• 专利附录
  • 专利说明
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    • 专利摘要:
    PURPOSE:Adducts of dipeptide esters and aminocarboxylic acid esters I(R1 is aliphatic oxycarbonyl, nuclear substd-benzyloxycarbonyl, etc.; R2 is methyl, isopropyl, etc.; R3 is lower alkoxy, benzyloxy, etc.; n is 1 or 2), e.g. adduct of Nbenzyloxycarbonyl-L-aspartyl-L-phenylalaninemethylester with L-phenylalaninemethylester.
    公开号:SU910117A3
    申请号:SU782570052
    申请日:1978-01-26
    公开日:1982-02-28
    发明作者:Изова Есиказу；Охмори Мунеки；Мори Каору；Исикава Тецуя；Нонака Юдзи；Кихара Кейити；Ояма Кийотака；Сато Хейидзиро；Нисимура Сигеаки
    申请人:Тойо Сода Мануфакчуринг Ко,Лтд (Фирма)；(Зайданходзин) Сагами Кемикал Рисерч Сентер (Фирма)；
    IPC主号:
    专利说明:

    39 where R enzyloxycarbonyl, para-toxybenzyloxycarbonyl; RJJ is benzyl; R-methoxy, ethoxy group; P - 1 or 2 N-substituted aminodicarboxylic acid of general formula .JS 1 and HOC- (CH2) p-CH-COH, where the values of Rf and n are shown above, interact with the amino carboxylic acid ester of the general formula HgT-CI-C-Bc (Iii) And, where the values of R and Rg are as indicated above. With a polar ratio of the initial compounds of the formulas II. and III 1: 1 - 1:: 3e75, in an aqueous medium, in the presence of metalloprotease at pH 5, temperature 10-90 C. The products obtained during the reaction, we have little solution in water and precipitate. In addition, the proposed method allows to obtain an optically active product of the addition of a dipeptide and an amino acid ester from the racemates, since the reaction for the formation of a peptide bond occurs only on L-isomers. On the other hand, the amino acid esters used to conduct the addition reaction to form the addition products can be either in the L-form, or in the D-form, or a mixture of them. When using the DL form of aminocarboxylic acid ester, the L isomer of the DL form of the amino carboxylic ester is consumed in the process of peptide synthesis in the preparation of the addition product, the residual amino acid ester is in the D form. amount of output. By using two moles of the DL form of the aminocarboxylic ester per 1 mole of the L-form of the N-substituted monoamino dicarboxylic acid, an addition product is obtained, consisting essentially of the LL dipeptide ester and 74 of the D-aminocarboxylic acid ester. The resulting addition product can be easily divided into two partial components, i.e., LL-dipeptide ester and D-amino acid ester. Thus, the proposed method can simultaneously provide a dipeptide ester and optical separation of the DL-aminocarboxylic ester. The separated D-form or the enriched D-form of the amino acid ester can be racemized in the usual way, and the resulting product can be used as a starting material in the described method. When using the DL form of N-substituted monoamino dicarboxylic acid and the L-form of the amino acid ester, the D-isomer from the DL form of the N-zammelyanny monoamino dicarboxylic acid, is inert, remains in an aqueous medium, so that the product can be obtained by adding LL-dipeptide ester and ester of L-aminocarbonic acid. Accordingly, if the D-form L of a substituted monoamino dicarboxylic acid is separated from an aqueous medium, then this moquette will simultaneously provide the addition product and the optical separation of the 1-substituted DL monoamino dicarboxylic acid. If the isolated N-substituted D-monoamino dicarboxylic acid is racemized in a known manner, then the resulting product can be used as the starting material. When using the DL form of N-substituted monoamino dicarboxylic acid and the DL form of an aminocarboxylic ester, an N-substituted DL-monoamino dicarboxylic acid as well as an adduct of LL-dipeptide ester and D-aminocarboxylic acid can be obtained from the aqueous medium, and the indicated product the attachments are divided into components as indicated above. At the same time, the formation of a dipeptide ester and the optical separation of the N-substituted DL-monoaminodicarboxylic acid and the DL-aminocarboxylic ester can be achieved. The proposed method makes it possible to eliminate the stages of introduction and removal of side-chain protecting groups that are considered unavoidable in the known 59 methods, which accordingly prevents the loss of the starting materials; and use the DL form of the starting compounds. In conventional ways, using the enzyme D-isomer, the 13B-form of the starting materials is not needed in the reaction, and although it does not affect the course of the reaction, it causes a loss of the starting materials. However, the D-form of the starting materials can be successfully used as an agent for sedimentation of the peptide and their subsequent reduction. Thus, when implementing the proposed method, the optical separation of N-substituted DL-aminodicarboxylic acid and DL aminocarboxylic ester can be simultaneously achieved. Addition products have characteristics that can be expected based on formula I. For example, a typical addition product obtained by the reaction M -benzyloxycarbonyl L-aspartic acid and L-phenylalanine methyl ester, gives absorption in the infrared range and the NMR spectrum indicated below. Infrared spectrum: 32bO cm (stretching tension of N-41 bond); 3000-3200 cm (1) stretching the bond C — H); G / h O (ester); 172U cm (urethane); 1bbO cm (1st amide absorption); 1BH0 cm (carboxylate); lijAOcM (2nd amide absorption 1; and I + SO cm (deformation vibration); 1390 cm (carboxylate); 1200 and 1290 cm (C – O – C coupling stretching and 3rd amide absorption); 1050 cm ( oscillation of phenyl in the plane; and and 69IJ cm (Oscillation of a monosubstituted benzene ring outside the plane). NMR spectrum, 6:. (1) 2.75 ppp (2H (2) 3i02 ppm (itH); (3) 3, 61 RRP SZN) 3.7 ppm (GH); (A) k, -k, Q ppm (GH); (5.) 5.05 ppm (2H); (6) 5.82 ppm (5H) (7 ) 7.3 ppm (15H). The results of the elementary analysis of the addition product turned out to be the same as the calculated values for formula I, where the values of R1, R2, Rj, and n are as above. If the attachment product is treated with a strong acid, such as hydrochloric acid, and the product obtained is extracted from an organic solvent, such as ethyl acetate, then the acidic compound is obtained from the organic 7.66 layer. and the properties corresponding to the compound of the formula; NOC- (CHg) p-CH SS-CHC-Cs 0) in the LL form, where R, Rg., RT, and n have the same meanings as in the adduct. The acidic compound obtained from the addition product in the course of catalytic reduction gives a known compound, methyl ester of para-phenyl alanine. The results of the analysis of the compounds obtained on the basis of infrared spectra, the NMR spectrum and the elementary analysis confirm the structure of the compound of formula I. Fully compatible. results were also obtained in the case when compounds with other values of R, Rj, b are selected. The proteases used in the proposed method are metalloproteases having a metal ion in the active center. Suitable metalloproteases are enzymes derived from microorganisms, such as neutral proteases from actinomycete, prolizin, thermolysin, collagenase, Crotuliis atrox protease, etc. Also, non-purified enzymes, such as thermoases, takinase-N, pronaz and t, can be used. , d. In order to suppress the action of esterase contained in crude enzymes, it is preferable to use an enzyme inhibitor, a potato inhibitor, together with the crude enzymes. Thiol proteases, papain or serine proteases, trypsin are used, however, they possess esterase activity. Therefore, during the 1; 1 reaction with the use of such enzymes, measures must be taken to prevent the esters from hydrolyzing. Example 1. 1335 mg (5 mmol) of L-aspartic acid H-benzyloxycarbonyl and 1078 mg (5 mmol) of L-phenylalanine methyl ester hydrochloride are placed in a 30 ml flask and 20 ml / WATER is added to dissolve them, the pH is adjusted to 6 via . ammonia
    791
    water. The resulting solution is mixed with 50 mg of thermolysin, the mixture shaken at 38-4 ° C overnight. The precipitate is filtered off and washed with BUT ml of water and dried. 1145 mg of fine needles are obtained, having a Y. pl. (adduct: no N-benzyloxycarbonyl methyl ester L-aspartyl-L-phenylalanine and L-phenylalanine methyl ester (1: 1); yield 75.5% based on methyl phenylalanine ester hydrochloride).
    After recrystallization from a solvent mixture of ethyl acetate and n-hexane, the physical properties and results; Elementary analysis of the product is the following: t. pl. 120-124C; (JL) + +7,} (, methanol).
    Found,%: C 63.15; H 6.15; N7.00
    Sag.Nz HjOg
    Calculated,%: C, 63.24; H 6.13; N 6.97.
    You are the IR spectrum and the 1 H-NMR spectrum of the product.
    1145 mg of the product obtained is dissolved in 40 ml of 1N. HC and extracted three times with 30 ml of ethyl acetate. The extracts are combined and washed. take portions of 20 ml (3 times), and dried over anhydrous magnesium sulphate. The solution is concentrated and the product is recrystallized from n-hexane, 640 mg is obtained. The physical properties and the results of the elementary analysis of the Product are as follows: t. Pl. 115-125 С; (J) -15.3 (methanol)
    Found,%: C 61.52; H 5.65; 146.57
    Saangangoog
    Calculated,%: C, 61.67; H 5.65; N6.54.
    The results of the infrared spectrum and the NMR spectrum give the characteristics expected in the methyl ester
    N-benziloxycabronyl-L-acpaptil-L-fe7. Nilalanine.
    The results obtained coincide with
    the results of the study of the compound obtained by benzyloxycarbonylation of the amino group of the methyl ester of L-acpartil-L-phenylalanine.
    L-phenylalanine methyl ester was isolated from a mixture of the hydrochloric acid phase and the washing water fraction separated during extraction from the ethyl acetate phase. It was confirmed that the compound obtained through the first reaction is an addition product of N-benzyl-hydroxy-parabonyl-L-ac-paraptil-L-phenylalanine methyl ester and L78 methyl ester;
    phenylalanine. SpectroH-NMR confirmed that their molar ratio was 1: 1.
    Example 2. 133 i mg (5 mmol) of L-benzyloxycaronyl: -b-aspartic acid and 1078 mg (5 mmol) of L-phenylalanine methyl ester hydrochloride are loaded into a 30 ml flask and 10 ml of water are added to the mixture until dissolved and pH the solution is made up to 6 with ammonia water. The resulting solution is mixed with 50 mg of thermolysin, and the mixture is continuously shaken at C overnight. The precipitate is filtered off, dried, to obtain 1504 mg of the product of the addition of methyl ester of M-benzylLcicaronyl-L-aspartyl-phenylalanine and methyl ester of L-phenylalanine 0-0 (yield per L-phenylalanine methyl ester hydrochloride, mp. 10} 113 0 .
    Example 3. The preparation of the product and the treatment of the reaction mass are carried out analogously to example 2, but the amount of N-benzyl-hydroxy-baseonyl-L-ac-paraginic acid and L-phenylalanine methyl ester is changed by 53 mg (2 mmol) and 863 mg (4 mmol), respectively, to obtain 1068 mg of the product of the addition of methyl ester of N-benzyloxycarbonyl-L-ac-para-n-L-phenylalanine and methyl ester of L-phenylalanine (1: 1). Yield 70.4% based on N-benzyloxycarbonyl-L-ac acid; m.p. 11b-119 S.
    Example 4. 534 mg (2 mmol) of N-benzyloxycarbonyl-L-ac-paraginic acid and 863 mg (4 mmop) of L-phenylalanine methyl ester hydrochloride are loaded into a 30 ml flask, dissolved in 8 ml of water, the pH is adjusted to 6.2 with the help of ammonia water. The resulting solution is mixed with 50 mg of thermolysin, and the mixture is shaken at 38-40 ° overnight. The precipitate is filtered off, separated from the solution and dried, to give T099 mg of the product of addition of N-benzyloxycarbonyl-L-ac-paraptil-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1: 1). Yield 90.5 based on N-benzyloxycarbonyl-L-ac arginic acid.
    Example 5. 2b7.2 mg (1 mmol) of N-benzyl-oxycarbonyl-L-ac-paracinic acid and 537 g 6 mg (3 mmol) of L-phenylslanine methyl ester are dissolved in 5 ml of Mac-Ilvay 9 buffer solution
    at pH 7.0, the resulting solution is mixed with 100 mg of thermoase and 100 mg of potato inhibitor, and the mixture is shaken for 20 hours at. The precipitate was filtered and washed with water and dried, yielding 580 ng of the crude crystalline addition product of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1: 1). Output .95,5 in the calculation of M-benzyloxycarbonyl-b-aspartic acid, so pl. 123-125 C.
    The product is dissolved in tO ml of a mixture of dimethylformamide and water (1: 1), and the H-form of the strongly acidic cation-exchange resin is added to the solution with stirring, which is then separated and the filtrate is concentrated under reduced pressure. The residue is dissolved in dimethylformamide and water is added to the solution, 330 mg of methyl 7H benzyloxycarbonyl-b-aspartyl-b-phenylalanine is precipitated, yield 77.0% based on M-benzyloxycarbonyl-b-aspartic acid, m.p. 123125 0.
    Example 6. 2b7.2 mg (1 mmol) of L-benzyloxycarbonyl-L-aspartic acid and 537.6 mg (3 mmol) of L-phenylalanine methyl ester are dissolved in 5 ml of McIlvayn buffer solution. (pH 7.0). The resulting solution is mixed with 100 mg of the takinase N and 100 mg of the potato inhibitor, the mixture is shaken for 6 hours at. The precipitate is filtered off, washed with water and dried. 120 ng of crude crystalline addition product of N-benzyloxycarbonyl-b-aspartyl-b-phenylalanine methyl ester and b-phenylalanine methyl ester (1: 1) yield 19.7, m.p. 119-123 C.
    According to Example 5, the product is treated with a strongly acidic cation-exchange resin in the H-form; 50 mg of the methyl ester of H-benzyloxycarbonyl-b-aspartyl-b-phenylalanine is obtained. yield 11.7%, so pl. 95-1054.
    Example 7. 1335 mg (5 mmol) of M-benzyloxycarbonyl-b-aspartic acid and 1078 mg (5 mmol) of L-phenylalanine methyl ester are loaded into a 30 ml flask, k CP of water is added, the pH of the resulting solution is adjusted to 6, 8 with triethylamine, 20 mg of thermolysin is added, the mixture is injected hp for 7-10.
    2 days. The precipitate is filtered off, washed with kQ ml of water, dried to obtain .475 kg of the product of the addition of methyl ester of N-benzyloxycarbonyl-1-aspartyl-b-phenylalanine and methyl ester of L-phenylalanine (1: 1), Output 31.3 per methyl chlorohydrate ether L-phenylalanine. After recrystallization from a mixture of ethyl acetate and n-hexane, the product is obtained with a mp. 120-124 С, (i-) J + 7.2 | С
    1, methanol)
    Elemental analysis Found,%: C 63.52; H 6.22; N 7, Q Calculated,%: C 63.2A; H 6.13; N 6.97
    Example 8. The process is carried out analogously to example 7, but the pH is adjusted. to 5.2, 753 mg of the product of addition of N-benzyloxycarbonyl-L-ac-paraptil-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1: 1) are obtained. Output, 5 in the calculation of the methyl ester of L-phenylalanine.
    Example 9. To a solution of 133.6 mg (0.5 mmol) of K-benzyl icarbonyl-β-aspartic acid and 89.2 mg (0.5 mmol) of L-phenylalanine methyl ester in 2.5 ml of a buffer solution
    Mac-Ilvain (pH 7.0) with 0.07 ml of triethylamine, having a pH of 6.7, 50 mg of thermoase and 50 mg of a potato inhibitor are added, the mixture is shaken at 20 hours. The precipitate is filtered off, washed with water, 130 mg are obtained. the crude product is attached to the methyl ester of N-benzyloxycarbonyl-L-ac-propyl-L-phenylalanine and methyl ester of L-phenylalanine (1: 1). Output 85.5 per methyl ester of L-phenylalanine, so pl. 115
    The product solution is dissolved in 20 ml of a mixture of dimethylformamide and water (1: 1), treated with strong acids.
    Noah cation exchange resin
    according to example 5, 75 mg of N-benzyloxycarbonyl-L-phenylalanine methyl ester are obtained, the total yield is 70% based on 50% of the amount of the starting
    methyl ester of L-phenylalanine.
    Example 10. The reaction is carried out similarly to Example 9, but using 0.05 ml of N-methylmrrpholine instead of 0.07 ml of triethylamine at an initial pH of 6, A. 120 mg of a crude crystalline addition product of N-benzyloxycaronyl-L-ac-propartyl-L-phenylalanine methyl ester and 119 L-phenylalanine methyl ester are obtained (1: 1 yield of 78.9, calculated on L-phenylalanine methyl ester, mp. LlS-IZiC Product treated with a strongly acidic cation-exchange resin in the H-form according to Example 9, 70 mg of N-benoyloxycarbonyl-b-phenylalanine crystalline methyl ester are obtained. The total yield is 66% based on the amount of the starting L-phenylalanine methyl ester. Example T, but the reaction is performed at pH 6.5 with shaking for 1 hour, 920 mg of the product of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1: 1) are obtained. Yield 75.8, Example 12. 52 mg (2 mmol) of M-benzyloxycarbonyl-L-asparagiNoic acid and 863 mg (k mmol); L-phenylalanine methyl ester hydrochloride is dissolved in 2 ml of water; 5.5 are added to the solution. mg of 1N US up to pH 7 The pactBop obtained is mixed with 50 mg of thermolysin and the mixture is shaken at 38 ° C for 2 4i The precipitate is filtered, dried to obtain 73 mg of the product of the addition of methyl ester of N-benzyloxycarbonyl-L-acpaptil-L-phenylalanine and methyl ester of L-phenylalanine (1: 1). Exit 60 ,. 10b-118 ° C. p. and measure 13. mg (2 mmol) of N-benzyl-oxycarbonyl-L-ac-paraginic acid and 863 mg (4 mmol) of L-phenylalanine methyl ester hydrochloride are dissolved in 7 ml of water, the pH is adjusted to 6 with ammonia water, 100 mg of thermoase is added to the solution, the mixture is shaken at 38 ° C for overnight. The precipitate is filtered off, washed with 70 ml of water, dried, and 550 mg of the N-benzyloxycarbonyl-L-paraptilyl-L-phenylalanine methyl ester (1: 1) are obtained. The output of 5.3%, so pl. 113-1164. EXAMPLE I. 270 ml (1 mmol) of N-benzyloxycarbonyl-L-acarginic acid and mg (2 mmal) of L-phenylalanine methyl ester are dissolved in 4 ml of water, the pH is adjusted to 6 with 7% ammonia water. 50 mg of thermoase is added to the resulting solution, the mixture is continuously shaken for 4 hours. The precipitate is filtered off and dried, 177 mg of the product Attachment 7. 12 of N-benzyloxycarbonyl-L-acnapgyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1: 1) are obtained. Exit 23, Pt. Pl. 103-112С. Example 15. The process is carried out analogously to Example H, but an additional 50 mg of a potato inhibitor is added to the solution. 381 mg of the same product are obtained. Exit 62, U, so pl. 105-117 ° C. Example 16. 53 mg (2 mmol) of N-benzyloxycarbonyl-L-acaparginic acid and 863 mg (k mmol) of DU-phenylalanine methyl ester hydrochloride are dissolved in 7 ml of water, the pH value is adjusted to 6.2 with 7% - ammonia water. The resulting solution is mixed with 50 mg of thermolysin and shaken overnight at. The precipitate is filtered off, dried, and mg of N-benzyloxycarbonyl-aspartyl-phenylalanine methyl ester and phenylalanine methyl ester (1: 1) are obtained. Chip 86.1 per N-benzoylcarbonyl-L-acaparginic acid, m.p. . After recrystallization from a mixture of ethyl acetate and n-hexane, a product is obtained. 127-135 0.14-1 - 6, (C 1, methanol). Found,%: C 63.52; H 6.19; N6.92 aCaaHvNjOg; Calculated: C 63.24; H 6.13; N6.97. The NMR method shows the identity of the obtained product with the product of addition of N-benzyl-hydroxycarbonyl-L-ac-aptil-L-phenylalanine methyl ester and D-phenylalanine methyl ester (1: 1), i.e. the compound of Example 1. 800 mg of the product are dissolved in 0 mg of 1N. carried and extracted with dichloromethane, (30 ml of X3) the organic phase is separated, washed, washed with water, dried over anhydrous sulfate, evaporated, the residue is recrystallized from ethyl-, mixture. acetate and n-hexane, get mg of crystalline product, etc. 12 1324, - 15.3 (C 1 methanol). . Found: C 61.38; H 5.58; N6.29 SgaIgs b. Calculated,%: C, 61.67; .H 5, B5; N 6.5A. The product is N-benzyl-oxycarbonyl-L-acapapyl-L-phenylalanine methyl ester.
    13: 9101
    After separation of the aqueous phase, the dichloromethane layer is mixed with sodium bicarbonate, the pH value is adjusted to 8.7 and the product is extracted three times with 30 ml of methylene chloride. The extract 5 is dried over anhydrous magnesium sulphate, hydrogen chloride gas 8 is fed into the extract for 10 minutes, then evaporated to dryness. The residue is recrystallized from ethyl ether; 29.0 mg of methyl hydrochloride are obtained.
    K-Z-DL-Asp
     . BUT
    B isome /} L isomer
    i ffWf /, N-Z-I, -Ap-L-Phe-No
    In the reaction
    Mr. Growth
    NzL-Asp-L-Phe-Me-L10b9 MrCt mmol) N-benzyloxy-carbonyl-Bb-aspartic acid and 1 863 mg (mmol) of L-phenylalanine methyl ester are dissolved in 2 ml of water, the pH is adjusted to 6c using ammonia water.
    The resulting solution is mixed with 50 .mg of thermolysin and shaken at 38-40 for 2 hours. The precipitate is filtered off, washed with 20 ml of water, dried, 787 mg of the product of N-benzyloxycarbonyl-L-aspargyl-b-phenylalanine methyl ester are obtained. methyl ester of L-phenylalanine
    N-Z-DL-Asp l
    B isomer 1, is the isomer ir-ZOGPS161LSL 8 reactionary pacmSope dissolved
    {The process is carried out similarly to Example 17, but using DL-phenylalanine methyl ester instead of methyl
    171
    air D-fvnilalanina, f, pl. NE 151 ° C, W - 1B, 1 (C 1, methanol); (the infrared spectrum and the NMR spectrum coincide with the spectra of the L-form),
    Thus, the resulting product is an addition product of N-benzyloxy-carbonyl-b-aspartyl-b-phenylalanine methyl ester and D-phenylalanine methyl ester (1: 1).
    Example 17. I
    L phe-me
    .one
    (1: 1) yield of 6.8 per N-benzyloxy | ebonyl-b-aspartic acid, so pl. 105-110 0.
    After recrystallization from a mixture of ethyl acetate and n-hexane, get the product of Art. square 121-125 0; ti-1 If “7.2 (C 1, methanol).
    N-benzyloxycarbonyl aspartic acid (mostly in the D-form) is recovered from the remaining reaction mixture. i.,. Pm and me 18. Use both dyuemeta
    DL - Phe - Me
    .
    It-isomer th isoper
    ether L-feyilalanina. 75b m1 of the L-benzyloxycarbonyl-L-Asp-L-Pbe-Me methyl ester addition product is obtained. H-Z-L-A p-L-Phe-Me-D-Phe-Me. residue 91-aspartyl-L-phenylalanine and D-phenylalanine methyl ester (1: 1). Yield 62.3% based on N-bvnzyloxycarbonyl | -aspartic acid, t. gsh. 105-111 C, The product is recrystallized from a mixture of ethyl acetate and n-hexane, m.p., - l g l lh g. O l l 1.1. XV f C- -; ; Ш - 6/5 (0 1, meta126-13 C / - .ratX zero). N-Benzyloxycarbonyl laslaparagic acid (mainly in the D-form) is recovered from the remaining reaction mixture. ;. After 19 g. (20 mmol) of K-benzyloxycarbonyl-L-aspartic acid and 7.32 g (2 mmol) of L-phenylalanine methyl ester are dissolved in 70 ml of water. Get a solution with a pH of 6.2-6.3. 200 ml of thermolysin is added to the solution and the mixture is continuously shaken for k hours at. The precipitate is filtered off, washed with 70 vs of water, dried, 10.11 g of product is obtained, (t. Pl. P7-120 ° C) .: The resulting compound is a product. addition of methyl ester of N-benzyloxycarbonyl-b-aspartyl-b-phenylalanine and methyl ester of L-phenylalanine (1: 1), after recrystallization from a mixture of acetate and H-hexane, mp, 120 12VC, + 7.2 (C 1, methanol). Found,% C 63, T6; H 6.14; N6.99 C..2Hi7N509 Calculated, G 63.24; Nb, 13 N 6.97 The infrared spectrum and the NMR spectrum showed the same characteristics as those indicated above for the product of the (1: 1) N-bey zyloxycarbonyl-L-aspargyl-b-phenylalanine methyl ester and L-phenylalanine methyl ester. After treatment of the product with a strong acid, extraction with ethyl acetate, and subsequent distillation of the organic solution, K-benzyl-xycarbonyl-b aspartyl-b-phenylalanine methyl ester is obtained. 1.00 g (5 mmol) of the obtained product of the addition of methyl ester H-benzyloxycarbonyl-b-aspartyl-b-phenylalanine and ester of L-phenylalanine are loaded into a 30 ml flask and 2 ml of water and 2.0 ml of 1N are added. HC, the mixture is stirred at room temperature for 10 minutes. The suspension is filtered, the precipitate is washed with 4 ml of water, dried, and 0.72 g of methyl ester of N-benzyl7 of sycronyl-b-aspargyl-b-phenylalanine is obtained. The yield is 98.8%. After being dissolved in ethyl acetate, the product is precipitated with n-hexane, m.p. 121-12tC, 1 -15.4 (C 1, mennol). Found,% C 61.58; H 5, N6.56. CrHN21, lHO Calculated,% C 61, b7; H 5.65; N 6.5. The infrared spectrum of the product coincides with the spectrum of the standard product. The identity of the product is confirmed by comparing the aqueous solution of the obtained product with the aqueous solution of the standard compound by high-speed liquid chromatography. The purity measured by this method is 100%. A device for carrying out high-speed liquid chromatography and the conditions for its performance are indicated below. This method is also used to assess the purity of the decomposition products, the addition products in the examples, unless otherwise specified. The same device and the same conditions are used in the examples when it comes to this method. High speed liquid chromatography device (TSK-HLC 801). Column: internal diameter 7.5 mm length 30 cm; filler: starch gel, particle size 5 μm (TSKGEL LS 170) V. Eluent: 0, sodium acetate aqueous solution. A flow rate of 0.8 ml / min. Pressure drop of 20 kg / cmj. The measurement temperature is room, the detector is a differential refractometer .V Approx 20. 20. 1.00 g, B5 mmol of the methyl ester addition product of M-benzyloxycarbonyl-b-aspartyl 1, -phenylalanine and I-phenyl-apanine methyl ester obtained in Example 19 is dissolved in a mixture of 2 ml of water and 1.32 MP I N. NA. The reaction Maqcy is stirred at room temperature for 10 minutes and treated under conditions as described in Example 19. 0.70 g of product is obtained in the form of fine prismatic crystals. The content of methyl ester of N-benzyloxycarbonyl-b-aspartyl-and-phenylalanine is 96.8%, mp. 100-.126С. PRI me R 21.
    17910117J8
    C-2-L-A5P .Di.-Phe-Me
    K-Z-L-Asp-L Phe-Ke
    N-Z-L-Asp-L-Flie-Me-D-Phe-Me D-Phe-Me does not react with N-Z-L-Asp, reacts with the protease with the dipeptide and precipitates as an additive compound. 0.53 g (2 mmol) of N-benzyloxycarbonyl-b-aspartic acid and 0.863 g (k mmol) of DL-phenyl-apanine methyl ester hydrochloride are diluted in 10 ml of water, the pH of the solution is adjusted to 6.0 with ammonia water. 50 mg of thermolysin are added to the solution and the mixture is shaken overnight at. The precipitate is filtered off, washed with 10 ml of water and dried, to obtain 0.90 g of crystalline product. T. pl. 120-126 0. After recrystallization from a mixture of ethyl acetate and n-hexane, get the product with so pl. 128-13 C - 6.3 (C 1, methanol). The infrared spectrum and the NMR spectrum of the product are identical to the addition spectra of N-benzyloxycarbonyl-L-aspartyl-L-phenylapanine methyl ester and L-phenylalanine methyl ester (1: 1). Found,%: C 63, A2; H 6.17; N6.95 CbaHvNiOs Calculated,% -. C 63.24; H 6.13; N 6.97. The product is treated with acid to give N-benzyloxycarbonyl-b-aspartyl-b-phenylalanine methyl ester and D-phenylalanine methyl ester in a molar ratio of 1: V. From the obtained results it was concluded that the compound is the addition product of L-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and D-phenylalanine methyl ester (1: 1). A mixture of 0.50 tons (0.82 mmol) of an adduct was mixed with 4 ml of water and 0.26 g of citric acid at room temperature for 10 minutes and treated under conditions of
    / -
    Ij isomer i isemer
    T
    -f
    I
    A precipitate of measure 19 gives 0.35 g of N-benzyloxycarbonyl-b-aspartyl-b-phenylalanine methyl ester crystals.  Purity yield 99.3%.  Example 22 .  To 0.50 g (0.82 mmol) of the product of the addition of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and methyl ester. of the L-phenylalanine ester prepared in Example 19, k ml of water and 0.24 g (1.2 mmol) of p-toluenesulfonic acid monohydrate are added, the mixture is treated as in Example 19, 0.33 g of N-benzyloxycarbonyl methyl ester is obtained -aspargil-b-phenylalanine.  Purity 100%; yield 93.6%.  Example 23  To a mixture of 0.45 g (0.82 mmol) of 85% formic acid and 8 ml of water, 0.50 g (0.82 mmol) of the product of N-benzyloxycarbonyl-b-aspartyl-b-phenylalanine methyl ester and methyl ester is added. L-phenylalanine obtained in example 19, the mixture is stirred for 20 min at room temperature, the product is filtered, washed. 10 ml of water are dried, and 0.312 g of white crystals of methyl ether, M-benzyloxycarbonyl-b-aspartyl-b-phenylalanine are obtained.  Purity yield 88.6%.  Example 24  0.47 g (0.82 mmol) of glacial acetic acid and 8 ml of water are placed in a 30 ml flask, 0.50 g (0.82 mmol) of the product of N-benzyloxycarbonyl-L-aspartyl-b-phenyl 1-methyl methyl ester and L-phenylalanine methyl ester, the mixture is stirred at room temperature for 10 minutes, the product is filtered, washed with 10 ml of water, washed, 0.308 g of white M-benaia methyl ester crystals are obtained.  199 Loxycarbonyl b aspartyl b-phenylalanine.  Purity 100 yield 8792%, Example 25.  1.00 g (1.65 mmol) of the addition product of the methyl ester of N-benzyloxycarbonyl-L-aspartyl of L-fluorylalanine and L-phenylalanine methyl ester obtained in Example 19. The flask is loaded into a 30 ml flask, 2 ml of water and 2 ml are added. 0 ml of 1, the mixture is stirred for 3 minutes, then treated in the same manner as described in Example 19, 0.35 g of H-benzyloxycarbonyl-b-aspartyl-b-phenylalanine methyl ester crystals are obtained.  Clean yield 100%.  Example 26  0.59 g (2 mmol H-P-methoxybenzyloxycarbonyl-b-asparate noah acid and 0.860 g (. k mmol methyl b-phenylalanine hydrochloride is dissolved in 1N. The NaOH pH value was adjusted to 6.0.  The resulting solution is mixed with 50 mg of thermolysin, shaken overnight.  The precipitate is filtered off, washed with 10 ml of water, dried, and 0.928 g of crystals are obtained having.   The results confirm the preparation of the addition product of N-P-methoxybenzyloxycarbonyl-L aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1 :. one).  After recrystallization of the product from a mixture of ethyl acetate and n-hexane.  .  T. square  72-76 C.  i-1 + 6.5 (C 1, methanol).  Found,% -.  C b1,85; H 6, N6, f Cb H NiOio Calculated: C 62.15; H 6.16; N 6.5: 9.  Infrared spectrum: 3280 cm. (oscillation stretching of N-H bond); 3020 and 2930 cm (fluctuation of stretching C – H); 1735 cm (ester C "0); 1700 cm (C O urethane,); (2nd amide absorption); cm (deformation oscillation); 1380 cm (carboxylate); 1210 to cm- (vibration of stretching of the bond G-0-C and 3rd amide absorption); 1030 cm (phenyl vibration in the plane), and 690, and 810 cm (phenyl vibration outside the plane).  NMR spectrum, t: (1) 2.7 ppm (2H); (2) 3.1 ppm (); (3) 3.6. ppn (sd; 3. 7 ppi (3N); (i) 3.8 ppm (3H); (5) 4.0 ppm (IH); (6) it, 3ppm (1H); (7) 720.  , 8 ppm (IH); (8) 5. 0ppm (2H); (9) 5.65 ppm (3H); (10) 5.65 ppm (IH); (11) b,); and (12) 6.8 to 7.3 ppm (YH).  The results show.  that the product is an adduct of formula I, where RI, Ra, R, and n mean p-methoxybenzyloxycarbonyl, benzyl, methoxy, and 1, respectively.  A mixture of 0.500 g (0.78 mmol) of the addition product of N-P-methoxybenzyloxycarbonyl-L-acnap-tyl-B-phenylalanine methyl ester and L-phenylalanine methyl ester, thus obtained, with 2 ml of water and 0.9 ml (0 , 9 mmol) 1 n. HC stirred at 60 ° C for 3 minutes  The resulting suspension is filtered off and washed with 6 ml of water, dried, and 0.32 g of crystals are obtained.  The following results underline that the product is N-P-methoxy-benzyloxycarbonyl-L-aspartyl-L-phenylalanine ester.  Purity 100, yield 89.1.  .  The crystals are dissolved in ethyl acetate and the product is precipitated with n-hexane to give the compound.  square  128-130 С, WQ - 15,1 (С 1 methanol) Found,%: С 60.30; H 5.7t; N5,99 GabHaeNtOe Calculated,%: C 60.25; H 5.72; N6.11.  Infrared spectrum: 3280 cm (N-H stretch); 2930 and 3020, (fluctuation of C – H stretching); 1735 cm (С О complex); 1700 cm (urethane); 1b50 cm (1st amide absorption); 1510 to cm (2nd amide absorption); JAO cm (C – H deformation); 1220 to 1270 cm (fluctuation of C – O – C stretch, 3rd amide absorption); 1030 and 1050 (fluctuation of phenyl in the plane); 690 and 810 cm (fluctuation of phenyl outside the plane).  NMR spectrum, 5: (1) 2.8 Rrga (2H); (2) 3.0 ppm (2U (3) 3.6 ppm (3N); (i) 3.8 ppm (3N) (5) 4.5 ppm (1H); (6), 8ppm (IH) ( 7) 6.6 ppm (2H); (8) 6.0 ppm (IH) (9) 6.6 ppm (IH); (10) 6.6 ppm (IH) (11) 6.8 to 7.3 ppm C9H).  These results indicate that the final product is a compound of formula IV, in KOtQpOM, RI, Rj, R, and n denote n-methoxybenzyloxycarbonyl, benzyl, methoxy, and 1, respectively.  219 0.2 parts by weight of the N-P-methoxybenzyloxy-carbonyl-L-aspargyl-L-phenylalanine methyl ester obtained is dissolved in 2 wt. h  acetone and to the resulting solution was added 1 weight. h  k N. HC2, the mixture is heated in a water bath with a weak countercurrent for 1.5 hours, until complete decomposition to form a solution containing. as. The main components are methyl ester L-asparti-L-phenylalanine, methyl ester, L-phenylalanine, and anise alcohol, and from this solution, methyl ester of L-aspartyl-L-phenylalanine is obtained. Example 27.  0, ii62 g (2 mmol of M-benzyloxycarbonyl-b-glutamic acid and 0.86 O g (4 mmol) of L-phenylalanine methyl ester chlorohydrate are dissolved in 1 H. NaOH, and. The pH is adjusted to 6.0. .  The resulting solution is mixed with 50 mg of thermolysin and the mixture is shaken at 38-40 ° C overnight.  The precipitate is filtered off, washed with 10 ml of water, dried, get 0.510 g of crystals, t.  square  80-85 0.  The following results confirm that the addition product of N-benzyloxycarbonyl-b-glutamyl-b-phenylalanine and L-phenylalanine methyl ester (1: 1) was obtained.  The product is recrystallized from a mixture of ethyl acetate and n-hexane t.  PP 92-97 ° C.    0.1 (C 1, methanol Found: C 63.61; H 6.31; N & 6 CbiHsgNiOs Calculated,%: C 63.75; H 6.32; N 6.76.  Infrared spectrum: cm (tension fluctuation of N-H bond); 2950 and 3030 cm (fluctuation of the tension of the C – H bond); 1730 and cm- (C 0 ester); 1bEO (C O ureta, n); IbbO cm (1st amide absorption); 1–20 cm (carboxylate); 1530 cm (2nd amide absorption); JAO cm (С – Н deformation oscillation); I40b cm (carboxylate); 12AA to 1,310 cm (C-0-C oscillation stretching and amide absorption); 1050 cm (fluctuation of phenyl in the plane); 700 and 750 cm (fluctuation of phenyl outside the plane).  NMR spectrum, S: (1) 2.0 ppm 2H); (2) 2.3 ppm (2H); (3) 3.0 ppm (iH); (k) 3.6 ppm (3H) and 3.7 RyuT ZN); (5) 3.8 ppm (1H); (6) ". З Ррт ПН); (7) i4.8 ppm (1n); (8) 5.0 Ppm (2H); (9) 722 5.6 ppm (3N); (10) 5.8 ppm (1H); (11) 7.2 ppm (1H); (12). 7G2 ppm (YUN); (1D) 7.3 ppm (5H).   These results indicate that the product is an addition of the formula I, where R, Rg, Rj, and n are benzyloxycarbonyl, benzyl, methoxy, and 2, respectively.  0.100 g of the addition product of N-benzyloxycarbonyl-b-glutamyl-b-phenylalanine methyl ester and L-phenylalanine methyl ester is added with stirring 2.3 ml (0.32 mmol) 0.  and the mixture is then stirred at room temperature for 15 minutes.  The white precipitate obtained is filtered off and washed with 3 ml of water, dried, and O 0683 g of crystals is obtained.  The following results are subject to the product being N-benzyl-oxycarbonyl-L-glutamyl-L-phenylalanine methyl ester. The clean output is 95.81.  The crystals are dissolved in ethyl acetate, -and the product is osedagot-and-hexane, t.  square  97-99 ° С, - 11.0 (С 1, methanol) Found, С 62.63; H 5, N6, Saj Ige NaO Calculated,%: C 62.3; H 5.92; N6.33.  Infrared spectrum: 3300 cm (N-H stretching variation); 2930 and 3030 (fluctuation of C – H stretching); 1735 cm (C O ester); 1x90 cm- (C O urethane); 1650 cm (1st amide absorption); 1530 cm (2nd amide absorption); cm (oscillation strain C – H); 1220 to 1280 cm (fluctuation of C – O – C stretch and amide absorption); 1050.  (oscillation of phenyl in the plane) and 695 and 735 cm (oscillation of phenyl outside the plane).  Nuclear Magnetic Resonance Spectrum, 5: (1) 2.0 r (2H); (2) 2, k ppm (2H); (3) 37 pTT2H); (it) 3.7 ppm (GH); (5), 3 ppm (-G); (6) k, 8 ppm (1H); (7) 5.1 ppm (2H); (8) 5.9 RRP (1H); (9) 7.2 ppm (1H); (10) 7.2 ppm (5H); (11) 7, Zrrt (5H); (12) 8.1 ppm (1H).  These results show that the final product is a compound of the formula IN, where Ri, Ra, Yaz and n means benzyloxycarbonyl, benzyl, methoxy and 2, respectively.  The resulting methyl ester of N-benzyl-oxycarbonyl-L-glutamyl-L-phenyl-23, 9 Nn is reduced by hydrogen to hydrogen into the methyl ester L of glutamyl-b-phenylalanine, and is converted by hydrolysis into N-benzyloxycarbonyl-L-glutamyl-gl-a-gl-a-amyl-methyl. Example 28  To a solution of 0.686 g (3.12 mmol) of lx hydrated L-phenylalanine methyl ester hydrate in 25 ml of water, with ice-cooling, add 2 H. NaOH aqueous solution with stirring, while bringing the pH to, then without stopping stirring, 0, ZbO g, (1.4 mmol) of N-benzyloxy-arbonyl-L-aspartic acid anhydride are gradually added, the pH is maintained within 7, 0-7.5 by adding 2 n. NaOH aqueous solution.  The mixture was stirred for 2 c, 1N was added to the reaction mixture. NSB aqueous solution to bring the pH to 6.  The precipitate obtained is filtered off, washed with 50 ml of water, dried, and O, A16 g of the product of N-benzyloxycarbonyl-L-aspartyl-phenyl-Nina methyl ester is obtained (mixture of 86 N-benzyloxycarbonyl-L-aspartyl (O -) - L- phenylalanine and.  H-Benzyloxycarbranyl-L-aspartyl- (Cus) -L-phenylalanine methyl ester; and L-phenylalanine methyl ester (1: 1) (t.  square  103-P5 ° C), A significant amount of i-benziloxycaponyl-b-aspartyl (04) -b-phenylalanine methyl ester is formed in the reaction mixture, but most of the compound remains in the filtrate and in the wash water.  The resulting addition product of N-benzyloxycarbo methyl ester.  nyl-b-aspartyl- (and one-b-phenylalanine and L-phenylalanine methyl ester are added to 1.7 ml (0.7 mol 0.4 N. HC2 while stirring, the mixture is continued to stir at room temperature for 15 minutes.  The resulting white precipitate was filtered off, washed with 3 ml of water, dried, and 0.136 g (yield, 96.3) of N-benzyloxycar bonyl-b-aspartyl-b-phenylalanine methyl esrtion crystals (containing 11% H-benzyloxy-carbonyl-b-aspartyl (C) -b-phenylalanine (t. square  110-118 ° C).  Example 29.  In a 30 ml flask 0, g (2 mmol) of N-benzyloxycarbonyl-b-aspartic acid and 0.918 g (k mmol) of L-phenylalanine ethyl ester hydrochloride are dissolved in 5 ml of water, and k H is added to the solution. The NaCH72 pH is adjusted to 6.  The resulting solution is mixed with 50 mg of thermolysin, and the mixture is shaken overnight at.  The precipitate is filtered off, washed with 30 ml of water, and it is obtained; 0.913 g of crystals is obtained, t.  square  85-90.  The product is identical with the product.  additions of N-benzyloxy-karbonyl-L-aspartyl-L-phenylalanine ethyl ester and L-phenylalanine ethyl ester (1: 1).  After recrystallization from a mixture of ethyl acetate and n-hexane, the product of st.  square  93-95 C, ti-lo + 6.0 (C 1, methanol).  Found,% 6.50; H 6.56; N 6.63 Calculated,%: C 6.23; H 6.50; N6.61. Infrared spectrum: 3300 cm (oscillation of the stretching of the bond N-H); 2900 to 3050 cm (fluctuation of stretching C – H); 1710 and cm (C O ester and C O urethane); 1640 cm (1st amide absorption); 1585 cm (carboxylate); 1520 cm (2nd amide absorption); IjfO cm (C – H deformation oscillation); 1380 cm (carboxylate); 1200, 1270: cm (fluctuation of C – O – C stretch and 3rd amide absorption); 1055 cm (fluctuation of phenyl in the plane); 700, 730 and 750 cm (oscillation of phenyl outside the plane).  NMR spectrum, in: (1) 1.1 rrp (6H); (2) 2.7 EPR (2H); (3) 3.0 ppm (H); (i. ) 4.0 ppm (4H); (5) 4.1 ppm (IH); (6) 4.5 PiJm (lH); (7) 4.7 ppm (IH); (8) 5.0 ppm (2H); (9) 6.2 ppm (IH) ;; (10) 6.7 ppm (3H); (11) 7.2 ppm (IH); (12) 7.2 ppm (10H); (13) 7.3 ppm (5H).  These results show that the product is an adduct of formula I, where R, R, Ri and n are benzyloxycarbonyl, benzyl, ethoxy and 1.  , In a 30 ml O flask, 125 g (0.197 mmol) of the resulting product of addition of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine ethyl ester and L-phenylalanine ethyl ester are mixed with 2 ml of water and ml of 1N.  HCf (0.24 mmol) and the mixture is stirred at room temperature for 30 NMH.  The resulting suspension is filtered, the precipitate is washed with 5 MP of water, dried, and 0.0807 g of crystals of ethyl ester of N-benzonoxycarbonyl-L-L-ac-para-ethyl-L-phenylalanine are obtained.  Purity 100%; yield 92.6%.  The crystals are recrystallized from a mixture of ethyl acetate and n-hexane, to obtain the product with t, pl.  128135c, cl - 17.3 (C 1, meta C 62.82; H 5.96; N6.1 Found;%: CabiiieiJiO%: C 62, t3; H 5.92; Calculated N6.33.  3300 CM Infrared spectrum: 2900 to 3100 cm; 1730 cm, -.  1690 1655 1530 cm; .  kkO cm; 1200-1280 1030 cm 690 NMR spectrum, b: 1.1 rrp; 2.8 ppm; 3 0ррт; , 1 ppm; +, 6 ppm; k, 8 ppm; five. 1ppm; 6.0 ri; 7.1 ppm; 7.3 ppn; 9.6 ppm.  Example 30  5.00 g (16.82 mmol of C-P-methoxy-benzyloxy carbonyl-L-aspartic acid and 7.26 g (33.6 mmol) of L-phenylalanine methyl ester hydrochloride are loaded into a 100 ml flask and 1N is added.  NaGH, pH of solution 6.0.  The resulting solution is mixed with 2.0 g of thermolysin and 0.4 g of potato inhibitor, the mixture is shaken at 5 hours.  The precipitate is filtered off, washed with 100 ml of water, dried, and 8.11 g of crystals have been obtained. square  88-92®C, 75.6% yield as an addition product of N-P-Me-Toxibenzyloxy-carbonyl-aspartyl-phenylalanine methyl ester and phenylalanine methyl ester (1: 1).  1 After recrystallization from a mixture of ethyl acetate and n-hexane, the physical characteristics and the results of elementary analysis are similar to the addition product obtained in Example 26.  . 0.3 g of the product of the addition of N-P-methoxy-benzyloxy-carbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester was diluted with 3 ml of acetone; 2 ml of 2.4N is added to the solution. HCE, the reaction is carried out with bOS for 1.  The reaction mixture is mixed with water, with an aqueous solution of NaHCO (j) and cyclohexanone, added as an internal standard.  Methyl ether preparation was confirmed by high-speed liquid chromatography. -L-aspartyl-b-phenylalanine.  You a stroke of 72.7%.  Example 31  0.3 product with N-P-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester compound and L-phenylalanine methyl ester obtained in Example 30, are decomposed and analyzed by co-.  For example 30, dioxane is used instead of acetone.  The yield of methyl ester is L-ac-partyl-b-phenylalanine.  Example 32J Similar to Example 31, but using methanol instead of dioxane.  The yield of methyl ether (-b-aspartyl-b-phenylalanine 63.3%.  Example 33  Similar to example 31, but instead of dioxane NI, N-dimethyl form MID.  The output of the methyl ester db-L-aspartyl-b-phenyl-.  alanine 28.1.  Example 3  Similar to Example 31, but using k ml of dioxane, 1 ml of 5.3 n dioxane solution, HC and triethylamine instead of 3 ml of dioxane, 2 ml of 2fk n as a neutralizing agent. NSE and 1.2 N.  an aqueous solution of mansose, respectively.  The yield of methyl ester-b-aspartyl-b-phenylalanine 98.6%.  PRI me R 35.  Similar to the example, but the reaction was carried out at 90 ° C for 20 minutes (instead of 60 ° C for 1 hour).  The yield of methyl ester is L-aspartyl-b of phenylalanine 88.5%.  Example 36  Similar to example 3, but using, 5 dioxane and 0.5 ml of dioxane solution 5.3 n.  HC 8 instead of ml and 1 ml of the above substances, respectively, and also conducts the reaction at 90 instead of 60 ° C.  The yield of methyl ester is L-aspartyl-L-phenylalanine 84,%.  PRI me R 37.  Similar to example 3, but using 3 ml of dioxane and 2 ml of dioxane solution 5.3 n. HCE instead of ml and 1 ml of these substances, respectively, and also conducts the reaction at 30 ° C for 120 minutes instead of 60 ° C for 1 hour.  The yield of methyl ester "L-L-aspartyl-b-phenylalanine 98.6%.  Example 38  Similar to the example but using, 5 ml of dioxane and 0.5 MP 60% perchloric acid instead of 4 ml of dioxane and 1 ml of dioxane solution 5.3 n. NA respectively.  The yield of methyl ester is L-aspartyl-L-phenylalanine.  Etc.  and m e r 39.  Similar to the example.  but 4.85 ml of dioxane and 0.15 ml of concentrated sulfuric acid are used instead of 4 ml of dioxane and 1 ml of a 279 dioxane solution of 5.3 n. NA.  The output of methyl-ether-L-aspartyl-L-femilanina.  Example fO.  0.3 g of the N-methoxy-isyloxycarbonyl L-asparty-L-phenylalanine methyl ester and L-phenylapanine methyl ester (1: 1) obtained in Example 30 are dissolved in 2 ml of dioxane and 3 ml of trifluoroacetic acid are added .  The reaction is carried out at for 1 h.  The reaction mixture is evaporated under reduced pressure, and then water, triethylamine and cyclohexanium are added to a part of this substance as an internal standard, and the sample obtained is analyzed by high-speed liquid chromatography. The yield of methyl -L-aspartyl-L-phenylalanine 96, A is.  Example 41  A mixture of 1,000 g of the M-P-methoxy-benzyloxy-carbonyl-L-aspartyl-L-phenyl-alaine methyl ester and L-phenylalanine methyl ester, (1: 1), obtained in. an example of DL, and ml of dioxane and 4 ml of dioxane solution / 5.3 n. NSE, loaded into a 650 ml flask;, and. stirred at, for 1 h, Dioxane was discharged from the reaction mixture under reduced pressure, 6 ml of water was added and 21} mp of diethyl ether to the remaining oily product, stirred, the mixture was divided into two phases, the aqueous phase was added 10 ml of diethyl ether, the product is extracted three times in the same way.  The ether extracts were combined, washed twice with 5 ml of a 5% aqueous solution of sodium bicarbonate and dried over anhydrous magnesium sulphate.  The ether is distilled off under reduced pressure, 0.176 g (yield 81.2) of crude anise alcohol is obtained.  The aqueous phase is neutralized with an aqueous solution of hydroxide to pH 6, incubated at about during the night.  The resulting crystals are filtered and washed with 2 ml of water, dried, and 0.316 g (yield: 68.5%) of crude L-aspartyl-L-phenyl alanine Na methyl ester is obtained.  The mother liquor and the aqueous washes are combined and alkalinized with an aqueous solution of sodium hydroxide to pH, the product is extracted with dichloromethane (3 x 15 ml), the combined extracts are washed with 5 ml of water, dried over anhydrous magnesium sulfate.  Dichloromethane was distilled off under reduced pressure of 728 to obtain 0.23 g (yield: 83.4%) of crude L-phenylalanine methyl ester.  EXAMPLE 1.189 g ((4 mmop) M-P-methoxybenzyloxycarbonyl-L-aspartic acid and 1.837 g (8 mmol) of L-phenylalanine ethyl ester hydrochloride are dissolved in 1N.  NaCH aqueous solution, pH 6.0.  Bring the volume of the reaction mixture with water to 15 ml.  The resulting solution is mixed with 100 mg of thermolysin.  A mixture of shake, sew at 38-40 C for 7 h.  The precipitate is filtered off, washed with 30 ml of water, dried, yielding 2.01 g (yield 90.2) of the product of addition of NP-methyl-benzyloxycarbonyl-L-aspartyl-L-fanylalanine ethyl ester and L-phenylalanine ethyl ether (1; 1j- THAT is confirmed by the following analysis results.  The product is recrystallized from a mixture of methanol and ether.  T.  square  82WU With.  CW n- 6.0 (C 1, methanol) Found,%: C 63.52; H 6.57; N6.5 CjsHiibNjOio Calculated,%: C b3, H 6.51; N 6.31.  The infrared absorption and NMR spectra give the following characteristic maxima.  Infrared spectrum: 3300 cm (N-H stretching variation); from 2,900 to 3,050 cm (fluctuation of C – H stretch); 1720, 1730 and (C O esters and urethane); 1650 cm (1st amide absorption) J from 1510 to cm (2nd amide absorption); H40 cm (C – H deformation oscillation); 1390 cm (carboxylate); 1220 to 1280 cm (C-O-C stretching and 3rd amide absorption); 1030 cm (deformation of phenyl in the plane); and 690, 760 and 810 cm (oscillation of phenyl outside the plane).  NMR spectrum: (1) 1.2 pim (6H); (2) 2.7 ppm (2Y); (3) 3.1 ppm (H); (4) 3.8 ppm (3N); (5) 4.0 ppm (4n); (6) 4.1 ppm (1H); (7) 4.5 ppm (1H); (8) 4.7 ppm (1H); (9) 5.0 ppm (1H); (10) 5.5 (4H); (11) 6.1 ppm (1H); (12) 6.8-7.4 {jprn (14H).  The results show that the product is an addition of the formula (1}, where R4, R2, Rj and and means p-methoxybenzyloxycarbonyl, benzyl, ethoxy and t, respectively.  The process is repeated as in Example 34, but using the resulting addition product instead of the Nrp-methoxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine ethyl ester, L-aspartyl-L-phenylalanine ethyl ester is obtained.  The yield of ethyl ether-L-L-aspartyl-b-phenylalanine 95.1)%.  Example {W.  1.89 g (.  mmol) M-p-methoxybenzyloxycarbonyl-b-asparaginoic acid and 1.725 g (8 mmol x of DL-phenylalanine methyl ester orhydrate are loaded into a 30 ml flask and dissolved with addition of 1N.  NaCH aqueous solution; pH 6.0.  The solution is diluted with water to 15 ml.  The resulting solution was mixed with 0.1 g of thermolysin, and the mixture was stirred at 38-LO C for 50 minutes.  The precipitate was filtered off, washed with 30 ml of water and dried, yielding 2.109 g of crystals having m.  square  119-123 C, then the product is recrystallized from a mixture of ethyl acetate and n-hexane, dried for 7 hours under reduced pressure. The compound is an adduct of methyl ester of H-p-methoxybenzyloxycarbonyl-L-partyl-b-phenylalanine and methyl ester 1 -Phenylalanine (1: 0 for uhydrate.  T.  square  131-138 °:, lJ-lf - i, 2 (C 1, methanol).  Found,%: C b1. 50g, H 6.12; N6, Sz MmNyOto- 1/2 NgR Calculated,%: C 61.29; H 6.23; N 6.50.  The infrared absorption and NMR spectra gave the same characteristic features as in the addition product of formula I obtained in Example 26, where R {, R, Ri, and h were p-met6 oxibenzyloxycarbonyl, benzyl, methoxy, and 1, respectively, with the exception that the absorption caused by the protons of water and the groups -NH- and MN | - shifted by 4.1 ppm due to the musculature due to the presence of water because the product included water of crystallization, as indicated below.  A portion of 1.502 g of the product is heated by microwave radiation for 12 minutes in a microwave oven at a frequency of 2, GHz and a power of 1.2 kW to dry the sample, whose weight was reduced to 1.8152 g after irradiation (loss on drying, O ,, 02091 g ).  An elementary analysis of the advanced sample gave the following results: 1730 Found,%: C 62.07; H 6.17; N6.69 CiiliisNjOw (I). Calculated: C, 62.15; H, 6.16; N 6.69.  The infrared absorption and NMR spectra of the irradiated sample gave the same characteristic features as in the addition product of formula I obtained in Example 26.  A portion of 1.0 g of the irradiated sample is mixed with 4 ml of water and 2 ml of 1N.  The HCt is then stirred at 3 minutes.  Filter, introduce N-p-methoxycarbonyl-b-phenylalanine methyl ester and D-phenylalanine methyl ester in a 1: 1 molar ratio from the solid phase and from the liquid phase, respectively.  The method is the same as Example 3, but 0.3 g of the obtained adduct is used (hemihydrate instead of the adduct obtained in Example 30).  The yield of methyl ester-L-aspartyl-b-phenylalanine and methyl ester of D-phenylalanine 95, Example 4. 0.3% of the addition product of N-p-methoxybenzyloxycarbonyl-L-acnaptil-L-phenylalanine methyl ester and L-phenylalanine methyl ester (1: 1) obtained in Example 30 was dissolved in 10 ml of a 0.3 N solution. NSV in chloroform, the reaction is carried out at 60 ° C for 2 hours.  The reaction mixture is evaporated under reduced pressure, water, triethylamine and cyclohexanone as an internal standard are added to the residue, the sample is analyzed by high-speed liquid chromatography.  The yield of methyl ester is A-L, -aspartyl-b-phenipalanine 9. 3  PRI me R kS.  53 g of N-benzyloxycarbonyl-L-acnapagic acid and 107.8 g (0.5 mol) of phenylalanine hydrochloride methyl ester are placed in a 1-liter flask and 300 ml of an aqueous solution of sodium hydroxide are added to the mixture to dissolve them and adjust the pH to 6.4.  0.3 g of thermoase and 0.26 g of calcium acetate monohydrate are added to the reaction mass, and the resulting solution is left at 20 ° C for 218 hours, while the additive methyl ester compound M-benzyloxycarbonyl-1-asparagyl is precipitated. 11-phenylalanine and 0-phenylalanine methyl ester.  The precipitate is filtered off.
    319101
    washed with water, dried by high-speed liquid chromatography show that the yield of the product is 92.7, calculated on the taken N-6 ene-xyloxy-base-L; - aspartic acid.5
    Example 6 The process and analysis of the reaction product was carried out analogously to Example 45, but using 161.8 g (0.75 mol) hydrochloride © hydrogen of D-phenylalanine, 500 ml of sodium hydroxide solution, pH 6.7 , 1.0 g of thermoase and 0.88 g of calcium acetate monohydrate, instead of 107.8 g, respectively, / 6.4, 0.3 g, and 0.26 g for 100 hours, instead of 21b, and -1 move 93.3, Considering the taken N-benzyloxycarbonyl-b-asparagi ovic acid.
    Example 47. The process is carried out under the conditions of example 45, but using 149.2 g (0.65 mmol) of DL-phenylalanine hydrochloride methyl ester, 50 ml of an aqueous solution of sodium hydroxide, pH 6.6, 0-, 7 g of thermoelectric 0.62 g calcium acetate monohydrate instead of 107.8 g, 300 ml, 6.4, 0.3 g and 0.26 g, respectively, for 195 hours instead of 20 ° C and 218 hours. Yield 90.9% starting from H-benzyl scicarbonyl-b-aspartate.
    G1 p and m-p -48. 0.534 g (2 mmol) of N-benzyloxycarbonyl-i-aspartate and 0.863 g (4 mmol) of L-phenylalanine hydrochloride methyl ester are sprayed into a 30 ml flask and added to the same
    7 ml of ammonium hydroxide to dissolve and bring the pH of the solution to 6.0.
    8 solution 50 mg thermolysin is added. The solution is stirred for 2 hours until precipitation of the addition product from N-bem-40-loxycarbonyl-B-aspart-B-phenipapa Nin methyl ester and L-phenylapan methyl ester. Large amount in (ed added
    into the reaction mixture containing precipitated impurity crystals -. 45 nor for their dissolution. The resulting homogeneous solution is analyzed by speed chromatography; yield of impurity compounds 54% .JQ
    P, and measure 49. 0.534 g (2 mmol) of L-benzyloxycarbonyl-L-aspartate and 0.853 g (4 mmol) of DL-phenylalanine hydrochloride methyl ester 1732:
    into a 30 ml flask and added thereto.
    5 ml of water along with triethylamine to dissolve and bring the pH to 5.0. 50 mg of thermolysin is added to the solution. The solution is stirred for 16 hours. The precipitated crystals of an adduct consisting of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and D-phenylalanine methyl ester are separated by filtration, dried. Get 0.836 1 crystals, t - pl. 96-113 0.
    Example 50, 0.53 g (2 mmol) of N-benZyloxycarbonyl-L-aspartate and 0.863 g (mmol) of DL-phenylalne methyl ester methyl hydrochloride are placed in a 30 ml flask and added thereto
    6ml ammonia water to dissolve
    and bringing the pH to 7.2. 50 mg of thermolysin are introduced into the solution and stirred for 4.5 hours. The precipitated crystals of the adduct consisting of N-benzyloxycarbogyl-b-aspartyl-b-phenylalanine methyl ester and DL-phenylalanine methyl ester are filtered and dried. 0.630 g of crystals are obtained. Exit 51.8. 10.7 g CtO (mmol) M-benzyloxycarbonyl-L-aspartate and 28.0 (130 mmol) of L-phenylalanine hydrochloride methyl ester are placed in a 260 ml flask and 10 ml of an aqueous solution of the hydroxide solution is added to dissolve and up to 6.6. 32 mg of thermolysin are added to the solution. The solution is held until the crystals of the addition product precipitate. Consisting of M-benzyloxycarbonyl-b-aspartyl-b-phenylalanine methyl ester and b-phenylalanine methyl ester. The resulting crystals are filtered and dried. High-speed XP (1 plots showed 82.5% yield, based on L-benzyloxycarbonyl-L-aspartate.
    Example 52: The process was carried out under the conditions of Example 9, but using 15 ml of ammonia water instead of water and tertsetilamine, the pH of the solution was adjusted to 6.0 instead of 5.0. Obtain 0.800 g of impurity compounds. Yield 65.8%.
    The invention is illustrated in table. 1 and 2.
    33
    : 3
    权利要求:
    Claims (1)
    [1]
    910117 Table. 39910P Claim of the invention it 1 Rj-C-CH-HHj-HOC- (CHi) where R is ben-yloxycarbonyl, n-to-toxibenzyloxycarbonyl Ri-benzyl; Rj is methoxy, ethoxy; n 1 or 2 ,. "Characterized in that the N-substituted aminodicarboxylic acid of the general formula RJ n" l20 LI V II I AND HOC-fCHjU-CH-SON where the values of R and n are indicated above, react with complex j. an aminocarboxylic acid ester of the general formula III of the diene of the dipeptide derivative and an amino acid of the general formula I 7 n ° n-CH-C-lM-ai-C-Rj i O P iHiK-CH-C-Kj, the molar ratio of compounds of formula II and III is 1: 1 - in an aqueous medium, in the presence of metalloprotease at pH b, 0-7.2, in-tint ® temperature. Sources of information taken into account during the examination 1. Josef S. Fnifon Advances in Protein chemistry, Academic Press, Jnc, New Jork, N. Y. ISjS, vol. 5, c. 33.
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    同族专利:
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP52007279A|JPS5745427B2|1977-01-27|1977-01-27|
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