HK1149249A - Polymorphic forms of n-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide - Google Patents

Description

Polymorphic form of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide

Technical Field

The present invention relates to two novel polymorphic forms of a compound of formula (I), named N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide, and their use in drug-dependent therapy, and in particular in the therapy of alcoholism.

Background

N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide as part of an amide group useful in the treatment of drug dependence and alcoholism has been disclosed for the first time in European patent EP0932597B 1.

According to the patent, N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide having a 4-trifluoromethylbenzyl residue shows the best properties in terms of neuropharmacological activity when compared with the salts of gamma-hydroxybutyric acid (GHB) which are well known in the treatment of alcoholism. In particular, it has shown potential and sustained action properties of action in the assessment of the effect on the locomotor behaviour of mice, which is better than GHB and also better than other amides with different residues in this structure.

Therefore, there is a need for N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in high purity and high yield for pharmacological use, based on its optimal neuropharmacological activity.

First, the inventors of the present invention have attempted to obtain N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide by the preparation method described in the above-mentioned patent.

In particular, according to EP0932597B1, this compound is prepared by the following conventional synthetic method described in this patent, page 8, comprising the following steps: A) at NH₄Reacting the 4-alkoxybutanoic acid ester with the appropriate amine in the presence of Cl at a temperature of 160-170 ℃ to obtain the crude product, B) chromatography of the crude product on silica gel eluting with cyclohexylamine/ethyl acetate and finally C) chromatography from CH₂Cl₂/Et₂And crystallizing the O.

However, according to this method, the inventors found that even if they obtained the desired compound, it had different physico-chemical parameters each time. The conventional synthesis method described in EP0932597B1 therefore shows itself to lack reproducibility and is very expensive due to the purification of step B) and is therefore not suitable for the preparation of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide on an industrial scale.

Disclosure of Invention

By attempting to solve the reproducibility problem in the preparation of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide, the present inventors have surprisingly found that this compound can be in different polymorphic forms. Specifically, in carrying out the preparation, distillation and analysis of the product obtained each time, they found two new polymorphic forms of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide with polymorphic form A and polymorphic form B stacked in different unit cells.

Accordingly, in one aspect, the present invention provides polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide of formula (I):

in the X-ray powder diffraction pattern. + -. 0.2, the following peaks are present in the diffraction degrees (2-. theta.):

9.7；12.0；18.0；24.1；25.9。

in another aspect, the present invention provides polymorph B of a compound of formula (I) having the following peaks in the X-ray powder diffraction pattern ± 0.2 at the degrees of diffraction:

11.7；19.8；22.3；23.6。

furthermore, the present inventors have found a process which makes it possible to produce the two novel polymorphic forms a and B consistently reproducible and stable and of high purity without the need for chromatographic purification steps.

Thus, in a further aspect, the present invention relates to a process for the preparation of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide, comprising the steps of:

i) reacting 4-trifluoromethylbenzylamine with methyl 4-methoxybutyrate in the presence of a catalyst, thereby obtaining crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide; and

ii) obtaining crystalline polymorph A from a solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in an organic solvent, said solution crystallizing out polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

In yet another aspect, the present invention relates to a process for preparing polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide, comprising the steps of:

i) reacting 4-trifluoromethylbenzylamine with methyl 4-methoxybutyrate in the presence of a catalyst, thereby obtaining crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide; and

ii) obtaining crystalline polymorph B from a solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in an organic solvent, said solution crystallizing out polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

Polymorphs A and B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide are useful in the treatment of drug dependence and in the treatment of alcoholism. More specifically, they are useful in reducing the spontaneous consumption of ethanol and in the treatment of withdrawal syndromes. In addition, polymorph a and polymorph B described above are also useful in the treatment of critical points of withdrawal from addictive drugs such as heroin, cocaine, morphine and neuroactive drugs.

The invention therefore also relates to the use of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide as a medicament and to the use of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide as a medicament.

In another aspect, the present invention also relates to a pharmaceutical composition comprising an effective amount of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide as an active agent and a pharmaceutically acceptable carrier, and another pharmaceutical composition comprising an effective amount of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide as an active agent and a pharmaceutically acceptable carrier.

Drawings

FIG. 1 shows an X-ray powder diffraction pattern of a crystalline polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide;

FIG. 2 shows an X-ray powder diffraction pattern of a crystalline polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide;

FIG. 3 shows an IR spectrum of crystalline polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide;

FIG. 4 shows an IR spectrum of crystalline polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide;

FIG. 5 shows a Differential Scanning Calorimetry (DSC) spectrum of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide;

FIG. 6 shows a Differential Scanning Calorimetry (DSC) spectrum of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide;

FIG. 7 shows the correlation between IR spectra of polymorph A and polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide;

FIG. 8A shows the results of a spontaneous alcohol intake model following administration of polymorph A in non-deprived (non-depleted) sP mice;

FIG. 8B shows the results of a model of the effect of alcohol inhibition in mice with unreleased sP after administration of high doses of polymorph A; and

fig. 8C shows the results of a model of the effect of alcohol inhibition in non-deprived sP mice after administration of low doses of polymorph a.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention relates to novel polymorph a and novel polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide, which have different associated peaks in the X-ray powder diffractogram at the diffraction degrees (angle ± 0.2 °, 2- θ), in particular polymorph a is 9.7; 12.0 of the total weight of the mixture; 18.0 of; 24.1; 25.9, polymorph B is 11.7; 19.8 of; 22.3; 23.6.

more specifically, polymorph a shows 18 peaks in the X-ray powder diffraction pattern depicted in fig. 1 at the diffraction degrees shown in table 1 below.

Table 1: peak value of polymorph A (Angle. + -. 0.2 °, 2-theta)

Peak value	2-θ	Strength (cps)	I/I0
				1	6.0	4082	40
2	9.7	797	8
				3	11.0	640	7
4	12.0	8297	80
				5	17.6	2032	20
6	18.0	2173	21
				7	18.7	2658	26
8	18.9	3293	32
				9	19.6	919	9
10	20.7	7158	69
				11	21.6	2730	27
12	22.2	2601	26
				13	23.4	3261	32
14	24.1	10380	100
				15	24.7	1663	17
16	25.9	5534	54
				17	26.2	1771	18
18	28.2	1889	19

According to the present invention, polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide can be prepared by a process comprising the steps of:

(i) reacting 4-trifluoromethylbenzylamine with methyl 4-methoxybutyrate in the presence of a catalyst, thereby obtaining crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide; and

(ii) crystalline polymorph A is obtained from a solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide in an organic solvent, which crystallizes to precipitate polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide.

The 4-trifluoromethylbenzylamine of step (i) may be prepared according to known arylamine synthesis methods. Preferably, 4-trifluoromethylbenzylamine can be prepared by reacting 4-trifluoromethylbenzaldehyde with hydroxylamine according to the following reaction scheme:

according to this reaction scheme, the yield of 4-trifluoromethylbenzylamine can reach 90%.

According to the invention, in step (i) 4-trifluoromethylbenzylamine is reacted with methyl 4-methoxybutyrate in the presence of a catalyst, preferably containing 30% sodium methoxide solution in methanol, although N, N-dimethylaminopyridine and ammonium chloride are also useful. Preferably, this step (i) is carried out at a temperature of from 95 ℃ to 135 ℃, more preferably from 110 ℃ to 120 ℃. At the end of the reaction, the crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide can be isolated by conventional separation techniques, for example by distillation using organic solvents. Advantageously, step (i) results in a yield of 70% of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

The crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide thus obtained is crystallized as polymorph a in step (ii) by first preparing a solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide in an organic solvent and subsequently precipitating the polymorph a by crystallization in the solution.

Such a solvent may be any suitable organic solvent capable of assisting in the crystallization of polymorph a. Preferably, this organic solvent may be selected from a mixture of toluene, ethyl acetate and n-hexane. More preferably, a solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide is prepared using a mixture of ethyl acetate and N-hexane. Advantageously, when the crude product is dissolved in a mixture of ethyl acetate and n-hexane, the ratio of ethyl acetate: the ratio of n-hexane is 1: 4 to 1: 2, more preferably 1: 3.

the solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in the solvent is preferably heated from 35 ℃ to 70 ℃, more preferably from 40 ℃ to 60 ℃ before crystallization of polymorph A. Precipitation of polymorph a preferably takes place between 0 ℃ and 35 ℃, more preferably between 10 ℃ and 20 ℃.

Advantageously, step (ii) results in a yield of about 95% of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

According to other embodiments of the present invention, polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide is provided. Polymorph B shows 16 peaks in the X-ray powder diffraction pattern depicted in figure 2 at the diffraction degrees shown in table 2 below.

Table 2: peak value of polymorph B (Angle. + -. 0.2 °, 2-theta)

Peak value	2-θ	Strength (cps)	I/I0
				1	5.9	5211	69
2	11.7	7402	98
				3	17.6	1845	25
4	19.0	3985	53
				5	19.8	4334	58
6	20.9	3405	45
				7	21.9	7127	94
8	22.3	6896	91
				9	23.6	7594	100
10	24.0	2689	36
				11	24.8	1434	19
12	26.0	1654	22
				13	27.0	1590	21
14	27.4	1089	15
				15	28.1	2695	36
16	29.6	1252	17

According to the present invention, polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide can be prepared by a process comprising the steps of:

(i) reacting 4-trifluoromethylbenzylamine with methyl 4-methoxybutyrate in the presence of a catalyst, thereby obtaining crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide; and

(ii) crystalline polymorph B is obtained from a solution of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in an organic solvent, which crystallizes to precipitate polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

The preferred aspects of the above mentioned process for the preparation of polymorph a, i.e. the preparation of benzylamine of step (i) and all the advantageous features of steps (i) and (ii), are the same for the process for the preparation of polymorph B and are hereby incorporated by reference.

According to the present invention, polymorph a and polymorph B are conveniently obtained by a simple process which also avoids the chromatographic methods used to obtain the pure forms and, more advantageously, which is reproducible and allows to obtain the desired crystalline form selectively in a stable form.

The crystalline polymorphs a and B can be distinguished by their X-ray powder diagrams shown in fig. 1 and 2, respectively, and they can also be distinguished by their infrared spectra in the experimental part.

Both crystalline polymorphs a and B are thermodynamically stable and do not transform from one crystalline form to another. Dissolution tests were performed for each polymorph and the two polymorphs a and B showed no difference in solubility characteristics. They also show surprising pharmacological activity in drug-dependent therapy and in particular in the treatment of alcoholism.

Due to such properties, crystalline polymorphs a and B may be used as medicaments.

Thus, according to the present invention, there is provided a pharmaceutical composition comprising polymorph a or polymorph B and suitable pharmaceutical excipients.

The composition according to the invention preferably comprises between 12.5% and 50% by weight of polymorph a or polymorph B.

Such compositions may be prepared using conventional diluents or excipients and techniques known in medicine. The pharmaceutical compositions comprising polymorphs a and B may be administered by any suitable route, for example orally or by injection.

Pharmaceutical compositions for oral administration are advantageously in solid form, such as powders, granules, tablets, optionally effervescent tablets, compressed or coated pills, dragees, sachets, hard or soft capsules, or in liquid form, such as solutions, suspensions or emulsions. Pharmaceutical compositions for administration by injection may be in the form of aqueous or non-aqueous solutions, suspensions or emulsions.

In solid compositions, polymorph a and polymorph B may be combined with any suitable solid excipient, for example selected from lubricants, dispersants, fillers and the like.

In liquid compositions, polymorph a or polymorph B may be dissolved in, for example, water, an organic solvent or alcohol.

Polymorph a or polymorph B may be advantageously used in the treatment of drug dependence and in the treatment of alcoholism.

For these purposes, the two polymorphs can be administered preferably at a dose of 5 to 50 mg/kg.

According to the present invention, although both polymorphs have similar properties and similar activities for use as a medicament, polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide is preferred. As a matter of fact, polymorph a exhibits optimal physical characteristics, such as compressibility and density, so that it has better workability and handling properties that are extremely important in formulation and product production.

Furthermore, as demonstrated from the experimental section, polymorph a surprisingly shows therapeutic activity on alcohol dependence at very low pharmacological doses of 5 to 10 mg/kg.

Polymorph a was also tested for safe pharmacology, toxicity and genotoxicity, and its results showed that it was safe and had very low toxicity and genotoxicity profiles as demonstrated below.

The invention will now be described in more detail by way of non-limiting examples to better characterize the characteristics of polymorph a and polymorph B and their chemical-physical and pharmacological properties.

Example 1: preparation of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide

A) Preparation of 4-trifluoromethylbenzylamine

In the reactor, 15kg of distilled water, 2.50kg of sodium acetate, 2.30kg of hydroxylamine hydrochloride and 4.00kg of methanol were charged. At room temperature, 5.0kg of trifluoromethylbenzaldehyde was added, and the mixture was first stirred for thirty minutes, and then 5kg of the solvent was distilled off under vacuum. Subsequently, 12.0kg of 80% acetic acid and subsequently 4.5kg of zinc were added in portions, so that the temperature rose to 60-80 ℃ due to the exotherm. This temperature is then maintained by cooling. At the end of this reaction, 10.0kg of toluene and 15.0kg of 30% ammonia were added to remove the zinc salts. The material obtained is stirred at 50-60 ℃ and the lower aqueous phase is subsequently removed.

After distillation to half volume under vacuum, the toluene solution containing 4-trifluoromethylbenzylamine was recovered and used in step (i) described below.

4.00kg of 4-trifluoromethylbenzylamine was obtained as determined by potentiometric titrator. Yield: 79.5 percent

B) Preparation of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide of step (i)

Step a) (4.00 kg of 4-trifluoromethylbenzylamine) was added to the reactor. After distillation until an oil residue was obtained, 3.20kg of methyl 4-methoxybutyrate, 0.40kg of 30% sodium methoxide were added. The solution was then heated to 110 ℃ and 120 ℃ and atmospheric distillation was carried out to remove all the methanol (i.e., no methanol in the reactor) and to maintain this temperature for at least two hours. Subsequently, the 110-. At the end of the reaction, 12.0kg of toluene, 2.0kg of water and 0.40kg of 80% acetic acid were added to the mass. After stirring, the lower aqueous phase was separated and removed. Subsequently, the organic phase is distilled under vacuum until an oil residue is obtained. To the oil residue obtained 4.00kg of ethyl acetate, 12.0kg of n-hexane were added and the final material was heated to 40-60 ℃ until a completely dissolved solution was obtained. Subsequently, this solution is cooled to 20-30 ℃ and kept at this temperature until a good precipitate is obtained. Subsequently, the material was cooled to 0-10 ℃ and centrifuged by washing with a mixture of 0.80kg of ethyl acetate, 4.00kg of n-hexane. The moist product obtained was used in the following procedure.

3.8kg of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide are obtained. Yield: 60.5 percent

C) (iii) crystallization of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide according to step (ii)

In the reactor, 3.8kg of crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide (corresponding moist product), 3.8kg of ethyl acetate and 11.4kg of N-hexane were added. The substance is heated to 40-60 ℃ until a completely dissolved solution is obtained, and subsequently the solution is cooled to 25-35 ℃. 0.038kg of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide crystallized out. The mass is kept at 25-35 ℃ for at least 1 hour and subsequently cooled to 10-20 ℃ and again kept at this temperature for at least 1 hour. Subsequently, the material was centrifuged by washing with a mixture prepared previously and containing 0.76kg of ethyl acetate, 2.28kg of n-hexane. The product obtained is dried at 40-50 ℃. 3.4kg of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide were obtained. Yield: 89.5% example 2: preparation of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide

The same procedure and the same amounts of the experimental parts A) and B) of example 1 were used to obtain crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

C) (iii) crystallization of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide according to step (ii)

In a laboratory flask, 34.0g of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide, 34.0g of ethyl acetate and 102g of N-hexane were added. The material was then heated to 40-60 ℃ until a completely dissolved solution was obtained. Subsequently, the solution was cooled to 25-35 ℃ and 0.35g of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide crystallized out. The mass is kept at 25-35 ℃ for at least 1 hour and subsequently cooled to 10-20 ℃ and again kept at this temperature for at least 1 hour. Subsequently, the material was centrifuged by washing with a mixture prepared previously and containing 6.8g of ethyl acetate, 20.4g of n-hexane. The product obtained is dried at 40-50 ℃. Approximately 31g of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide were obtained.

Example 3: analysis of polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide

First, the crystalline product of example 1 was analyzed to confirm that it was N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

The sample of example 1 was analyzed as follows:

mass spectrometry was performed by (+) ESI (electrospray ionization) technique using a Thermo-Finnigiam LCQ-Advantage Instrument.

The molecular weight is 275 and the results of the proton/proton fragment diagram are shown in table 3 below:

table 3: results from proton/proton fragment diagrams

The molecular weight and proton/proton fragmentation patterns confirm the structure of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

CDCl passage using a Variant Geminy model 200 instrument operating at 200MHz₃And CDCl₃+D₂In an O solvent¹H-NMR analysis.

The NMR spectrum confirmed the structure of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide according to the following results:

table 4: n- [4- (trifluoromethyl) benzyl]-4-methoxybutanamide¹H-NMR spectrum

δ(ppm)	Multiplicity of properties	(H)	J(Hz)	Determining
					1.91	d-triplet	(2)	6.2;7.3	CH2
2.33	Triple-fold	(2)	7.3	CH2
					3.28	Singleweight	(3)	-	CH3
3.40	Triple-fold	(2)	6.2	CH2
					4.46	Double	(2)	5.9	CH2
6.31	Broad peak	(1)	-	NH
					7.37	AA 'BB' system	(2)	8.1	H;H
7.56	AA 'BB' system	(2)	8.1	H;H

Elemental analysis

The samples provided the following elemental values, corresponding to the calculated values:

table 5: c₁₃H₁₆NO₂F₃Value of (2)

Example 4: analysis of polymorph B of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide

Mass spectrometry, 1H-NMR and elemental analysis were repeated on a sample of the crystalline product obtained according to example 2 by using the same technique and instrument.

All the data obtained confirm that the product of example 2 is N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

Example 5: determination of polymorphism of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutanamide

Two samples of the crystalline products of examples 1 and 2 were analyzed by the following technique:

using a Rigaku Miniflex apparatus and using Cu-. alpha.₁-radiation and Cu-alpha₂-the radiation undergoes X-ray powder diffraction;

-DSC analysis using a Perkin-Elmer DSC6 instrument and using a scanning speed of 10 ℃/min at a temperature of 50-260 ℃;

infrared analysis was carried out using a Perkin-Elmer FT-IR Spectrum-one, where the sample analyzed was a suspension in KBr.

The X-ray powder diffraction pattern of a sample of polymorph a of example 1 is shown in fig. 1, and the 2-theta and intensity values (cps) are shown in table 1 above.

The X-ray powder diffraction pattern of a sample of polymorph B of example 2 is shown in figure 1, and the 2-theta and intensity values (cps) are shown in table 2 above.

The IR spectrum of polymorph a (example 1) is shown in figure 3. IR band (cm) as depicted in FIG. 3^-1) The following were used: 3308.68, 3067.42, 2997.56, 2971.26, 2935.88, 2882.97, 2834.85, 2740.58, 1924.26, 1642.03, 1541.19, 1480.60, 1446.91, 1424.54, 1373.53, 1330.93, 1253.96, 1234.94, 1209.04, 1165.17, 1114.08, 1068.67, 1047.11, 1031.50, 1020.76, 954.22, 916.98, 886.86, 835.75, 815.71, 757.32, 722.82, 692.97, 639.65, 590.06, 531.17, 508.96, 482.28.

The IR spectrum of polymorph B (example 2) is shown in figure 4. IR band (cm) as depicted in FIG. 4^-1) The following were used: 3305.52, 3076.93, 2989.08, 2932.17, 2869.83, 2839.20, 2817.11, 2752.21, 2651.07, 2296.64, 2069.08, 1931.29, 1642.82, 1542.23, 1482.83,1452.36, 1417.48, 1383.87, 1341.05, 1247.47, 1122.67, 1071.56, 1017.95, 955.01,884.95，872.11，853.35，817.02，764.69，722.01，642.51，590.87，536.59，490.57，465.16。

DSC profiles of polymorph a (obtained from example 1) and polymorph B (obtained from example 2) are shown in figures 5 and 6, respectively, where the initial and peak temperatures are shown. As can be seen from all of fig. 1-6, both polymorph a and polymorph B apparently include isomorphic crystals. However, polymorph a and polymorph B have different crystal structures depending on the values obtained in the different analyses.

To better emphasize their structural differences, the IR spectrum of the example 1 sample (polymorph a) depicted in fig. 3 and the IR spectrum of the example 2 sample (polymorph B) depicted in fig. 4 were superimposed as represented in fig. 7, and their correlation was calculated. The correlation value was 65.26%. This result and figure 7 confirm that polymorph a and polymorph B are two different polymorphs of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide. Example 6: evaluation of the physico-chemical Properties of samples of polymorph A and of samples of polymorph B

A sample of polymorph a obtained in example 1 was analysed after micronisation to less than 10 microns.

The results of the analysis are shown in table 6 below.

Table 6: physico-chemical characteristics of polymorph A and polymorph B

Both polymorphs appear as highly pure crystals. In addition, polymorph a is completely a crystalline powder. Because of this property, polymorph a is considered to be the best candidate for the preparation of pharmaceutical compositions from the viewpoint of better workability and handling.

Samples of the two powders (polymorph a and polymorph B) were used to assess the solubility of the two polymorphs at different pH values.

The two samples were tested under the following pH conditions:

hydrochloric acid buffer at pH =1.2

-acetic acid at pH =3.0

Phosphate buffer pH =3.0

Phosphate buffer pH =4.6

Phosphate buffer at pH =6.0

Phosphate buffer pH =7.4

-alkaline phosphate buffer at pH =8.0

0.5g of each sample was dissolved in a glass flask using 100ml of a suitable buffer. Both samples were then stirred for 30 minutes. If both samples were completely dissolved, an additional 1g of sample was added to the flask and stirred for 30 minutes. This procedure was repeated until undissolved product appeared at the bottom of the flask. After this dissolution step, the samples were stored for 24 hours and subsequently the amount of dissolved polymorph a and polymorph B was determined separately by HPLC assay. The results are shown in table 7 below:

table 7: solubility of polymorph A and polymorph B at different pH values

The two polymorphs showed no clear difference in solubility.

The solubility of these two compounds is not affected by the pH and in this case the slight difference is not important. The low solubility confirms the high stability of the two polymorphs. After 24 hours, the solubility increased by 10-30% at different pH values. According to this test, both polymorph a and polymorph B can be used for the preparation of a medicament.

Example 7: pharmacological testing

A) Evaluation of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in the treatment of alcohol dependence.

The experiments were performed with Sardinian drinking mice (sP), i.e. genetically selected animals of the order rodentia with spontaneous alcohol consumption. These animals were used as the main specific animal model in the study program, since they consumed a large amount of alcohol (6-7 g/kg/day) in a freely chosen manner between water and 10% ethanol solution, and had a large preference for the compounds tested (80-100%).

Under this criterion, these mice obtained two bottles of freely selected means between 10% ethanol and water (24 h/day) in animal cages without restriction, and they showed the following characteristics: ingesting 6g/kg of alcohol per day; preference ratio higher than 80% (alcoholic solution vs total liquid); the intake of alcohol is about 3-4 times per day; BAL (blood alcohol concentration) is higher than 50mg% every time of bingge drinking; by concentrating, enjoying the effects (sedation, motor stimulation) and regularity of drugs induced by site-specific mechanisms.

These tests were carried out according to the following model:

1. spontaneous alcohol intake in non-deprived sP mice

2. Alcohol deprivation Effect in deprived sP mice

1. Spontaneous alcohol intake in non-deprived sP mice

According to this model, the alcohol intake in both bottles in a freely chosen manner represents the "active drinking" state of alcoholism in humans. Therefore, the active compounds to be tested should reduce the preference of the mice for alcohol consumption. All results in this model demonstrate that polymorph a reduced alcohol intake after acute gavage administration at a wide range of doses (10-100 mg/kg). It is important to emphasize that the effect of alcohol is especially pronounced, as evidenced by the compensatory increase in water intake and by normal food intake in sP mice (data not reported), whose reduced alcohol intake is not associated with sedation. In fig. 8A, the results of one experiment are shown. In particular, 10, 20, 25 and 50mg/kg doses of polymorph a were administered and alcohol intake was assessed. Surprisingly, a low dose of 10mg/kg significantly reduced alcohol intake.

2. Alcohol deprivation Effect in deprived sP mice

To further illustrate the anti-hangover effect of polymorph a, the Alcohol Deprivation Effect (ADE) of this compound was evaluated. ADE well confirms the temporary increase in alcohol intake that occurs after a period of abstinence, and for the compulsive, uncontrolled type of alcohol demand and intake behavior, indicates that alcoholics are again dependent on alcohol. According to this model, sP mice ingesting alcohol underwent a two-week abstinence period during which no ethanol was exposed. After this period, 30 minutes before the start of the test, the animal was dosed with polymorph a and subsequently re-exposed to alcohol. Intake was measured 1 hour after the start of the test.

The results are depicted in fig. 8B and 8C for the high and low doses of crystalline polymorph a, respectively. Polymorph a completely inhibited the ADE effect, showing very good activity at a dose range of 5-100 mg/kg. In addition, a low dose of 5mg/kg completely consumed additional alcohol.

Example 8: evaluation of safe pharmacology, toxicity and genotoxicity of polymorph A

1. Safe pharmacology

The potential side effects of polymorph a on CNS, cardiovascular, respiratory and immune functions were evaluated in different test modes, both in vitro and in vivo (mice and dogs). In vivo testing, the compounds to be tested are usually administered orally at doses of 100, 300 and 1000mg/kg alone.

A) CNS system

Irwin test and body temperature in mice

The effect of neuronal behavior of polymorph a was studied by using a standard observation battery according to the Irwin test, evaluating the activity of the peripheral and central nervous system (e.g. motor activity, motor coordination, somatosensory/motor reflex response, autonomic response); body temperature was measured by an electronic thermometer. The compound induces a transient decrease in spontaneous locomotor activity at 100 mg/kg; at higher doses, the effect on exercise was more pronounced and longer lasting and correlated with the miorelaxant effect and diminished consciousness. At a dose of 1000mg/kg, mice showed toe positioning, ataxia and subsequent lying or lying position; all effects are reversible. Body temperature was unaffected at the 100mg/kg dose, while at higher doses body temperature dropped significantly for up to 4 hours.

Hexobarbital sleep time in mice

The test involves determining the duration of hexobarbital-induced sleep; substances with sedative or refreshing effects increase or decrease respectively the duration of hexobarbital-induced sleep. At the lowest dose (100 mg/kg), no significant statistical effect was seen on the time to sleep or the duration of sleep. At the intermediate dose, a slight decrease in sleep duration was recorded, whereas at the highest dose, a significant decrease in time to sleep and sleep duration was observed.

Spasmodic behaviour of mice

In this study, the potential spasmodic effects of polymorph a after combined administration with pentylenetetrazole inducing a spastic response were explored; pretreatment with a substance having spasmodic properties rapidly initiates a spastic response. Polymorph a had no statistically significant spasmodic effects when taken at all doses, whereas doses of 100 and 300mg/kg induced an increase in the time to onset of spasticity, which means a potential antispasmodic effect.

B) Cardiovascular instrument

In vitro

HERG cells (human methyl ether-related gene cells)

The potential blocking effect of polymorph A on the Herg tail currents recorded from HEK-293 cells stably transfected with HERGG-1cDNA (human embryonic kidney cells) was investigated. The method involves measuring HERG tail current in all cell structures using the patch clamp technique. Compounds that inhibit HERG current were shown to prolong myocardial action potential and increase QT interval.

The results obtained show that polymorph A is at 10^-7M does not induce a significant statistical inhibition of HERG tail current; at 10^-6M and 10^-5M concentration, a slight and dose-independent reduction was observed, and at the highest concentration tested 10^-4M, a 50% reduction occurred. It is emphasized that this inhibition never reached a value of 70%, which is considered to be the critical value for the activity of the compound in this test.

-Purkinje fibres

The potential adverse effects induced by polymorph a on myocardial action potential were evaluated in isolated purkinje fibers of canine. Measuring transmembrane action potential by an intracellular microelectrode technology; this method is recommended to determine the ability of a substance to induce a delay in the QT interval. Concentration 10^-7，10^-6And 10^-5Polymorph a of M has no significant statistical effect on action potential parameters in normal or low-stimulated mice; 10^-4A significant reduction in the action potential duration of repolarization was observed with very high concentrations of M. No advance or delay was noted after repolarization at all concentrations tested.

These results show that, according to the electrophysiological profile of polymorph a, no TDP (ventricular tachycardia) or QT delay occurs; polymorph a can be included in a medicament that does not induce TDP or QT delay in humans.

In vivo

Cardiovascular assessment of conscious dogs

On conscious, free-moving dogs, which had previously been equipped with a telemetric transmitter, any potential effect of polymorph a on blood pressure, heart rate and electrocardiogram taken orally at doses of 100, 300 and 1000mg/kg was assessed. In the first part of the study, only telemetric values are recorded; the parameters recorded began at least 24 hours prior to compound administration and continued at the dose for 24 hours. In the second part, only the highest dose of 1000mg/kg is taken, and measurements such as 6-lead electrocardiogram (lead i, i, aVL, aVR and aVF), blood sample and observation of the animals are supplemented before and 3 hours after the treatment.

A first part: 100mg/kg of polymorph A did not induce relevant changes in blood pressure, heart rate and electrocardiography (especially in the T waveform). When at doses of 300 and 1000mg/kg, a slight increase in arterial blood pressure (mean, systolic and diastolic arterial blood pressure), a slight decrease in the interval time between PR and PQ, and a slight increase in the QT interval time for heart rate correction using the Sarma method were recorded. The changes observed at 300mg/kg were very subtle and isolated and therefore were not due to the pharmacologically relevant effects of polymorph a resulting from the observed changes in PR and PQ intervals at 1000mg/kg, while the increase in heart rate corrected QT intervals was clearly correlated with the effects of polymorph a, which means an increase in ventricular repolarization time. At all experimental doses, no interference was observed in the electrocardiogram (lead II), and no changes were observed in the T waveform in particular.

A second part: no interference in 6-lead ECG was observed before and 3 hours after taking polymorph A at a dose of 1000 mg/kg. Emesis occurred in all animals between 0.5 and 17 hours after administration of this dose. Plasma analysis confirmed the presence of polymorph a in plasma 3 hours after administration.

These results show that by oral administration of polymorph a at doses of 100, 300 and 1000mg/kg, only a dose of 1000mg/kg induces slight hypertension associated with increased ventricular repolarization time.

C) Respiratory system

-respiratory system assessment in conscious mice

Evaluation of the effect of polymorph a on respiratory parameters (respiratory rate, peak inspiratory and expiratory flow, inspiratory and expiratory time, airway resistance index, breaths per minute and tidal volume) was performed on conscious mice after single oral administration. Respiration was measured by whole body plethysmography. 100mg/kg of polymorph A had no relevant effect on respiratory parameters, and 300 and 1000mg/kg induced tachypnea associated with transient decreases in tidal volume. No statistically significant changes were observed in the inspiratory and expiratory flow peaks, respiratory volumes per minute or airway resistance index, which means that the test compound did not produce any respiratory or bronchoconstrictive effects.

D) Immune system

PCF test in mice

The potential effect of polymorph a on the immune system was evaluated systematically by a method using mouse lysoplaque-forming cells (PCF) 28 days after oral administration of doses of 150, 250 and 500mg/kg in mice. This method is based on stimulation of the immune system with an antigenic agent (sheep's red blood cells) and evaluation of the effect of the test substance on the immune response. The immune response was assessed by measuring the proportion of splenocytes against antibodies raised against the antigen agent (plaque forming cells) in the presence of complement. The results obtained in the study showed that polymorph a had a slight and dose-independent immunosuppressive activity; the lower dose (150 and 250 mg/kg) tests are marginal effects of the control, which only became clear at the highest dose (500 mg/kg). In fact, statistical analysis showed 150mg/kg to be significant, rather than 250 mg/kg; these findings suggest that the observed effect at these doses is due to the variability normally present in this assay, as heterogeneity is often seen in the immune response and may be associated with individual differences in immune sensitivity.

All the above studies on safe pharmacology were performed according to the GLP regulation and the ICH S7A guidelines for safe pharmacology.

2. Toxicity and genetic toxicity

Single and repeated dose toxicity studies were performed in rodents and non-rodents to support clinical trials of the test compound, polymorph a. Single toxicity studies were performed in both mice and mice by injection and oral forms. Repeated oral studies (28 days of administration followed by a 14 day recovery period) were performed on mice and dogs.

An Ames test and micronucleus test were performed to determine the potential genotoxicity of the compounds.

Table 8 below summarizes the studies performed using polymorph a.

Table 8: toxicity and genotoxicity Studies of polymorph A

NOEL: no level of effect observed

Genotoxicity assessment in mice using Ames and micronucleus assays

Detection of the Return mutation of Salmonella typhimurium (Ames test)

The mutation potential of polymorph a was evaluated in vitro in the detection of reversion mutations in salmonella typhimurium according to EC guidelines. The compounds tested did not induce genetic mutations by base pair change or frameshifting of the strain genes of the tested Salmonella typhimurium (TA 1535, TA1537, TA98, TA100 and TA 102) at concentrations of 50-3000. mu.g/ml with and without metabolic activation. Thus, polymorph a is considered to be free of mutations in the salmonella typhimurium back-mutation assay.

Micronucleus test

Any fragmentation or spindle toxin activity of polymorph a was determined by detecting micronuclear polystained erythrocytes in the bone marrow of treated mice. The study was performed according to EC guidelines. The method involves looking for the presence of chromosome fragments, or the number of chromosomes, in the polychromatic red blood cells of the bone marrow, which are due to knockout or mitotic spindle toxin effects. The cleavage products can produce chromosomal breaks during mitosis, and spindle toxins interfere with the structure of the mitotic spindle. Centromere-free fragments of chromosomes that have not been able to move normally are not retained in the nucleus of daughter cells, but appear in the cytoplasm. It is called a Haowell-Jazz corpuscle or micronucleus. This micronucleus can be detected in polychromatic erythrocytes, because these cells expel their nuclei shortly after a sustained mitosis, and the micronucleus remains in the red blood cells. Male and magnetic Sprague Dawley mice were allowed to orally administer polymorph A at a dose of 1000-500mg/kg per 2000-and after 24 and 48 hours of treatment, their femur was extracted and bone marrow cells were extracted.

At all doses and times tested, polymorph a did not induce fragmentation behaviour.

Claims (12)

1. Polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide of formula (I):

wherein the polymorph A has the following peaks in the X-ray powder diffraction pattern + -0.2 at diffraction angles 2-theta:

peak value 2-θ 1 6.0 2 9.7 3 11.0 4 12.0 5 17.6 6 18.0 7 18.7 8 18.9 9 19.6 10 20.7 11 21.6 12 22.2 13 23.4 14 24.1 15 24.7 16 25.9 17 26.2 18 28.2

And said polymorph a has an X-ray powder diffraction pattern as depicted in figure 1.

2. A process for preparing polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide of claim 1, comprising the steps of:

i) reacting 4-trifluoromethylbenzylamine with methyl 4-methoxybutyrate in the presence of a catalyst to obtain crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide; and

ii) preparing a solution containing crude N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide in an organic solvent selected from toluene or a mixture of ethyl acetate and N-hexane, heating the solution from 35 ℃ to 70 ℃ to cause precipitation of polymorph A between 0 ℃ and 35 ℃, and crystallizing polymorph A of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide.

3. The process according to claim 2, wherein the 4-trifluoromethylbenzylamine is prepared by reacting 4-trifluoromethylbenzaldehyde with hydroxylamine hydrochloride by the following route:

。

4. a process according to claim 2 or 3, wherein in step (i), the catalyst is a methanol solution containing 30% sodium methoxide.

5. The process of claim 2, wherein the organic solvent is a mixture consisting of ethyl acetate and n-hexane.

6. The process of claim 5, wherein the ratio of ethyl acetate to n-hexane in the mixture is 1: 4 to 1: 2.

7. a pharmaceutical composition, wherein said composition comprises an effective amount of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide of any one of claims 1 to 3 as active agent and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein the polymorph A is present in an amount of 12.5% to 50% by weight.

9. Use of the polymorphic form a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide according to claim 1 for the preparation of a medicament for the treatment of drug dependence and alcoholism.

10. Use according to claim 9, for the preparation of a medicament for reducing spontaneous ethanol consumption and/or for the treatment of withdrawal syndrome.

11. Use according to claim 9 or 10, wherein the dosage of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide is between 5 and 50 mg/kg.

12. The use according to claim 11, wherein the dosage of polymorph a of N- [4- (trifluoromethyl) benzyl ] -4-methoxybutyramide is from 5 to 10 mg/kg.