxide solution. This 
was cooled in an ice bath where upon the 5-nitroimidazole product 
crystallized (2.3 g). The crystalline material (2.0 g, 0.011 mole) was 
dissolved in glacial acetic acid (15 ml) and treated with 30% hydrogen 
peroxide solution (3.1 ml, 0.027 mole). The reaction mixture was heated at 
100.degree. C. for 60 minutes. After cooling to room temperature, the 
reaction mixture was added to water (150 ml) and adjusted to pH 7 with 
concentrated ammonium hydroxide solution. This was then extracted with 
ether (3.times.100 ml), the ether layers combined, dried over sodium 
sulfate and evaporated to give 2.06 g of sulfone. The sulfone (1.9 g, 8.7 
mmole) was dissolved in dimethylformamide (20 ml) and potassium iodide 
(1.59 g, 9.6 mole) was added. The reaction mixture was heated gently at 
reflux for 60 minutes. After cooling to room temperature, the reaction 
mixture was added to water (125 ml) with stirring. The flask was cooled in 
ice and the crystalline product filtered off to give 1.46 g of 
4-nitroimidazole product. 
EXAMPLE 3 
1-Methyl-2-ethylsulfinyl-4-nitroimidazole 
1-Methyl-2-ethylthio-4-nitroimidazole (200 mg, 1.07 mmole) was stirred in 
methylene chloride (5 ml). A solution of m-chloroperbenzoic acid (250 mg, 
1.45 mmole) in methylene chloride (5 ml) was added dropwise over 40 
minutes. The reaction was stirred at room temperature for a further 2 
hours and then diluted with methyl chloride and washed successively with 
solutions of sodium sulfite, sodium carbonate, and water. The organic 
layer was dried over sodium sulfate and evaporated to give an oil. This 
was purified on a silica gel column eluted with 1% methanol in methylene 
chloride and 2% methanol in methylene chloride to give 100 mg of product.