WO1996011191A1 - Substituted 3-phenyl-1,2,3,4-oxatriazole-5-imines useful for the treatment of asthma and thrombosis - Google Patents

Abstract

3- and 5-substituted 1,2,3,4-oxatriazole-5-imine compounds of general formula (I), wherein R1 is the same or different groups and represents alkyl or alkoxy groups having 1 to 3 carbon atoms, halogen, trifluoromethyl, nitro, cyano, phenyl or alkylsulphonyl groups; n is 1 to 3; X is -C(O)NH-; -SO¿2?-; -C(O)- or a direct bond; Y means -S-Z; -SO-Z; -S?+(Z)¿2, A?-; N+(Z)¿3, A-; -C(Z) = CZ¿2?; -C C-Z or a group (a), wherein b = c = N or C-Z; d = N-Z, O or S, and A?-¿ is any pharmaceutically acceptable anion; and Z is the same or different groups and represents H, cyano, nitro, trifluoromethyl, -CH = CH¿2?, -C CH, straight or branched C1-C6-alkyl, C3-C7-cycloalkyl, phenyl or optionally substituted phenyl; may be used for the preparation of a medicament for the treatment of asthma, a medicament for the treatment of blood clots (thrombosis), a medicament being effective against impotence and a medicament being effective against pre-eclampsia.

Description

Substituted 3-phenyl-l,2,3,4-oxatr1azole-5-1ιτι.nes useful for the treatment of asthma and thrombosis.

Technical Field

5 The present invention relates to hitherto unknown 3- and 5-substituted 1,2,3,4- oxatriazole-5-imine compounds which have proved to have pharmacological effects making them very suitable for effective treatment of thrombotic and asthmatic disorders, a process for the preparation thereof and a pharmaceutical preparation containing said compounds. Furthermore, the invention relates to the use of said 10 compounds for the preparation of medicaments.

Thrombotic and asthmatic disorders have become more and more common and serious along with the industrial development. Mankind has needed effective drugs for treatment of these serious welfare diseases.

The most fatal thrombotic conditions are cerebral and coronary thrombosis. These 15 conditions cannot be treated with blood pressure lowering drugs (i.e. hypotensive agents) alone as, for example, those stated below. The same applies to the different classes of hypotensive drugs (e.g. 0-blockers, calcium antagonists and ACE-inhibi- tors). The thrombotic disorders must be treated with drugs directly attacking the thrombi (blood clots), that is inhibition of the formation of thrombi, the adhesion 20 of the thrombus to the vessel wall and dissolution (fibrinolysis - thrombolysis) of already established thrombi.

Asthmatic conditions are characterised by narrowing of the airways caused by bronchoconstriction and inflammation (swelling) of the mucosa in the respiratory system. An effective treatment of asthmatic disorders would be the use of drugs 25 combining bronchodilatation with an anti-inflammatory effect in the lung mucosa. The established asthma therapy mainly deals with bronchodilatatory drugs alone. Only steroids exert an anti-inflammatory activity as well. However, long lasting asthma treatment with steroids is connected with serious local and in particular systemic side effects, which make such effective anti-asthmatics less attractive.

Background Art

N.G. Finnegan et al., J. Org. Chem. 30, pages 567-575 (1965) discloses the compound 3-cyclohexyl-l,2,3,4-oxatriazole-5-imino-hydrochloride. However, no biological effect of said compound is mentioned.

K. Masuda et al., Chem. Pharm. Bull. 19 (3) pages 559-563 (1971) discloses 3-aryl- l,2,3,4-oxatriazole-5-imine compounds and acyl derivatives thereof, wherein the aryl group may be monosubstituted by methyl or halogen. Even though these compounds were synthesized in the hope of finding new hypotensive agents, no biological effects of the compounds are described.

C. Christophersen et al. , Acta Chemica Scandinavica, 25, pages 625-630 (1971), discloses 3-substituted l,2,3,4-oxatriazole-5-imino compounds, wherein the 3- substituent may be propyl or phenyl or cyclohexyl. However, no biological effects of said compounds are described.

Hanley et al. , J.C.S. Perkin Trans I, 736-740 (1979), discloses 3-aryl-l,2,3,4- oxatriazole-5-imine compounds, wherein the aryl group may be monosubstituted by methyl or halogen. However, no biological effects of the compounds are described.

The JP Patents Nos. 20904/70 and 21102/70 disclose 3-substituted 1,2,3,4-oxatria- zole-5-imine salts and acyl derivatives thereof, wherein the 3-substituent may be aryl, optionally monosubstituted by chlorine or methyl. The vasodepressor activity of said compounds is stated as a biological effect.

GB patent specification No. 2 015 878 discloses 3-phenyl-l,2,3,4-oxatriazole-5- imine compounds, for which a pesticidal and/or pest ovicidal and/or herbicidal acti- vity has been found. US patent specification No. 4,329,355 discloses anhydro-5-imino-l,2,3,4-oxatria- zolium hydroxides of a structure similar to the structure of the compounds of the present invention. However, the compounds known from this patent specification are only mentioned as being useful in the treatment of cancer.

Furthermore, from J.C.S. Perkin Trans I, 747-751 (1979) compounds of a structure similar to the structure of the compounds of the present invention are known. However, no biological effects of said compounds have been stated.

WO 92/13847 and WO 94/03442 disclose 3- and 5-substituted l,2,3,4-oxatriazole-5- imine compounds of a structure similar to the structure of the compounds of the present invention and having biological effects of the same type as the compounds of the present invention. However, it has surprisingly become apparent that the new compounds of the present invention have both a bioavailability and a stability being significantly superior to the bioavailability and stability of the compounds disclosed in the above WO publications which make them usable for preparing pharmaceutical compositions for the treatment of various diseases and in particular thrombotic and asthmatic disorders.

Disclosure of the Invention

The present invention relates to hitherto unknown 3- and 5-substituted 1,2,3,4- oxatriazole-5-imine compounds of the general formula I

being characterised in that R¹ is the same or different groups and represents alkyl or alkoxy groups having 1 to 3 carbon atoms, halogen, trifluoromethyl, nitro, cyano, phenyl or alkylsulphonyl groups, n is 1 to 3;

X is -C(O)NH-, -SO₂-, -C(O)- or a direct bond;

Y means -S-Z; -SO-Z ; -S⁺(Z)₂, A^" ; N⁺(Z)₃, A^" ; -C(Z) = CZ^; -C -≡ C-Z or a group

wherein b = c = N or C-Z ; d = N-Z, O or S,

and A^" is any pharmaceutically acceptable anion; and

Z is the same or different groups and represents H, cyano, nitro, trifluoromethyl, -CH = CH₂, -C = CH, straight or branched C,-C₆-alkyl, C₃-C₇-cycloalkyl, phenyl or optionally substituted phenyl.

The compounds according to the invention differ from the above prior art com¬ pounds by their chemical constitution, as they have a different substitution in the 5- position of the oxatriazole ring, and they differ from the compounds known from the above patents with respect to their biological effect, as they inhibit the blood pla¬ telet aggregation and have a relaxation effect on the trachea. Compared with the compounds known from WO 92/13847 and WO 94/03442, the compounds of the present invention are superior with respect to bioavailability and stability irrespective of the fact that the pharmacological activity of the compounds is of the same nature as that of the compounds disclosed in the above two WO publications. The compounds according to the invention differ from the compounds known from the above earlier patents with respect to their biological effect, as in addition to inhibition of the blood platelet aggregation (the first step in thrombus formation), they desaggregate already aggregated platelets and decompose the fibrin content in an established thrombus (the incorporation of fibrin in the aggregated platelets is the second step in thrombus formation), i.e. a fibrinolytic/thrombolytic activity.

Furthermore, the compounds according to the invention both relax the precontracted trachea, inhibit the inflammatory cell infiltration, and inhibit the non-allergic as well as the allergic bronchoconstriction in whole animals (guinea pigs). The latter is very similar to asthma in humans.

The compounds according to the invention are, in addition to their unique phar¬ macological profile, characterised by combining good stability with satisfactory pharmacokinetic properties, i.e. the compounds are well-absorbed subsequent to oral administration. As mentioned above, in this respect they are superior to the com- pounds known from WO 92/13847 and WO 94/03442.

The invention further relates to a pharmaceutical preparation being characterised in that it comprises as an active ingredient a compound of formula I together w ith a pharmaceutically acceptable carrier or diluent.

Moreover, the invention relates to a process for the preparation of said 3- and 5- substituted l,2,3,4-oxatriazole-5-imine compounds of the general formula I, said process being characterised by ring closing a 1-arylthiosemicarbazide derivative of the general formula II

wherein R¹ and n have the same meaning as in formula I, by treatment with alkyl nitrite having 1 to 6 carbon atoms or alkali metal nitrite under acidic conditions at 0 to 10°C, whereafter the resulting salt is converted into the corresponding free compound, which is subsequently reacted with a compound of the type ClSO₂-CH₂Y or O = C = N-CH₂-Y, wherein Y has the same meaning as in formula I.

In the process according to the invention it is preferred to use ethyl nitrite as alkyl nitrite having 1 to 6 carbon atoms, and sodium nitrite is preferred as an alkali metal nitrite.

It is known per se to cyclize 1 ,4-disubstituted thiosemicarbazides with nitrous acid (sodium nitrite and acid) to form 3-substituted l,2,3,4-oxatriazole-5-imines. The yields at this reaction are stated to be between 18 and 57%.

For reacting 1 equivalent of the 1-aryl-thiosemicarbazide derivative with alkyl nitrile having 1 to 6 carbon atoms, it is preferred to use 2 to 2.5 equivalents of alkyl nitrite in a suitable solvent, such as alkyl alcohol having 1 to 6 carbon atoms, to obtain a 3-arylsubstituted 1 ,2,3,4-oxatriazole-5-imine salt in a substantially quantitative yield After filtration of the precipitated sulphur and evaporation of the solvent, the pnxl uct is, if necessary, recrystallized from for instance alkyl alcohol having 1 to ts carbon atoms, acetonitrile or nitromethane, whereby the yields of the pure product obtained are usually between 60 and 95%.

As alkyl alcohol having 1 to 6 carbon atoms methanol or ethanol is preferred.

The necessary starting compounds of the general formula II may be prepared in a manner known per se by reacting the corresponding arylhydrazine hydrochloride with an alkali thiocyanate or an ammonium thiocyanate in a suitable solvent, such as alcohol or water, using reflux for 6 to 18 hours, for instance as described by Houben-Weyl: "Methoden Der Organischen Chemie E4", page 513. PREPARATION OF THE STARTING MATERIALS

Preparation of l-(2-trifluoromethylphenyl)thiosemicarbazide

63.8 g ( 0.3 mole) of 2-trifluoromethylphenylhydrazine-hydrochloride were dis¬ solved in 360 mil of absolute ethanol. 32.3 g (0.33 mole) of potassium thiocyanate were added to the solution, and the mixture was heated during reflux for 8 hours. The mixture was then cooled and subsequently evaporated to dryness on a rotary evaporator. The product was then boiled with 150 ml of water, separated by filtration and then thoroughly washed with water and dried under vacuum.

Yield: 69.9 g = 99J % Melting point: 205-206 °C Elemental analysis: CgH^^S:

Calculated: C: 40.85% H: 3.43% N: 17.87%

Found: C: 40.66% H: 3.35% N: 17.59%

Preparation of 3-(2-trifluoromethylphenyl)-l .2.3.4-oxatriazole-5-imine hvdrochloride

8.23 g (0.35 mole) of l-(2-trifluoromethylphenyl)thiosemicarbazide were dissolved in 125 ml methanol and 5 ml of 37% hydrochloric acid while being stirred at room temperature. The mixture was cooled to 0 to 5 °C by means of an ice bath, and 5.25 g (0.07 mole) of ethyl nitrite were subsequently added in small quantities over a period of 5 minutes. The mixture was stirred for one hour, and additional 3 ml of ethyl nitrite were then added, and the reaction was then left for about 15 minutes while being stirred. The sulphur was separated by filtration and the mixture was evaporated on a rotary evaporator at a bath temperature of 30°C. The product was recrystallized from 75-100 ml of acetonitrile and washed with diethyl ether and dried. An extra crop of crystals was collected after addition of 150 to 200 ml diethyl ether to the mother liquor which was separated by filtration and washed with diethyl ether and dried. Yield: 7.70 g = 82.5% Melting point: 162-164 °C Elemental analysis: C₈H₆ClF₃N₄O:

Calculated: C: 36.04% H: 2.27% N: 21.02% Found: C: 35.82% H: 2.34% N: 20.77%

Preparation of l-(3-chloro-2-methylphenyl)thiosemicarbazide

19.3 g (0J mole) of 3-chloro-2-methylphenylhydrazine-hydrochloride were dis¬ solved in 200 ml of absolute ethanol. 11.64 g (0.12 mole) of potassium thiocyanate were added to the solution, and the mixture was heated during reflux for 16 hours. The mixture was then cooled, whereby the product was partially precipitated, and the mixture was subsequently evaporated to dryness on a rotatory evaporator. The product was recrystallized from 200 ml water and 250 ml methanol, separated by filtration and washed thoroughly with water.

Yield: 17.8 g = 82.5% Melting point: 192-193°C.

Elemental analysis: C₈H₁₀C1N₃S:

Calculated C: 44.54% H: 4.67% N: 19.48% SJ4.86%

Found C: 44.22% H: 4.58% N: 19.60% S: 14.67%

500 MHz 1H NMR (d₆-DMSO): δ 9.33 (br s, IH, NH), δ 7.80 (br s, IH, NH), δ 7.72 (br s, IH, NH), δ 7.52 (br s, IH, NH), δ 6.80 (m, 3H, ArH), δ 2J8 (s, 3H, CH₃).

Preparation of 3-(3-chloro-2-methylphenyl)-l ,2.3.4-oxatriazole-5-imine hvdrochloride

8.6 g (40 mmole) of l-(3-chloro-2-methylphenyl)thiosemicarbazide were dissolved in 100 ml of methanol and 5 ml of 37% hydrochloric acid while being stirred at room temperature. The mixture was cooled to 0 to 5°C by means of an ice bath, and 6.3 g (7 ml) of ethyl nitrite were subsequently added in small quantities over a period of approximately 5 minutes. The mixture became dark coloured by the nitrous vapours, but turned light after a few minutes at the same time as free sulphur precipitated. The mixture was stirred for 10 minutes, and additional 0.9 g (1 ml) of ethyl nitrite was then added, and the reaction mixture was then left for about 20 minutes while being stirred. The sulphur was separated by filtration and the mixture was evaporated on a rotary evaporator at a bath temperature of 30°C. If necessary, the mixture was dehydrated by evaporation together with toluene/etha- nol. The crystals were stirred with diethyl ether, separated by filtration and washed further with small amounts of diethyl ether.

Yield: 9.2 g = 94% Melting point: 194-195°C (decomposes) IR: 1700 cm^"1. Elemental analysis C₈H₇C1N₄0, HC1, '4H₂0:

Calculated C: 38.19% H: 3.41 % N: 22.28% C1:28J8%

Found C: 38.07% H: 3.19% N: 22.30% Cl:28.58%

500 MHz 1H NMR (D₂0): δ 7.52 (m, 3H, ArH), δ 2.38 (s, 3H, CH₃)

Example 1

3-(2-trifluoromethylphenyl)-1.2.3.4-oxatriazole-5-(N-2-propenylcarbamoyl)imine

5.3 g (20 mmole) of 3-(2-trifluoromethylρhenyl)- 1,2,3, 4-oxatriazole-5-imine hydro- chloride were dissolved in 60 ml of water and 2J g (25 mmole) of sodium hydro- gencarbonate were subsequently added while being stirred. After terminated deve- lopment of carbon dioxide 60 ml of dichloromethane were added, whereafter the precipitated substance dissolved. Under vigorous stirring 1.66 g (20 mmole) of allyl isocyanate were added to the mixture and stirring was continued for 30 minutes. The dichloromethane phase was then separated and washed twice with IN hydrochloric acid and twice with water and then evaporated. The substance was stirred with a small quantity of diethyl ether and separated by filtration and dried.

Yield: 2.90 g = 75.3% Melting point: 162-163 °C

Elemental analysis C₁₂H₁₀F₃N₅O₂:

Calculated: C: 46.01 % H: 3.22% N: 22.36%

Found: C: 45.82% H: 3.18% N: 22.05%

IR: 1665 cm ¹, 1620 cm^"1

Example 2

3-(3-chloro-2-methylphenyl)-1.2.3,4-oxatriazole-5-(N-2-propenylcarbamoyl)imine

7.35 g (30 mmole) of 3-(3-chloro-2-methylphenyl)-l,2,3,4-oxatriazole-5-imine hydrochloride were dissolved in 90 ml of water and 2.7 g (31.5 mmole) of sodium hydrogencarbonate were subsequently added while being stirred. After terminated development of carbon dioxide 90 ml of dichloromethane were added, whereafter the precipitated substance dissolved. Under vigorous stirring 2.49 g (60 mmole) of allyl isocyanate were added to the mixture and stirring was continued for 30 minu¬ tes. The dichloromethane phase was then separated and washed twice with IN hydrochloric acid and twice with water and then evaporated. The substance was stirred with a small quantity of diethyl ether and separated by filtration and dried.

Yield: 6.9 g = 77.9% Melting point: 139-141 °C Elemental analysis C₁₂H₁₂ClN₅O₂:

Calculated: C: 49.07% H: 4.12 % N: 23.84% Cl: 12.07% Found: C: 48.41 % H: 3.96% N: 23.48% Cl: 12.07%

IR: 1670 cm ¹, 1620cm-¹ Example 3

3-(2-trifluoromethylphenvI)-1.2.3.4-oxatriazole-5-(N-2-propynylcarbamoyl)imine

5.3 g (20 mmole) of 3-(2-trifluoromethylphenyl)-l, 2, 3,4-oxatriazole-5-imine hydro¬ chloride were dissolved in 70 ml of water and 1.8 g (21 mmole) of sodium hydro- 5 gencarbonate were subsequently added while being stirred. After terminated deve¬ lopment of carbon dioxide 70 ml of dichloromethane were added, whereafter the precipitated substance dissolved. Under vigorous stirring 1.62 g (20 mmole) of propargyl isocyanate were added to the mixture and stirring was continued for 4 hours. The precipitated substance was separated by filtration and washed twice with 10 IN hydrochloric acid and twice with water. The substance was stirred with a small quantity of diethyl ether and then separated by filtration and dried.

Yield: 5.2 g = 83.60% Melting point: 191-192°C dec. Elemental analysis C₁₂H_gF₃N₅O₂: 15 Calculated: C: 46.31 % H: 2.59% N: 22.51 %

Found: C: 45.95% H: 2.55% N: 22.10%

IR: 1700-1610 cm ¹ br.

Example 4

3-(3-chloro-2-methylphenyl)- 1.2.3.4-oxatriazole-5-(N-2-Dropynylcarbamoyl)imine

20 24.7 g (100 mmole) of 3-(2-trifluoromethylphenyl)-l,2,3,4-oxatriazole-5-imine hydrochloride were dissolved in 300 ml of water and 8.9 g (106 mmole) of sodium hydrogencarbonate were subsequently added while being stirred. After terminated development of carbon dioxide 300 ml of dichloromethane were added, whereafter the precipitated substance dissolved. Under vigorous stirring 8.0 g (100 mmole) of

25 propargyl isocyanate were added to the mixture and stirring was continued for 30 minutes. The precipitated substance was separated by filtration and washed twice with IN hydrochloric acid and twice with water. The substance was stirred with a small quantity of diethyl ether and then separated by filtration and dried.

Yield: 16.8 g = 57.9% Melting point: 165-166°C dec. Elemental analysis C₁₂H₁₀C1N₅O₂:

Calculated: C: 49.24% H: 3.79% N: 23.93% Cl: 12.11 %

Found: C: 48.98% H: 3.51 % N: 23.89% Cl: 12.52%

IR: 1680-1620 cm ¹

Example 5

3-(2-trifluoromethylphenyl)-1.2.3.4-oxatriazole-5-(N-methylthiomethylcarbamoyl)- imine

5.3 g (20 mmole) of 3-(2-trifluoromethylphenyl)-l , 2, 3,4-oxatriazole-5-imine hydro¬ chloride were dissolved in 70 ml of water and 1.8 g (21 mmole) of sodium hydro¬ gencarbonate were subsequently added while being stirred. After terminated deve- lopment of carbon dioxide 70 ml of dichloromethane were added, whereafter the precipitated substance dissolved. Under vigorous stirring 2.06 g (20 mmole) of methylthiomethyl isocyanate in 45 ml benzene were added to the mixture and stirring was continued for 75 minutes. The precipitated substance was separated by filtration and washed twice with IN hydrochloric acid and twice with water . The substance was stirred with a small quantity of diethyl ether and then separated by filtration and dried.

Yield: 6.41 g = 96,17% Melting point: 179-180°C dec. Elemental analysis C_nH₁₀F₃N₅O₂S: Calculated: C: 39.64% H: 3.02% N: 21.02% S: 9.62%

Found: C: 39.28% H: 2.87% N: 20.65% S: 9.23%

IR: 1665cm^"1 , 1620cm^"1 Example 6

3-(2-trifluoromethylphenyl)- 1.2.3.4-oxatriazole-5-(N-methylsulfinylmethylcarba- movDimine

2.0 g (6 mmole) of 3-(2-trifluoromethylphenyl)-l ,2,3,4-oxatriazole-5-(N-methylthio- methylcarbamoyl)-5-imine were dissolved in 1000 ml of dichloromethane, while stirring at 0-5 °C, whereafter 1.50 g (7 mmole) of m-chloroperoxybenzoic acid slowly were added. After stirring for 40 minutes, the mixture was washed with 100 ml of a saturated aqueous potassium carbonate solution. The organic phase was then separated end evaporated, and the resulting compound was stirred with a small quantity of diethyl ether and then separated by filtration and dried.

Yield: 1.90 g = 90.7% Melting point: 143-144°C dec. Elemental analysis C₁₁H₁₀F₃N₅O₃S:

Calculated: C: 37.82% H: 2.89% N: 20.05% Found: C: 37.34% H: 2.83% N: 19.36%

IR: 1700-1600 cm ¹ br.

PHARMACOLOGICAL TESTS

1. Inhibition of blood platelet aggregation

Compounds according to the invention were tested for their inhibition of clumping together (aggregation) of blood platelets (thrombocytes), which is the first phase of the formation of blood clots (thrombi). Such an inhibition may prevent the formation of blood clots and inhibit the development of new thrombi after a diagnosed throm¬ bus.

The method of demonstrating this effect is a so-called aggregometer measurement, which was first described by Born (Nature (LondJ 194, 927-929, 1962). Citrate stabilized ( 0.38% of sodium citrate, final concentration) venous blood from healthy volunteers is used, who have not used medicine for at least 8 days. Slight centrifu- gation (160 x g for 10 minutes) results in PRP (blood plasma rich in platelets) which is pipetted. PPP (blood plasma poor in platelets) is obtained by an intense centrifu- gation (3000 x g for 10 minutes) of the remaining blood. The light transmission is measured by the aggregometer (CHRONOLOG). PRP allows nearly no light transmission, while PPP allows complete transmission of light. The PRP is placed in the aggregometer at 37 °C while being stirred by a magnet. Addition of a pro- aggregating substance causes the PRP to aggregate gradually and an increasing light transmission takes place at the same time. At complete aggregation a light trans¬ mission corresponding to PPP is obtained. Adenosine diphosphate (ADP) is used as pro-aggregating substance, said substance representing a basic biochemical mecha¬ nism for aggregation of blood platelets. The test substances are incubated for three minutes in PRP placed in the aggregometer at 37 °C during magnetic stirring. A predetermined positively aggregating dosage of adenosine diphosphate (ADP) (2 to 8 μM) is then added. At least three different concentrations of the test substances are tested to demonstrate dosage-dependent inhibition of the aggregation. A so-called IC₅₀- value (that is the concentration inhibiting the aggregation by 50% relative to the control aggregation) is calculated for each test substance by linear regression analysis (log concentration μM as constant ad abscissa and % inhibition as variable ad ordinate). The following known reference substances have been used: nitroglyce¬ rine (GTN), sodium nitroprusside (NNP) and SIN-1 (the active metabolite of molsidomine).

The results for five compounds according to the invention, ten prior art compounds and three reference substances appear from Table 1.

It appears from Table 1 that the compounds according to the invention in general are potent inhibitors of platelet aggregation. Table 1 Anti-platelet activity

Inhibition of platelet aggregation induced by ADP (2-8 μM) in human PRP.

Compound prepared according to IC₅₀, μM

Example No.

1 1.7

2 4.9

3 33

4 2J

6 3J

Prior art compounds

VIII 0.6

X 1.7

XI 0.34

III 1.22

XII 0.39

. I 2.27

VII 2.4

VI 1.95

II 0.46

GTN > 100

NNP 2.8

SIN-1 3.9

Prior Art Compounds relating to the above Table 1 and subsequent Tables 2-9

I 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-2,5-dimethoxy- phenylcarbamoyl)imine .

II 3-(2-trifluoromethylphenyl)- 1 ,2,3 ,4-oxatriazole-5-(N-cyclohexylcarbamoyl)- imine.

Ill 3-(3-chloro-2-methylphenyl)-l,2,3,4-oxatriazole-5-(N-4-methoxyphenyl- sulfonyl)imine. IV 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-4-methoxyphenyl- carbamoyl)imine.

V 3-(3-chloro-2-methylphenyl)-l,2,3,4-oxatriazole-5-(N-l-adamantylcarba- moyl)imine.

VI 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-cyclohexylcarba- moyl)imine.

VII 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-l-methylcyclohexyI carbamoyl)imine.

VIII 3-(2-trifluoromethylphenyl)-l,2,3,4-oxatriazole-5-(N-methylsulfonylmethyl- carbamoyl)imine.

IX 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-carbethoxy-carba- moyl)imine.

X 3-(3-chloro-2-methylphenyl)-l,2,3,4-oxatriazole-5-(N-cyanomethylcarhj moyl)imine.

XI 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-4-methylbenzene sulfonyl)imine.

XII 3-(3-chloro-2-methylphenyl)-l ,2,3,4-oxatriazole-5-(N-3,4,5-trimethoxy- benzoyl)imine.

2. Desaggregation of platelet aggregates

Compounds according to the invention were tested for their ability to split (i.e. des¬ aggregate) the ADP-induced platelet aggregates when added to the test tubes at the time of complete aggregation. The results for five compounds according to the invention and one prior art com¬ pound are seen in Table 2.

Table 2 Desaggregation of platelet aggregates induced by ADP (2-8 μM) in human PRP.

Compound prepared according to Example No. μM % desaggregation

1 3 60

2 1 55

3 3 24

4 3 53

4 30 61

6 1 75

6 30 78

Prior art compound

XI 1 35

It appears form Table 2 that the compounds according to the invention strongly desaggregate already established platelet aggregates.

3. Desaggregation and fibrinolvsis in vitro

Compounds according to the invention were tested for desaggregation and fibrino- lysis of thrombin (THR)-induced aggregation and platelet-rich fibrin clot formation in human PRP.

A modification of the method described by Lam et al. (J. Pharm. Exp. Ther. 259. 1371-1378, 1991) was employed. PRP was prepared as stated above. THR was added to the PRP in the minimal amount (0.2-0.6 U/ml PRP) causing platelet aggregation (a gradual increase in light transmission as above) and fibrin formation and polymerization (a rapid increase in light transmission). The end result is a distinct white fibrin clot with incorporation of the platelet aggregates allowing an almost 100% light transmission (like PPP). This platelet-rich fibrin clot is the second phase in the formation of blood clots (thrombi). A desaggregation and fibrinolysis together with an inhibition of platelet aggregation will improve throm¬ botic disorders considerably.

Compounds according to the invention were added to the test tubes with or without the known fibrinolytic agent streptokinase (SK, 2000 U/ml PRP) subsequent to the formation of the platelet-rich clot. Desaggregation and fibrinolysis was recorded as a decrease in light transmission following dissociation of the platelet-rich fibrin clot and the released platelet aggregates. When the decrease in light transmission was maximum, the percentage of desaggregation/fibrinolysis was calculated as compared to the preceding total increase in light transmission. The EC₃₀-value (i.e. the concentration inducing 30% desaggregation fibrinolysis) was calculated by linear regression analysis.

The results for five compounds according to the invention, two prior art compounds and two reference substances are seen in Table 3.

Table 3 Desaggregation/fibrinolysis

Aggregation and fibrin formation were induced by THR (0.2-0.6 U/ml PRP). Test compounds were added after SK (2000 U/ml PRP).

Compound prepared according to

Example No. IC₃₀ μM

1 5.8

2 6J 3 28 4 11 6 1.9

Prior art compounds vm 1.9

X 2.8

GTN 74 SIN-1 4.8

It appears from Table 3 that the compounds according to the invention in combi¬ nation with SK induce desaggregation and fibrinolysis of platelet-rich fibrin clots. SK alone had no effect in this test. Higher doses (100 μM) of the compounds according to the invention induced desaggregation and fibrinolysis without SK. This was not the case for the reference substances.

4. Fibrinolysis in rats ex vivo

Compounds according to the invention were tested for fibrinolytic activity one hour after oral administration of 10 mg/kg to male rats.

The euglobulin clot lysis time (ECLT) method as described by von Kaulla & Schultz (Am. J. Clin. Pathol. 29, 104-112, 1958) was employed. Blood samples were taken from the carotid artery into tri-sodium citrate (3.15% w/v) in a ratio of 9: 1 and centrifuged at 15000 x g for one minute to produce PPP. Distilled water was added (14 ml per ml PPP) and the pH adjusted to pH 5.4 by bubbling with CO₂ gas (approx. 3 min.) This procedure causes precipitation of the euglobulin fraction, while the acidity destroys the remaining plasminogen activator inhibitor (PAI) activity. After a second centrifugation at 15000 x g for one minute, the supernatant was discarded and the euglobulin precipitate dissolved in 1 ml sodium/potassium phosphate buffer.

At time 0, two units of human thrombin (THR) (10 μl; 200 units of THR per ml 0.05 M CaCl₂) were added to a 0.3 ml euglobulin fraction to induce clot formation.

Euglobulin clots in duplicates were incubated at 37 °C and the time for complete lysis to occur recorded.

The results for three compounds according to the invention and one prior art compound are seen in Table 4.

Table 4 Fibrinolytic effect of four compounds in rats ex vivo, as measured by shortening of ECLT one hour after administration of 10 mg/kg p.o.

Compound prepared % Fibrinolytic effect according to Example No. (% shortening of ECLT)

3 70 2 70 4 45

Prior art compound

VIII 60

It appears from Table 4 that the compounds according to the invention possess potent fibrinolytic properties after oral administration to rats. 5. Stability in vitro and relative bioavailability

The stability at 25 °C and 37 °C and the oral absorption % in rats for five com¬ pounds according to the invention and nine prior art compounds are shown in Table 5.

Table 5

Compounds prepared Stability Percentage according to Example No. 25 °C 37°C Oral Absorption

1 OK OK 51

2 OK OK 40

3 OK OK 60

4 OK OK 60

6 OK OK 64

Prior Art Compounds

XI OK OK 0

III OK OK 0

XII OK OK 0

I OK OK 15

VI ÷ ÷ 33

II ÷ ÷ 19

IV OK OK 0

V OK OK 0

VIII OK OK 20

It appears from Table 5 that the compounds according to the invention in general are both stable in vitro and well-absorbed orally.

6. Effect on blood pressure in rats after oral administration

Compounds according to the invention were tested for effect on arterial blood pressure in anaesthetized rats after oral administration of 10 mg/kg.

The results for five compounds according to the invention and five prior art com¬ pounds are seen in Table 6. Table 6 Effect on blood pressure in rats after 10 mg/kg p.p.

Compounds prepared according Hypotensive activity to Example No. mm Hg

1 10-15 2 15-25 3 25-30 4 25-30 6 15-25

Prior art compounds

VIII 15-25

XII 0

I 0

XI 0 III 0

It appears from Table 6 that the compounds according to the invention lower the arterial blood pressure after oral administration to rats. This is an indirect proof of oral absorption. The duration of the hypotension was 60 to 90 minutes.

7. Relaxation effect on the trachea

Compounds according to the invention were tested for their ability to relax a pre- contracted trachea. A contraction of the respiratory passages in combination with a swelling of the mucous membrane therein represents an important factor for asthma¬ tic conditions. Relaxation or dilation of the contracted respiratory passages will improve the asthmatic condition.

A modification of the method described by Emmerson & MacKay (J. Pharm. Pharmacol 31, 798, 1979) was employed. An isolated trachea from a guinea pig is used. After preparation of a strip which has maintained the circular musculature, the organ strip is divided into two parts of equal size. The two tracheal strips are suspended in a superfusion-cascade system superfused with Krebs buffer at 37°C, constantly bubbled with carbogen (95% ^ and 5% CO₂). The strips are connected to a transducer recording the contraction and relaxation of the organs by means of a recorder. After an equilibration time of about 3 hours the tracheal strips are tested for their sensitivity (contractility) to histamine, a bolus (3.13 μg) being added right above the organ strips. If the contraction is satisfactory, histamine (17 μM) is added to the Krebs buffer. The tracheal strips are now constantly susperfused with hista¬ mine and slowly develop a permanent contraction ("asthma"). The test compounds are added right above the organ strips in bolus form. After maximum relaxation, the strips automatically revert to the previous contraction state. At least three different doses of the test compounds are tested to demonstrate a dose-dependent relaxation of the tracheal strips. An ED₅₀- value (that is the dose relaxing the organ by 50% relative to the maximum relaxation) is calculated for each test compound by means of a linear regression analysis (log dose (nM) as a constant ad abscissa and % rela¬ xation as variable ad ordinate). SIN-1 and SNAP are used as reference compounds.

The results for five compounds according to the invention, five prior art compounds and for two reference compounds are listed in Table 7.

Table 7 Relaxation of guinea pig tracheal strips precontracted with his¬ tamine (17 μM). Test compounds were added in bolus form.

Compound ED50 mmole

SIN-1 1298 SNAP 2293

Compounds prepared according to Example No.

1 194 2 240 3 209 4 263 6 418 Table 7 continued

Compound ED₅₀ nmole

Prior art Compounds

VIII 497

XI 82

III 94

XII 256

I 26230

It appears from Table 7 that the compounds according to the invention possess a strong relaxing effect on the pre-contracted tracheal strips. The potencies are higher than those of SIN-1 and SNAP. In addition, the compounds according to the invention have a longer lasting effect than the reference substances.

The compounds according to the invention both inhibit inflammatory cell infiltration (PMN's) and bronchoconstriction in guinea pigs (data not shown).

8. Pharmacological conclusion

The compounds according to the invention have pharmacological properties making them suitable for the treatment of thrombosis (blood clots) and asthma.

Thrombosis

The inhibition of platelet aggregation and adhesion to vessel walls, the desaggre- gation of platelet aggregates, the desaggregation pjus fibrinolysis of platelet-rich fibrin clots and the fibrinolytic activity ex vivo make the compounds interesting for the prevention of thrombosis as well as for the dissolution (treatment) of already established thrombi (blood clots). The stability of the compounds according to the invention together with their satisfactory oral absorption make them further suitable. Asthma

The relaxation of the respiratory passages in vitro and in vivo together with the inhibition of the leucocyte function make the compounds interesting for the treat¬ ment of asthma, whereby the latter property will inhibit the inflammatory swelling of the mucous membrane in the respiratory passages.

Beyond the pharmacological properties already mentioned the compounds of the invention also have a relaxing effect on other smooth muscle cells in for instance arteries, veins and cavernous tissue.

9. Bioavailability

If the compounds according to the invention shall become important pharmaceuticals in the future, they must be administrated orally, as for instance a tablet or a capsule. Therefore, the compounds according to the invention were tested in male rats for the extent of absorption (oral bioavailability, %F) from the gastrointestinal tract (stomach, small intestine and large intestine). The single dose given orally (p.o.) was compared to a single dose given intravenously (i.v).

Intravenous dosing: One rat per blood sampling point was intravenously dosed with a dose of 10 mg/kg of one of the compounds. A blood sample was taken approximately 2, 5, 10, 15, 20, 30 or 45 minutes or 1, 1,5, 2, 3, 4 or 6 hours after i.v.-administra- tion.

Per oral dosing: One rat per blood sampling point was orally dosed with a dose of 100 mg/kg given as a suspension. A blood sample was taken approximately 10, 20, 30 or 45 minutes or 1, 1,5, 2, 3, 4, 5, 6, 7, 8 or 18 hours after oral administration.

Plasma was separated from each blood sample immediately. All the plasma samples were analyzed by UV-HPLC after a solid-phase extraction procedure. The concen¬ tration (μg/ml plasma) of the intact compound was calculated from a standard curve. The limit of detection in the analysis was 0.05 μg/ml plasma for each of the tested compounds. The area under the concentration versus time curve (AUC) was calcu- lated according to trapezoidal rule for both routes of administration. The oral bioavailability, %F compared to intravenous administration was calculated as (AUC_po*dose_iv/AUV_iv*dose_po)*100%. After oral administration, the plasma sample containing the highest concentration of intact compound (C_max) and the time for this concentration (T_max) were noticed.

The in vitro stability of the compounds according to the invention in rat plasma at 37 °C was measured to make sure that it is reasonable to look for intact compounds in plasma after oral and intravenous administration. If the compounds are destroyed rapidly in plasma (T^ <0.2 hours), it is impossible to determine the oral bioavail¬ ability, if any, because the intact compound will be destroyed in any of the plasma samples, before the analysis has taken place.

A volume of rat plasma was spiked with one of the compounds according to the invention and kept at 37 °C. At different points of time, samples were drawn for analysis for intact compounds. The in vitro half life (T^) was calculated.

Table 8 shows the result from these experiments. Besides the compounds according to the invention, five prior art compounds were tested in the same way.

It appears from Table 8 that the compounds according to the invention in general are superior to the prior art compounds with respect to oral bioavailability. For three of the prior art compounds, none of the plasma samples taken after oral administration contained measurable amounts of intact dosed compound. Table 8 Bioavailability and in vitro plasma stability

Δ

Route of AUC c T in

Compound administration (μg/ml)h (μg/ml) (h) % vitro

F (h)

Compound prepared according to Example No.

1 i.v. 2.34 p.o. 12.0 9J0 1.0 51 5.6

2 i.v. 8.86 p.o. 33.9 17.8 0.33 40 11.5

3 i.v. 2.13 p.o. 17.7 3.63 1.5 60 4.3

4 i.v. 4J1 p.o. 24.2 2.72 2 60 9.7

Prior art compound

I i.v. 4.23 p.o. 6.42 1.1 1.5 15 5.7

II i.v. 4.37 p.o. 8.24 2.35 2.0 19 4.3

III i.v. 0.40 p.o. N.D. N.D.* - 0 4.0

IV i.v. 3.82 p.o. N.D. N.D.* ~ 0 6.5

V i.v. 0.81 p.o. N.D. N.D* - 0 23.0

N.D.* = Not detectable, all plasma samples contained less than 0.05 μg/ml 10. Stability

Stability of the compounds according to the invention were checked at 25 °C and 37 °C. A sample of each of the compounds was placed in a freezer, in an oven at 25°C and in an oven at 37°C. After some time and at each temperature, a small portion of each of the compounds was analyzed at UV-HPLC. The content in the samples stored at 25 °C and 37 °C was compared to the content in the same sample stored in the freezer at -18°C. The compounds were stable at -18°C. The time it takes to break down 10% of the compounds is listed in Table 9. Three prior art compounds were also tested for their stability at 25 °C and 37°C. All the results are shown in Table 9.

Table 9 Stability at 25 °C and 37 °C (time for 10% breakdown)

COMPOUND 25 °C 37°C

Compound prepared according to Example No.

1 > 50 days > 50 days

2 > 50 days > 50 days 3 > 100 days > 100 days 4 > 100 days > 100 days 6 > 42 days > 42 days

Prior art compounds

VI 3 days 1 day VII 4 days 1 day IX 1 day < 1 day

It appears from Table 9 that the compounds according to the invention are much more stable compared to the prior art compounds. The stability is a very importent parameter from a practical pharmaceutical point of view. In order to be able to produce dispensable oral forms, Le. tablets with acceptable shelf-life at stores and at the patients' site. The invention has been described with reference to preferred embodiments. Many modifications may, however, be carried out without thereby deviating from the scope of the invention.

Claims

Claims

1. 3- and 5-substituted l,2,3,4-oxatriazole-5-imine compounds of the general formula I

c h a r a c t e r i s e d in that R¹ is the same or different groups and represents alkyl or alkoxy groups having 1 to 3 carbon atoms, halogen, trifluoromethyl, nitro, cyano, phenyl or alkylsulphonyl groups, n is 1 to 3;

X is - C(O)NH-, -SO₂-, -C(O)- or a direct bond;

Y means -S-Z; -SO-Z; -S⁺(Z)₂, A^"; N⁺(Z)₃,A^"; -C(Z) = CZ₂; -C ≡ C-Z or a group

wherein b = c = N or C-Z; d = N-Z, O or S, and A^" is any pharmaceutically acceptable anion; and

Z is the same or different groups and represents H, cyano, nitro, trifluoromethyl, -CH = CH₂, -C -s CH, straight or branched C_rC₆-alkyl, C₃-C₇-cycloalkyl, phenyl or optionally substituted phenyl. 2. A compound as claimed in claim 1, characterised in that it is 3-(2- trifluoromethylphenyl)-l,

2,3,4-oxatriazole-5-(N-2-propenylcarbamoyl)imine.

3. A compound as claimed in claim 1, characterised in that it is 3- (3-chloro-2-methylphenyl)-l,2,3,4-oxatriazole-5-(N-2-propenylcarbamoyl)imine.

4. A compound as claimed in claim 1, characterised in that it is 3-

(2-trifluoromethylphenyl)-l,2,3,4-oxatriazole-5-(N-2-propynylcarbamoyl)imine.

5. A compound as claimed in claim 1, characterised in that it is 3- (3-chloro-2-methylphenyl)-l,2,3,4-oxatriazole-5-(N-2-propynylcarbamoyl)imine.

6. A compound as claimed in claim 1, characterised in that it is 3- (2-trifluoromethylphenyl)- 1,2,3, 4-oxatriazole-5-(N-methylthiomethylcarbamoyl)- imine.

7. A compound as claimed in claim 1, characterised in that it is 3- (2-trifluoromethylphenyl)-l,2,3,4-oxatriazole-5-(N-methylsulfinylmethylcarbamoyl)- imine.

8. A pharmaceutical preparation, characterised in that it comprises as an active ingredient a compound of the general formula I as claimed in claim 1 together with a pharmaceutically acceptable carrier or diluent.

9. A process for the preparation of 3- and 5-substituted 1 ,2,3,4 oxatriazole-5- imine compounds of the general formula I as claimed in claim 1, characte- r i s e d by ring closing a 1-arylthiosemicarbazide derivative of the general formula π

wherein R¹ and n have the same meaning as in formula I, by treatment with alkyl nitrite having 1 to 6 carbon atoms or alkali metal nitrite under acidic conditions at 0 to 10°C, whereafter the resulting salt is converted into the corresponding free compound, which is subsequently reacted with a compound of the type ClSO₂-CH₂- 5 Y or O=C=N-CH₂-Y, wherein Y has the same meaning as in formula I.

10. The use of 3- and 5-substituted l,2,3,4-oxatriazole-5-imine compounds of the general formula I as claimed in claim 1 for the preparation of a medicament for the treatment of asthma.

11. The use of 3- and 5-substituted l,2,3,4-oxatriazole-5-imine compounds of 10 the general formula I as claimed in claim 1 for the preparation of a medicament having an inhibiting effect on the blood platelet aggregation.

12. The use of 3- and 5-substituted l,2,3,4-oxatriazole-5-imine compounds of the general formula I as claimed in claim 1 for the preparation of a medicament being effective against impotence.

15 13. The use of 3- and 5-substituted l,2,3,4-oxatriazole-5-imine compounds of the general formula I as claimed in 1 for the preparation of a medicament being effective against pre-eclampsia.