GB2088370A - 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 -NHR<1> group, wherein R<1> is 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 --NHR' group, wherein R is 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 compound 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol (shortly called THIP in the following) has recently been described as having GABA-related activity, for example in European Patent Application No. 78 100 191.2. It is, however, relatively shortacting and has also some side effects.

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, ma leates, succinates, fuma rates, 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 R2 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, at reflux temperature, whereupon the protecting group R2 is split off and the compound of Formula I isolated as the free base or a pharmaceutically acceptable acid addition salt thereof.

When R is a --NHR' group it has proved especially advantageous to use as a reactive derivative an isocyanate of the formula R1-N=C=O.

As protecting groups R2 are preferably used groups which may subsequently be split off quite easily such as alkyloxy-carbonyl 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 N-acylation according to the method of the invention is preferably carried out in conventional manner in an organic solvent at reflux temperature.

Some of the starting materials of Formula II are conveniently prepared by reaction of 4,5,6,7tetrahdroisoxazolo [5,4-c] -pyridine-3-ol (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 R2 may be removed under mild conditions such as controlled hydrolysis. When R2 is a tertiary butyloxy carbonyl group it may conveniently be removed by reaction with anhydrous trifluoroacetic acid at about OOC Centigrade.

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

Example 1 3Hydroxy6(tbutyloxycarbonyl)4,5,6,7te:rahydrnisoxazolo[5,4-cipyridine (t-BOC THIP) 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 di-carbonate (11.0 g) was added and the temperature was raised to 250 C. The mixture was vigorously stirred for another 1.5 hour. Ethyl acetate (300 ml) and water (1 00 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. 134-1 360 C, IR (KBr): VOH 2400-3200 (broad, complex bands), vco 1 690 cm-1.

Example 2 2-Benzoyl-4,5,6,7-tetrahydroisoxazolo[5,4-cjpyridine-3-one and its Oxalate t-BOC THIP (2.4 g) was refluxed with benzoic acid anhydride (2.5 g) in 40 ml of CHCI3 for 1 3 hours. The organic solution was washed with ice cooled 0.1 M K2CO3-solution (2x25 ml), dried over anhydrous MgSO4 and CHCI3 evaporated. The remaining oil was dissolved in 20 ml of a 1:1 mixture of ether and n-hexane. After stirring and cooling for a while, 2-benzoyl-6-(t-butyloxycarbonyl)-4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridine-3-one precipitated. Yield: 1.3 g (38%). M.P. 126-1 280C.

This product was added to 10 ml of ice cooled anhydrous trifluoroacetic acid. The mixture was stirred until the CO2-evolution had stopped. Excess trifluoroacetic acid was evaporated and the resulting oil dissolved in a few milliliters of dry acetone. Anhydrous oxalic acid (2 grams) in 10 ml of dry acetone was added. Under stirring and cooling the oxalate of 2-benzoyl-4,5,6,7-tetrahydroisoxazolo[5,4-c]-3-one precipitated. Yield: 89%. M.P 210-21 30C (dec.) (Found: C 53.22; H 4.45; N 8.08; C,5H,4N207 requires: C 53.89; H 4.23; N 8.38%).

Example 3 2-Acetyl-4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridine-3-one, and its Oxalate t-BOC THIP (7.2 g) was refluxed with acetic acid anhydride (5.0 g) in 50 ml of CHCI3 for 2 hours.

2-Acetyl-6-(t-butyloxywarbonyl)-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-one was isolated as an oil in 6.4 g (76%) yield by column chromatography (Silicagel 60 "Merck") (eluted with ether/CH2CI2 (1 :3)). The t-BOC group was split off as above, yielding 73% of the oxalate. M.P. 177-1 780C (dec.) (Found: C 44.32; H 4.70; N (10.58); C1oH12N207 requires C 44.12; H 4.45; N 10.29%).

Example 4 2-lsobutyryl-4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridine-3-one, and its Oxalate The intermediate, 2-isobutyryl-6-(t-butyloxyca rbonyl)-4,5,6,7-tetrahydroisoxazolo[5,4- c]pyridine-3-one was prepared and isolated as described in Example 3 in 82% yield. M.P. 11 8- 11 90C. The t-BOC group was split off as above, yielding 94% of the oxalate. M.P. 174-1 760C (dec.) (Found: C 47.72; H 5.50; N 9.45; Ct2Ht6N207 requires C 47.99; H 5.38; N 9.33%).

Example 5 2-Carbamoyl-4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridine-3-one, and its Oxalate To a suspension of potassium cyanate (3.2 g) in 50 ml of CH2CIz was added a solution of trifluoroacetic acid (4.0 g) in 50 ml of CH2CI2 at OOC. t-BOC THIP (4.8 g) dissolved in 100 ml of CH2CI2 was slowly added under cooling. The mixture was further stirred at room temperature for 0.4 hour. The whole reaction mixture was submitted to column chromatography without evaporating CH2Cl2. 2- Carbamoyl-6-(t-butyloxycarbonyl)4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-one was eluted with 10% methanol in ether. Yield: 4.6 g (81%). M.P. 129-131 OC. The t-BOC group was split off as above, yielding 87% of the oxalate. M.P. 209--21 OOC (dec.) (Found: C 39.48; H 4.24; N 15.31; CgH11N307 requires: C 39.56; H 4.07; N 15.38%).

Example 6 2-( N-methylcarbamoyl )-4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-one, and its Oxalate t-BOC THIP (4.8 g) was refluxed with methylisocyanate (3.0 g) in 50 ml of CH2CI2 for 1.5 hours.

The solvent was evaporated. The remaining solid was stirred with cold isopropylether yielding 5,4 g (91 %) of 2-( N-methylcarbamoyl)-6-(t-butyloxycarbonyl)-4,5,6 ,7-tetrahydroisoxazolo[5 ,4-c] pyridine-3- one. M.P. 134-1 350C. The t-BOC group was split off as above yielding 90% of the oxalate. M.P.

168-1 690C (dec.) (Found: C 41.60; H 4.74; N 14.43; C10H,3N307 requires: 41.81; H 4.57; N 14.63%).

Example 7 2-(N-cycloheXylcarbamoyl)-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-one, and its Oxalate t-BOC THIP (3.6 g) was refluxed with cyclohexylisocyanate (1.9 g) in 50 ml ofCH2Cl2for2 hours.

Most of the CH2CI2 was evaporated and by addition of n-hexane 2-(N-cyclohexylcarbamoyl)-6-(t buWloxycarbonyl)-4,5,6,7-tetrnhydrnisoxazolo[5,4-c]pyddine-3one precipitated. Yield: 4.8 g (88%).

M.P. 92-940C. The t-BOC group was split off as above, yielding 87% of the oxalate. M.P. 2042050C (dec.) (Found: C 50.53; H 6.17; N 11.45; C15H2,N307 requires: C 50.69; H 5.97; N 11.83%).

Example 8 2-(N-phenylcarba moyl )-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-one, and its Oxalate t-BOC THiP (4.8 g),triethylamine (2.1 g) and phenylisocyanate (2.5 g) were refluxed in 50 ml of dry tetrahydrofuran for 1.5 hours. Volatile material was evaporated and the remaining oil was dissolved in 1 00 ml of ether. 2-(N-phenylcarba moyl)-6-(t-butyloxycarbonyl) -4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-one precipitated from the solution. Yield: 4.9 g (68%). M.P. 1 52-1 540 C.

The t-BOC group was split off as above yielding 90% of the oxalate. M.P. 21421 80C (dec.) (Found: C 52.02; H 4.40; N 12.01; C15Hl5N307 requires: C 51.57; H 4.34; N 12.03%).

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.

Macrolong cages type II Dosage and Procedure The test compound is injected i.p. in the doses or 1/2, 1/8 and 1/32 of the determined "i.v. LD 50". In case of insoluble substances the doses 0, 1/4, 1/16 and 1/64 of the determined "i.p. LD 50" 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. LD 50" 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. LD 50", 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. LD 50".

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 ofthe control value. The registration can also be done as an on-line 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.

3H-GABA Binding to Rat Brain Membranes Rats 125--200 g 0.32 M sucrose (made fresh every day) 10% Triton X-1 00 0.05 N Tris-citrate buffer (pH--6.8) 6.05 gTrisma(R? (base) 3.402 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 MM 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 1 7000 g (40 C). 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 (40 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 (giass 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-1 00 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-citrage 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 ul of drug dissolved in water, 20 yl of 1,uM 3H-GABA (final concentration of 3H-GABA in tubes=1 OnM) 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 for THIP are indicated as references.

Test Isoniazide Mouse substance antagonism grid shock 3H-GABA binding lC50 Example 2 2.0 1 0-7 Example 3 weak effect no effect Example 5 5.8 1.9 1 0-7 Example 6 1.3 2.7.1 o-6 Example 7 2.1 4.3 10-7 THIP 1.3 + 1.7.10-

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 nonacylated compound.

Typical examples of formulas for compositions containing 2-carbamoyl-4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridine-3-one (called Lu 1 8-028 for short) as the active ingredient are as follows: 1. Tablets containing 10 milligrams of Lu 1 8-028 calculated as the free base in the form of the oxalate; Lu 18-028: 10 mg lactose: 37 mg potato starch: 74 mg gelatine: 2 mg talcum: 8 mg 2. Capsules containing per capsule: Lu 18-028: 25 mg 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 bitartrates, 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 of exact compound or compositions shown and described, as obvious modifications and equivaients will be apparent to one skilled 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 -NHR1 group, wherein Ra is 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 --NHR' group, wherein R1 is hydrogen, lower alkyl, phenyl or cyclohexyl, as well as pharmaceutically acceptable acid addition salts thereof, which comprises reacting a compound of the formula: :

wherein R2 is an amino-protecting group readily removable with a reactive derivative of an acid of the formula R COOH, wherein R is as defined above, at reflux temperature, whereupon the protecting group R2 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 containing as an active ingredient a compound of formula I as defined in Claim 1, or a pharmaceutically acceptable acid addition salt thereof together with a pharmaceutical carrier or excipient.

4. A pharmaceutical composition of Claim 3 containing further a minor tranquillizer or a neuroleptic.