METHOD OF CRYSTALLIZING N?2 -((S)-1-ETHOXYCARBONYL-3-PHENYLPROPYL)-N?6 -TRIFLUOROACETYL-L-LYSYL-L-PROLINE

Abstract: A method of crystallizing N²-((S)-1-ethoxycarbonyl-3-phenylpropyl)-N⁶-trifluoroacetyl-L-lysyl-L-proline by using at least one compound represented by the general formula CR¹R²R³R⁴ as the solvent for crystallization and, if required, a subsidiary solvent for controlling the crystallization condition.

Description: TECHNICAL FIELD The present invention relates to a process for crystallizing N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline which is useful as an intermediate for the preparation of an inhibitor of angiotensin converting enzyme (ACE), Lisinopril (N 2 -((S)-1-carboxy-3-phenylpropyl)-L-lysyl-L-proline). BACKGROUND ART N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline can be prepared by a method described in JP-A-61036297 which corresponds to EP-A-0 168 769, and the like. As a method for isolation and purification of thus obtained N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, the crystallizing method is extremely easy. EP-A-0 168 769 discloses a crystallizing method using methyl tert-butyl ether/cyclohexane (650ml/240ml). EP -A- 0 336 368 discloses crystallization using methyl tert-butyl ether / cyclohexane (5.5 ml / 2 ml). US -A- 4,786,737 discloses crystallization using methyl tert-butyl ether / cyclohexane (300 ml / 110 ml). Also, in Journal of Organic Chemistry, 53 , 836-843 (1988), there is disclosed a crystallizing method using methyl tert-butyl ether/cyclohexane or methyl tert-butyl ether/hexanes (C 6 H 14 ) (225ml/85ml cyclohexane). However, as to the methods using these solvents as crystallizing solvents, there are problems that the solvents are highly hazardous and that the crystal properties of the resulting N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline is unfavorable. Therefore these methods are not necessarily preferable. Due to the unfavorable crystal properties, solid-liquid separation takes a lot of time and mother liquor cannot be sufficiently replaced when washing the cake. Further, once obtained crystal easily melts or becomes oily (syrupy). So, during the solid-liquid separating step to drying step, there arise following problems. That is the molten or oily state of some or most of the crystal results in unsuccessful separation and collection thereof. Also, the extreme difficulty of the removal of solvents from the obtained oily matter or molten matter, requires a lot of time for the drying step or results in stopping of the drying step halfway. Preliminary research of the present inventors for crystallizing solvents other than methyl tert-butyl ether/cyclohexane or methyl tert-butyl ether/hexanes (C 6 H 14 ) has shown problems that N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline is not crystallized or is obtained only in an oily state, or even if gelled matter is obtained, collection by filtration cannot be done due to unsuccessful crystallization. Thus it has been proved that the crystallization is an extremely difficult technical subject. An object to be solved by the present invention is to find out solvents which enable the crystallization of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline with favorable crystal properties, instead of methyl tert-butyl ether/cyclohexane or methyl tert-butyl ether/hexanes (C 6 H 14 ). DISCLOSURE OF THE INVENTION As a result of continuous effort and earnest investigation of the present inventors with respect to solvents for crystallization of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, it has been found that solvents which enable the crystallization with favorable crystal properties are methyl tert-butyl ether as the crystallizing solvent, and methylcyclohexane as the auxiliary solvent for controlling the crystallizing condition such as the solubility, the crystallization concentration, the yield and the crystal properties of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline. BRIEF EXPLANATION OF THE DRAWINGS Fig. 1 is a chart showing the result of FT-IR measurement with respect to a sample containing 1 wt % of 1,1,1-trichloroethane in Reference Example 17. Fig. 2 is a chart showing the result of FT-IR measurement with respect to a sample containing 5 wt % of 1,1,1-trichloroethane in Reference Example 17. Fig. 3 is a chart showing the result of FT-IR measurement with respect to a sample containing 15 wt % of 1,1,1-trichloroethane in Reference Example 17. BEST MODE FOR CARRYING OUT THE INVENTION Control for crystallizing conditions such as solubility, crystallization concentration, yield and crystal properties of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline is sometimes unsuitable. In such case, a suitable amount of an auxiliary solvent can be added for controlling the above-mentioned condition. As such auxiliary solvent, there can be exemplified diethyl ketone, dioxane, ethyl acetate, nitromethane, methylene chloride, tert-butanol, benzene, n-hexane, cyclohexane, methylcyclohexane, and the like. Particularly, there is preferably employed a method wherein the crystallization concentration is increased by improving the solubility to some extent, using methylene chloride or ethyl acetate, or a method wherein yield and crystal properties are improved by using methylcyclohexane. In case of using, for example, methylcyclohexane as the auxiliary solvent, due to the effect thereof on phase transition property and amount of solvent present inside crystal, operability in drying step is more excellent and crystal which hardly melts can be obtained, compared with the case of using cyclohexane, while the effects on a solvent for crystallizing N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline are similar to each other. These auxiliary solvents can be effectively used together with a crystallizing solvent in a preferred ratio, according to characteristic of each auxiliary solvent based on known properties such as melting point, boiling point, vapor pressure property and phase transition, the amount of solvent present inside crystal mentioned above, and effect on solubility of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline. Now, it has been found that the action mechanism of methyl tert-butyl ether, which has been known as an excellent crystallizing solvent for N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, is based on its molecular structure. Therefore, it can be also combined with an auxiliary solvent to control the crystallization condition of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline. Particularly, in case of using methyl tert-butyl ether together with methylcyclohexane as the auxiliary solvent, the characteristic of methylcyclohexane as mentioned above can be shown, compared with the combination with cyclohexane described in JP-A-61036297. In this case, the ratio of methyl tert-butyl ether and methylcyclohexane can be substantially the same degree ratio as in the combination with cyclohexane described in JP-A-61036297. The weight ratio of methyl tert-butyl ether/methylcyclohexane is, however, practically 1/1 to 1/0.1, preferably 1/0.7 to 1/0.2, more . preferably 1/0.4 to 1/0.25, at the end point of the crystallization. When the ratio of methyl tert-butyl ether/methylcyclohexane is at most 1/1, there deposit impurities and the quality of the resulting crystal deteriorates, or there tends to be collected oily matter or crystal having worse crystal properties. When the ratio is larger than 1/0.1, the yield lowers or melting preventing effect for crystal is low. Thus such ratio is not preferable. As a crystallizing method using the above-mentioned compound as a solvent, there can be employed a usual crystallizing method such as cooling crystallization, concentrating crystallization or a crystallizing method wherein a solution comprising the other solvents is replaced with solution comprising the above-mentioned compounds. On the crystallization, seed crystal can be added. In order to improve the crystal properties, handling property or yield of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline crystal to be obtained, mixture solvent of at least two kinds can be used, or a suitable amount of an auxiliary solvent such as n-hexane, cyclohexane or methylcyclohexane can be added previously or during the crystallization. In case that the crystallization cannot be easily carried out owing to the effect of coexistent impurities, it is preferably employed that firstly the crystallization is carried out by using only a crystallizing solvent, and then an auxiliary solvent is successively added. Thus obtained N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyI-L-lysyl-L-proline crystal can be subjected to a usual solid-liquid separation, cake washing and then drying. As a method for solid-liquid separation, there can be employed a usual method such as pressure filtration, filtration under reduced pressure or centrifugation. As a drying method, drying under reduced pressure (vacuum drying) at a low temperature of about 20°C to about 50°C is preferably employed from the viewpoint of suppression of melting or becoming oily. In order to improve the crystal properties or the handling property of wet crystal, the wet crystal can be washed or replaced with the compounds usable in carrying out the present invention. Hereinafter, the present invention is explained by means of Examples and Reference Examples. Reference Example 1 N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline was dissolved with heating in a concentration of at most 100 w/v % into a compound in Table 1 as a solvent, to give each transparent solution. The resulting solution is cooled to room temperature and observed the presence of crystal deposition. No. Compound used as a solvent Presence of crystal deposition 1 Water - 2 Methanol - 3 Ethanol - 4 Isopropanol - 5 tert-butanol - 6 tert-pentyl alcohol - 7 neopentyl alcohol - 8 acetonitrile - 9 propionitrile - 10 acrylonitrile - 11 acetone - 12 methyl ethyl ketone - 13 diethyl ketone - 14 methyl n-propyl ketone - 15 methyl n-butyl ketone - 16 methyl isobutyl ketone - 17 methyl tert-butyl ketone + 18 acetophenone - 19 benzene - 20 toluene - 21 xylene - 22 n-butylbenzene - 23 sec-butylbenzene - 24 tert-butylbenzene + 25 phenyltrichloromethane + 26 thiophen - 27 methyl acetate - 28 ethyl acetate - 29 isopropyl acetate + 30 isobutyl acetate - 31 tert-butyl acetate + 32 γ-butyrolactane - 33 nitromethane - 34 nitroethane - 35 diethyl ether - 36 dioxane - 37 anisole - 38 methyl tert-butyl ether + 39 2,2-dimethoxypropane + 40 trimethyl orthoacetate + (slight crystallization) 41 tetramethyl orthocarbonate + 42 formic acid - 43 acetic acid - 44 n-hexane - 45 cyclohexane - 46 methylcyclohexane - 47 neopentane - 48 methylene chloride - 49 1,2-dichloroethane - 50 n-butyl chloride - 51 isobutyl chloride + 52 tert-butyl chloride + 53 2,2-dichloropropane + 54 1,1,1-trichloroethane + 55 carbon tetrachloride + 56 1,1,2-trichloroethane + 57 neopentyl chloride + 58 2,2-dichlorobutane + 59 bromotrichloromethane + 60 dimethylformamide - 61 dimethylsulfoxide - Reference Example 2 Into 100 ml of 1,1,1-trichloroethane was dissolved 12 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline with heating. Then the solution was cooled to room temperature and the deposited crystal was collected by filtration. The obtained crystal was washed with 1,1,1-trichloroethane and dried under reduced pressure, to give about 9 g of the crystal. The melting point of the dry crystal was 78°C. Reference Example 3 A mixture of 15 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-Iysyl-L-proline, 31 g of carbon tetrachloride, and 20 g of isopropyl acetate was dissolved with heating, followed by filtering off an insoluble matter. After cooling with ice, the deposited crystal was collected by filtration and sufficiently washed with n-heptane. The resulting crystal was about 15 g. Reference Example 4 A mixture of 15 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 31 g of carbon tetrachloride and 17 g of methyl tert-butyl ether was dissolved under heating, followed by filtering off an insoluble matter. After cooling with ice, the deposited crystal was collected by filtration and washed sufficiently with cyclohexane. The resulting crystal was 12 g. Reference Example 5 A mixture of 15 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 64 g of carbon tetrabromide and 17 g of methyl tert-butyl ether was dissolved under heating, followed by filtering off an insoluble matter. After cooling with ice, the deposited crystal was collected by filtration and washed sufficiently with cyclohexane. The resulting crystal was 10 g. Reference Example 6 A mixture of 20 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 14 g of 1,1,1-trichloroethane and 8 g of diethyl ketone was dissolved with heating, followed by filtering off an insoluble matter. The resulting solution was cooled to deposit crystal. The deposited crystal was 17 g. Reference Example 7 A mixture of 30 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 10 g of carbon tetrabromide and 8 g of diethyl ketone was dissolved with heating, followed by filtering off an insoluble matter. The resulting solution was cooled to deposit crystal. The deposited crystal was 23 g. Example 1 A mixture of 15 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 27 g of 1,1,1-trichloroethane and 17 g of methyl tert-butyl ether was dissolved with heating, followed by filtering off an insoluble matter. Then the resulting solution was cooled with ice to deposit crystal. Thereto was added 10 g of methylcyclohexane. The obtained crystal was collected by filtration and washed with a mixed solvent of 8 g of 1,1,1-trichloroethane, 5 g of methyl tert-butyl ether and 3 g of methylcyclohexane, then with 10 g of methylcyclohexane. The obtained crystal was 12 g. The melting point of the dry crystal was 78°C. Example 2 A mixture of 3 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 3 g of methylcyclohexane and 12 g of methyl tert-butyl ether was dissolved with heating, followed by filtering off an insoluble matter. The resulting solution was cooled to deposit crystal The deposited crystal was about 2 g. Example 3 A mixture of 6 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline and 20 g of methyl tert-butyl ether was dissolved with heating, followed by filtering off an insoluble matter. Then thereto was added seed crystal, and the mixture was cooled to deposit crystal. After stirring overnight, thereto was added dropwise 13 g of methylcyclohexane, followed by cooling with ice. After filtration, the residue was washed with cooled methyl tert-butyl ether/methylcyclohexane (weight ratio 5 : 2). The deposited crystal was about 6 g. Reference Example 8 Each crystal was collected by filtering the suspension obtained in Reference Example 1 with No. 38, 39 or 41. Then the crystal was sufficiently washed with n-hexane. The amount of solvent present inside the crystal (mole % of the solvent used for the crystallization per N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, hereinafter the same) was determined with respect to the above-mentioned sample. The results are shown in Table 2. No. Amount of solvent present inside crystal (mole %) 38 57 39 25 41 14 Reference Example 9 Each crystal was collected by filtering the suspension obtained in Reference Example 1 with No. 52, 53, 54 or 55. Then the crystal was sufficiently washed with n-hexane. The amount of solvent present inside crystal was determined with respect to the above-mentioned sample. The results are shown in Table 3. No. Amount of solvent present inside crystal (mole %) 52 48 53 69 54 66 55 54 Reference Example 10 The amount of solvent present inside crystal was determined with respect to the sample obtained in Reference Example 3. There were found 48 mole % of carbon tetrachloride and 6 mole % of isopropyl acetate. Reference Example 11 The amount of solvent present inside crystal was determined with respect to the sample obtained in Reference Example 4. There were found 50 mole % of carbon tetrachloride and 31 mole % of methyl tert-butyl ether. Reference Example 12 The amount of solvent present inside crystal was determined with respect to the sample obtained in Reference Example 5. There were found 70 mole % of carbon tetrabromide and 23 mole % of methyl tert-butyl ether. Example 4 The amount of solvent present inside crystal was determined with respect to the sample obtained in Example 2. There were found 55 mole % of methyl tert-butyl ether and 22 mole % of methylcyclohexane. The wet crystal was vacuum dried at room temperature, to give the dry crystal having a melting point of 78°C. Reference Example 13 A mixture of 6 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline and 20 g of methyl tert-butyl ether was dissolved with heating, followed by filtering off an insoluble matter. Then thereto was added seed crystal, and the mixture was cooled to deposit crystal. After stirring overnight, thereto was added dropwise 8 g of cyclohexane, followed by cooling with ice. After the filtration, the residue was washed with cooled methyl tert-butyl ether/cyclohexane (weight ratio 5 : 2). The deposited crystal was about 6 g. The amount of solvent present inside crystal was determined and there were found 74 mole % of methyl tert-butyl ether and 18 mole % of cyclohexane. The wet crystal was vacuum dried at room temperature, to give the dry crystal having a melting point of 77°C. With respect to the wet crystal which was allowed to stand for 8 hours in an atmosphere at 20°C there was observed a partial melting thereof. Reference Example 14 A mixture of 6 g N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline and 20 g of methyl tert-butyl ether was dissolved with heating, followed by filtering off an insoluble matter. Then thereto was added seed crystal, and the mixture was cooled to deposit crystal. After stirring overnight, thereto was added dropwise 8 g of n-hexane, followed by cooling with ice. After the filtration, the residue was washed with cooled methyl tert-butyl ether/n-hexane (weight ratio 5 : 2). However, the contractibility of cake was large and therefore the washing operability was worse. The deposited crystal, of which deposited amount was about 6 g, seems transparent. The amount of solvent present inside crystal was determined. As a result, 56 mole % of methyl tert-butyl ether was detected, but n-hexane was not detected. The wet crystal, which easily melts, was immediately vacuum dried at room temperature, to give the dry crystal having a melting point of 74°C. Example 5 The amount of solvent present inside crystal was determined with respect to the sample obtained in Example 3. There were found 56 mole % of methyl tert-butyl ether and 16 mole % of methylcyclohexane. The wet crystal was vacuum dried at room temperature, to give the dry crystal having a melting point of 78°C. With respect to the wet crystal which was allowed to stand for 24 hours in an atmosphere at 20°C, there was not observed any melting indication. Example 6 A mixture of 3 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, 17 g of methylcyclohexane and 12 g of methyl tert-butyl ether was dissolved with heating, followed by filtering off an insoluble matter. The resulting solution was cooled to give an oily matter. Reference Example 15 Into 30 ml of ethanol was dissolved 10 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-prolin. To this solution was added gradually cyclohexane, to give an oily matter. Reference Example 16 Into 30 ml of methylene chloride was dissolved 10 g of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline. To this solution was added gradually cyclohexane, to give an oily matter. Reference Example 17 FT-IR was measured with respect to samples in Reference Example 2 obtained on various drying times. The results of FT-IR measurement are shown in Fig. 1 to Fig. 3. Apparatus: FTS-15E/D (trade name, made by Bio Rad Digilab, USA) Attached Apparatus: Diffuse reflectance spectrometer (trade name, made by Spectra tech, USA) (Condition) Light source: Special ceramics Detector: MCT Resolution: 4 cm -1 Integrating times: 500 times Sample dispersing medium: none As a result, the absorption peaks of each sample around at 3300 cm -1 and 3100 cm -1 were found at positions as shown in Table 4. As clearly shown in Fig. 1 to Fig. 3, there were observed changes in the intensity of peak at 1724.4 cm -1 . From the above-mentioned, it can be considered that the solvent interacts with the substrate, namely N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, and that this interaction participates in the crystallization. INDUSTRIAL APPLICABILITY According to the present invention, N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, which is very hard to be crystallized, can be stably obtained as a crystal with favorable crystal properties.

Claims: A process for crystallizing N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl-N 6 -trifluoroacetyl-L-lysyl-L-proline by using methyl tert-butyl ether as a crystallizing solvent, characterized in that methylcyclohexane is used as an auxiliary solvent in combination with said methyl tert-butyl ether. The process of claim 1, wherein the crystallization is carried out by solvent replacement of a solution comprising other solvents to form a solution comprising methyl tert-butyl ether and methylcyclohexane. The process of claim 1 or 2, wherein the weight ratio of methyl tert-butyl ether/methylcyclohexane is 1/1 to 1/0.1. The process of claim 1 or 2, wherein the weight ratio of methyl tert-butyl ether/methylcyclohexane is 1/0.7 to 1/0.2. The process of claim 1 or 2, wherein the weight ratio of methyl tert-butyl ether/ methylcyclohexane is 1/0.4 to 1/0.25. The process of any one of claims 1 to 5, wherein methylcyclohexane is successively added in the presence of both N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline and methyl tert-butyl ether. The process of any one of claims 1 to 5, wherein N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline is dissolved in methyl tert-butyl ether and crystallized, and methylcyclohexane is added thereto. The process of any one of claims 1 to 7, wherein the obtained crystals of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline are dried under reduced pressure at a temperature of about 20°C to about 50°C. A process for crystallizing N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline, wherein a mixture of four parts by weight of methyl tert-butyl ether and one part by weight of methylcyclohexane is used as a crystallizing solvent. The process of claim 9, wherein the obtained crystals of N 2 -((S)-1-ethoxycarbonyl-3-phenylpropyl)-N 6 -trifluoroacetyl-L-lysyl-L-proline are dried under reduced pressure at a temperature of about 20°C to about 50°C.

Inventors: MANABE HAJIME, UEDA YASUYOSHI, MANABE, HAJIME, UEDA, YASUYOSHI
Applicants: KANEGAFUCHI CHEMICAL IND, KANEGAFUCHI KAGAKU KOGYO KABUSHIKI KAISHA
Application year: 1993
Main classification: C07K5
IPC codes: C07K5