CA1308726C - Imidazole derivatives having therapeutical activity, process for their preparation and pharmaceutical compositions containing them - Google Patents

Abstract

"Novel imidazole derivatives having therapeutical activity, process for their preparation and pharmaceutical compositions containing them"
ABSTRACT
The econazole and miconazole sulfosalicylates show in vitro a relevant anti-fungine activity with wide spectrum and, if locally applied, are well tolerated and more effective than the corresponding nitrates for the control, in vivo and clinically, of the mycosis as induced from dermatophytes and from Candida albicans.

Description

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1 The present invention relates t~ novel imidazole derivatives, more specifically to two novel salts with sulphosalicylic acid of econazole, namely 1-(2-((4-chlorophenyl)-methoxy)-2-~2,4-dichlorophenyl)-ethyl)-1-H-imidazole, and of miconazole, namely 1-t2-((2,4-dichlorophenyl)-2-(2,4-dichlorophenyl)methoxy)-ethyl)-lH-imidazole, having therapeutical actLvity, particularly anti-mycotic activity.
The novel derivatives of the present invention have following general formula:

~ C O OH
CH2--C ~ --O--CH2~ ~ OH

~ R

wherein R represents H, Cl.
The cutaneous mycosis are infections mainly induced from dermatophytes and from Candida Albicans. The dermatophytes are fungi found as parasites in the horny layer, in the hair and in the nails owing to the presence of keratolitic enzymes, capable of hydrolizing the long polypeptidic chains of keratin.
Three genuses (Trichophyton, Microsporum and Epidermophyton) are mainly responsible of the cutaneous pathologies. The infections induced from dermatophytes are generally defined as tinea. Depending on the part af-fected there are several clinical pictures : tinea capitis, tinea barbae, tinea corporis, tinea cruris, tinea pedis, tinea manuum, tinea faciei, tinea un~ _um.
The Candida albicans is an ubiquitary, non keratinophylic fungus normally being saprophyte of cutis and of mucosae, which becomes pathogenic when its spronting and reproduction are promoted by a particularly suitable environnment or by the weakening of the organic defences.

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l The clinical evidences induced from Candida albicans vary according to the part involved: intertriginis, vulvovaginities, oral candidiases ("thrush"), boccheruolo, paronichia.
Among the drugs useful for the topical treatment of surface mucosae the imidazole derivatives have acquired, in the last years, a relevant importance.
More particularly the active principles known as econazole (DCI) and miconazole (DCI), correspond to the above indicated chemical names, are anti-mycotic drugs with wide activity spectrum; they sre endowed with a powerfull activity against dermatophytes and Candida albicans, as well as against some gram-positive germs. Their action takes place by selective inhibition of the purine and glutamine fixing onto the membrane of myce-tes.
The miconazole and the econazole are used as nitrates in several pharmaceutical forms (cream, powder, ovuli, etc.).
The imidazole compounds according to the present invention are chemically defined in the following manner:
a) 1-(2-((4-chlorophenyl)-methoxy)-2-(2,4-dichlorophenyl)-ethyl)-1~-imi-dazole 5-sulfosalicylate (econazole SSA) corresponding to the formula C25H20C14N207S.
Its molecular weight is 599.89.
Its centesimal elemental analysis i8 the following:
C 50.05 (found 50.25) H 3.53 (found 3.47) N 4.67 (found 4.81) 0 18.67 (found 18.54 S 5.34 (found 5.4~) Cl 17.74 (found 17.44, by difference).
The substance is in form of a white fine powder, is soluble in methanol, ethanol and dimethylsulfoxide, poorly soluble in water, insoluble in the common organic solvents.
It crystallize from methyl or ethyl alcohol and melts at 176-178C (at 1 J ~ ~.7 ~

1 Kofler ).
The saturated aqueous solution of the salt has a pH of 3.15, as measured with a glass electrode.
b) 1-(2-(2,4-dichlorophenyl)~2-((2,4-dichlorophenyl)-methoxy)ethyl)-lH-i-midazole 5-sulfosalicylate (miconazole SSA) corresponding to the formula C25H20C14N207S .
Its molecular weight is 634.3.
Its centesimal elemental analysis is the following:
C 47.34 (found 47.49) H 3.18 (found 3.32) N 4.42 (found 4.59) 0 17.66 (found 17.51) S 5.05 (found 4.92j Cl 22.35 (found 22.17, by difference) The substance is in form of a white fine powder, is soluble in methanol, ethanol and DMS0, poorly soluble in water, insoluble in the common organic solvents. It crystallizes from ethyl or methyl alcohol and melts at 181-183C. (Kofler). The saturated aqueous solution of the salt has a pH of 3.20 as measured with a gla~s electrode.
The compounds according to the present invention are produced by reacting in ethanol solution under boiling miconazole and respectively econazole with 5-sulphosalicylic acid in equimolar pro portions; the reaction mix-ture is concentrated up to dryness, and thereafter the residue is taken with ethyl ether and crystallized from ethyl alcohol. The process ~hall be better understood from the following examples, having purely illustrative and non limiting purposes.

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1 Further objects and advantages of the invention will appear from the following description taken together with the accompanying drawings in which:
Figure 1 is an ultra violet spectrum confirming the structure of the product obtained by the process of Example l;
Figure 2 is an infrared spectrum confirming the structure of the product obtained by the process of Example l;
Figure 3 is an ultra violet spectrum confirming the structure of the product obtained by the process of Example 2;
Figure 4 is an infrared spectrum confirming the structure of the product obtained by the process of Example 2;
Figure 5 shows a differential thermal analysis confirming the structure of the product obtained by the process of Example 2; and Figure 6 shows a differential thermal analysis confirming the structure of the product obtained by the process of Example 2.

1.82 g of 5-sulfosalicylic acid (7.2 mmoles) are dissolved at room temperature in 40 ml of absolute ethyl alcohol.
Then 3 g of miconazole base (7.2 mmoles), are added, it being likewise known as 1-(2-((2,4-dichlorophenyl)-2-(2-(2,4-dichlorophenyl)methoxy)ethyl)-lH-imidazole, and the mixture is refluxed.

1 3 ~ i 7 ~ .i 1 After complete solubilization of the miconazole, the solution is maintained under refluxing for 10 minutes, then it is charged in a rota-ting evaporator under vacuum, and it is concentrated up to dryness in the water bath of the apparatus. The residue is taken with ethyl ether, the mixture being cooled from outside with a refrigerating mixture, filtered under vacuum and crystallized with ethyl alcohol. In this way with a quantitative yield, the final product is obtained, it having appearance of a white powder having melting point 181-183C (Kofler).
The UV and IR spectra confirm the foreseen structure (figures 1-2).

1.98 g of 5-sulfosalicylic acid (7.8 mmoles) are dissolved at room temperature in about 40 ml of abso]ute ethyl alcohol. Then 3 g of econa-zole base (7.8 mmoles), also known as 1-(2-((4-chlorophenyl)-methoxy-2-(2,4-dichlorophenyl)-ethyl)-lH-imidazole, are added and the mixture is refluxed. After complete solubilization of the econazole, the solution i8 maintained at reflùxing for 10 minute~; then it is placed into a rotating evaporator under vacuum and concentrated up to dryness in the water bath of the apparatus.
The residue is taken with ethyl ether by cooling it from outside with a refrigerating mixture, filtered under vacuum and crystallized from ethyl alcohol.
In this manner there is obtained, with quantitative yields, the final product having the appearance of a white fine powder and having melting point of 176-178C (Kofler).
The UV and IR spectra and the differential thermal analysis have con-firmed the foreseen structure (fig. 3-6).
The compounds of the present invention have been the subject of pharmacological investigation, both in vitro and in vivo, the results of which, for reporting clarity, shall be separately illustrated for each compound.
I- a In vitro antimycotic activity.
The antimycotic activity of miconazole SSA has been evaluated against -6- l J ~ ~, 7 ~

l Candida albicans 73/079 tYMA and SAB), Cryptococcus neoformans 451, Saccharomyces cerevisiae, Aspergillus niger, Trichophyton mentagrophyties 569~, Hendersonula toruloidea TH65 and Pacilomyces varioti.
The microorganisms have been incubated onto agar-glucose medium, (Sabourauds medium) at 30 or 37C, depending on the species, in the presence of miconazole SSA.
The incubation was of 24 hours for Candida and Cryptococcus, of 48-72 hours for the other mycetes. The miconazole SSA has been solubilized in dimethylsulfoxide (DMSO) , and then diluted in phosphate buffer at pH 6.6 up to a concentration of 100 /ug/ml.
The diameters of the inhibition areas are reported in table l.
Under these experimental conditions the results indicate a relevant anti-mycotic activity in vitro of miconazole SSA.
Since for some imidazole derivatives a discrepancy has been detected, sometimes of relevant magnitude , between the antimycotic activity in vitro and that in vivo (Richardson et al., Antimicrob Agents Chemother 27: 832, 1985), and since both the tests of antimycotic activity in vivo and the tests of therapeutical activity in the human being su~fering from skin mycosis are considered a~ more reliable than those in vitro (Odds et al. J. Ant{microb. Chemoter. 18, 473, 1986), the antimycotic activity of miconazole SSA has been controlled both in the experimental animals and in the human being.
I-b Antimycotic activity in vivo.
The antimycotic activity of miconazole SSA has been investigated in vivo in an infection model induced from Candida albicans in comparison with miconazole nitrate.
The experiment has been carried out according to the model of Van Cutsem and Thienpont (J. Van Cutsem and D. Thienpont. Experimental cutaneous Candida Albicans infections in guinea pigs. Sabouraudia 9:17, 1971), A group of 40 guinea pig8, previously treated with alloxan, has been in~
oculated with 3 x 10 blastospores and C. Albicans in a shaved cutis area. The hair has been shaved at beginning of the experiment and, sub-1 3~ ~,72~ ~

l sequently, every seven days.
Immediately after the inoculation, the animals have been divided into four groups: the first group did not received any treatment and was used as the contro~; the second group has been treated with placebo; the third group has been treated with 2% miconazole nitrate cream; the fourth group has been treated with ~ miconazole SSA cream. All the treatments have been carried out with two cutaneous applications per day at 12 hours in-terval between them.
The lesion entity has been evaluated after 7, 15 and 25 days from the in-oculation according to a rating score scale: O) no lesions; 1) slight le-sion; 2) moderate lesion; 3) marked lesion; 4) heavy lesion.
The results are reported in table 2.
The placebo application (cream devoid of the active principle) had no in-fluence on the lesion behaviour, as demonstrated from the possibility of superimposing the scores of the first group (no treatment) and of the second group of animals (placebo).
On the contrary, relevant ~lighter le~ions and a quick recovery have been ob~erved in animals treated in comparison with 2% miconazole nitrate cream, as demonstrated from the lower score recorded for the third group at each of the three ob~ervation~ times.
La~tly, for the animals treated with 2% miconazole SSA cream a relevantly faster recovery has been observed with respect to the animals treated with 2% miconazole nitrate cream, as demonstrated from difference of the scores recorded for the third and fourth groups of animals.
The results of the experiments demonstrated that miconazole SSA is endowed with an in vivo anti-mycotic activity higher than that of miconazole nitrate, thus leading to the recovery of the lesion induced from Candida albicans in shorter time~ and in a more efficacious way.
I-c Anti-mycotic activity in the human being.
The antimycotic activity of miconazole SSA in the human being has been evaluated in comparison with miconazole nitrate in the case of 20 patients suffering from vulvo vaginalis candidiasis, diagnostically "
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1 assessed by means of cultural and microscopical examination.
The patients have been randomized and treated, 10 with miconazole nitrate and 10 with miconazole SSA, in form of vaginal ovuli, each containing the equivalent of 100 mg of miconazole base. The posology was of 2 ovuli per day for 14 days. The cultural and microscopic examinations have been repeated after 7 and 14 days from the beginning of the treatment and the results have been recorded and reported in the table 3.
From the results of clinical experiments a greater therapeutical effec-tiveness of the miconazole SSA with respect to the miconazole nitrate is shown as evidenced from the percentages of positivity at the culture e-xaminat on (10 vs 40h at the seventh day; 0 vs 10% at 14 day (p < 0.01) and at the microscopical examination for Candida albicans (10 vs 3~/0 at 7 days; 0 vs 20/~ at 14 days) (p ~ 0.01).

Dlameter o~ the inhibition area for some microorganisms in the presence of miconazole SSA and miconazole nitrate.
Microorganism M.S. M.N.
mm Candida Albicans 23.5 23.8 73/079 (YMA) Candida Albicans 25.4 20.0 73/079 (SAB) Cryptococcus 451 22.4 30.4 Saccharomyces cer. 30.0 24.9 Aspergillus niger 22.1 17.0 Trychopyton 569A 43.9 31.4 Hendersonula toruloidea 19.2 23.1 M.S. = miconazole SSA
M.S. = miconazole nitrate :.

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l TABLE 2 Antimycotic activity of miconazole SSA in comparison with miconazole nitrate in a cutaneous infection model induced from Candida Al~icans.

Gro~lp N.of gui- t (days) Lesions scores nea pigs 0 1 2 3 4 No treatment 10 7 0 0 1 4 5 Placebo 10 7 0 0 2 4 4 Miconazole nitra-te 2% 10 7 1 3 4 2 0 Miconazole SSA
2% 10 7 3 3 4 0 0 _ _ _ _ _ _ _ _ Scores of the lesions: 0) absent; 1) slight; 2) moderate; 3) marked; 4) heavy.

-10- 1 7r~7?-' `
I

Antimycotic activity of miconazole SSA and miconazole nitrate in women suffering from vulvovaginalis candidiasis.
a) Percentage of poæitivity at culture examination for Candida Albicans Treatment days Miconazole nitrate 100 40 10 Miconazole SSA 100 10 0 b) Percentage of positivity at microscopical examination for Candida ~bicans Treatment days Miconazole nitrate 100 30 20 Miconazole SSA 100 10 0 II- a In vitro antimycotic activity.
l'he antlmycotic activlty of econazols SSA has been evaluated against Candida Albicans 73/07g (YMA and SAB), Cryptococcus neoformans 451, Saccharomyces cerevisiae, Aspergillus niger, Trichophyton mentagrophytes 569A, Hendersonula toruloidea TH65 and Pacilomyces varioti.

The microorganisms have been incubated onto agar-glucose medium, (Sabourauds medium) at 30 or 37C, depending on the species, in the presence of econazole SSA.

The incubation was of 24 hours for Candida and Cryptococcus, of 48-72 hours for the other mycetes. The econazole SSA has been solubilized in dimethylsulfoxide (DMS0) , and then diluted in phosphate buffer at pH 6.6 up to a concentration of 100 /ug/ml.
The diameters of the inhibition areas are reported in table 1.
Under these experimental conditions the results indicate a relevant anti-mycotic activity in vitro of econazole SSA.
Since for some imidazole derivatives a discrepancy has been detected, so=eeimes of relevdnt ==gnit de , between the ~nti=ycotic ~ctivity In .

1 vitro and that in vivo (~ichardson et al., Antimicrob Agents Chemother 27: 832, 1985), and since both the tests of antimycotic activity in vivo and the tests of therapeutical activity in the human beings suffering from skin mycosis, are considered as more reliable than those in vitro (Odd~3 et al. J. Antimicrob. Chemoter. 18, 473, 1986) the antimycotic ac-tivity of econazole SSA has been controlled both in the experimental an-imals and in the human beings.
I-b Antimycotic activity in vivo.
The antimycotic activity of econazole SSA has been investigated in vivo in an infection model induced from Candida albicans in comparison with econazole nitrate.
The experiment has been carried out according to the model of Van Cutsem and Thienpont (J. Van Cutsem and D. Thienpont. Experimental cutaneous Candida Albicans infections in guinea pig. Sabouraudia 9:17, 1971).
A group of 40 guinea pigs, previously treated with alloxan, has been in-oculated with 3 x 10 boastospores and C. Albicans in a shaved cutis area. The halr has been shaved at be~lnning of the experiment and, sub-sequentl~, every seven days.
Immediate]y after the inoculation, the animals have been divided into four groups: the first group did not received any treatment and was used as the contro~; the second group has been treated with placebo; the third group has been treated with 2% econazole nitrate cream; the fourth group has been treated with 2~o econazole SSA cream. All the treatments have been carried out with two cutaneous applications per day at 12 hours in-terval between them.
The lesion entity has been evaluated at 7, 15 and 25 days from the inocu-lation according to a rating scale: O) no lesions; 1) slight lesion; 2) moderate lesion; 3) marked lesion; 4) heavy lesion.
The results are reported in table 2.
The placebo application (cream devoid of the active principle) had no in-fluence on the lesion behaviour, as demonstrated from the possibility of superimposing the scores of the first group (no treatment) and of the ' ~ ~

-12- l J ~ ~,7 l second group of animals (placebo).
On the contrary, relevant slighter lesions and a quick recovery have been observed in animals treated with 2% econazole nitrate cream, as demonstrated from the lower scores recorded for the third group at each of the three observations times.
Lastly, for the animals treated with 2% econazole SSA cream a relevantly faster recovery has been observed with respect to the animals treated with 2Yo econazole nitrate cream, as demonstrated from difference of the ~cores recorded for the third and fourth groups of animals.
The results of the experiments demonstrated that econazole SSA is endowed with an in vivo anti-mycotic activity higher than that of econazole nitrate, thus leading to the recovery of the lesion induced from Candida Albicans in shorter times and in a more efficacious way.
.
I-c Anti-mycotic activity in the human being.
The antimycotic activity of econazole SSA in the human being has been ev-aluated in comparison with econazole nitrate in the case Of 20 patients ~uffering from vulvovaginalis candidiasis, assessed by means of cultural and microscopical examinatlon.
The patients have been randomized and treated, 10 with econazole nitrate and 10 with econazole SSA, in form of vaginal ovuli, each containing the equivalent of 100 mg of econazole base. The posology was of 2 ovuli per day for 14 days. The cultural and microscopic examinations have been repeated after 7 and 14 days from the beginning of the treatment and the results have been recorded and reported in the table 6.
From the results of the clinical experiments a greater therapeutical ef-fectiveness of the econazole SSA with respect to the econazole nitrate is shown as evidenced from percentages of positivity at the culture examina-tion (10 vs 40YO at the seventh day; 0 vs lOYo at 14 day) (p ~O.O1) and at the microscopical examination fGr Candida albicans (10 v~ 30% at 7 days;
0 vs 20Yo at 14 days) ~p ~ 0.01).

Diameter of the inhibition area for some microorganisms in the presence '-, -13- 1 3 ,7 ` ~

l of econa~.ole SSA and econazole nitrate (100 ug/ml) Microorganism E.S. E.N.
mm Candida Albicans 27.3 23.8 73/079 (YMA) Candida Albicans 23.4 20.0 73/079 (SAB) Cryptococcus 451 25.3 30.4 Saccharomyces cer. 31.6 24.9 Aspergillus niger 29.4 17.0 Trychophton 569A 45.1 31.4 Hendersonula TH65 33.9 23.1 -Paecilomyces 16.2 12.5 E.S. = econazole SSA
E.N. = econazole nitrate -14- ~ ? ~ '~

Antimycotic activity of econazole SSA in comparison with econazole nitrate in a cutaneous infection model induced from Candida Albicans.

Group N.of gui- t (days) Lesion scores nea pigs 0 1 2 3 4 No treatment 10 7 0 0 1 5 4 0 2 3 2 ~ 3 Placebo 10 7 0 0 1 5 4 Econazole nitra-te 2% 10 7 1 4 3 2 0 Econazole SSA
2% 10 7 3 3 4 0 0 -Scores of the lesions: 0) absent; 1) slight; 2) moderate; 3) marked; 4) heavy.

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-15- l ~ ~ j7 2 l TABLE 6 Antimycotic activity of econazole SSA and econazole nitrate in women suf-fering from vulvovaginalis candidiasis.
a) Percentage of positivity at culture examination for Candida albicans Treatment days Econazole nitrate 100 50 20 Econazole SSA 100 20 0 b) Percentage of positivity at microscopical examination for Candida albicans Treatment days Econazole nitrate 100 40 10 Econazole SSA 100 20 0 The compounds of the present invention are used for the preparation of pharmaceutical forms ~uitable for the topical use and thus for the foreseen therapeutical application, such as creams, ointments, lotions gels, milk, tinctures, powders, ovuli, foams, vaginal capsules, vaginal washings, oral gels.
For their preparation the normal excipients, solvents, vehicles and ad-ditives are used, according to the standard pharmaceutical techniques.
As regards the dosage of the active principle in the aforesaid pharmaceutical formulations and as regards the use posology, the dosage and posology already known and used for the corresponding nitrates still hold~ true.

Claims (8)

1. Imidazole derivatives having the formula :

wherein R represents H, Cl.

2. 1-(2-((4-chlorophenyl)-methoxy)-2-(2,4-dichlorophenyl)-ethyl)-1H-imi-dazole 5- sulfosalicylate according to claim 1.

3. 1-(2-((2,4-dichlorophenyl)-2-(2,4-dichlorophenyl)methoxy)-ethyl)-1H-imidazole 5-sulfosalicylate according to claim 1.

4. Pharmaceutical composition for topical use, characterized by contain-ing, as the active ingredient a compound according to claim 1, together with the normal excipients, vehicles, solvents and diluents.

5. Pharmaceutical composition according to claim 4 in form of cream, ointment, lotions, gels, milk, tincture, powder, ovuli, foam, vaginal capsules, vaginal washings.

6. Pharmaceutical composition according to claim 4 for the therapy of cutaneous mycosis.

7. A process for the preparation of the compounds of claim 1, charac-terized in that equimolecular amounts of sulfosalicylic acid and of a compound selected respectively among miconazole and econazole are reacted under hot conditions, as a solution in alcoholic solvent.

8. A process according to claim 7, characterized in that said reaction is carried out at the boiling point and in ethanolic solution.