NO124600B - - Google Patents

Description

Analogy method for the preparation of therapeutically active pyrazole derivatives.

This invention relates to an analogue method for the preparation of therapeutically active pyrazole derivatives.

The pyrazole derivatives produced according to the invention have the general formula:

where R1 and R2, which may be the same or, different, mean? hydro-

gen, methyl, ethyl, n-propyl, isopropyl or n-butyl, and X

means hydrogen, hydroxy, methyl, methoxy, chlorine or bromine.

The invention also encompasses the preparation of the pharmaceutically acceptable acid addition salts of the compounds of formula I.

The compounds of formula I are prepared according to the invention by a 3-benzofuryl ketone with the general formula:

where R.j and X have the meanings given above, is heated with hydrazine hydrate or with an alkylhydrazine, the corresponding pyrazole derivative that is formed is isolated, and optionally converted into the desired non-toxic salt.

The starting materials represented by formula II can, for example, be prepared by reacting the hydrochloride of isonicotinoyl chloride with a 2-alkyl-benzofuran, optionally substituted in the 5-position, by the method described in Chimie Therapeutique 2, 119, 1967, or, when 2 The -alkyl group is substituted with a hydrogen atom, by the method described in Bull.Soc. Chim. France 1952, 1056-1060.

The compounds produced according to the invention have been found to possess biological activity in animal bodies. In particular, it has been found that compounds of formula I

are in possession of the unusual property that they exert a poly-valent antiviral. effect in that they have been shown to be effective against both RNA and DNA viruses and more particularly against myxoviruses, adenoviruses, rhinoviruses and various viruses of the Herpes group. It has also been found that compounds included in formula I tend to affect cell division and metabolism. Examples of such compounds are 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole, its 5-methyl-, 5-n-propyl-, 5-isopropyl- and 5-n-butyl homologues , 3-(4-pyridyl)-4-(2-hydroxy-5-methoxyphenyl)-5-methylpyrazole, 2-methyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole and its 2-isopropyl and 2-n-butyl homologues. Biological studies have been carried out to determine the activity of the compounds that fall within the definition

of formula I, with respect to certain viruses of the RNA and DNA groups. The viruses were given in lethal dilutions made from multiples of the concentration necessary to kill 50 # of untreated animals (LD5Q)o

Regarding the RNA class of viruses, three types of experiments (experiments 1, 2 and 3) were performed on mice using different influenza viruses.

Experiment No. 1 (Mouse survival test).

The mice were first divided into two groups. The animals in one group were given, either by the intraperitoneal (IP) or oral (PO) route, the compound to be investigated, suspended in 0.25$ > carboxymethyl cellulose. An hour later, they were infected with a dilution of the virus in an aerosol. Additional doses of the test compound, at the same concentrations as the first, were then given by the same route at 3, 24, 48 and 72 hours post-infection. The animals of the second group, which constituted the control group, were infected in the same manner as the treated animals, but did not receive any compound of formula I.

During the experiment, which lasted for 15 days, the following data were recorded in a) Average death day (GDD) expressed in days for both treated and control groups. b) The ratio between the GDD of treated animals and the GDD of control animals, which gives the survival index (Ol), 01 was considered as

significant if it exceeded 1.24.

c) The percentage of treated animals that survived at the end of the experiment.

The compounds prepared according to the invention, which were used in the first series of investigations, were 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole (Compound A), its 5-methyl- (Compound B), 5-n-propyl-(Compound C), 5-isopropyl-(Compound D) and 5-n-butyl-(Compound E) homologues, 3-(4-pyridyl)-4-(2-hydroxy- 5-methoxyphenyl)-5-methyl-pyrazole (Compound F), 2-methyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-ethylpyrazole (Compound G) and its 2-isopropyl- (Compound H) and 2-n-butyl (Compound I) homologues.

The following results were recorded:

In another series of investigations with compounds A and D, but with other influenza viruses, the following results were obtained:

If 1.24 is taken as the minimum survival index number of significance, the preceding tables show that the compounds tested were effective in extending the lifespan of mice exposed to lethal dilutions of various influenza viruses.

Experiment #2 (48-hour mouse test).

This experiment was undertaken to study the effects of the compounds prepared according to the invention on various viruses during the early stages of the virus growth cycle in mice.

The mice were divided into three groups. The first two groups received a dose of the test compound, one group by the intraperitoneal route (IP) and the other by the oral route (PO). One hour later, all three groups were infected with a dilution of the virus in aerosol. Further doses of the compound under test, at the same concentrations as in the first, were then given to the first two groups by the same route three hours and 24 hours after infection. The third, untreated group constituted the control group. 48 hours after infection, the treated groups were euthanized at the same time as the control group. The lungs from each group were removed, pooled separately for each group and ground in a casein hydrolyzate to form three 10 µl homogenates. Two series of dilutions, in which each dilution was 1/10 the concentration of the preceding dilution, were prepared from the homogenates obtained from the treated animals. These dilutions were then injected into 11-day-old embryonated chicken eggs (0.2 ml/egg) to titrate the egg infectivity of the virus from the mouse lung material. 6 eggs were infected with each dilution of the virus. After incubation for 48 hours at 37°C, the eggs were cooled and the allantoic fluid from the eggs was collected. A hemagglutination pattern was obtained by adding 0.5 ml of a fresh 0.5 # suspension of chicken erythrocytes to an equal volume of allantoic fluid from each egg. The titer at which the 50 # endpoint of egg infectivity (EID^q) occurred was calculated on the basis of the results of the hemagglutination test. These results were compared with the results obtained by the same process using the lung material from the control animals. A reduction of EID^Q of one log compared to the control value was considered significant.

The compound produced according to the invention that was used was 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole. The results obtained with this compound against the different viruses used are reproduced below:

Experiment No. 3 (intranasal).

Trials were conducted to determine effectiveness

against an influenza virus of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole Compound A) administered intranasally to mice. The test compound was given at different concentrations at different times before the infection by aerosol with an appropriate dilution of the virus. The results obtained with a 10 µl suspension of the test compound compared to the results obtained with the diluent used for the suspension, in mice infected with 25 multiples of the LD^q d°sen of the virus, are reproduced below:

The results showed that the test compound had a clear protective effect at any time of administration. Between 40 and 70% of the animals survived the trial period, compared to 100% mortality for the untreated control animals. The survival period for the treated animals that died was increased to approximately twice the corresponding period for the control animals. Duration of the effect was marked since no significant differences were observed between the results obtained with the different . trial connection administration times.

A similar intranasal test performed with different concentrations of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-isopropyl-pyrazole (Compound D) in a suspension administered 30 minutes before aerosol infection with influenza virus Jap 305, they gave the following results:

Other experiments were performed to determine the inhibitory effect of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole (Compound A) and of 3-(4-pyridyl)-4-(2-hydroxy -phenyl)-5-isopropyl-pyrazole (Compound D) on different types of rhinovirus.

For each test, two cultures of human fetal lung were prepared, one of which was treated with the above compound, while the other served as a control. The cultures were then infected with a strain of rhinovirus. Compound A

was used at concentrations of 50 and 75 micrograms/ml and compound D at a concentration of 50 micrograms/ml, and the compounds were considered effective if the cytopathogenic effect of the rhino virus was reduced by at least 75% compared to the control culture. This cytopathogenic effect was determined by counting the number of plaques formed by destroyed cells.

For compound A, seven human types of rhinovirus were used, and the following results were recorded:

For compound D, 25 types of rhinovirus of human origin were used, and the following results were obtained:

Another series of experiments was performed in which the inhibitory concentrations of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole against different human types of rhinovirus were determined by the gradient plate-plaque reduction technique. Cultures of HeLa cancer cells were used, and two were made for each sample. One culture was treated with the above compound applied in a gradient, while the other culture served as a control. Both cultures were then infected with a human type of rhinovirus. The inhibitory concentration was determined by measuring the zone of inhibition on the cell sheet. Six types of rhinovirus were used and the following results were recorded.

Finally, human trials were conducted with 3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-isopropyl-pyrazole to determine the effectiveness of this compound against infection by a rhinovirus strain, type 39. 12 volunteers were divided into two equal groups. They were all given a bottle of nasal drops. For one group, the bottle contained the above compound. Treatment began 24 hours before infection.

For 6 days, such symptoms as sneezing, runny nose, congestion of the respiratory organs, sore throat, coughing, headache and tremors were noted. To enable measurement of the preventive effect of the compound, a value was assigned to each symptom according to the degree of severity, ranging from 0 for lack of symptom to 4 for strong degree of symptom. Based on this assessment, it was observed that the severity of the symptoms among the volunteers who had been infected reached a total of 24, while the number for the volunteers who had received the compound prepared according to the compound was only 10.

In the case of viruses of the DNA group, tissue culture tests were performed to determine the activity of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole against Herpes Simplex. The experimental material used was rabbit kidney tissue. In a test involving pre-treatment of virus with the compound, equal volumes of the compound and of the virus were incubated together in test tubes for one hour at room temperature. This mixture was then inoculated into a flask containing the rabbit kidney tissue. The flask was incubated at 36°C for two hours, after which the liquids were discarded and 5 ml of a coating material was added. After 4 days the coating material was removed and crystal violet was added for 5 minutes. The plaques appeared as clear areas on a blue background. A plaque reduction of 50% was considered significant. The same process was followed in another experiment, with the exception that the rabbit kidney tissue was infected first and the compound was introduced into the coating material. It was found that a dose of 250 micrograms caused 60% plaque reduction in the first study and 100% plaque reduction in the second.

Another experiment was carried out with the same compound to assess its effect against Herpes-induced keratitis in the rabbit eye. The compound was applied topically at concentrations of 2, 1 and 0.5% and it was found that for all three dose levels the compound inhibited the formation of geographic keratitis lesions compared to the infected control animals.

Preliminary biological investigations have shown that compounds falling within the definition of formula i exert an action against adenoviruses, parainfluenza viruses and rhinoviruses.

It is to be understood that for therapeutic use the compounds produced according to the invention will normally be administered in the form of pharmaceutical preparations containing as a substantially active ingredient at least one such compound together with a pharmaceutical carrier. The carrier can be a diluent or excipient of the type normally used for the production of ready-to-use medicinal products, for example lactose, potato starch, corn starch, talc, magnesium stearate, polyvinyl pyrrolidone, an ointment base or an emulsifying medium"

The preparation is made in a form suitable for the desired method of administration, which may be oral or topical. The term "topical" shall here be understood to mean all routes of administration other than per os, per rectum and by injection» The preparation can conveniently be made in separate dose units that suit the desired form of administration. The dosage unit can thus be, for example, a tablet, pill, wrapped powder or capsule for oral administration, drops or spray for intranasal administration, or a sterile ointment packed in a suitable container such as a tube or jar for topical application. The amount of active ingredient in each dosage unit for oral administration will be such that one or more units are required for each therapeutic administration. The dose unit can, for example, contain from 50 mg to 10 g of the active ingredient depending on the type of administration.

The following examples shall serve to further illustrate the invention.

Example 1

Preparation of 2-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole and its hydrochloride

1455 g (5.80 mol) of 3-ethyl-3-isonicotinoylbenzofuran and 10 l of isopropanol were placed in a 20 liter, three-necked reactor equipped with a riser cooler, a dropping funnel and a mechanical stirrer.

The resulting solution was stirred, and 447 ml

98# hydrazine hydrate dissolved in 1455 ml of isopropanol was slowly added. During the operation, the temperature of the reaction medium rose gradually, so that the temperature had reached 60°C when all the hydrazine hydrate solution had been added. The solution was refluxed for 15 minutes and then cooled to room temperature. The formed precipitate was centrifuged out and washed with 500 ml of isopropanol.

In this way, a first fraction of 1175 g of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole was obtained.

The isopropanol mother liquor was evaporated under vacuum until new crystals formed, which were then centrifuged out and taken up in two liters of isopropanol. The suspension was centrifuged and another fraction of 250 g of the desired product was obtained.

The total amount of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole thus prepared was 1425 g, representing a yield of 92.8%, m.p. 243-244°C.

In the previous example, ethanol or methanol can be used instead of isopropanol, and the final product can be purified by crystallization from ethanol or methanol instead of isopropanol.

To prepare the hydrochloride, 53 g (0.2 mol) of 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole were suspended in 300 ml of isopropanol. To this suspension were added 20 ml of concentrated hydrochloric acid (37 $>) and 20 ml of distilled water, and the suspension was heated until the pyrazole was completely dissolved. The hot solution was then passed through a filter, and the filtrate was cooled with slow stirring. The hydrochloride crystals formed were filtered out, washed over a filter with isopropanol and dried under vacuum. Yield: 51.8 g (86

Melting point: Cleavage at approx. 260°C.

By the same method as described in the preceding example, the following compounds were prepared from the indicated starting compounds: From 2-methyl-3-isonicotinoyl-benzofuran, m.p. 80°C was prepared 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-methylpyrazole, m.p. 260°C.

From 2-n-butyl-3-isonicotinoyl-benzofuran, m.p. 52°C was prepared 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-n-butylpyrazole, m.p. 238°C. From 2-n-propyl-3-isonicotinoyl-benzofuran, b.p. 145-150°C/0.005

mm Hg was prepared 3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-n-propyl-pyrazole, m.p. 250°C.

From 2-methyl-3-isonicotinoyl-5-methoxy-benzofuran, m.p. 45°C

was prepared 3-(4-pyridyl)-4-(2-hydroxy-5-methoxyphenyl)-5-methylpyrazole, m.p. 232°C.

From 2-ethyl-3-isonicotinoyl-5-chloro-benzofuran, m.p. 98°C pentasyllated 3-(4-pyridyl)-4-(2-hydroxy-5-chlorophenyl)-5-ethylpyrazole, m.p. 298°C.

From 2-ethyl-3-isonicotinoyl-5-methyl-benzofuran, m.p. (Hydrochloride) 170°C (dec.) 3-(4-pyridyl)-4-(2-hydroxy-5-methylphenyl)-5-ethylpyrazole was prepared, m.p. 276°c. Pra 2-ethyl-3-isonicotinoyl-5-bromo-benzofuran, m.p. 90°C was prepared 3-(4-pyridyl)-4-(2-hydroxy-5-bromophenyl)-5-ethylpyrazole, m.p. 290°C.

Pre 2-isopropyl-3-isonicotinoyl-benzofuran, m.p. (Hydrochloride) 165°C (dec.) was prepared

3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-isopropylpyrazole, m.p. 222°C. Pra 3-isonicotinoyl-benzofuran, m.p. 145°C was prepared 3-(4-pyridyl)-4-(2-hydroxyphenyl)-pyrazole, m.p., 220°C.

Example 2

Preparation of 2-methyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole

45 g (0.18 mol) of 2-ethyl-3-isonicotinoylbenzofuran, 400

ml of absolute methanol and 27 g of methylhydrazine. The solution was refluxed for 10 hours and then cooled to room temperature. The precipitate formed was centrifuged, washed with a small amount of absolute ethanol and dried under vacuum. The resulting product was recrystallized from absolute ethanol.

In this way, 33.9 g of 2-methyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole were obtained, m.p. 236°C. Yield: 68

To prepare the hydrochloride, 50 g (0.18 mol) of 2-methyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-ethylpyrazole were suspended in 250 ml of isopropanol. 16.2 was added to this suspension

ml of concentrated hydrochloric acid (37 µl), and the suspension was heated until the pyrazole was completely dissolved. The hot solution was then passed through a filter and the filtrate was cooled with slow stirring. The formed hydrochloride crystals were filtered out, washed over a filter with isopropanol and dried under vacuum. Yield: 45g(80.3#)«

Melting point: Cleavage at approx. 230°C.

9 By the same procedure as described above, the following compounds were prepared from the indicated starting materials: From 2-methyl-3-isonicotinoyl-benzofuran, m.p. 80 C and methylhydrazine was prepared 2,5-dimethyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)pyrazole, m.p. 213°C.

From 2-isopropyl-3-isonicotinoyl-benzofuran, m.p. (HCl) 165 C and methylhydrazine was prepd

2-methyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-isopropylpyrazole, m.p. 247°C.

Pra 2-n-butyl-3-isonicotinoyl-benzofuran, m.p. 52°C and methylhydrazine was prepared 2-methyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-n-butylpyrazole, m.p. 126°C.

Pra 2-methyl-3-isonicotinoyl-benzofuran, m.p. 80°C and ethyl hydrazine was prepared 2-ethyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-methylpyrazole, m.p. 158°C.

Pra 2-ethyl-3-isonicotinoyl-benzofuran, m.p. 58°C and ethyl hydrazine was prepared 2,5-diethyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-pyrazole, m.p. 191°C.

Pra 2-n-butyl-3-isonicotinoyl-benzofuran, m.p. 52°C and ethyl hydrazine was prepared 2-ethyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-n-butylpyrazole, m.p. 163°C.

Pra 2-ethyl-3-isonicotinoyl-benzofuran, m.p. 58°C and n-propyl-hydrazine was prepared 2-n-propyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-ethylpyrazole, m.p. 175°C.

Pra 2-methyl-3-isonicotinoyl-benzofuran, m.p. 80°C and isopropylhydrazine was prepared 2-isopropyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-methylpyrazole, m.p. 217°C.

Pra 2-ethyl-3-isonicotinoyl-benzofuran, m.p. 58°C and isopropylhydrazine was prepared 2-isopropyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-ethylpyrazole, m.p. 172°C.

Pre 2-isopropyl-3-isonicotinoyl-benzofuran, m.p. (HCl) 165°C

and isopropylhydrazine was prepared

2,5-diisopropyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)pyrazole, m.p. 177°C.

From 2-n-butyl-3-isonicotinoyl-benzofuran, m.p.52°C and isopropylhydrazine, 2-isopropyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-n- butylpyrazole m.p. 164°C.

Pra 2-ethyl-3-isonicotinoyl-benzofuran, m.p. 58°C and n-butylhydrazine was prepared 2-n-butyl-3-(4-pyridyl)-4-(2-hydroxy-phenyl)-5-ethylpyrazole, m.p. 170°C.

Pre 2-isopropyl-3-isonicotinoyl-benzofuran, m.p. (HCl) 165°C

and n-butylhydrazine was prepared

2-n-butyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-5-isopropylpyrazole, m.p. 212°C.

Pra 2-n-butyl-3-isonicotinoyl-benzofuran, m.p. 52°C and n-butylhydrazine was prepared

2,5-n-dibutyl-3-(4-pyridyl)-4-(2-hydroxyphenyl)-pyrazole, m.p. 121°C„

Tablets were prepared by granulating and pressing the following ingredients according to known methods:

An ointment suitable for topical administration was prepared according to known pharmaceutical methods from the following ingredients:

Suspensions for nasal instillation containing respectively 5 io and 10 io of active compound were prepared according to known pharmaceutical methods from the following ingredients:

A 1 io solution for nasal administration was prepared from the following ingredients:

Claims (2)

1. Analogous method for the preparation of therapeutically active pyrazole derivatives with the formula: wherein R^ and R^, which may be the same or different, are hydrogen, methyl, ethyl, n-propyl, isopropyl or n-butyl, and X is hydrogen, hydroxy, methyl, methoxy, chlorine or bromine, and their pharmaceutically acceptable acid addition salts, characterized in that a 3-benzofuryl ketone with the general formula: where and X have the meanings given above, is reacted with a hydrazine compound of the general formula H2N-NHR2, where R2 has the meanings given above, to form the desired substituted pyrazole, which can then be reacted with the appropriate acid to form the desired salt. 2. Method as stated in claim 1, characterized in that 2-isopropyl-3-isonicotinoyl-benzofuran is reacted with hydrazine hydrate.