GB2088371A - Tetrahydroisoxazolopyridine Compounds - Google Patents

Abstract

Novel compounds of the formula: <IMAGE> wherein R is an alkyl group, branched or unbranched, having from one to seventeen carbon atoms inclusive, a phenyl group optionally substituted with one or two groups selected from lower alkyl, lower alkyloxy and halogen, a phenylalkyl group, lower alkyloxy group or a -N R<1>R<2> group wherein R<1> and R<2> each are hydrogen, lower alkyl, phenyl or cyclohexyl, as well as pharmaceutically acceptable acid addition salts thereof, are shown to have gamma-aminobutyric acid (GABA) related activity and are indicated for use in treating GABA- system malfunction-related diseases such as epilepsy, parkinsonisme, schizophrenia, Huntington's chorea, diseases involving malfunction of the pituitary hormones, and cerebral arterioschlerosis. They moreover show strong analgesic and myotonolytic effects.

Description

SPECIFICATION Heterocyclic Compounds The present invention relates to hitherto unknown compounds of the formula

wherein R is an alkyl group, branched or unbranched, having from one to seventeen carbon atoms inclusive, a phenyl group optionally substituted with one or two groups selected from lower alkyl, lower alkyloxy and halogen, a phenylalkyl group, lower alkyloxy group or a --NR'R2 group wherein R' and R2 each are hydrogen, lower alkyl, phenyl or cyclohexyl, as well as pharmaceutically acceptable acid addition salts thereof, which are shown to have gamma-aminobutyric acid (GABA) related activity and are indicated for use in treating GABA system malfunction-related diseases such as epilepsy, parkinsonisme, schizophrenia, Huntington's chorea, diseases involving malfunction of the pituitary hormones, and cerebral arterioschlerosis.

They moreover show strong analgesic and myotonolytic effects.

The compounds of Formula I have been broadly disclosed in for example European Patent Application No. 78 100 191.2 where they are said to serve as intermediates for the preparation of the compound THIP (4,5,6,7-tetrahydro isoxazolo [5,4-c] pyridine-3-ol) which has been shown to possess GABA-related activity. There are, however, no actual examples of such intermediates.

In accordance with the present invention it has now surprisingly been found that compounds of Formula I as well as their pharmaceutically acceptable acid addition salts show GABA-related activity at the same level as does the compound THIP, and some of the novel compounds of the invention also show a prolonged effect compared with THIP. They moreover show pronounced analgesic and myotonolytic effects.

The invention further relates to novel pharmaceutical compositions containing as an active ingredient a compound of Formula I or a pharmaceutically acceptable acid addition salt thereof.

The present invention also comprises a method for the preparation of compounds of Formula I, and methods for the treatment of GABA system malfunction-related diseases, a method for alleviating pain of varying aetiology and a method of treating myotonic conditions (e.g. in inducing muscle relaxation or in treating muscle spasm or muscular spasticity.

The terms "lower alkyl" and "lower alkyloxy" groups comprise such groups having from one to six carbon atoms inclusive.

As examples of pharmaceutically acceptable salts of the compounds of the Formula I may be mentioned salts with inorganic acids, e.g. hydrochlorides, hydrobromides, nitrates, sulfates, phosphates and the like, or with organic acids, such as acetates, propionates, glycolates, malonates, maleates, succinates, fumarates, tartrates, citrates, oxalates, benzoates, pamoates, methane sulfonates, ethane sulfonates, benzen sulfonates, toluene sulfonates and the like, which salts may be prepared by procedures known per se, e.g. by adding the acid in question to the base, preferably in a solvent.

According to the method of the invention a compound of the formula

wherein R3 is an amino-protecting group readily removable, is acylated with a reactive derivative of an acid of the formula R COOH, wherein R is as defined above, whereupon the protecting group R3 is split off and the compound of Formula I isolated as the free base of a pharmaceutically acceptable acid addition salt thereof.

As protecting groups R3 are preferably used groups which may subsequently be split off quite easily such as alkyloxycarbonyl groups. Preferably tertiary butyloxy carbonyl-substitution was used according to the method described by Tarbell et al, Proc. Nat. Acad. Sci. 69(3), p. 730, 1 972.

The O-acylation according to the method of the invention is carried out in conventional manner in an organic solvent, preferably in the presence of a tertiary amine, at low temperatures.

Some of the starting materials of Formula II are conveniently prepared by reaction of 3-hydroxy4,5,6,7-tetrahydroisoxazolo [5,4-c]-pyridine (in the following called THIP for short) with a dialkyldicarbonate, preferably ditertiary butyl dicarbonate.

After the acylation according to the method of the invention the protecting group R3 may be removed under mild conditions such as controlled hydrolysis. When R3 is a tertiary butyloxy group it may conveniently be removed by reaction with anhydrous trifluoroacetic acid at about 0 Centigrade.

The method of the invention is illustrated by the following working examples, which may not be construed as limiting.

Example 1 3-Hydroxy-6-(t-butyloxycarbonyl)-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine THIP,HBr (11.1 g) was suspended in 50 ml of dioxane and 25 ml of water. By addition of NaOH (4.0 g) in 25 ml of water under ice cooling a clear solution was obtained. Di-t-butyl dicarbonate (11.0 g) was added and the temperature was raised to 250C. The mixture was vigorously stirred for another 1.5 hour. Ethyl acetate (300 ml) and water (100 ml) were added and the pH of the aqueous solution was adjusted to --3.0 with a KHSO4 solution. The ethyl acetate phase was separated and washed with 2x25 ml of water, dried over anhydrous MgSO4 and the solvent evaporated leaving 11.8 g (98%) of product.M.P. 134-1 360C, IR (KBr):v,, 2400-3200 (broad, complex bands), Ic=o 1690 cm-1.

Example 2 3-Qcetyloxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine, Oxalate.

The tertiary butyloxycarbonyl protected THIP (4.8 g) was dissolved in 100 ml of dry tetrahydrofuran, triethylamine (2.1 g) added and the solution cooled to 0C. A solution of acetylchloride (1.8 g) in 20 ml of dry tetrahydrofuran was added dropwise (t < 50C). The solution was finally stirred for 1 5 minutes at room temperature, triethylammonium hydrochloride filtered off and tetrahydrofuran evaporated. Yield: 4.2 g (75%) of 3-acetyloxy-6-(t-butyloxycarbonyl)-4,5,6,7- tetrahydroisoxazolo [5,4-c]pyridine. M.P. 93-97 0C.

This product (3.0 g) was added to 10 ml of ice cooled anhydrous trifluoroacetic acid. The mixture was stirred until the C02-evolution had stopped. Excess trifluoroacetic acid was evaporated and the resulting oil dissolved in a few ml of dry acetone. Anhydrous oxalic acid (2.5 g) in 10 ml of dry acetone was added. Under stirring and cooling the oxalate precipitated. Yield: 2.2 g (76%). M.P. 217-21 90C (dec.) (Found: C 44.43; H 4.59; N 10.07; C,oH,2N207 requires: C 44.12; H 4.45; N 10.29%).

Example 3 3-Benzoyloxy-4,5,6,74etrahydrnisoxazolo[5,c] pyridine, Oxalate.

The intermediate 3-benzoyloxy-6-(t-butyloxy carbonyl)-4,5,6;7-tetrahydroisoxazoloL5,4- c]pyridine was prepared as an oil in 74% yield according to the procedure of Example 2. The t-BOC group was split off as described above yielding 71% of the oxalate, M.P. 1 95--1 97 C (dec.) (Found: C 53.51; H 4.35; N 8.64: C15H,4N207 requires: C 53.89; H 4.23; N 8.38%).

Example 4 3-(2,6-dimethylbenzoyloxy)-4,5,6,7-tetrahydroisoxazolo [5,4-cl pyridine, Oxalate.

The intermediate 3-(2,6-dimethylbenzoyloxy)-6-(t-butyloxycarbonyl)-4,5,6,7- tetrahydroisoxazolo[5,4-c] pyridine was prepared in 80% yield according to the procedure of Example 2. M.P. 147-1 490C. The t-BOC group was split off as described above yielding 84% of the oxalate.

M.P. 188-1 900C (dec.). (Found: C 55.51; H 5.16; N 7.62; Cr7Hr8N207 requires: C 56.34; H 5.02; N 7.73%).

Example 5 Ethyl 3-(4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridyl)carbonate, Oxalate.

The intermediate ethyl 6-(t-butyloxycarbonyl)-3-(4,5,6,7-tetrahydroisoxazoio [5,4-ci pyridyl) carbonate was prepared in 68% yield from t-BOC THIP and ethyl chloroformiate according to the procedure of Example 2. M.P. 95-970C. The t-BOC group was split off as described above yielding 97% of the oxalate. M.P. 154-1 570C (dec.). (Found: C 43.76; H 4.84; N 9.27; C,1H,4N208 requires: C 43.71; H 4.68; N 9.27%).

Example 6 3-(4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridyl)N,N-dimethylcarbamate Oxalate.

t-BOC THIP ( 4.8 g) was dissolved in 100 ml of dry tetrahydrofuran and triethylamine (2.1 g) was added. Under ice cooling N,N-dimethylcarbamoylchloride (2.2 g) in 20 ml of dry tetrahydrofuran was added. The mixture was refluxed for 2 hours and finally the tetrahydrofuran was evaporated. The residue was dissolved in 200 ml CH2CI2 and washed with 2x250 ml ice cooled 0.1 m K2CO3 solution.

The CH2CI2-phase was dried (MgS04) and the solvent evaporated. The remaining oil was submitted to column chromatography (Silicagel 60 "Merck"), 6-(t-butyloxycarbonyl)-3-(4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridyl)N,N-dimethylcarbamate was eluted with ether. Yield; 2.4 g (39%). M.P. 68-720C. The t-BOC group was split off as described above yielding 94% of the oxalate. M.P. 240-2420C (dec.).

(Found: C 43.79; H 5.35; N 13.35; C,1H,5N307 requires: C 43.85; H 5.03; N 13.95%).

Example 7 In the same manner as described in Example 6 was prepared 3-(4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridyl)-N,N-diethylcarbamate, oxalate melting at 21 6- 21 8 degrees Centigrade, and 3-(4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridyl)-N-methyl-N-phenylcarbamate, oxalate melting at 1 83-1 84 degrees Centigrade by substituting N,N-dimethylcarbamoylchloride with N,Ndiethylcarbamoylchloride and N-methyl-N-phenylcarbamoylchloride.

The compounds of Example 7 were tested for analgetic activity according to a hot-plate test in mouse described by Chen and Beckmann, Science, vol. 13, page 631, 1951, and showed ED50 figures of 1 6 and 1 8 mg/kg p.o. respectively, and further against arthritis pain in the rat according to a method of A. W. Pircio et al, Eur. J. Pharmacoi., vol. 31, pg. 207215, 1975, showing ED50 figures of ca. 56 (1 hour), 42 (3 hours), 42 (5 hours) and 25 (1 hour), 25 (3 hours), 15 (5 hours) respectively, by peroral administration.

In the same manner were prepared 3-Butanoyloxy-4,5,6,7-tetrahydroisoxazoío[5,4-c]pyridine citrate.

3-Capryloxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine succinate.

3-Palmitoyloxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine maleate.

3-(4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridyl)-N-methyl-N-cycloheXyl carbamate-acetate.

The compounds of Formula I were tested according to standard reliable test methods, which are described in the following: Isoniazide Antagonism Mice, male, 20-25 g Isoniazide 300 mg/kg s.c.

Macrolon cages type II Dosage and Procedure The test compound is injected i.p. in the doses 0, 1/2, 1/8 and 1/32 of the determined "i.v.

LD50". In case of insoluble substances the doses 0, 1/4, 1/16 and 1/64 of the determined "i.p. LD50" are used.

Five mice are used for each dose level.

Immediately after administration of test substance, isoniazide 300 mg/kg is injected s.c. This dose of isoniazide induces intermittent tonic clonic seizures within 60 minutes.

The calculations are performed as an "on line procedure" on the EDP-terminal. The results are recorded as % increase in time until convulsions occur and in addition the least dose (MED) which shows significant effect (minimal effective dose, calculated by means of Van der Waerden-test).

ED50 is determined as the dose in mg/kg which increases the time 50%.

Mouse Grid Shock Mice, male, 20-23 g.

The mouse grid consists of a perspex cage with wire grid bottom and a perspex lid, on which is placed a microphone sensitive to the frequency of a mouse-squeak. A stimulator with motordriven potentiometer applies a sequence of square wave impulses of continuously increasing milliamperage to the grid. Frequency of impulses 20 cycles/sec., duration 5 msec. Milliamperage is recorded on a digital amperemeter connected to the stimulator. Activation of the microphone by a mouse-squeak cuts off the current and the final milliamperage appear on the meter.

Dosage and Procedure The test substance is given i.p. in the doses 1/2, 1/4 and 1/8 of the determined "i.v. LD50". For insoluble substances the doses 1/4, 1/8 and 1/16 of the "i.p. LD50" are used.

Five mice are used for each dose level. Each mouse serves as its own control Prior to the administration of test substance the animals are placed on the grid one at a time and the pain threshold is determined by increasing the current intensity until the mouse squeaks. The pain threshold may be read on the milliamperemeter.

Fifteen minutes and 30 minutes after administration of test substance the mice are tested again and the pain thresholds recorded. Furthermore the test substance may be tested after oral administration in the doses 1/1, 1/2 and 1/4 of "the i.v. LD50". and the pain threshold is determined before and 30 min. after the administration. Insoluble test substances are tested orally in the doses of 1/2, 1/4 and 1/8 of the "i.p. LD50".

Analgesic effect is present when the pain threshold is increased over the pre-dosing value (control value). The results are stated as % increase in pain threshold calculated on the basis of the control value. The registration can also be done as an online procedure. In this case the punching instruction and punching cards will be provided automatically and the results will be registered as a minimal effective dose (MED) determined after van der Waerden's X-test.

'H-GABA binding to rat brain membranes Rats 125-2O0g 0.32 M sucrose (made fresh every day) 10% Triton X-1 00 0.05 N Tris-citrate buffer (pH--6.8) 6.05 g Trisma (R) (base) 3.502 citric acid, H20 to 1 liter of water 3H-GABA=aminobutyric acid, y-2,3-3H(N) approx.

35 Ci/mmol, from New England Nuclear (diluted daily to 1 ,uM in water) Procedure A. Preparation of Membranes Rats are killed by a blow to their head, exsanguinated and their brains removed and cooled in icecold saline. After rinsing and weighing two brains are pooled and homogenized in 40 ml of icecold 0.32 M sucrose using a motordriven homogenizer with teflon pestle (6 strokes up and down, slow speed rotation).

The samples are centrifuged for 10 min. at 900 g, and thereafter the supernatants are centrifuged for 20 min. at 17000 g (40C). To each pellet are added 20 ml of water, and the samples are homogenized (Ultra Turrax) for 30 sec. After addition of further 10 ml water the samples are centrifuged for 20 min. at 8500 g (4 C). Two thirds of the supernatant are discarded, and the light brown upper part of the pellet is whirled up by hand. This supernatant is centrifuged for 30 min. at 37500 g (40C). The pellet is homogenized (Ultra Turrax) for 1 min. in 10 ml of water. The sample is divided into two glasses and a further 25 ml water are added to each (each glass now contains membranes from one brain in 30 ml water).These samples are centrifuged for 30 min. at 37500 g (40 C), and the pellets are frozen in acetone/dry ice, corked and stored frozen until use (at least one night).

B. Pretreatment of Membranes On the day of measurements the pellet is frozen in acetone/dry ice for 20 min., and 25 ml of water are added before homogenization (Ultra Turrax) for 1 min. 25 ml of 0.05 N Tris-citrate buffer and 250 yl of 10% Triton X-100 are added, and the sample is incubated for 30 min. at 370C, and centrifuged at 37500 g for 20 min. (40 C). The supernatant is discarded and the pellet is suspended in 0.05 N Tris-Citrate buffer (Ultra Turrax, 75 sec.) to a concentration of 30 mg original wet weight/ml.

The suspension is placed at room temperature for 20 min. and thereafter kept on ice.

C. Binding Assay Incubation tubes in duplicate receive on ice 780,ul of water, 200 yl of drug dissolved in water, 20 pl of 1,aM 3H-GABA (final concentration of 3H-GABA in tubes=10 nM) and 1000,ul of the membrane suspension. After incubation on ice for 5 min., the samples are centrifuged at 31,000 g for 1 8 min. The supernatant is discarded and the pellet is carefully flushed with 3x5 ml icecold water. Additional water are carefully wiped away with soft paper. One ml of Soluene 350 is added, and the samples are incubated for 30 min. at 370C. Ten ml of Instagel or Lumagel containing 10 ml of acetic acid per 1 are added, and the radioactivity are determined by liquid scintillation counting. The unspecific binding of 3H-GABA is determined by incubating the samples with 1 mM of GABA.

Each series consist of 8 duplicates (1 control, 1 containing 1 mM GABA and one to two series of test compounds in 3 to 6 concentrations).

The means of controls and 1 mM GABA samples are calculated. The measured cpm are plotted against drug concentration on semilogarithmic paper, and the best fitted s-shaped curve drawn. The IC50-values are determined as the concentrations, at which the binding is 50 per cent of the total binding minus the unspecific binding.

The results obtained appear from the following table, where the test substances are indicated by the number of the Example describing the preparation of the substance in question.

The corresponding test results forTHIP are indicated as references.

Table 1

Test Isoniazide Mouse substance antagonism grid shock 3H-GABA binding 1C50 Example 2 2.9 + 2.8 . 10-7 Example 3 3.2 + 3.3. 10-7 Example 4 8.6 + 5.3. 10-6 Example 5 10.8 + 8.2 . 10-7 Example 6 1.2 + 5.5 . 10-5 THIP 1.3 + 1710-7 The compounds of formula I and the non-toxic acid addition salts thereof may be administered to animals such as dogs, cats, horses, sheeps or the like, including human being, both orally and parenterally, and may be used for example in the form of tablets, capsules, powders, syrups or in the form of the usual sterile solutions for injection. Results upon administration to human beings have been very gratifying.

Most conveniently the compounds of Formula I are administered orally in unit dosage form such as tablets or capsules, each dosage unit containing a non-toxic acid addition salt of one of the said compounds in an amount of from about 5 to about 100 mg, most preferably, however, from about 10 to 50 mg, calculated as the free amine, the total daily dosage usually ranging from about 20 to about 200 mg. The exact individual dosages as well as daily dosages in particular case will, of course, be determined according to established medical principles under the direction of a physician.

When preparing tablets, the active ingredient is for the most part mixed with ordinary tablet adjuvants such as corn starch, potato starch, talcum, magnesium stearate, gelatine, lactose, gums, or the like.

When the compound of Formula I is a free amine, preferably R is a higher alkyl group having from 8-1 7 carbon atoms inclusive, the composition may advantageously be an oily solution for injection, and such solutions often have a very prolonged effect when compared with the corresponding unesterified compound.

Typical examples of formulas for compositions containing 3-acetoxy-4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine (called Lu 18-013 for short) as the active ingredient are as follows: 1. Tablets containing 10 milligrams of Lu 18-013 calculated as the free base in the form of the oxalate; Lu 18-013: 10mg lactose: 37 mg potato starch: 74 mg gelatine: 2 mg talcum: 8 mg 2. Capsules containing per capsule: Lu 18-013:25mg lactose: 40 mg magnesium stearate: 0.5 mg Any other pharmaceutical tableting adjuvants may be used provided that they are compatible with the active ingredient, and additional compositions and dosage forms may be similar to those presently used for neuroleptics such as thiothixene, clopenthixol or flupenthixol.Also combination of the compounds of Formula I as well as their non-toxic acid salts with other active ingredients, especially other neuroleptics, thymoleptics, tranquilizers or the like, fall within the scope of the present invention.

As previously stated, when isolating the compounds of Formula I in the form of an acid addition salt, the acid is preferably selected so as to contain an anion which is non-toxic and pharmacologically acceptable, at least in usual therapeutic doses. Representative salts which are included in this preferred group are the hydrochlorides, hydrobromides, sulphates, acetates, phosphates, nitrates, methanesulphonates, ethanesulphonates, lactates, citrates, tartrates, or bi-tartrates, embonates and maleates of the amines of Formula I. Other acids are likewise suitable and may be employed if desired.

For example; fumaric, benzoic, ascorbic, succinic, salicyclic, bismethylenesalicyclic, propionic, gluconic, malic, malonic, mandelic, cinnamic, cintraconic, stearic, palmitic, itaconic, glycolic, benzenesulphonic, and sulphamic acid may also be employed as acid addition saltforming acids. When it is desired to isolate a compound of the invention in the form of free base, this may be done according to conventional procedure as by dissolving the isolated or unisolated salt in water, treating with a suitable alkaline material, extracting the liberated free base with a suitable organic solvent drying the extract and evaporating to dryness or fractionally distilling to effect isolation of the free basic amine.

The invention also comprises a method for the alleviation, palliation, mitigation or inhibition of the manifestations of certain physiological-psychological abnormalies of animals by administering to a living animal body, including human beings, an adequate quantity of a compound of Formula I or a nontoxic acid addition salt thereof. An adequate quantity would be from about 0.5 mg to about 20 mg per kg of body weight per day and from about 20 milligrams to about 200 milligrams per day for oral administration.

It is to be understood that the invention is not limited to the exact details of operation or exact compound or compositions shown and described, as obvious modifications and equivalents will be apparent to one skiiled in the art.

Claims (4)

Claims

1. A compound of the formula:

wherein R is an alkyl group, branched or unbranched, having from one to seventeen carbon atoms inclusive, a phenyl group optionally substituted with one or two groups selected from lower alkyl, lower alkyloxy and halogen, a phenylalkyl group, a lower alkyloxy group or a --NR'R2 group wherein R1 and R2 each are hydrogen, lower alkyl, phenyl or cyclohexyl, as well as pharmaceutically acceptable acid addition salts thereof.

2. A method for the preparation of a compound of the formula:

wherein R is an alkyl group, branched or unbranched, having from one to seventeen carbon atoms inclusive, a phenyl group optionally substituted with one or two groups selected from lower alkyl, lower alkyloxy and halogen, a phenylalkyl group, a lower alkyloxy group or a -NR1R2 group wherein R1 and R2 each are hydrogen, lower alkyl, phenyl or cyclohexyl, as well as pharmaceutically acceptable salts thereof, which comprises reacting a compound of the formula: :

wherein R3 is an aminoprotecting group readily removable, with a reactive derivative of an acid of the formula R.COOH, wherein R is as defined above, whereupon the protecting group R3 is split off and the compound of Formula I isolated as the free base or a pharmaceutically acceptable acid addition salt thereof.

3. A pharmaceutical composition comprising as an active ingredient a compound of the general formula I as stated in Claim 1, or a pharmaceutically acceptable salt thereof together with a pharmaceutical carrier or excipient.

4. A pharmaceutical composition according to Claim 3 which additionally contains a minor tranquillizer or a neuroleptic.