Formula 1

Pg is a commonly used protecting group, particularly t-butyloxycarbonyl (Boc), benzyloxykarbonyl (Cbz), acetyl or trifluoroacetyl, and

R is NR^ ² , wherein R ¹ and R ² is the same or different substituent, which may be hydrogen, a substituted or unsubstituted alkyl, or R ¹ together with R ² form a substituted or unsubstituted heterocyclic containing at least one nitrogen atom, particularly a piperazine derivative.

Background Art

Derivatives of 3-amino-4-(2,4,5-trifluorophenyl)-butanoic acid of general formula 1 are especially useful as advanced intermediates of some inhibitors of dipeptidyl peptidase-4 (DPP- 4), such as 7-(l-oxo-3[(R)-ammo]-4-(2,4,5-1xifluorophenyl)-butyl)-3-trif luoromethyl-5,6,7,8- tetrahydro- 1 ,2,4-triazolo[4,3-a]pyrazine (SITAGLIPTIN), 7- [ 1 -oxo-3 [(R)-amino]-4-(2,4,5- trifluorophenyl)-butyl]-2-trifluoro or 7-[l- oxo-3[(R)-amino]-4-(2,4,5-trifluorophenyl)-butyl]-4-(pyrid-2 -ylcarbonyl)-piperazine.

An inhibitor of dipeptidyl peptidase-4 (DPP-4) is obtained from the corresponding derivative of 3-amino-4-(2,4,5-trifluorophenyl)butanoic acid of formula 1 by removal of the protecting Pg group and can be further converted to a pharmaceutically acceptable salt suitable for preparation of a therapeutical. The intermediates of the above mentioned DPP-4 inhibitors are prepared by condensation of 3- amino-4-(2,4,5-trifluorophenyl)butanoic acid, whose amino group is protected with a suitable protecting group, with the corresponding organic amine. This reaction is carried out by means of a condensation agent.

Condensation agents include the expensive and, as generally known, poorly stable carbonyldiimidazole (CDI), which reacts with water in seconds, producing carbon dioxide and two equivalents of imidazole. For these reasons it must be stored in places where air humidity is excluded and reactions using CDI require anhydrous reaction conditions and expensive anhydrous solvents.

Another compound that can be used as the condensation agent is the expensive and potentially dangerous (explosive) 1-hydroxybenzotriazole (HOBT) in combination with l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC), as described e.g. in the patent document US6699871.

The condensation reaction can also be performed with the use of 2-chloro-4,6-dimethoxy- 1,3,5-triazine in the presence of a tertiary organic amine. This option is described in the patent document WO2012099381.

All the above mentioned methods require (dry) aprotic solvents such as THF, DMF, dichloromethane, toluene.

Verification of hitherto known techniques has shown that if CDI is used, an intermediate product is separated, namely the active amide of formula 2

Formula 2

in the form of a suspension that is very difficult to stir. This leads to poor contact of the reagents and subsequently to lower conversion of the reaction and worse quality of the product.

Using the HOBT-EDC reagent has resulted in achieving a low yield, which has already been described in US6699871. And in all the cases, i.e. also with the use of 2-chloro-4,6- dimethoxy-l,3,5-triazine (in accordance with WO2012099381), the reaction mixture has to subsequently be processed by extraction and the product has to be isolated by complete concentration from an organic solvent, followed by purifying by crystallization or other purification method.

The necessity of extraction and crystallization of the obtained product considerably increases the volumes of the solvents used and waste quantities. These operations, crystallization and extraction, make the production process considerably longer and require additional technological equipment.

Naturally, isolation of the product by complete concentration from an organic solvent, as described in WO2011099381 , is absolutely unsuitable for industrialization of the process.

The above mentioned material technological drawbacks have represented the reason for looking for more suitable reaction conditions.

Disclosure of Invention

The present invention provides a new, unexpectedly highly economical method for the preparation of 3-amino-4-(2,4,5-trifluorophenyl)butanoic acid derivatives of general formula 1, useful for the preparation of DPP-4 inhibitors using a very cheap reagent, 4-(4,6-dimethoxy- l,3,5-triazin-2-yl)-4-methylmorpholinium halide of general formula 3,

wherein X is a halide anion, particularly a CI anion or Br anion. This method is especially characterized by the use of a low volume of cheap auxiliary raw materials and solvents, a short reaction time, easy isolation of the product and a high yield and quality of the obtained product. These advantages make this method very suitable for industrial production. Detailed description of the invention

The present invention provides a highly efficient method for the preparation of 3-amino-4- (2,4,5-trifluorophenyl)-butanoic acid derivatives of general formula 1,

wherein Pg is a commonly used protecting group, particularly t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), acetyl or trifluoroacetyl and R is NR^ ² , wherein R ¹ and R ² is the same or different substituent, which may be hydrogen, a substituted or unsubstituted C _1-6 alkyl, or R ¹ together with R ² form a substituted or unsubstituted 4-, 5-, or 6-membered heterocyclic containing at least one nitrogen atom, particularly a piperazine derivative.

The derivatives of the amino acid include its pure R- or S- enantiomers.

The method is based on a reaction of 3-amino-4-(2,4,5-trifluorophenyl)butanoic acid of formula 4,

(4) wherein Pg is a commonly used protecting group, particularly t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), acetyl or trifluoroacetyl, with the corresponding organic amine of general formula 5, wherein R ¹ and R ² is the same or different substiruent, which may be hydrogen, a substituted or unsubstituted C _1-6 alkyl, or R ¹ together with R ² form a substituted or unsubstituted 4-, 5-, or 6-membered heterocyclic containing at least one nitrogen atom; in particular a piperazine derivative,

using a condensation agent of general formula 3,

wherein X is a halide anion, particularly a CI anion or Br anion.

The organic amine generally means a secondary amine, particularly one of the derivatives 3-trifluoromemyl-5,6,7,8-tetrahydro-l,2,4-triazolo[4,3-a]pyr azine, 2-trifluoromethyl-5, 6,7,8- tetrahydropyrido[3,4-d]pyrimidine or 4- pyrid-2-ylkarbonyl)-piperazine, or a suitable salt thereof with an organic or inorganic acid. The 3-trifluoromemyl-5,6,7,8-tetrahydro-l,2,4- triazolo[4,3-a]pyrazine base of formula 7 or its salts with hydrochloric acid are used.

The reaction is carried out in an organic solvent selected from the group of polar solvents, particularly solvents miscible with water such as some ethers, ketones, nitriles or alcohols. Especially suitable solvents belong to the group of lower alcohols such as methanol, ethanol, propan-l-ol or propan-2-ol.

The reaction can be carried out at a temperature in the range of -15°C to the boiling point of the solvent used corresponding to the pressure used. Preferred is the range of 15°C to the boiling point of the solvent used at the normal atmospheric pressure. The present invention further relates to a method for the preparation of 3 -[(1,1- dimethylethoxycarbonyl)amino]-4-(2,4,5-trifluorophenyl) butanoyl] -3-(trifluoromethyl)- 5,6,7,8-tetrahydro-l,2,4-triazolo[4,3-a]pyrazine of formula 8

with 3-(trifluoromemyl)-5,6,7,8-tetrahydro-l,2,4-triazolo[4,3-a]p yrazine, or a salt thereof with hydrochloric acid of formula 7 the reaction being carried out using the condensation agent 4- (4,6-dimethoxy-l,3,5-triazm-2-yl)-4-methylmorfolinium chloride of formula 9,

in an environment of methanol or propan-2-ol. The preparation method in accordance with the invention is characterized by easy isolation of the product, by commonly used methods for separation of a suspension, such as filtration, centrifugation or decanting. Filtration of the product that spontaneously crystallizes from the reaction mixture is especially convenient. Before isolation of the product from the reaction mixture the reaction mixture can be diluted with another solvent that supports crystallization of the product. The product is conveniently isolated by filtration after dilution of the reaction mixture with water.

Brief Description of Drawings

Fig. 1 shows an X-ray powder diffraction pattern of the crystalline salt of hydrochloric acid with 7-(l-oxo-3[(R)-amino]-4-(2,4,5-trifluorophenyl)-butyl)-3- trifluoromethyl-5,6,7,8-tetrahydro-l ,2,4-triazolo[4,3-a]pyrazine (sitagliptin hydrochloride), prepared in accordance with Example 3. Fig. 2 shows the DSC curve of the crystalline salt of hydrochloric acid with 7-(l-oxo- 3 [(R)-amino]-4-(2,4,5-trifluorophenyl)-buty^^ 1 ,2,4- triazolo[4,3-a]pyrazine (sitagliptin hydrochloride), prepared in accordance with Example 3.

Overview of analytic methods HPLC - system with UV/VIS

Column: size: length: 0.25 m, inner diameter 4.6 mm

stationary phase: X-Terra RP18 (5 μπι)

temperature: 55°C

Mobile phase: A: 1 ml of ammonia R diluted with water R to 1000 ml, pH is adjusted to

10.0 using sodium hydroxide.

B: acetonitrile R

Elution: gradient: Time Mobile phase A Mobile phase B

(min) (% V/V) (% V/V)

0 85 15

20 40 60

22.5 60 40

25 85 15

30 85 15

Flow rate: 1.0 ml/min

Detection: spectrophotometric detector 210

Injection: 15 μΐ

Auto-sampler temperature: 15°C

XRPD measurement parameters: The diffraction pattern was measured using an X ERT PRO MPD PANalytical diffractometer with a graphite monochromator, radiation used CuKa (λ=1.542 A), excitation voltage: 45 kV, anode current: 40 mA, measured range: 2 - 40° 2Θ, increment: 0.01° 2Θ. The measurement was carried out using a flat powder sample that was placed on an Si plate. For the primary optic setting programmable divergence diaphragms with the irradiated sample area of 10 mm, Soller diaphragms 0.02 rad and an anti-dispersion diaphragm ¼ were used. For the secondary optic setting an X'Celerator detector with the maximum opening of the detection slot, Soller diaphragms 0.02 rad and an anti-dispersion diaphragm 5.0 mm were used.

The Differential Scanning Calorimetry (DSC) records were measured with a DSC Pyris 1 device made by Perkin Elmer. The charge of the sample in a standard Al pot was between 3-4 mg and the heating-up rate was 10°C/min. The temperature program used consists of 1 minute of stabilization at the temperature of 50°C and then of heating to 250°C at the heating-up rate of 10°C/min. As the carrier gas 4.0 N ₂ was used at the flow of 20 ml/min.

Working Examples

The examples given only serve as illustration of the present invention and should not be considered as limiting the scope or sense of the present invention. Preparation of 3-[(l , 1 -dimethylethoxycarbonyl)amino]-4-(2,4,5-trifluorophenyl)buta noyl]-3- (trifluoromethyl)-5,6,7,8-tetrahydro-l,2,4-triazolo[4,3- ]pyrazine.

Reference Example 1 (use of CDI)

(3i?)-3-[(l,l-dimethylethoxykarbonyl)amino]-4-(2,4,5-trifluo rophenyl)butanoic acid (1.74 g) is mixed with carbonyldiimidazole (CDI; 1.1. g) in dry THF (20 ml). The reaction mixture passes through a nearly clear solution and becomes virtually solid again in its entire volume within 3 minutes. 3-(Trifluoromethyl)-5,6,7,8-tetrahydro-[l,2,4]triazolo[4,3-a ]pyrazine (1 g) is added to the resulting gel-like suspension and the suspension is heated up to 60°C. The reaction mixture is stirred for 8 hours at this temperature. Gradually, a white stirrable suspension is formed. After cooling, the reaction mixture is extracted with ethyl acetate (20 ml) and distilled water (30 ml) and the phases are separated. The aqueous phase is extracted with ethyl acetate (20 ml) and then the combined organic phases are washed with brine (10 ml). The extract is dried and concentrated.

The desired product is obtained in the yield of 99% and HPLC quality of < 90%.

Reference Example 2 (use of CDI)

The reaction when carried out in methyltetrahydrofuran, acetonitrile, dichloromethane, toluene or cyclopentyl methyl ether virtually exhibits the same course and results.

Reference Example 3 (use of 2-chloro-4,6-dimethoxy-l,3,5-triazine)

(3i?)-3-[(l,l-Dimethylemoxycarbonyl)ammo]-4-(2,4,5-trifluoro phenyl)butanoic acid (3g) is dissolved in tetrahydrofuran (30 ml) and N-methylmorpholine (3g) is added to the resulting solution. The obtained suspension is cooled to 0-5 °C and 2-chloro-4,6-dimethoxy- 1,3,5 - triazine is added at this temperature and the suspension is stirred at the same temperature for another hour. Then, 3-(trifluoromemyl)-5,6,7,8-te1rahydro-[l,2,4]triazolo[4,3-a] pyrazine hydrochloride is added and the mixture is stirred until the temperature reaches the laboratory temperature. When complete conversion is achieved, the reaction mixture is diluted with dichloromethane (30 ml) and extracted with water (30 ml). The organic phase is separated and washed with a carbonate solution (30 ml) and brine (30 ml). Evaporation of organic solvents provides the crude product. After crystallization from an ethyl acetate - propan-2ol mixture (2: 1 ; 18 ml) the desired product is obtained in the yield of 94 and HPLC quality of > 99%.

Reference Example 4 (use of l-ethyl-3-(3-dimethylammopropyl)carbodiirnide hydrochloride (EDC) with 1-hydroxybenzotriazole (HOBT))

A solution of (3i?)-3-[(l,l-dimethylethoxycarbonyl) amino]-4-(2,4,5-trifluorophenyl)butanoic acid (8 g) in N,N-dimethylformamide (32 ml) is added dropwise to a solution of 3- (trifluoromethyl)-5,6,7,8-tetrahydro-[l,2,4]triazolo[4,3-a]p yrazine hydrochloride (5.6 g), diisopropylethylamine (6.2 g), 1-hydroxybenzotriazole (4.4 g) and EDC*HC1 (5.6 g) inN,N- dimethylformamide (32 ml) at 0-5°C. The resulting solution is stirred at the laboratory temperature for another 12 hours. Then, most of the N,N-dimethylformamide is removed by distillation at a reduced pressure. The brown residue is stirred up in a 10% aqueous solution of sodium carbonate (20 ml) and the product is extracted with ethyl acetate (3x 20 ml). The combined extracts are washed with water (15 ml) and filtered through a layer of activated carbon and concentrated until dry. Crystallization of the concentrated residue from an ethyl acetate - propan-2ol solution (2:1; 40 ml) provides the desired product in the yield of 80% and HPLC quality of <97%.

Example 1

(3i-)-3-[(l,l-Dimemylethoxycarbonyl)ammo]-4-(2,4,5-trifluoro phenyl)butanoic acid (10 g) is mixed with 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10 g) in methanol (100 ml) at the laboratory temperature. 3-(Trifluoromethyl)-5,6,7,8-tetrahydro- [l,2,4]triazolo[4,3-a]pyrazine hydrochloride (7 g) is added to the resulting solution and triethylamine (4.7 ml) is added to the stirred suspension. During 2-3 minutes a solution is obtained that is further stirred at the laboratory temperature. After approximately 1 hour, white suspension of the product starts to separate. The reaction mixture is heated up to 50°C and distilled water (35 ml) is added to the resulting solution and the reaction mixture is cooled down to 15°C under stirring during 2 h. The separated product is filtered off and washed with 15 ml of a methanol/water (2/ 1 ) mixture.

After drying the desired product is obtained with the yield of 96% and HPLC quality of >99%. Example 2

(3i?)-3-[(l,l-Dimethylethoxycarbonyl)arnino]-4-(2,4,5-triflu orophenyl)butanoic acid (10 g) is mixed with 4-(4,6-climethoxy-l,3,5-triazm-2-yl^ chloride (10 g) in propan-2-ol (100 ml) at the laboratory temperature. 3-(Trifluoromethyl)-5,6,7,8-tetrahydro- [l,2,4]triazolo[4,3-a]pyrazine hydrochloride (7 g) is added to the obtained suspension and triethylamine (4.7 ml) is added to the stirred suspension. The reaction mixture is heated up to 60°C. The reaction mixture is maintained at this temperature for another 10 minutes and then distilled water (35 ml) is added to the resulting solution and the reaction mixture is cooled down to 15 °C under stirring during 2 hours. The separated product is filtered off and washed with 15 ml of a propan-2-ol/water (2:1) mixture.

After drying the desired product is obtained in the yield of 94% and HPLC quality of >98%. Example 3

Preparation of 7-(l-oxo-3 [(R)-amino] -4- (2,4,5-trifluorophenyl)-butyl)-3-trifluoromethyl- 5,6,7,8-tetrahydro-l ,2,4-triazolo[4,3-a]pyrazine hydrochloride.

3 - [( 1 , 1 -Dimethylethoxycarbonyl)amino] -4-(2,4, 5 -trifluorophenyl)butanoyl] -3 - (trifluoromemyl)-5,6,7,8-tetrahydro-l,2,4-triazolo[4,3-a]pyr azine, prepared in accordance with Example 1 (14.7 g), is dissolved in methanol (150 ml) and concentrated hydrochloric acid (15 ml) is added. The reaction mixture is moderately refluxed until complete conversion is achieved. Then, approximately 4/5 of methanol is removed by distillation and propan-2-ol (180 ml) is added to the reaction mixture at 55°C and the reaction mixture is slowly cooled down to the laboratory temperature. A crystalline product is obtained in the yield of 92% with the melting point of 165-167.5°C.

The obtained salt exhibits the following characteristic peaks in an X-ray diffraction pattern: 6.6; 8.0; 13.7; 15.9; 18.1; 22.6; 27.1° 2Θ ± 0.2° 2Θ.