JPH0616694A - Reproduction of calcitonin - Google Patents

Abstract

PURPOSE:To obtain calcitonin having high quality and useful as an agent for treating steoporosis, etc., in high yield by synthesizing plural osubfragments, condensing the subfragments to synthesize a plurality of protected salmon calcitonin fragments having specific amino acid sequences and condensing the obtained fragments. CONSTITUTION:A C-terminal amino acid having protected carboxyl group is condensed to a protected amino acid positioned next to the C-terminal and having protected alpha-amino group by a process for the liquid-phase synthesis of peptide, the alpha-amino-protecting group is removed, the product is made to react with the next amino-protected amino acid and the procedures are repeated. If necessary, a disulfide bond is formed in the molecule by oxidation to synthesize four kinds of protected salmon calcitonin fragments of formula I (Boc is t-butyloxycarbonyl; tBn is t-butyl), formula II (Fmoc is fluorenylmethyloxycarbonyl), formula III and formula IV, the four kinds of fragments are successively condensed with each other and the protecting groups are removed to obtain the objective calcitonin having high quality in high yield.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing calcitonin, and more particularly to a method for producing calcitonin with high quality and high yield.

Demand for pharmaceutical grade salmon calcitonin continues to grow worldwide. Although current manufacturing methods are based on automated solid-phase methods, this method has advantages and disadvantages. The advantage is speed. The disadvantages are that errors in the condensation process are difficult to see during production and that current equipment limits the batch size with the capacity of the reaction vessel (ie, to increase the production unit, run at the same time). You have to increase the number of devices). One solution to this capacity problem is recombinant DN
Probably method A. However, this method is accompanied by other problems such as proof of purity and removal of trace impurities derived from residual microorganisms.

For these reasons, a 4-protection-fragment liquid phase synthesis method has been developed for a long time. In this method, fragments are stored at 5 ° C. and used as a raw material for fragment condensation. The protected fragment method was applied to the liquid phase synthesis of human calcitonin (B. Kamber, H. Brueckne.
r et al, Helv. Chim. Acta, 53 , 556-564 and P. Sieber, B. Riniker et al, 53 , 2135.
~ 2150). The conventional synthetic method of human calcitonin is
First, 11 sub-fragments are prepared, they are fragment-condensed into 6 fragments, and fragment-condensation is performed 5 times between these fragments to synthesize.

[0004]

SUMMARY OF THE INVENTION According to the present invention for salmon calcitonin, it is possible to prepare 7 sub-fragments, fragment-condense them into 4 fragments and synthesize them by performing fragment-condensation 3 times between the fragments. I knew I could do it. In general, the lower the number of fragment condensations, the less the risk of racemization of the final product and thus the less activity loss. According to the present invention, synthesis is performed with a larger batch size than the conventional method, and
A product with high enzyme activity is obtained with a purity of not less than%.

The solution phase synthesis method of the present invention involves the preparation of four protected fragments (1-10, 11-16, 17-23 and 24-32), which are 5 fragments.
It can be stored stably at ℃ and can be used as a raw material for fragment condensation to produce a product with a purity of 99% or more and an activity of about 5500 IU / mg in a larger batch size without racemization. Can be done. Incidentally, the condensation reaction used in the present invention, E. Schroeder and
The procedure was similar to that described by K. Luebke, The Peptides, Volume 1 (1965), Academic Press, New York.

The present invention will be described below with reference to specific examples. 1. Preparation of protected fragment (1-10) [C (Boc-Cys-
Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys) -Val-Leu-Gl
y-OH] FIG. 1 illustrates a synthetic route. Subfragment (1-4) : ZA using PyBOP reagent
Z-Asn-Leu-OMe is condensed by condensing sn-OH and H-Leu-OMe.HCl.
And deprotecting the Z group by catalytic reduction to give H- Asn-Leu-
Use OMe and HCl. Next, add Z-Ser (tBu)-to this dipeptide.
OH is reacted to form Z-Ser (tBu) -Asn-Leu-OMe, which is removed by catalytic reduction to remove the Z group to form H-Ser (tBu) -Asn-Leu-OMe. Further, this tripeptide is condensed with Boc-Cys (Trt) -OH to give Boc-Cys (Trt) -Ser (tBu) -Asn-Leu-OMe. This protected tetrapeptide was treated with hydrazine hydrate to give Boc
And -Cys (Trt) -Ser (tBu) -Asn-Leu-N 2 H 3. Subfragment (5-10) : Z-Leu-OH and H-Gly-OMe
-HCI is condensed to Z-Leu-Gly-OMe-H, which is catalytically reduced to obtain H-Leu-Gly-OMe-HCl. Next, Z-Val-OH is condensed with this dipeptide to give Z-Val-Leu-Gly-OMe, which is catalytically reduced to H-Val-Leu-Gly-OMe.HCl. Further, Boc-Cys (Acm) -OH is condensed with this tripeptide to give Boc-Cys (Acm) -Val-Leu-Gly-OMe. H-Cys (Acm) -Val-Leu-Gly-OMe after removal of Boc group using TFC
・ Get TFA.

Next, Fmoc-Thr (tBu) -OH is reacted with the above protected tetrapeptide to produce Fmoc-Thr (tBu) -Cys (Acm) -Val-Le.
u-Gly-OMe. H-Thr (tBu) -Cys (Acm) -Val-Leu was deprotected using diethylamine to deprotect the base-labile Fmoc.
-Get Gly-OMe. Fmoc-Ser (tBu) -OH was condensed with the protected pentapeptide to give Fmoc-Ser (tBu) -Thr (tBu) -Cys (Ac
m) -Val-Leu-Gly-OMe. Deprotection of the base-labile Fmoc with diethylamine to remove H-Ser (tBu) -Thr (tBu)
-Cys (Acm) -Val-Leu-Gly-OMe is obtained.

The above two subfragments are condensed by azide coupling to give the decapeptide Boc-Cys (Tr
t) -Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (Acm) -Val
-Leu-Gly-OMe. The C-terminal methyl ester is saponified and removed with caustic soda in trifluoroethanol. Cyclization of this protected decapeptide with I ₂ (in the form of disulfide bonds)

[0009]

[Chemical 2] And The pure and homogeneous compound is obtained by silica gel chromatography.

[0010]

[Table 1]

2. Preparation of protected fragment (11-16) [Fmoc-Lys (BOC) -Leu-Ser (tBu) -Gln-Glu (OtBu) -Leu-O
H] synthetic route is shown in FIG. Z-Glu (OtBu) and H-Leu-OMe ・ HC
l is condensed to Z-Glu (OtBu) -Leu-OMe, which is catalytically reduced to give H-Glu (OtBu) -Leu-OMe.HCl. This dipeptide was then condensed with Z-Gln-OH using PyBOP as a condensing agent to give Z-Gln-Glu (OtBu) -Leu-OMe, which was catalytically reduced to give H-Gln-Glu (OtBu). -Leu-OMe.HCl is obtained. Further, the above tripeptide is condensed with Z-Ser (tBu) -OH to form Z
-Ser (tBu) -Gln-Glu (OtBu) -Leu-OMe is catalytically reduced to H-Ser (tBu) -Gln-Glu (OtBu) -Leu-OMe.HCl.

Z-Leu-OH is condensed with the above tetrapeptide to give Z-Leu-Ser (tBu) -Gln-Glu (OtBu) -Leu-OMe. The methyl ester was then removed by hydrolysis and the amino protecting group was removed by catalytic reduction to remove H-Leu-Ser (tBu) -Gln-Gl.
u (OtBu) -Leu-OH. The sodium salt of this pentapeptide is reacted with Fmoc-Lys (BOC) -OSu to neutralize it, and then the heptapeptide Fmoc-Lys (BOC) -Leu-Ser (tBu) -Gln-Glu (OtBu)-
Leu-OH.

Table 2.11-16 data structure of process Fragment [α] _D Purity (%) Yield (%) Z-Glu (OtBu) -Leu-OMe −22.6 ° 65 ( Videl-Gomez et al, JHChem. 12: 273, 1975) H-Glu (OtBu) -Leu-OMe.HCl-4.3 99 <82 Z-Gln-Glu (OtBu) -Leu-OMe-3999 <80. H-Gln-Glu (OtBu) -Leu-OMe.HCl -2995 Z-Ser (tBu) -Gln-Glu (OtBu)-32.8 99 <91 Leu-OMe H-Ser (tBu) -Gln- Glu (OtBu)-36.8 99.6 100 Leu-OMe-HCl Z-Leu-Ser (tBu) -Gln-Glu (OtBu)-100 88 Leu-OMe Z-Leu-Ser (tBu) -Gln- Glu (OtBu)-98 Leu-OH H-Leu-Ser (tBu) -Gln-Glu (OtBu) -80 Leu-OH.HCl Fmoc-Lys (Boc) -Leu-Ser (tBu) -Gln-Glu (OtBu ) -Leu-OH [α] ²⁵ _D : -12.2 ° Purity: 98% Moisture content: 0.62% * Purity was determined by thin-layer chromatography.

3. Preparation of protected fragment (17-23) (His-Lys-Leu-Gln-Thr-Tyr-Pro-OH) The synthetic route is shown in FIG. Subfragment (19-23) : Z-Tyr (tBu) was added to H-Pro-
Dipeptide Z-Tyr (tBu) -Pro-OM condensed with OMe ・ HCl
Then, H-Tyr (tBu) -Pro-OMe.HCl is obtained by catalytic reduction. This dipeptide is reacted with Z-Thr (tBu) -OH to give Z
-Thr (tBu) -Tyr (tBu) -Pro-OMe and catalytic reduction of this give H-Thr (tBu) -Tyr (tBu) -Pro-OMe.HCl.

Further, the above tripeptide is used as a condensing agent in P
Condensed with Z-Gln-OH using yBOP to produce Z-Gln-Thr (tBu)-
Tyr (tBu) -Pro-OMe was prepared by catalytic reduction of H-Gln-T
Obtain hr (tBu) -Tyr (tBu) -Pro-OMe.HCl.

Next, Z-Leu-OH is reacted with the above tetrapeptide to obtain Z-Leu-Gln-Thr (tBu) -Tyr (tBu) -Pro-OMe. The methyl ester was removed by hydrolysis and then the amino protecting group was removed by catalytic reduction to remove H-Leu-Gln-Thr (tBu)-
Tyr (tBu) -Pro-OH. Subfragment (17-18) : Using PyBOP reagent
Z-His (Bum) and H-Lys (BOC) -OMe
-His (Bum) -Lys (Boc) -OMe. This was treated with hydrazine hydrate in methanol to give a hydrazide compound, which was then azidated to form the above subfragment (19-23).
Z-His (Bum) -Lys (Boc) -Leu-Gln-Thr (tBu)-
Obtain Tyr (tBu) -Pro-OH.

[0017] Table 3 17 over 23 steps of the data configuration Flag e n t Yield (%) Z-Tyr (tBu ) -Pro-OMe 98 H-Tyr (tBu) -Pro-OMe · HCl 93 Z- Thr (tBu) -Tyr (tBu) -Pro-OMe 96 H-Thr (tBu) -Tyr (tBu) -Pro-OMe ・ HCl 100 Z-Gln-Thr (tBu) -Tyr (tBu) -Pro-OMe 74 H-Gln-Thr (tBu) -Tyr (tBu) -Pro-OMe.HCl 98 Z-Leu-Gln-Thr (tBu) -Tyr (tBu) -Pro-OMe 91 H-Leu-Gln-Thr (tBu) -Tyr (tBu) -Pro-OH / AcOH 100 Z-His (Bum) -Lys (Boc) -OMe 100 Z-His (Bum) -Lys (Boc) -N ₂ H ₃ 91 Z-His (Bum)- Lys (BOC) -Leu-Gln-Thr (Bu)-58 Tyr (tBu) -Pro-OH [α] ²⁵ _D : -41.4 ° Purity (thin-phase chromatography): 95%

4. Preparation of protection fragment (24-32) to (Arg-Thr-Asn-Thr -Gly-Ser-Gly-Thr-Pro-NH 2) synthesis pathway shown in FIG. Subfragment 29-32 : active ester Z-Gly-OSu
Reacts with H-Thr (tBu) -OH to dipeptide Z-Gly-Thr (t
Bu) -OH and isolated as a cyclohexyl ammonium salt by crystallization. The isolated dipeptide is catalytically reduced to H-Gly-Thr (tBu) -OH. Furthermore, this was reacted with the active ester Fmoc-Ser (tBu) -OSu to give the tripeptide Fmoc
-Ser (tBu) -Gly-Thr (tBu) -OH.

Next, using DCC and HOBT as a condensing agent, this tripeptide is condensed with H-Pro-NH ₂ to produce Fmoc-Ser.
(tBu) -Gly-Thr (tBu) -Pro-NH ₂ . Further, this was treated with diamine to remove the Fmoc group, and H-Ser (tBu) -Gly-
Thr (tBu) -Pro-NH ₂ . Subfragment 25-28 : Z-Thr (tBu) -OH to H-Gly-
It is reacted with OMe to give Z-Thr (tBu) -Gly-OMe, which is catalytically reduced to give H-Thr (tBu) -Gly-OMe.HCl. This dipeptide was reacted with Z-Asn-ONp to produce Z-Asn-Thr (tBu) -Gl.
y-OMe and catalytic reduction of H-Asn-Thr (tBu) -Gl
Obtain y-OMe.HCl.

Further, Z-Thr (tBu) -OH is reacted with the above tripeptide to give Z-Thr (tBu) -Asn-Thr (tBu) -Gly-OMe. Z-Thr (tBu) -Asn-Thr (tBu) -Gly-OH is obtained by hydrolyzing the tetrapeptide methyl ester.

Subfragments 25-28 and 29-32
Was condensed with PyBOP reagent to give octapeptide Z-Thr
(tBu) -Asn-Thr (tBu) -Gly-Ser (tBu) -Gly-Thr (tBu) -Pro-N
H ₂ and H-Thr (tBu) -Asn-Thr (tB
u) -Gly-Ser (tBu) -Gly-Thr (tBu) -Pro-NH ₂ .

Using DCC / HOBT as the condensing agent Z
-Arg (Pmc) -OH is condensed with the above octapeptide to give Z-Arg
(Pmc) -Thr (tBu) -Asn-Thr (tBu) -Gly-Ser (tBu) -Gly-Thr (t
Bu) -Pro-NH ₂ · HCl.

[0023] Table 4 24 over 32 data configuration Flag main emission step preparative yield (%) Z-Gly-Thr (tBu) -OH · CHA 83 H-Gly-Thr (tBu) -OH · H 2 O 91 Fmoc-Ser (tBu) -OSu 81 Fmoc-Ser (tBu) -Gly-Thr (tBu) -OH 100 Fmoc-Ser (tBu) -Gly-Thr (tBu) -Pro-NH ₂ 98 H-Ser ( tBu) -Gly-Thr (tBu) -Pro-NH ₂ 96 Z-Thr (tBu) -Gly-OMe 90 H-Thr (tBu) -Gly-OMe · HCl 100 Z-Asn-Thr (tBu) -Gly- OMe 98 H-Asn-Thr (tBu) -Gly-OMe.HCl 91 Z-Thr (tBu) -Asn-Thr (tBu) -Gly-OMe 89 Z-Thr (tBu) -Asn-Thr (tBu) -Gly -OH 93 Z-Thr (tBu) -Asn-Thr (tBu) -Gly-Ser (tBu)-86 Gly-Thr (tBu) -Pro-NH ₂ H-Thr (tBu) -Asn-Thr (tBu)- Gly-Ser (tBu)-100 Gly-Thr (tBu) -Pro-NH ₂ · HCl Z-Arg (Pmc) -Thr (tBu) -Asn-Thr (tBu) -Gly- 86 Ser (tBu) -Gly- Thr (tBu) -Pro-NH ₂ H-Arg (Pmc) -Thr (tBu) -Asn-Thr (tBu) -Gly- 96 Ser (tBu) -Gly-Thr (tBu) -Pro-NH ₂ HCl fragment (29-32) white powder, decomposing at _{^{170-190 ℃ [α] 25 D:}} -8.0 confirmed by ° ± 1.5 elemental analysis purity / HP C (AUC): 95% fragments (24-32) mp: 191 ° C. (with _{^{decomposition) [α] 25 D = -4.8}} (c = 1% MeOH) confirmed by elemental analysis purity: 95% Moisture: 1 .1% * Purity was determined by thin layer chromatography.

5. Production of salmon calcitonin by fragment condensation Basic condensation pathway:

[0025]

[Table 2]

Analysis data of protection (1-32): Melting point: 170 ° C. (with decomposition) [α] ²⁵ _D : -46.7 (c = 1% TFE) Confirmed by elemental analysis Purity: 93% ( Purity was determined by thin-layer chromatography) Moisture content: 1.74% Analytical data for (1-32) salmon calcitonin: Melting point: 220 ° C. <[α] ²⁵ _D : -18.2 (c = 1% TFE) Elemental analysis Confirmed by: Purity: 99% (Purity was determined by thin phase chromatography) Moisture: 1.32% Activity according to British Pharmacopoeia (1988) is about 5500 IU / m
It was g.

Example Preparation of protected (1-32) calcitonin (salmon calcitonin = SCT) Cyclized Boc (1-32) -SCT-OH 15.7 g (1
2.4 mmol) and H- (11-32) -SCT-NH
239.0 g (11.3 mmol) were dissolved in 400 ml of dimethylformamide (analytical grade) ice-cooled to approx. 0 ° C. in a 2 liter round bottom flask and 0 ° C.
Maintained at. 2.3 ml of EDPA (13.5
HOBT, 2.1 g (13.5 mmol) and TBTU, 4.3 g (13.5 mmol). After stirring sufficiently, the flask was left overnight to return to room temperature. When the reaction was confirmed by thin layer chromatography,
H- (11-32) -SCT-NH ₂ spot was observed. Next, after concentrating under reduced pressure with a rotary evaporator to reduce the volume, the residue was returned to room temperature over one and a half hours, treated with 300 ml of hydrogen gas, and then dried. The solid content was filtered off and washed 4 times with water. Compound thus obtained

[0028]

[Chemical 3] Was heated to 35 ° C. under high vacuum and dried over calcium chloride. The yield was 54.0 g, and the yield was 100%.

The main spot was observed at Rf0.5 by thin layer chromatography analysis, and in addition, trace amount of P was observed.
BOC- (1-32) -S unreacted with yBOP degradation products
CT-OH was observed. The phase development of thin layer chromatography was carried out using a Poly reagent and was detected by ultraviolet rays.

Purification of (1-32) -SCT-Ac 6.0 g of freeze-dried crude SCT was purified by medium pressure reverse phase liquid chromatography (MPLC) at room temperature. Developing solvent is acetonitrile, water, acetic acid and 85%
Phosphoric acid was used.

The MPLC column used is 800 × 80 m
m, filler: Vydac C18, 20-30 μ, wide pores. The eluent was 0.08M phosphoric acid (pH adjusted to 2.25 with triethylamine) and acetonitrile 74:
A mixture of 26 (v / v) was used.

[Brief description of drawings]

FIG. 1 depicts a synthetic route to the protected fragment (1-10).

FIG. 2 depicts a synthetic route to the protected fragment (11-16).

FIG. 3 depicts a synthetic route to the protected fragment (17-23).

FIG. 4 depicts a synthetic route for protected fragments (23-32).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C07K 7/36 7306-4H // C07K 9/00

Claims (7)

[Claims] 1. Four protected fragments of salmon calcitonin. A method of manufacturing. 2. The method according to claim 1, wherein the four fragments are condensed to produce a protected salmon calcitonin. 3. The method according to claim 2, wherein the protecting group of the protected salmon calcitonin is removed to produce a deprotected, cyclized (1-32) salmon calcitonin. 4. The method according to any one of claims 1 to 3, wherein a substance having a pharmacological activity effective for osteoporosis is produced. 5. A method according to claims 1 to 3 for producing a substance having a pharmacological activity effective against Paget's disease. 6. The method according to any one of claims 1 to 3, wherein a substance having a pharmacological activity effective for calcemia is produced. 7. The method according to any one of claims 1 to 3, wherein a substance having a pharmacological activity effective against bone-destructive tumor is produced.