KR20010024155A - Amino acid derivatives - Google Patents

Abstract

PURPOSE: Provided is a novel amino acid derivative which is used in manufacturing stably a desired peptide, such as calcitonin, having a high biological activity, with a high yield. CONSTITUTION: Provided is a side chain protected amino acid derivative as described in general formula (I), wherein R1 may represent -CH2CH2CH2CH2NH-, -CH2CO- or -CH2CH2CO-, and R2 may represent phenylthioethyloxycarbonyl(Ptc), phenylsulfonylethyloxycarbonyl(Psc), phenylthioethyloxy(OPte) or phenylsulfonylethyloxy(OPse), and R3 may represent hydrogen or alpha -amino protecting group; and pharmaceutically acceptable salt thereof, which have high stability but also good reactivity under the process for preparing peptides.

Description

Amino acid derivatives

Peptides can be obtained by i) natural extraction, ii) DNA recombination, and iii) chemical synthesis. Among these methods, i) and ii) have low yields of the desired peptide compounds, The method of demonstrating the purity of the peptide compound is unclear and has a limitation that it is difficult to purify impurities generated in the microorganism. Thus, chemical synthesis has been used to produce large-scale commercial peptides, and at the same time, advanced new methods are being developed.

Generally, chemical synthesis is largely divided into a liquid phase process in a solution phase and a solid phase method using a polymerization support. The latter method is not suitable for producing large amounts of peptides for the following reasons; i) protecting the side chain protecting groups of the constituent amino acids as much as possible, ii) using an excess of amino acid derivatives, carrying out the reaction in a limited condensation method, iii) difficult to purify intermediates during synthesis, and iv) possibly growing peptides. Coupling ends unexpectedly due to the folding of ⅴ) when the peptide is fragmented from the polymerization support after completion of the polypeptide chain, yield is greatly reduced; It is a residue and difficult to load, and i) polymerization supports, amino acid derivatives, reaction reagents, and solvents applied in solid phase technology are very expensive.

On the other hand, in the former liquid phase method, i) the protection of amino acid side chain groups constituting the peptide compound can be reduced to a minimum according to the nature and synthesis process of the peptide compound, and ii) various condensation methods can be freely selected. It is possible to purify intermediates generated during the synthesis process, and to easily increase or decrease the reaction amount according to the amount of the desired peptide compound, and thus it is widely used to obtain a large amount of high purity peptide compound.

However, especially when the long-chain (25-50 amino acids) peptides are synthesized by the liquid phase method, the reaction process requires a long time in several stages, and there is a high possibility of side reactions during the reaction. It is difficult to separate and purify impurities by.

In this regard, it is important to research and develop a process for producing a large amount of high purity and high yield peptides using the liquid phase method, and to develop a new material that can be efficiently used in the process and is urgently required in practice.

[Summary of invention]

The present invention provides a high-quality peptide such as calcitonin effectively by liquid phase synthesis by conventional segmental condensation of fragments obtained by the stepwise binding of Boc-protected amino acids using condensing agents (coupling reagents; DCC, HOBT) or active esters. It provides a new method of manufacturing.

The present invention also provides a novel method for the preparation of peptides using novel side chain protecting groups or carbon terminal protecting groups based on liquid phase synthesis.

It is an object of the present invention to provide a method for preparing calcitonin using novel side chain protected amino acid derivatives having the general formula (I) and salts thereof.

(I)

Wherein R ₁ is -CH ₂ CH ₂ CH ₂ CH ₂ NH-, -CH ₂ CO- or -CH ₂ CH ₂ CO-, and R ₂ is phenylthioethoxycarbonyl (Ptc), phenylsulfonylethoxy Carbonyl (Psc), phenylthioethyloxy (OPte) or phenylsulfonylethyloxy (OPse), and R ₃ represents hydrogen or an α-aminoprotecting group.

Another object of the present invention is to provide a new method for preparing a peptide using the amino acid derivatives mentioned above.

The present invention relates to novel side chain protected amino acid derivatives having the general formula (I) and their use in preparing peptides by classical liquid phase synthesis.

(I)

Wherein R ₁ is -CH ₂ CH ₂ CH ₂ CH ₂ NH-, -CH ₂ CO- or -CH ₂ CH ₂ CO-, and R ₂ is phenylthioethoxycarbonyl (Ptc), phenylsulfonylethoxy Carbonyl (Psc), phenylthioethyloxy (OPte) or phenylsulfonylethyloxy (OPse), and R ₃ represents hydrogen or an α-aminoprotecting group.

According to the present invention these amino acid derivatives can be used for the synthesis of various peptides because of their high stability and good reactivity in the reaction process.

Segment condensation used in the present invention is a method useful for long-chain peptide synthesis. However, this method almost always results in racemization at the activated carbon end of the carbonyl component. Reducing the degree of such racemization is recommended as follows; Iii) appropriate selection of activated carbonyl residues: glycine and proline are suitable, ii) control of condensation reaction conditions: low polarity solvent, neutral pH, low temperature is preferred, and iii) proper selection of carboxyl activation conditions. iv) in the condensation method The use of an alkoxycarbonyl protected amino acid protecting group.

In the present invention, it is planned to obtain a reaction process useful for preparing calcitonin in consideration of the above. As a result, it provides a production method useful for large scale calcitonin synthesis.

Specifically, the method for preparing salmon calcitonin is described below to demonstrate the superiority of the present invention, and the newly improved amino acid derivatives will now be described.

H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg- Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ (1-7-disulfide bond) (salmon calcitonin)

H-Ser-Gly-Thr-Pro-NH ₂ (29-32) Peptide 1

Boc-Arg-Thr-Asn-Thr-Gly-OH (24-28) Peptide 2

Boc-Leu-Gln-Thr-Tyr-Pro-OH (19-23) Peptide 3

Boc-Leu-His-Lys (Psc) -OH (16-18) Peptide 4

Boc-Lys (Psc) -Leu-Ser-Gln-Glu (OPse) -OH (11-15) Peptide 5

H-Ser-Thr-Cys (Acm) -Val-Leu-Gly-OY TFA (5-10) Peptide 6

Boc-Cys (Acm) -Ser-Asn-Leu-OH (1-4) Peptide 7

According to the present invention, the fragments 1 to 7 used for the synthesis of salmon calcitonin by the segmental condensation reaction are universal, inexpensive, and have only t-butyloxycarbonyl (Boc) group which can be simply protected and deprotected. Manufactured alone, it is a temporary shield It is different from other methods (eg JP Hei 6-16694 A) where various protecting groups should be used for amino protecting groups. The size or number of segments has been optimized to reduce the sensitivity of racemization; Glycine or Florin were preferentially selected from each segment as carbon-terminal residues. In particular, new side chain protecting groups have been introduced in the synthesis of fragments for the production of high-purity salmon calcitonin. The process for producing salmon calcitonin by segmental condensation reaction based on this invention is shown in Scheme 1 below.

Scheme 1

In general, in the case of lysine, glutamic acid, arginine, histidine, asparagine, and the like, the active side chains must be protected to eliminate branching of the chains in the synthesis of the peptide. And the protecting group applied for this purpose should not change in the elimination of the α-aminoprotecting group or in the condensation step, usually deprotected at the end of the chain condensation process.

Since the production of salmon calcitonin involves glutamic acid with carboxyl groups and amino acids with lysine which must be protected semipermanently until the peptide chain is completed, the choice of such side chain protecting groups is important in the overall synthesis process. Play a role. Among the known protection methods, t-butyl and benzyl (Bz) groups are used to protect the side chain carboxyl groups of glutamic acid, and benzyloxycarbonyl (Z) and t-butyloxycarbonyl (Boc) groups are used to protect the side chain amino groups of lysine. Trityl (Trt) groups are mainly used, and these protecting groups are removed under catalytic reduction or acidic conditions, resulting in complex reaction conditions during the reaction and requiring a long reaction time.

In order to solve this problem, the present invention has developed a new base-labeled side chain protecting group which is removed under soft and selective conditions in the bond and stable during condensation reaction together with Boc used for the temporary protection of the α-amino group. As a result, a phenylsulfonylethyl (Pse) group and a phenylsulfonylethoxycarbonyl (Psc) group were typically used to protect the side chain of glutamic acid or lysine. That is, in the present invention, by introducing a new phenylsulfonyl group which excludes the negative inductive effect of the para-position substituent of the phenyl group, it is very effective even in the condensation process proceeding in acidic, catalytic reduction reaction and weak basicity. A new stable phenylsulfonyl group was developed and used as a side chain protecting group, but they are still removed by adding piperidine in a short time. Protecting groups of these amino acid derivatives such as Boc-Lys (Psc) -OH DCHA (5), Boc-Asp (OPse) -OH DCHA, (8) Boc-Glu (OPse) -OH DCHA (9) Sea calcitonin was synthesized. These compounds were prepared by the procedure shown in Scheme 2. The ε-amino group of lysine is protected by addition of phenylthioethylchloroformate (3) while the α-groups are combined with copper (II) chelate, followed by oxidation. Β- and Glutamic Acid and Aspartic Acid The carboxyl group was protected by phenylthioethanol and oxidized.

Scheme 2

(n = 1; 6, 8, n = 2; 7, 9)

According to the present invention, para-nitrophenylsulfonylethyl (Nse) group was newly used as a carbon terminal protecting group in the synthesis of fragments of peptides 6 and 7 used as intermediates. The choice of carbon-terminal protecting group does not break under condensation conditions, like the side chain protecting group, and retains the protecting group properties. It should be considered that it is stable when removing the amino protecting group, that it can be easily removed under conditions that do not change the peptide chain, and that the side chain protecting group and the carbon terminal protecting group should be removed under different conditions. For this reason, the present invention is very stable under acid and catalytic reduction conditions, and can be easily removed from basic. A p-nitrophenylsulfonylethyl (Nse) group known as an aminoprotecting group (see Samukov, VV Tetrahedron Lett. 35 1994, 7821; US pat. 5,527,881) was newly introduced to the protection of the carbon terminus.

In the present invention, for the purpose of description without limitation, the following examples are given.

Example

In the following description all amino acids represent L-form unless otherwise indicated.

Melting points were measured using a Buch apparatus. Non-photon measurements were performed with a Jasco DIP-1000. HPLC: Hewlett pakard 1100. FT-IR: Jasco 300 F. Atomic Absorption Spectrometer (AAS): AA-6601F. UV: Shimadzu UV-265.

Thin layer chromatography (TLC) was used Merck ₆₀ F ₂₅₄ silica gel.

Solvent:

1) EPAW-1: Ethyl acetate / pyridine / acetic acid / water = 42/14 / 6.6 / 1

2) BWA-1: n-butanol / water / acetic acid = 4/1/1

3) AAW-1: acetonitrile / acetic acid / water = 8 / 0.2 / 2

4) AWT-1: acetonitrile / water / tripulouroacetic acid = 20/5 / 0.1

5) CMA-1: Chloroform / Methanol / Acetic acid = 95/5/3

6) CMA-2: Chloroform / Methanol / Acetic acid = 90/10/3

The abbreviation of a substituent, a reagent, etc. is as follows.

Acm: acetamide methyl

Boc: t-butyloxycarbonyl

Bz: Benzyl

DCC: Dicyclohexylcarbodiimide

DCHA: Dicyclohexylamine

DCU: N, N'-dicyclohexylurea

DMAP: Dimethylaminopyridine

DMF: Dimethylformamide

EDTA: Ethylenediaminetetraacetic acid

Et: Echil

HOBT: hydroxybenzotriazole

NMM: N-Methylmorpholine

Nse: p-nitrophenylsulfonylethyl

OPse: Phenylsulfonylethyloxy

OPte: Phenylthioethyloxy

pfp: pentafluoro

Psc: Phenylsulfonylethoxycarbonyl

Pse: Phenylsulfonylethyl

Ptc: Phenylthioethyloxycarbonyl

Pte: Phenylthioethyl

Su: Succinimide

Tce: Trichloroethyl

Tcp: Trichlorophenyl

TEA: Triethylamine

TFA: trifluoroacetic acid

Example 1 Preparation of Amino Acid Derivatives

a) H-Lys (Ptc) -OH (4)

20.1 g of H-Lys-OH.HCl and 12 g of copper carbonate were added to 250 ml of water, and the mixture was stirred for 30 minutes and then boiled and filtered while hot. After washing with 30 ml of water, 125 ml of dioxane and 55 ml of 2N sodium hydroxide solution were added to the collected filtrate and cooled in an ice container. 50 ml of dioxane solution and 50 ml of 2N sodium hydroxide solution in which 21.7 g of 2-phenylthioethyl chloroformate is dissolved in the cooling solution are added dropwise for 1 hour 30 minutes. After the dropwise addition, the mixture is stirred for 2 hours, the mixture is filtered and washed sufficiently with 150 ml of water, 150 ml of acetone, and 50 ml of ether. Collect the precipitate and dissolve in 500 ml of 2N hydrochloric acid. A solution of 30 g of EDTA in 1 L of water is added to the reaction solution with stirring. The mixture was acidified to pH 4, washed with water and dried to yield 26 g (77% yield) of the desired compound 4 as a white powder.

m.p. : 255-259 ℃

TLC: Rf = 0.65 (developing solvent 3)

[α] ^D : +12.9 (20 ° C, 0.5N HCl)

HPLC: 100% purity, Rt = 19.5min

IR (KBr, cm ^-1 ): 3343, 2947, 1683, 1578, 1533, 1448, 1325, 1236, 734

b) Boc-Lys (Psc) -OH (5)

19.6 g of compound 4, 12.5 g of potassium carbonate, 350 ml of a water-isopropyl alcohol-DMF (4: 2: 1, v / v) mixture, and 16 ml of Boc ₂ O were added with stirring at 45-50 ° C., followed by further stirring for about 3 hours. do. The mixture is concentrated to 1/2 volume and diluted with 500 ml of water and then washed with 150 ml of ether. 200 ml of ethyl acetate is added to the aqueous layer and acidified to pH = 1.5. After 150 ml of ethyl acetate is added, the oil layer is separated from the mixture, washed with 150 ml of water and 100 ml of brine and dried over anhydrous sodium sulfate. The filtrate is concentrated to give 26 g of emulsion. This emulsion is dissolved in 300 ml of acetone. 15 ml of 0.3 M Na ₂ MoO ₄ solution and 14 ml of hydrogen peroxide are added to this solution. After 1 hour the mixture is stirred at 50 ° C. for 5 hours and distilled. The residue is washed thoroughly with 300 ml of water, 150 ml of 0.5N hydrochloric acid and 100 ml of brine. The solution is dried over anhydrous sodium sulfate, concentrated and crystallized by addition of DCHA. When these precipitates form, it is filtered and 36.5 g (95%) of compound 5 is obtained as a white powder in vacuo.

m.p. : 109-112 ℃

TLC: Rf = 0.40 (Developed Solvent 5)

[α] ^D : +7.2 (20 ° C, 10% AcOH)

HPLC: purity 100%, Rt = 21.4 min

IR (KBr, cm ^-1 ): 3393, 2933, 2858, 1698, 1636, 1560, 1398, 1318, 1254, 1149,

1051, 728

c) H-Asp (OPte) -OH (6)

13.3 g of asphaltic acid is dissolved in 90 ml of dimethoxyethane, and then 10 ml of concentrated sulfuric acid and 40 ml of 2-phenylthioethanol are added. After stirring until a clear solution, it is left at room temperature for 2 days. After distillation, 300 ml of an aqueous solution in which 30 g of sodium acetate is dissolved is added and stirred vigorously. When white precipitate is formed, it is filtered, washed and dried to obtain Compound 6 (14.6 g, yield 54%).

m.p. : 215-217 ℃

TLC: Rf = 0.80 (2 solvents)

[α] ^D : +21.2 (22 ° C, 1N HCl)

HPLC: purity 100%, Rt = 17.6min

IR (KBr, cm ^-1 ): 3123, 2379, 2306, 1739, 1636, 1416, 1343, 1135, 730, 688

d) Boc-Asp (OPse) -OH DCHA (8)

15 ml of Boc ₂ O was added to a mixture of 14.0 g of Compound 6, 8.4 ml of TFA, and 50 ml of DMF with stirring at 45-50 ° C., followed by further stirring for 3 hours. The mixture was diluted with 500 ml of water and extracted with 500 ml of 5% potassium hydrogen sulfide. The organic layer was washed twice with 250 ml of 5% potassium hydrogen sulfide and 200 ml of brine and dried over anhydrous sodium sulfate. After filtration, the filtrate is distilled to obtain an emulsion. 250 ml of acetone is added to this emulsion to dissolve it. And 13 ml of 0.3 M Na ₂ MoO ₄ solution and 12 ml of hydrogen peroxide were added to this solution. After one hour the mixture is stirred at 50 ° C. for 5 hours and distilled. The residue is diluted with 250 ml of water and 250 ml of 250 ml of acetic acid is added to separate the oil layer, washed with 150 ml of water and 150 ml of 0.5N hydrochloric acid and finally with 100 ml of brine. The solution is then dehydrated with anhydrous sodium sulfate, distilled and crystallized by addition of DCHA. When a precipitate formed, it was filtered and dried under vacuum to give 28.8 g of compound 8 as a white powder.

m.p. : 142-144 ℃

TLC: Rf = 0.20 (5 solvents)

[α] ^D : -5.4 (23 ° C, 10% AcOH)

HPLC: purity 100%, Rt = 26.5 min

IR (KBr, cm ^-1 ): 3397, 2937, 2866, 1740, 1708, 1584, 1489, 1397, 1318, 1149

e) H-Glu (OPte) -OH (7)

Compound 7, a white powder, was obtained in a yield of 55% by the procedure described in c).

m.p. : 182-183 ℃

TLC: Rf = 0.55 (developing solvent 2)

[α] ^D : +17.65 (at 20 ° C, 0.5N HCl)

HPLC: purity 100%, Rt = 18.7 min

IR (KBr, cm ^-1 ): 2955, 1726, 1586, 1508, 1421, 1202, 733

f) Boc-Glu (OPse) DCHA (9)

Compound 9, a white powder, was prepared in the same manner as described in d) and obtained in a yield of 92%.

m.p. : 155-157 ℃

TLC: Rf = 0.36 (developing solvent 5)

[α] ^D : -5.8 (20 ° C, 10% AcOH)

HPLC: purity 100%, Rt = 21.5min

IR (KBr, cm ^-1 ): 2938, 2857, 1733, 1701, 1637, 1560, 1449, 1399, 1297, 1141,

1085, 727, 686

Example 2 Synthesis of Salmon Calcitonin

a) H-Ser-Gly-Thr-Pro-NH ₂ (Segment 1, Compound 10)

Condensation was carried out by the active ester method to obtain White powder 1 (purity 99% or more, Rt = 6.54 min: HPLC) in a yield of 75%.

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-Thr-OPfp 17.0g H-Pro-NH ₂ HCl 6.2g Active ester method / TFA HTP-NH ₂ TFA17 93% (12.4 g) 1) 0.13 Boc-Gly-OPfp 13.6 g 17 12.0 g Active ester method / TFA HGTP-NH ₂ TFA18 95% (14.0 g) 1) 0.09 Boc-Ser-OPfp 14.8g 18 14.0 g Active ester method / TFA HSGTP-NH ₂ TFA10 85% (13.0 g) 2) 0.17

b) Boc-Arg-Thr-Asn-Thr-Gly-OH (Segment 2, Compound 11)

Synthesis stepwise using active ester method and well-known addition of condensing agents (DCC, HOBT), and deprotection of benzyl groups by catalytic (Pd-C) reduction method. 99% or more and Rt = 12.6 min: HPLC).

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-Thr-OH 9.6g H-Gly-OBz TsOH 13.6 g DCC, HOBT / TFA H-T-G-OBz TFA19 90% (14.0 g) 1) 0.32 Boc-Asn-OH 9.4g 19 13.8 g DCC, HOBT / TFA H-N-T-G-OBz TFA20 86% (15.6 g) 1) 0.17 Boc-Thr-OPfp 12.4g 20 14.6 g Active ester method / TFA H-T-N-T-G-OBz TFA21 90% (15.6 g) 1) 0.20 Boc-Arg-OH 8.2 g 21 15.6 g Active ester method Boc-R-T-N-T-G-OBz TFA22 100% (22.6 g) 1) 0.40 22 22.6 g Pd-C, H ₂ Boc-R-T-N-T-G-OH TFA11 84% (14.4 g) 1) 0.17

c) Boc-Leu-Gln-Thr-Tyr-Pro-OH (Segment 3, Compound 12)

Condensation stepwise using a condensing agent (DCC, HOBT) method, followed by base catalyzed hydrolysis and catalytic (Pd-C) reduction reaction to segment 3 of the white powder (purity above 99%, Rt = 18.9 min: HPLC) Got.

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-Tyr (Bz) -OH 15.0 g H-Pro-OEt 9.0g DCC, HOBT / TFA H-Y (Bz) -P-OEt TFA23 Quantitative (26.0 g) 1) 0.10 Boc-Thr-OH 9.2g 23 26.0 g DCC, HOBT / TFA H-T-Y (Bz) -P-OEt TFA24 Quantitative (30.0 g) 1) 0.10 Boc-Gln-OH 9.8g 24 30.0 g DCC, HOBT / TFA H-Q-T-Y (Bz) -P-OEt TFA25 70% (21.0 g) 1) 0.37 Boc-Leu-OH 10.0g 25 21.0 g DCC, HOBT / TFA Boc-L-Q-T-Y (Bz) -P-OEt26 85% (20.4 g) 1) 0.37 26 20.4 g NaOH, water, MeOH Boc-L-Q-T-Y (Bz) -P-OH27 90% (17.6 g) 5) 0.27 27 17.6 g Pd-C, H ₂ Boc-L-Q-T-Y-P-OH12 92% (14.6 g) 3) 0.58

d) Preparation of Boc-Leu-His-Lys (Psc) -OH (Segment 4, Compound 13)

Segment 4 was finally obtained in a yield of 81% by the following process; First of all, the new side-chain protected compound Boc-Lys (Psc) -OH was protected with carbon by trichloroethyl group, then deprotected Boc, then condensed stepwise and finally by catalytic hydrogenation (Zn). Deprotection yielded the compound 13 (purity 99% or more, Rt = 23.6min: HPLC) of a white powder.

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-His (Boc) -OTcp 10.2g H-Lys (Psc) -OTce 10.5g Active ester method Boc-H-K (Psc) -OTce28 94% (15.5 g) 4) 0.63 Boc-Leu-OSu 8.7g 28 15.5 g Active ester method / HCl, AcOH Boc-L-H-K (Psc) -OTce29 93% (14.6 g) 5) 0.33 29 14.6 g Zn, AcOH Boc-L-H-K (Psc) -OH13 93% (11.5 g) 1) 0.30

e) Preparation of Boc-Lys (Psc) -Leu-Ser-Gln-Glu (OPse) -OH (Segment 5, Compound 14)

Segment 5 (purity> 99%, Rt = 23.5 min: HPLC) was finally obtained in the following steps with a yield of 58%. ; First, the α-carboxyl group of Boc-Glu (OPse) -OH is protected with trichloroethyl group, followed by step condensation by the method shown below after deprotection of Boc, and finally deprotection by catalytic hydrogenation. Compound 14 was obtained as a white powder.

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-Gln-OH 7.4 g H-Glu (OPse) -OTce TFA 14.5g DCC, HOBT Boc-Q-E (OPse) -OTce30 93% (16.3 g) 5) 0.42 Boc-Ser-OPfp 10.0g 30 16.3 g TFA / DCC, HOBT Boc-S-Q-E (OPse) -OTce31 90% (16.5 g) 5) 0.32 Boc-Leu-OTcp10.3g 31 16.5 g TFA / active ester method Boc-L-S-Q-E (OPse) -OTce32 88% (16.7 g) 5) 0.42 Boc-Lys (Psc) -OTcp 13.8g 32 16.7 g TFA / active ester method Boc-K (Psc) -L-S-Q-E (OPse) -OTce33 93% (21.5 g) 5) 0.33 33 21.5 g Zn, AcOH Boc-K (Psc) -L-S-Q-E (OPse) -OH14 85% (16.0 g) 5) 0.22

f) H-Ser-Thr-Cys (Acm) -Val-Leu-Gly-ONse TFA (Segment 6, Compound 15)

Segment 6 (purity 99% or more, Rt = 19.6 min: HPLC) as a white powder was finally obtained in a yield of 67% by the following procedure; The α-carboxyl group of Boc-Giy-OH was protected with an Nse group, and then condensed stepwise by a method using a condensing agent (DCC, HOBT) or an active ester to obtain a white powder of Compound 15.

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-Leu-OH 5.9g H-Gly-ONse8.3g DCC, HOBT / TFA H-L-G-ONse34 94% (10.1 g) 1) 0.47 Boc-Val-OH 5.2g 34 10.1 g DCC, HOBT / TFA H-V-L-G-ONse35 89% (10.6 g) 1) 0.48 Boc-Cys (Acm) -OPfp 5.9g 35 10.6 g Active ester method / TFA H-C (Acm) -V-L-G-ONse36 94% (12.8 g) 1) 0.47 Boc-Thr-OPfp 7.1g 36 12.8g Active ester method / TFA H-T-C (Acm) -V-L-G-ONse37 98% (14.2 g) 1) 0.27 Boc-Ser-OPfp 5.9g 37 14.2 g Active ester method / TFA H-S-T-C (Acm) -V-L-G-ONse15 87% (13.7 g) 1) 0.38

g) H-Cys (Acm) -ser-Asn-Leu-OH (Segment 7, Compound 16)

Segment 7 was obtained by the following procedure; First, the α-carboxyl group of Boc-leu-OH is protected by adding Nse-OH in the presence of DMAP, and then the Boc-group is deprotected to obtain quantitatively H-Leu-ONse. Then, condensation stepwise by a method using a condensing agent (DCC, HOBT) or an active ester, and finally deprotected with piperidine to give compound 16 (purity 99% or more, Rt = 16.4 min: HPLC) as a white powder.

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf Boc-Asn-OH 8.2 g H-Leu-ONse TFA 14.5g DCC, HOBT / TFA H-N-L-ONse TFA38 85% (15.4 g) 1) 0.36 Boc-Ser-OPfp 11.1g 38 15.4 g Active ester method / TFA H-S-N-L-ONse TFA39 94% (16.6 g) 1) 0.23 Boc-Cys (Acm) -OPfp 9.1g 39 16.6 g Active ester method Boc-C (Acm) -S-N-L-ONse40 93% (19.1 g) 5) 0.43 40 19.1 g piperidine Boc-C (Acm) -S-N-L-OH16 87% (12.3 g) 2) 0.57

h) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro- Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ (salmon calcitonin)

Segmented condensation was performed as shown in Scheme 1 to prepare salmon calcitonin with high bioactivity of white powder. The synthesis was started by condensing the tetrapeptides, segment 1 and Boc-protected segment 2, corresponding to the carboxamide end of the salmon calcitonin sequence in a typical manner using DCC (condensing agent) and HOBT (anti-racematization agent). Stepwise condensation of Boc-protected fragments 3,4,5 and removal of Boc groups resulted in moderate yields of newly branched protected docosapeptides 45 under DCC / HOBT and TFA. Acm-protected segment 6 and Acm-protected segment 7 are condensed by conventional methods for the preparation of cyclic decapeptide 46. And the Nse group to protect the carbon terminus is deprotected with piperidine. And two Acm groups for protection of dentifrices are automatically deprotected and oxidized with iodine under AcOH. After the final condensation of docosapeptide 45 and decapeptide 46, the resulting peptide was treated with TFA and piperidine in DMSO to obtain salmon calcitonin, which was shown to be 66% to 70% pure by analytical HPLC in a shorter time. do. Purification was performed by purified HPLC to obtain at least 98% high purity salmon calcitonin.

mp: 220 ^o C

[ _D : -55.0 (20 ^o C, 50% AcOH)

_max : 275nm

LC / Mass: (1mm, C-18 column, 40μl / min, water / Acetonitrile / TFA buffer)

m / z 3431.8 (M ⁺ ) 1717.2 ([M + 2H] ²⁺ ) 1144.8 ([M + 3H] ³⁺ ) 859.0 ([M = 4H] ⁴⁺

Rt = 22.90min

AAA: Arg (0.83) Asp (0.97) Cys / 2 (1.0) Glu (0.90) Gly (1.0) His (0.92) Leu (1.05) Lys (1.0) Pro (0.62) Ser (0.91) Thr (0.98) Tyr ( 1.1)

Reactant 1 Reactant 2 Condensation Law / Deprotection Law product Yield Rf 11 8.6 g 10 10.5 g DCC, HOBT / TFA HRTNGSGTP-NH ₂ 2TFA42 83% (15.0 g) 1) 0.13 12 10.4 g 42 14.3 g DCC, HOBT / TFA HLQTYPRTNGSGTP-NH ₂ 2TFA43 79% (17.4 g) 1) 0.13 13 7.7 g 43 17.0g DCC, HOBT / TFA HLHK (Psc) -LQTYPRTNGSGTP-NH ₂ 3TFA44 90% (21.5 g) 4) 3) 0.24 14 12.0 g 44 20.1 g DCC, HOBT / TFA HK (Psc) -LSQG (OPse) -LHK (Psc) -LQTYPRTNGSGTP-NH ₂ 3TFA45 75% (21.2 g) 6) 0.38 15 13.6 g 16 10.6 g DCC, HOBT / i) piperidin ii) I ₂ , AcOH Boc-C-S-N-L-S-T-C-V-L-G-OH46 77% (12.0 g) 4) 3) 0.32 45 20g 46 10.0 g DCC, HOBT / i) TFAii) piperidin Salmon Calcitonin 74% (18.0 g)

Example 3: Determination of Biological Activity of Synthetic Salmon Calcitonin

Hypocalcaemic effect of synthetic salmon calcitonin was measured in comparison with the standard (NIBSC, WHO). First, 30 rats of the same sex of about 225 g were divided into six groups. After fasting for one day, three groups received a standard (1, 3, 9 mIU / 0.25 ml albumin solution, rat / 100 g body weight), and the other three groups were injected with a synthetic product. Blood was taken exactly 1 hour later. After blood collection, plasma was separated and the amount of calcium was measured by an atomic absorption spectrophotometer. The relationship between the concentration of calcium and the logarithmic value of the dose was calculated by standard statistical methods.

Subcutaneous injection concentration (mIU / 100g body weight) Biological Activity Compared to Standards 9 99.75% 3 98.65% One 98.75%

According to the present invention, it will be described that a target peptide having high yield, high purity and high biological activity can be prepared using a novel amino acid derivative which is condensed and deprotected in a very stable state.

Claims (3)

Side chain protected amino acid derivatives described by general formula (I) or their pharmaceutically acceptable salts (I) Wherein R ₁ is -CH ₂ CH ₂ CH ₂ CH ₂ NH-, -CH ₂ CO- or -CH ₂ CH ₂ CO-, and R ₂ is phenylthioethoxycarbonyl (Ptc), phenylsulfonylethoxy Carbonyl (Psc), phenylthioethyloxy (OPte) or phenylsulfonylethyloxy (OPse), and R ₃ represents hydrogen or an α-aminoprotecting group. The method of claim 1 wherein the amino acid derivative is H-Asp (OPte) -OH, H-Glu (OPte) -OH, H-Lys (Ptc) -OH, Boc-Lys (Psc) -OH, An amino acid derivative selected from the group consisting of Boc-Glu (OPse) -OH, Boc-Asp (OPse) -OH. Use in preparing the calcitonin of a side chain protected amino acid derivative according to claim 1 or a pharmaceutically acceptable salt thereof.