JPH0352479B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel human parathyroid hormone (h-
PTH) derivatives. More specifically, the present invention provides a therapeutic agent for hypoparathyroidism, or
[Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ]-h-PTH (1-34) useful as a therapeutic agent involving PTH or as a raw material for labeled compounds for testing parathyroid function
NH ₂ , i.e. the formula H−S ¹ _er −V _2al −S ³ _er −G ⁴ _lu −I ⁵ _le −G ⁶ _lo −L ⁷ _eu −N ⁸
_is −
A ¹⁰ _so −L ¹¹ _eu −G _12ly −L ¹³ _ys − _14is −L _15eu −A ¹⁶ _so −S ^1
7 _er −N ¹⁸ _le −
G ¹⁹ _lu −A ²⁰ _rg −V ²¹ _al −G ²² _lu −T ²³ _rp −L ²⁴ _eu −A ²⁵ _rg −L
²⁶ _ys −L ²⁷ _ys
−L ²⁸ _eu −G ²⁹ _lo −A ³⁰ _sp −V ³¹ _al −H _32is −A ³³ _so −T ³⁴ _yr
−NH2
...[) (In the formula, Ser is L-serine, Val is L-valine,
Glu is L-glutamic acid, Ile is L-isoleucine, Gln is L-glutamine, Leu is L-leucine,
Nle is L-norleucine, His is L-histidine,
Asn is L-asparagine, Gly is glycine, Lys
is L-lysine, Arg is L-arginine, and Trp is L-lysine.
-Tryptophan, AsP is L-aspartic acid,
Tyr represents L-tyrosine) or a salt thereof. h-PTH is a peptide hormone consisting of 84 amino acids, and its biological activity is
-34 N-terminal fragment, namely h-PTH(1-
34) [Proc. Nat.
Acad.Sci.USA, 68 , 63-67 (1971)]. However, h-PTH is unstable due to the presence of L-methionine (Met), and its hormone activity is lost when labeled with ^I125 [Recent Prog.
Res., 18 , 269-295 (1962)]. In the conventional method for quantifying h-PTH, in order to quantify only the portion that has h-PTH activity,
A specific antibody using (1-34) as an antigen has now been prepared. However, h-PTH(1-34)
is unstable due to the presence of L-methionine (Met), and when labeled with ^I125 , Met present at positions 8 and 18 is oxidized, resulting in inactivation of the hormone activity. Therefore, as an h-PTH derivative that not only has PTH activity and immunoactivity against PTH antibodies, but also has stable hormonal activity even when labeled with ^I125 , and stable radioactivity, 8 Met at position and 18th position were replaced with L-norleucine, and L-phenylalanine at position 34 was replaced with L-tyrosine [N ⁸ _le , N ¹⁸ _le , T ³⁴ _yr ³⁴ ]-h-PTH (1-34 ) was discovered [JP-A-55-113753]. However, this [N ⁸ _le , N ¹⁸ _le , T ³⁴ _yr ]−h−
Since PTH (1-34) does not have Met in its molecule, it is not inactivated even when labeled with I ¹²⁵ , but its hormonal activity is at most the same as that of natural h-PTH (1-34). It had no more than a The target compound [] has the known h-PTH (1-34) and [N ⁸ _le , N ¹⁸ _le ,
T ³⁴ _yr ]-h-PTH (1-34) has a stronger affinity and not only has h-PTH activity approximately 1.5 to 2 times higher, but also has immunoactivity against PTH antibodies. However, labeling with ^I125 did not reduce hormone activity;
[N ⁸ _le , N ¹⁸ _yr , T ₃₄ yr ]-h-PTH (1-34) about 2
It has twice the radioactivity and does not lose its biological activity even during long-term storage, so it has twice the radioactivity of the known h-PTH.
(1-34), [N ⁸ _le , N ¹⁸ _le , T ³⁴ _yr ]-h-PTH (1-
34) Demonstrate more extreme and superior effects. Therefore, the object compound [ ] is an extremely useful peptide as a raw material for a therapeutic agent for hypoparathyroidism, a therapeutic agent for bones involving PTH, and a labeled compound for testing parathyroid function. The peptide [] of the present invention has the formula []
Individual protected amino acids and/or protected lower peptides are condensed to the amino acid order shown by liquid phase synthesis, and in the final step of the condensation reaction, a protecting group for the N-terminal amino group and a functional group for the side chain are added. It can be obtained by removing the protective group of by acid decomposition. The condensation reaction itself is carried out by repeating the attachment/detachment of a protecting group and the condensation reaction according to a conventional method for peptide synthesis. That is, the various protecting groups used in the production of the raw materials of this peptide [] and all intermediates are those known in peptide synthesis;
Protecting groups are therefore used which are easily removable by known means such as hydrolysis, acid hydrolysis, reduction, aminolysis or hydrazinolysis. Such protecting groups are described in the literature and reference books in the field of peptide synthetic chemistry. For example, as a protecting group for an amino group,
Acyl groups such as formyl group, trifluoroacetyl group, phthaloyl group, p-toluenesulfonyl group, o-nitrophenylsulfenyl group, benzyloxycarbonyl group, o (or p)-bromobenzyloxycarbonyl group, o (or benzyloxycarbonyl group such as p)-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, trichloroethyloxycarbonyl group, t
-Aliphatic oxycarbonyl groups such as butyloxycarbonyl group, t-amyloxycarbonyl group, diisopropylmethyloxycarbonyl group, 2
-phenyl-isopropoxycarbonyl group, 2-
Tolyl-isopropoxycarbonyl group, 2-p-
Examples include aralkyloxycarbonyl groups such as diphenyl-isopropoxycarbonyl groups. These amino groups can also be protected by forming enamines obtained by reacting with 1,3-diketones such as benzoylacetone and acetylacetone. Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the amide group is substituted with a 3,4-dimethoxybenzyl group, a bis-(p-methoxyphenyl)methyl group, or the like. The hydratide group is substituted with a benzyloxycarbonyl group, a trichloroethyloxycarbonyl group, a trifluoroacetyl group, a t-butyloxycarbonyl group, a trityl group, a 2-p-diphenyl-isopropoxycarbonyl group, and the like. Ester groups include methanol, ethanol,
Alkanols such as t-butanol and cyanomethyl alcohol, benzyl alcohol, p-bromobenzyl alcohol, p-chlorobenzyl alcohol, 2,6-dichlorobenzyl alcohol, p
- aralkanols such as methoxybenzyl alcohol, p-nitrobenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol, p-chlorobenzoylmethyl alcohol, 2,4,6
- substituted with phenols such as trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, thiophenols such as thiophenol, p-nitrothiophenol, etc. Ru. The hydroxyl groups of serine and tyrosine can be protected, for example, by esterification or etherification. Groups suitable for this esterification include, for example, acetyl group, benzoyl group, benzyloxycarbonyl group, and ethyloxycarbonyl group. Examples of groups suitable for etherification include benzyl group, 2,6-dichlorobenzyl group, tetrahydropyranyl group, and t-butyl group. To protect these hydroxyl groups, 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group, 2,2,2-trifluoro-1-
Benzyloxycarbonylamino groups are also suitable. However, it is not necessary to protect these hydroxyl groups. Groups used to protect the amino group in the guanidino group of arginine include, for example, a nitro group, a tosyl group, a benzyloxycarbonyl group, a mesitylene-2-sulfonyl group, but the guanidino group is not necessarily protected. There's no need. Examples of the group used to protect the imino group of histidine include benzyl group, trityl group, benzyloxycarbonyl group, tosyl group,
2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group, 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, etc., but this imino group is not necessarily protected. do not have to. In the present invention, t-
A butyloxycarbonyl group or a t-amyloxycarbonyl group is used, an o-chlorobenzyloxycarbonyl group is used to protect the side chain amino group, i.e. the ξ-amino group of lysine, and a benzyl ester group is used to protect the α-carboxyl group. , an ethyl ester group, a phenacyl ester group, a benzyl ester group is used to protect the side chain carboxyl group of glutamic acid and aspartic acid, a benzyl group is used to protect the hydroxyl group of serine, and a benzyl group is used to protect the hydroxyl group of tyrosine. It is preferable to use a 2,6-dichlorobenzyl group for the protection of arginine, and to use a tosyl group or a mesitylene-2-sulfonyl group for the protection of the amino group in the guanidino group of arginine. In the synthesis of the target compound [], condensation of individual amino acids and/or lower peptides is performed, for example, with an amino acid or peptide having a protected α-amino group and an activated terminal carboxyl group, and a free α-amino group and An amino acid or peptide with a protected terminal carboxyl group is reacted with an amino acid or peptide with an activated α-amino group and a protected terminal carboxyl group with a free terminal carboxyl group and a protected α-amino group. This can be carried out by reacting amino acids or peptides with groups. In this case, the carboxyl group is, for example, an acid azide, an acid anhydride, an acid imidazolide or an active ester, such as cyanomethyl ester, thiophenyl ester, p-nitrothiophenyl ester, p
-Nitrophenyl ester, 2,4-dinitrophenyl ester, 2,4,5-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, It can be activated by converting it into N-hydroxyphthalic acid imide ester or the like. Condensing agents such as carbodiimides, such as N,N'-dicyclohexyl-carbodiimide, N-ethyl-N'-3-dimethylaminopropyl-carbodiimide, N,N'-carbonyl-diimidazole or isoxolium salts, such as Woodward reagents, may also be used. It can be activated by using and reacting. Preferred condensation methods in the present invention are the azide method, active ester method and carbodiimide method. In each step of the condensation, it is desirable to use a method that does not cause racemization or a method that minimizes racemization, preferably the azide method, active ester method, Biunsch method [Z.Naturforsch., 21b, 426 (1966)] or Geiger method [Chem Ber., 103, 788 (1970)]
In particular N-ethyl-N'-3-dimethylaminopropyl-carbodiimide (WSC) as condensing agent.
It is appropriate to use a modified method using . The condensation order can be synthesized in any order as long as it is the amino acid order shown by the formula [], but C-
It is preferable to connect amino acids and/or peptides sequentially from the terminal side. For example, it is preferable to condense a C-terminal fragment consisting of amino acids 29 to 34 with a peptide fragment consisting of amino acids 23 to 28. Condensation of this C-terminal fragment with hexapeptide 23-28 is suitably carried out by a modified Geiger method using WSC. A peptide fragment consisting of amino acids 18 to 22 is linked in front of the C-terminal fragment 23-34 obtained, which is suitably carried out by a modified Geiger method using WSC. The obtained C-terminal fragment 18-34 was preceded by a peptide fragment consisting of amino acids 13 to 17, a peptide fragment consisting of amino acids 8 to 12, and a peptide fragment consisting of amino acids 1 to 7. It is preferable to connect them. A protecting group for α-amino group in the above condensation reaction, such as t-butyloxycarbonyl group, t-
The amyloxycarbonyl group is removed with trifluoroacetic acid. Protecting groups for α-carboxyl groups, such as ethyl ester, can be decomposed with dilute sodium hydroxide solution or converted into protected hydratides such as hydratide or trichloroethoxycarbonyl hydratide, and phenacyl ester groups can be removed with Zn powder in acetic acid. The benzyl ester group can also be decomposed by anhydrous hydrogen fluoride decomposition, hydrogenolysis, or converted to a hydratide. The N-terminal α-amino group thus protected, ξ-
A tetratriacontapeptide having an amino group, a side chain carboxyl group, a guanidino group and/or a hydroxyl group is obtained. These protecting groups are preferably removed in one step by acid decomposition, such as with anhydrous hydrogen fluoride or trifluoromethanesulfonic acid, to yield the target compound of formula []. The peptide [] thus obtained can be separated and purified by known means for purifying peptides or proteins. For example, it can be carried out by gel filtration using a gelling agent such as Cephadex G-25, Cephadex G-50, Cephadex LH-20, column chromatography using carboxymethyl cellulose, ion exchange resin, etc., high performance liquid chromatography, etc. can. The peptide [] of the present invention can be obtained in the form of a base or its salt depending on the conditions of the method, including inorganic acid salts, formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, citric acid. It is a salt with an organic acid such as. The present peptide [ ] can be added with certain inorganic or organic substances to form a complex. This complex refers to a compound of unknown composition that is formed upon addition and that confers a sustained effect on the peptide. Such substances include, for example, inorganic compounds derived from metals such as calcium, magnesium, aluminium, cobalt or zinc, especially slightly soluble phosphates, pyrophosphates or polyphosphates of these metals. salts and hydroxide compounds,
Alternatively, alkali metal polyphosphates can be mentioned. Furthermore, the peptide [] of the present invention preferably has
Used as a labeling reagent for RIA. For example, a ¹²⁵ I-containing phosphate buffer (PH
Add the peptide of the present invention [] and chloramine T to 7.1), stir, then add sodium bisulfite, and then add a small amount of potassium iodide and serum albumin to perform chromatography.
By collecting the ¹²⁵ I-labeled fractions, ^{the 125} I-labeled radioactive substance is obtained. Next, the peptide of the present invention [] and radioactive parathyroid hormone (PTH) labeled with ¹²⁵ I
Let's talk about activity. <Preparation of ¹²⁵ I-labeled substance> Contains ¹²⁵ I-NaI with 2 mCi of radioactivity
h-PTH in 50μl of 0.5M phosphate buffer (PH7.1)
(1-34), h-PTH( _1-34 ) _NH2 , [ ^H8le ,
N ¹⁸ _le , T ³⁴ _yr ]−h−PTH(1−34) and [N ⁸ _le ′,
After adding 10 μl of a solution containing 2 μg of N ¹⁸ _le , T ³⁴ _yr ]-h-PTH(1-34)NH ₂ and 20 μl of a solution containing chloramine T (3.5-mg/ml) and stirring for 30 seconds, bisulfite was added. The reaction was stopped by adding 50 μl of a solution containing sodium (4.5 mg/ml). After adding 0.5 ml of 0.1N acetic acid solution containing 5% human serum albumin to this, it was charged to a Sephadex G-10 column (1 x 50 cm).
Elution was performed with the above acetic acid solution to obtain fractions containing each test product labeled with ¹²⁵ I. <PTH activity measurement method> (1) Preparation of PTH receptor SD male rats (weighing 200-250 g) were decapitated and exsanguinated. After laparotomy, the kidney was removed, its surface membrane was removed, and the renal cortex was cut out. Cool on ice. The following operations are performed at as low a temperature as possible (0 to 4°C).
Add 0.25M sucrose and
10mM Tris-HCl buffer containing 1mM MEDTA (PH
7.5) (hereinafter referred to as Solution A), and homogenize the kidney cortex by adding 3 times the volume (ml) of Solution A to its wet weight (g) using a glass jacket tube using Teflon petals. The homogenate is centrifuged at 150Xg for 10 minutes, and the supernatant is further centrifuged at 2200Xg for 15 minutes. Discard the supernatant, suspend the upper cloudy part of the precipitate in Solution A, and add this suspension to 2200X.
g, washed by centrifugation for 155 minutes, resuspended and dispensed into containers, frozen at -70°C and stored at -20°C. (2) Reaction between PTH and PTH receptor Test product was mixed with ATPMg 2mM, MgCl ₂ 10mM, KCl 60mM,
GTP 20μM, isobutylmethylxanthine 1mM,
Dissolve in 100mM Tris-HCl buffer (PH7.5) containing 8mM creatine phosphate and 0.2% bovine serum albumin (BSA) (hereinafter referred to as solution B), and perform the same procedure on the standard bovine PTH (1-84). Dispense 50 μl of each of the four solutions into glass test tubes, and prepare 8 tubes of each. The sample is kept in ice water to prevent the decomposition of other substances such as ATP. The PTH receptor preparation stored at −20°C was thawed at room temperature.
Dissolve creatine kinase in Solution A in advance, add creatine kinase, and add 0.1mg/creatine kinase in Solution A.
ml, PTH receptor preparation protein amount is 1.4 mg/ml and kept on ice. After placing the above dispensed sample solution in a constant temperature bath at 37℃ for several minutes,
Add 50 μl of the above PTH receptor-creatine kinase solution and incubate at 37°C for 10 minutes. Next, add 100 μl of 0.1M acetate buffer (PH4.0) and immediately place the test tube in ice water. Immediately heat the test tube in boiling water for 1 minute to stop the reaction. (3) Measurement of produced C-AMP The above reaction-stopped sample is diluted 10 to 30 times with distilled water, and protein is removed by centrifugation at 2000XG for 15 minutes. The amount of C-AMP in the supernatant is measured using an RIA kit (manufactured by Yamasa Soy Sauce Co., Ltd.). (4) Measurement of PTH titer Convert the measured value of C-AMP to the unit of PM/mg PTH receptor protein/min, use this as the reaction value,
Test the test product against the values obtained with the standard product using a parallel line test 2 x 2 point method. (5) PTH activity results (U/mg)

[Table] The abbreviations described herein have the following meanings. Ser; L-serine Val; L-valine Glu; L-glutamic acid Ile; L- _isoleucine Gln; L-glutamine L eu; L-leucine _Nle ; L-norleucine His; L-histidine Asn; L-asparagine Gly ; Glycine Lys; L-lysine Arg; L-arginine Trp; L-tryptophan Asp; L-aspartic acid Tyr; L-tyrosine Boc; t-butyloxycarbonyl Aoc; t-amyloxycarbonyl Z-Cl; o-chlorobenzyl Oxycarbonyl Bzl; benzyl Bzl-Cl ₂ ; 2,6-dichlorobenzyl Tos; tosyl OEt; ethyl ester OBzl; benzyl ester ONP; p-nitrophenyl ester OPAC; phenacyl ester TFA; trifluoroacetic acid TosOH; p-toluene Sulfonic acid Et ₃ N; triethylamine NMM; N-methylmorpholine TBA; t-butylamine DCHA; dicyclohexylamine NaOH; sodium hydroxide THF; tetrahydrofuran DMF; dimethylformamide DMSO; dimethylsulfoxide ether; diethyl ether DCC; N,N'-dicyclohexyl Carbodidiimide WSC; N-ethyl, N'-3-dimethylaminopropyl-carbodiimide HOBt; 1-hydroxybenzotriazole PEE ( ); PF means protected amino acid or peptide fragment, numbers in parentheses are formula The order of amino acids in [] is shown. Next, production examples of the present invention will be specifically explained with reference to Examples. The carrier and developing solvent system for thin layer chromatography (TLC) used in the Examples and the conditions for amino acid analysis are as follows. <TLC> Support: Silica gel G Developing solvent system: 1 Chloroform-methanol-acetic acid (95:5:
3), 2 Chloroform-methanol-acetic acid (85:15:
5), 3 Chloroform-methanol-acetic acid (80:25:
2), 4 Chloroform-ethanol-ethyl acetate (5:2:5), 5 Hexane-ethyl acetate (1:1) Support: Cellulose (manufactured by Merck & Co., DC-
Alufolien) Developing solvent system; 6 Butanol-pyridine-acetic acid-water (5:3:
0.1:11) Upper layer <Amino acid analysis> Unless otherwise specified, samples were prepared with 6N hydrochloric acid at 110℃, 24~
Hydrolyzed in a sealed tube for 48 hours. Example 1 [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ]-h-PTH (1-34)
Production of NH ₂ 1) PF (34); Boc−Tyr(Bzl−Cl ₂ )−NH ₂
[] Boc-Tyr (Bzl-Cl ₂ )-OH52.84g (0.12M)
and 16.69 g (0.12 M) of p-nitrophenol were dissolved in dry TH and cooled at -5°C.
A dry THF solution of 24.76 g (0.12 M) of DCC was added dropwise, and the mixture was stirred overnight. After the reaction, remove the precipitate,
The liquid was saturated with NH ₃ gas and stirred for 5 hours. A precipitate was formed, which was dissolved by adding DMF and then concentrated under reduced pressure. After crystallizing the residue from ether,
The product was collected and dried to obtain the desired product [1]. Yield: 44.77g (yield 84.9%) Melting point: 214-216℃ TLC; Rf ₁ = 0.62 Elemental analysis [as C ₂₁ H ₂₄ O ₄ N ₂ Cl ₂ ] C% H% N% Calculated value 57.41 5.51 6,38 Measured value 57.42 5.59 6.51 [α] ²⁵ _D −5.54゜ (C=1, DMF) 2) PF (33−34); Boc−Asn−Tyr (Bzl−Cl ₂ )
-NH2[ ₂ ] 26.36g (60mM) of compound [1] was dissolved in methylene chloride, and 100ml of TFA was added thereto under ice-cooling, followed by stirring at room temperature for 30 minutes. After the reaction, methylene chloride and TFA were distilled off under reduced pressure, and the residue was crystallized with ether, then taken and dried. the obtained crystals,
Boc-Asn-OH13.93g (60mM) and
8.1g (60mM) of HOBt was dissolved in DMF, and 10.98ml (60mM) of WSC was added thereto under cooling at -15°C, followed by stirring overnight. After the reaction, remove the precipitate and
% sodium bicarbonate solution (1 time), water (2 times), methanol (1 time)
The crystals were washed in the following order and dried to obtain crystals of the desired product [2]. DMF was distilled off from the mother liquor under reduced pressure, and the obtained crystals were washed with water and methanol in that order and dried to obtain compound [2], which was combined with the target product [2]. Yield: 28.64g (yield 86.25%) Melting point: 240-242℃ [α] ²⁵ _D -24.06゜ (C = 1, DMF) Elemental analysis [as C ₂₄ H ₃₀ O ₆ N ₄ Cl ₂ ] C% H% N% Calculated value 54.25 5.46 10.13 Measured value 54.46 5.38 10.31 3) PF(32−34);Boc−His(Tos)−Asn−
Tyr(Bzl-Cl ₂ )-NH ₂ [3] Compound [2] 22.14g (40mM) was dissolved in a small amount of methylene chloride, 100ml of TFA was added under ice cooling, and after stirring at room temperature for 30 minutes, TFA was removed under reduced pressure. The residue was distilled off to obtain a Boc-free product. On the other hand, Boc-His(Tos)-OH・DCHA28.36g
(48mM) was suspended in 500ml of ethyl acetate, washed twice with 1N sulfuric acid and twice with water, dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off under reduced pressure. Dissolve the residue in dry DMF and add the dried Boc-removed product to this.
Add DMF solution and NOBt6.48g (48mM),
Next, 8.78 ml (48 mM) of WSC was added while cooling at -15°C, and the mixture was stirred at room temperature overnight. After reaction, under reduced pressure
DMF was distilled off, and the residue was washed once with 5% aqueous sodium bicarbonate and twice with water, and then dried to obtain a crude product. This was crystallized from methanol-ether to obtain the desired product [3]. The crystal mother liquor was concentrated under reduced pressure, and the residue was crystallized from methanol-hexane to obtain compound [3], which was combined with the target compound [3]. Yield: 28.81 g (yield 85.1%) Melting point: 170-175°C TLC: Rf ₃ =0.68, 0.42 A product was obtained in which Tos was partially eliminated. Elemental analysis [as C ₃₈ H ₄₄ O ₉ N ₇ Cl ₂ S] C% H% N% Calculated value 53.96 5.24 11.59 Measured value 53.03 5.65 12.04 4) PF (31−34); Boc−Val−His−Asn−
Tyr(Bzl-Cl ₂ )-NH ₂ [4] Compound [3] 28.81g (34.06mM) was dissolved in a small amount of methylene chloride, 120ml of TFA was added under ice cooling, and after stirring at room temperature for 30 minutes, TFA was dissolved. It was distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected, dried, and then dissolved in 140 ml of DMF. This solution was neutralized with NMM, and 8.14 g of Boc-Val-OH was added to it.
(37.47mM) and HOBt5.06g (37.47mM) dissolved in 60ml of dry DMF were added, and then -
Add 6.86ml (37.47mM) of WSC while cooling at 15℃.
Stir overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, and the residue was washed once with 5% aqueous sodium bicarbonate and three times with water, and dried to obtain the target product [4]. Yield 27.76g (yield 103.2%) Melting point: 164-166℃ TLC; Rf ₃ = 0.65 [α] ²⁵ _D -28.38゜ (C = 1, DMF) Elemental analysis [as C ₃₆ H ₄₆ O ₈ Cl ₂ ] C % H% N% Calculated value 54.75 5.87 14.19 Measured value 53.37 5.87 13.43 5) PF(30−34);Boc−Asp(OBzl)−Val−
His-Asn-Tyr(Bzl- _Cl2 ) _-NH2 [5] Compound [4] 27.76g (35.15mM) was suspended in a small amount of methylene chloride, 110ml of TFA was added under ice-cooling, and the mixture was stirred at room temperature for 30 minutes. After that, TFA was distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected, dried, and then dissolved in 120 ml of DMF. Neutralize this solution by adding 10ml of NMM, and add Boc-
Asp(OBzl)-OH12.5g (38.67mM) and
A solution of 5.22 g (38.67 mmM) of HOBt dissolved in 80 ml of dry DMF was added and then cooled at -15℃.
After adding 7.08 ml (38.67 mM) of WSC, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was washed once with 5% aqueous sodium bicarbonate and twice with water, suspended in methanol, and recrystallized by adding ether to obtain the target product [5]. Yield: 31.42g (yield 89.8%) Melting point: 214-215℃ TLC: Rf ₃ = 0.6 [α] ²⁵ _D -23.28゜ (C = 1, DMF) Elemental analysis [C ₄₇ H ₅₇ O ₁₁ N ₉ Cl ₂ ] C% H% N% Calculated value 56.74 5.78 12.67 Measured value 56.19 5.79 12.07 6) PF (29−34); Boc−Gln−Asp(OBzl)−
Val-His-Asn-Tyr(Bzl- _Cl2 ) _-NH2 [6] Compound [5] 31.11g (31.27mM) was suspended in methylene chloride, and 120ml of TFA was added under ice-cooling.
After stirring at room temperature for 30 minutes, TFA and methylene chloride were distilled off under reduced pressure. The residual ether was added, and the precipitated crystals were collected, dried, and then dissolved in 100 ml of dry DMF. Neutralize this solution with 8 ml of NMM and add Boc
-Gln-ONP12.64g (34.4mM) and
A solution of 0.42 g (3.13 mmol) of HOBt dissolved in 100 ml of dry DMF was added, and then 3.78 ml of NMM was added under ice cooling, followed by stirring overnight. After reaction, DMF under reduced pressure
was distilled off, and the residue was washed once with 5% aqueous sodium bicarbonate and twice with water, suspended in methanol, and recrystallized with ether to obtain the desired product [6]. Yield: 33.19g (yield 94.5%) Melting point: 81-83℃ TLC: Rf ₃ = 0.47 [α] ²⁴ _D -23.98゜ (C = 1, DMF) Elemental analysis [C ₅₂ H ₆₅ O ₁₃ N ₁₁ Cl ₂ ] C% H% N% Calculated value 55.61 5.83 13.72 Measured value 54.81 5.96 13.07 Amino acid analysis; Asp2.19(2), Glu1.05(1), Val/
(1), Tyr0.73(1), His0.85(1) 7) PF(27−28); Boc−Lys(Z−Cl)−Leu−
OEt [7] Boc-Lys(Z-Cl)-OH・TBA97.6g
(0.2M) was suspended in 500ml of ethyl acetate, and this was added to 1N
After washing with hydrochloric acid and water in that order and drying with anhydrous sodium sulfate,
Concentration under reduced pressure gave an oil. Dry this with THF500
ml and add 39.14 g of H-Leu-OEt・HCl to this.
(0.2M) and 27.0g (0.2M) of HOBt were added, and then 36.6ml (0.2M) of WSC was added while cooling to -15°C, followed by stirring at room temperature overnight. After reaction, THF under reduced pressure
was removed. Dissolve the remaining residue in 600ml of ethyl acetate,
The mixture was washed with 5% sodium bicarbonate solution, water, 1N hydrochloric acid, and water in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was left in the cold to crystallize. Hexane was added and removed to obtain the desired product [7]. Yield: 110.62g (yield 99.5%) Melting point: 77-80℃ TLC; Rf ₅ = 0.48 [α] ²⁹ _D -19.08゜ (C = 1, DMF) 8) PF (26-28); Boc-Lys ( Z−Cl)−Lys
(Z-Cl)-Leu-OEt [8] 110.62 g (0.199 M) of compound [7] was added to 50 ml of methylene chloride, and to this was added 250 ml of TFA under ice cooling, followed by stirring at room temperature for 1 hour. After reaction, under reduced pressure
TFA and methylene chloride were distilled off to obtain an oily de-Boc product. On the other hand, Boc-Lys(Z-Cl)-OH・TBA97.1g
(0.199M) in 500ml of ethyl acetate and 1N hydrochloric acid.
Wash with 300 ml of water and dry with anhydrous sodium sulfate, then concentrate under reduced pressure to obtain an oil. Dry this with THF150ml
and add the above-mentioned de-Boc compound and
A solution of 26.9 g (0.199 M) of HOBt dissolved in 250 ml of dry THF was added and then cooled to -15℃.
After 36.4 ml (0.199 M) of WSC was added dropwise, the mixture was stirred at room temperature overnight. After the reaction, THF was distilled off under reduced pressure to precipitate agar-like crystals. Dissolve this in ethyl acetate, wash with 5% sodium bicarbonate solution, water, 1N hydrochloric acid, and water in this order.
After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The resulting precipitate was treated with hexane and then collected. This was recrystallized from ethyl acetate, ether, and hexane to obtain the desired product [8]. Yield: 156.52g (yield 92.2%) Melting point: 114-116℃ TLC; Rf ₂ = 0.78 [α] ²⁸ _D -20.72゜ (C = 1, DMF) 9) PF (25-28); Aoc-Arg ( Tos)−Lys(Z
−Cl)−Lys(Z−Cl)−Leu−OEt

[9] 156.5 g (184 mM) of compound [8] was added to 50 ml of methylene chloride, and to this was added 250 ml of TFA under ice cooling, followed by stirring at room temperature for 1 hour. After concentrating the reaction solution under reduced pressure and dissolving the residue in 300 ml of dry DMF, NMM
It was neutralized. Add this to Aoc−Arg(Tos)−OH86.0g
(202mM) dissolved in 100ml of dry DMF and 27.3g of HOBt (202mM) were added, and then -15℃
After dropping 37.0ml (202mM) of WSC under cooling into
Stir overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 1 liter of ethyl acetate. This solution was mixed twice with 5% sodium bicarbonate solution, twice with saturated saline solution, and twice with 1N hydrochloric acid.
The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Add ether to the residue and collect the target product.

[9] was obtained. Yield: 217.91g (yield 100.6%) TLC: Rf ₁ = 0.09, Rf ₂ = 0.67 Melting point: 75-78°C [α] ^D ₂₈ -14.02° (C = 1, DMF) 10) PF (24-28) ;Boc−Leu−Arg(Tos)−
Lys(Z-Cl)-Lys:(Z-Cl)-Leu-OEt[10] Compound

[9] Add 100 ml of methylene chloride and 250 ml of TFA to 217.9 g (0.185 M), stir at room temperature for 80 minutes, and then add methylene chloride and TFA under reduced pressure.
was removed. The resulting oil was dissolved in 300 ml of dry DMF and neutralized by adding NMM. in this solution
Boc−Leu−OH・H ₂ O50.9g (0.204M) and
A solution of 27.6g (0.204M) of HOBt dissolved in 100ml of dry DMF was added and then cooled at -15°C.WSC37.3
ml (0.204M) was added dropwise, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was added to water to collect the precipitated product. Methanol-
The desired product [10] was obtained by crystallization twice from ether-hexane. Yield: 213.63g (yield 90.5%) Melting point: 157-160℃ TLC; Rf ₁ = 0.28, Rf ₂ = 0.77 [α] ²⁷ _D −18.68° (C = 1, DMF) 11) PF (23−28) ;Boc−TrP−Leu−Arg
(Tos)−Lys(Z−Cl)−Lys:(Z−Cl)−Leu−
OEt [11] To 153.17 g (0.12 M) of compound [10] were added 100 ml of methylene chloride and 250 ml of TFFA, and after stirring at room temperature for 80 minutes, methylene chloride and TFA were distilled off under reduced pressure. Dissolve the residue in 250ml of dry DMF,
Neutralized to PH7 with NMM. HOBt17.84 in this solution
g (0.132M) and Boc−Trp−OH40.17g
(0.132M) and then cooled at -15℃.
After 24.2 ml (0.1321 M) of WSC was added dropwise, the mixture was stirred at room temperature overnight. After the reaction and after reducing the pressure, DMF was distilled off under reduced pressure, and the residue was poured into 5% aqueous sodium bicarbonate solution to collect the precipitated product. This was suspended in water and recrystallized twice from methanol-ether to obtain the desired product [11]. Yield: 142.57g (yield 81.2%) Melting point: 168-170℃ TLC; Rf ₁ = 0.31, Rf ₂ = 0.82 [α] ²⁸ _D −18.64° (C = 1, DMF) 12) PF (23−28) ;Boc−Trp−Leu−Arg
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Leu−
OH[12] Compound [11] 140.64g (96.16mM) was dissolved in 1200ml of hot ethanol, cooled, a small amount of precipitate was separated, 288ml of 1N-NaOH aqueous solution (3xM) was added, and the solution was dissolved at room temperature for 140.64g (96.16mM). Stir for hours. 1N− to the reaction solution
After adding 192 ml of TosOH aqueous solution (22 times M), the mixture was separated and ethanol was distilled off under reduced pressure. 1N in concentrate
- After adding 96 ml (equal M) of TosOH and then 2 portions of water, the resulting precipitate was collected. After washing twice with water, the product was dried to obtain the desired product [12]. Yield: 142.98g (yield 101.1%) TLC: Rf ₂ = 0.71 Melting point: 125-130°C [α] ²⁷ _D −37.24° (C = 1, DMF) Elemental analysis [C ₆₉ H ₉₄ O ₁₅ N ₁₂ SCl ₂・As 2H ₂ O] C% H% N% Calculated value 56.35 6.72 11.43 Measured value 56.03 6.62 11.85 Amino acid analysis; Leu2(2), Lys2.08(2), Arg1.10
(1), Trp0.83(1) 13) PF (23−34); Boc−Trp−Leu−Arg
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Leu−
Gln−Asp(OBzl)−Val−His−Asn−Tyr(Bzl
_-Cl2 ) _-NH2 [13] 1.68g (1.5mM) of compound [6] was suspended in a small amount of methylene chloride, and then 7ml of TFA was added under ice-cooling, followed by stirring at room temperature for 30 minutes. After reaction, under reduced pressure
TFA was distilled off, ether was added to the residue, and the precipitated crystals were collected and dried. Dry this crystal
It was dissolved in 30 ml of DMF and neutralized with a small amount of NMM.
In this solution, 2.43g (1.65mM) of compound [12],
HOB; 0.22g (1.65mM) and dry DMF 20ml
was added, and then 0.3 ml of WSC (1.1 times M) was added while cooling at -15°C, followed by stirring at room temperature overnight. After the reaction,
DMF was distilled off under reduced pressure, and the residue was diluted once with 5% sodium bicarbonate solution.
After washing twice with water, it was suspended in methanol, ether was added, and the mixture was taken and dried to obtain the desired product [13]. Yield: 3.62g (yield 99.1%) Melting point: 260-270℃ [α] ²⁵ _D -4.66゜ (C=0.3, DMF) Amino acid analysis: Asp1.94(2), GGlu0.96(1),
Val0.71(1), Leu2.00(2), Tyr0.98(1), Lys2.09(2),
His0.58(1), rg0.91(1), Trp0.78(1) 114) PF(22); Boc-Glu(OBzl)-OPAC[14] Boc-Glu(OBzl)-OH128.2g(0.38 M)
After dissolving in 600ml of DMF and adding 113.5g (0.57M) of phenacyl bromide under ice-cooling,
79.3 ml (0.57 M) of Et ₃ N was added dropwise. After dripping, 30
The mixture was stirred at ℃ for 4 hours, then 30 g of potassium acetate was added, and after stirring for 45 minutes, DMF was distilled off under reduced pressure. Add 600 ml of ethyl acetate to the residue, wash twice with 5% aqueous sodium bicarbonate and twice with water, dry the ethyl acetate layer over anhydrous sodium sulfate, and then evaporate the solvent under reduced pressure.
Crystals were precipitated, and hexane was added thereto to obtain the desired product [14]. Yield: 156.19g (yield 90.2%) TLC; Rf ₅ = 0.73 15) PF (21−22); Boc−Val−Glu(OBzl)−
OPAC [15] 50 ml of methylene chloride was added to 147.88 g (0.325 M) of compound [14], and 300 ml of TFA was added thereto under ice cooling. After stirring at room temperature for 1 hour, methylene chloride and TFA were distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected and dried. The crystals were dissolved in 300 ml of dry DMF and neutralized to pH 7 with NMM. Add 35.14 g (0.26 M) of HHOBt and 56.49 g (0.26 M) of Boc-Val-OH to this solution, and -15
After dropping WSC47.6ml (0.26M) under cooling at °C,
Stirred at room temperature for 2 days. After reaction, DMF under reduced pressure
was distilled off, the residue was dissolved in 500 ml of chloroform,
It was washed in the following order: 5% sodium bicarbonate solution, water, 1N hydrochloric acid, and water.
The chloroform layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, hexane was added to the obtained crystals, and the crystals were recrystallized from ethyl acetate-ether to obtain the desired product [15]. Yield: 106.97g (yield 74.2%) TLC: Rf ₃ = 0.63 Melting point: 139-141°C [α] ²⁹ _D -18.92° (C = 1, DMF) 16) PF (20-22); Aoc-Ars ( Tos）−Val−
Glu(OBzl)-OPAC [16] Add 50 ml of methylene chloride to 99.83 g (0.18 M) of compound [15], add 200 ml of TFA under ice cooling,
After stirring at room temperature for 1 hour, methylene chloride and TFA were distilled off under reduced pressure. After treating the residue with hexane and removing the hexane by decanting,
After treatment with ether, the ether was distilled off under reduced pressure. The resulting oil was dissolved in 200 ml of dry DMF and neutralized with NMM. In this solution
HOBt24.33g (0.18M), Aoc−Arg(Tos)−
Add 76.60 g of OH (0.18 M) and 200 ml of dry DMF, and add 32.94 ml of WSC (0.18 M) while cooling at -15°C.
was added dropwise, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate. This solution was mixed with 5% sodium bicarbonate, water, 1N hydrochloric acid,
After washing with water and drying with anhydrous sodium sulfate, ethyl acetate was distilled off under reduced pressure. The process of crystallizing the obtained oil from ethyl acetate-ether and suspending the obtained crystals in ether was performed three times to obtain the desired product [16]. Yield: 149.75g (yield 94.6%) TLC: Rf ₁ = 0.74, Rf ₄ = 0.81 Melting point: 110-114°C [α] ²⁹ _D −11.5° (C = 1, DMF) 17) PF (19-22) ;Boc−Glu(OBzl)−Arg
(Tos)-Val-Glu(OBzl)-OPAC [17] Add 50 ml of methylene chloride to 149.40 g (0.170 M) of compound [16], add 300 ml of TFA under ice cooling, stir at room temperature for 1 hour, and then reduce the pressure. Methylene chloride and TFA were distilled off at the bottom. The residue was treated with ether and the ether was distilled off under reduced pressure.
The resulting oil was dissolved in 200 ml of dry DMF. To this, HOBt25.27g (0.187M) and Boc-Glu
Add (OBzl)-OH63.09g (0.187M) and dry.
Add 100ml of DMF and 34.22ml of WSC under cooling at -15℃
(0.187M) and stirred at room temperature overnight. After the reaction, the solvent was distilled off, the residue was poured into 6 liters of water, and the precipitated crystals were collected. These crystals were suspended in methanol and ether, collected, dissolved in hot methanol, precipitated when cooled, and further suspended in methanol to be collected. These steps were repeated three times to obtain compound [17]. Ta. The solvent was distilled off from the crystal mother liquor, and the desired product (25.02 g) was obtained by crystallization from methanol-ether. Yield: 141.44g (yield 76.7%) TLC: Rf ₁ = 0.56, Rf ₄ = 0.82 Melting point: 119-121°C [α] ²⁹ _D −129° (C = 1, DMF) 18) PF (18-22) ;Boc−Nle−Glu(OBzl)−
Arg(Tos)-Val.Glu(OBzl)-OPAC [18] Compound [17] 6.51g (6mM) Methylene chloride and TFA 24ml were added under ice cooling, and after stirring at room temperature for 40 minutes, methylene chloride and TFA were added under reduced pressure. Distilled away. The residue was crystallized by adding ether and dried. Dissolve the crystals in dry DMF and cool on ice.
Neutralized to PH7 with NMM. In this solution, Boc−Nle
−OH1.67g (7.2mM) and HOBt0.97g
(7.2mM) dissolved in 40ml of dry DMF was added, and after cooling to -15°C, 1.3ml (7.2mM) of WSC was added thereto, followed by stirring overnight. After the reaction, DMF was distilled off under reduced pressure, water was added to the residue, and the resulting precipitate was collected.
Washing was performed in the following order: 5% sodium bicarbonate solution, water (3 times), 1N hydrochloric acid solution (3 times), and methanol. Next, reprecipitation was performed from methanol-ether to obtain the desired product [18]. Yield: 5.61g (yield 78%) TLC; Rf ₁ = 0.56 19) PF (18−22); Boc−Nle−Glu(OBzl)−
Arg(Toc)-Val-Glu(OBzl)-OH [19] Compound [18] 5.03 g (4.2 mM) was dissolved in 30 ml of acetic acid, 8 g of zinc powder was added thereto, and the mixture was stirred at room temperature for 5.5 hours. After the reaction, the zinc dust was removed and the acetic acid was distilled off under reduced pressure. Ether was added to the precipitated crystals to obtain the desired product [19]. Yield: 4.42g TLC: Rf ₁ = 0.18, Rf ₂ = 0.67 Melting point: 210°C (decomposition) Amino acid analysis: Nle1.01(1), Glu2.05(2),
Arg0.98(1), Val/(1) 20) PF(18−34); Boc−Nle−Glu(OBzl)−
Arg(Tos)−Val−Glu(OBzl)−Trp−Len−
Arg(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Leu
−Gln−Asp(OBzl)−Val−His−Asn−Tyr
(Bzl-Cl ₂ )-NH ₂ [20] Compound [13] 8.9g (3.5mM) with 0.5 skatole
g (3.5mM), 25ml of dimethyl sulfide, 2.5ml of ethanedithiol and 25ml of TFA, and incubated at 0°C.
After stirring for 10 minutes and at room temperature for 45 minutes, the reaction solution was concentrated under reduced pressure. Add ether to the residue, take the resulting precipitate, dry it, and dissolve it in 100 ml of dry DMF.
Neutralized to PH7 with NMM. HOBt0.54 in this solution
g (4mM) and compound [19] 4.3g (4mM)
After adding 0.73ml of WSC while cooling to -15℃,
Stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium bicarbonate was added to the residue, and the resulting precipitate was collected and thoroughly washed with water. The desired product [20] was obtained by dissolving this product in ethanol and adding ether to precipitate it twice. Yield: 11.12g (yield 94%) TLC: Rf ₃ = 0.72 Melting point: 250℃ (decomposition) [α] ²⁸ _D -4.73゜ (C = 0.53, DMF) Amino acid analysis: Asp1.98(2), Glu3. 04(3),
Val1.69(2), Len2(2), Tyr1.07(1), Lys1.93(2),
His0.59(1), Arg1.97(2), Trp0.35(1), Nle1.07(1) 21) PF(17); Boc−Ser(Bzl)−OPAC[21] Boc−Ser(Bzl )-OH88.6g (0.3M)
Dissolve in 400ml of DMF, add 89.6g (0.45M) of phenacyl bromide, and add to this under ice cooling.
3.5 at 30℃ after dropping 62.6ml (0.45M) of _Et3N
Stir for hours. Next, potassium acetate was added to this reaction solution.
22.1g (0.225M) was added and stirred at room temperature for 1 hour. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 500 ml of ethyl acetate and washed successively with 5% aqueous sodium bicarbonate and water. After drying the ethyl acetate layer with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was left in the refrigerator to crystallize, and hexane was added to obtain the desired product [21]. Yield: 122.97g (yield 99.1%) TLC: Rf ₅ = 0.82 [α] ^29.5 _D −11.88° (C = 1.0, DMF) Melting point: 45-47°C 22) PF (16-17); Boc-Asn- Ser(Bzl)−
OPAC [22] 50 ml of methylene chloride was added to 119.9 g (0.29 M) of compound [21], and to this was added 250 ml of TFA under ice cooling, followed by stirring at room temperature for 1 hour. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were collected and dried. The crystals were dissolved in 400 ml of dry DMF and neutralized to pH 7 with NMM. In this solution
HOBt31.35g (0.232M) and Boc−Asn−
53.88 g (0.232 M) of OH was added, and 42.46 ml (0.232 M) of WSC was added dropwise to this while cooling to -15°C, followed by stirring at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure.
The residue was dissolved in 500 ml of ethyl acetate and washed with 5% aqueous sodium bicarbonate. During the liquid separation, crystals were precipitated, so the crystals were taken and washed with water, and then with ether to obtain 41.79 g of crystals of the desired product [22]. The liquid ethyl acetate layer was concentrated under reduced pressure, and the residual oil was crystallized from ethyl acetate-ether to obtain 6.22 g of crystals of the desired product [22]. Yield: 48.01g (yield 39.2%) TLC: Rf ₂ = 0.61, Rf ₄ = 0.62 Melting point: 174-176°C [α] ^29.5 _D −5.54° (C = 1.0, DMF) Amino acid analysis: Asp1.22(1 ), Ser1.00(1) 23) PF (15-17); Boc−Leu−Asu−Ser(Bzl)
-OPAC [23] 50 ml of methylene chloride was added to 80.91 g (0.153 M) of compound [22], and then 150 ml of TFA was added under ice cooling, followed by stirring at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the resulting oil was separated by a decanting method, and then dissolved in 150 ml of dry DMF.
Neutralized to PH7 with NMM. This solution HOBt22.7g
(0.168M), Boc-Leu-OH・H ₂ O41.9g
(0.168M) and 100ml of DMF were added, and 30.7ml of WSC (0.168M) was added dropwise while cooling to -15°C. The reaction mixture was stirred at room temperature and turned into a gel. After leaving it in an ice room for 3 days, water was removed. In addition, the resulting precipitate was collected, washed with 5% sodium bicarbonate solution and water in that order, and dried to obtain the desired product [23]. Yield: 88.52g (yield 90.3%) TLC: Rf ₂ = 0.80, Rf ₃ = 0.88 Melting point: 192-193°C Elemental analysis [as C ₃₃ H ₄₄ O ₉ N ₄ ] C% H% N% Calculated value 61.86 6.92 8.75 Measured value 61.81 7.05 8.56 24) PF (14−17); Boc−His(Tos)−Leu−
Asn-Ser(Bzl)-OPAC [24] To 87.55 g (0.137 M) of compound [28] was added 100 ml of methylene chloride, then 200 ml of TFA was added under ice cooling, and the mixture was stirred at room temperature for 70 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was collected, dried, and dissolved in 200 ml of dry DMF.
A Boc-free solution was obtained by neutralizing to pH 7 with NMM. On the other hand, Boc-His(Tos)-OH・DCHA89.2g
(0.151M) was suspended in ethyl acetate and 1N sulfuric acid was added.
It was washed with 500 ml and the precipitated crystals were separated. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. Dry the resulting oil
Solution dissolved in DMF150ml and HOBt20.4g
(0.151M) was added to the above-mentioned Boc-depleted solution, and -
After cooling to 15° C., 27.6 ml of WSC (0.151 M) was added dropwise, and the mixture was stirred at room temperature for 3 days. After the reaction, the solvent was distilled off under reduced pressure, the residue was added to water, and the resulting precipitate was collected, washed with 5% sodium bicarbonate solution and water in that order, and dried to obtain the target product [24]. Yield: 108.63 g (yield 85.51%) TLC: Rf ₂ =0.20, 0.79 Rf = 0.55, 0.87 A product was obtained in which Tos was partially eliminated. Melting point; 154-156℃ [α] ^29.5 _D −18.58゜ (C=1.0, DMF) 25) PF (13-17); Boc−Lys(Z−Cl)−His−
Leu-Asn-Ser(Bzl)-OPAC [25] Compound [24] 107.96 g (0.1161 M) was added with 100 ml of methylene chloride, and then 200 ml of TFA was added under ice-cooling, followed by stirring at room temperature for 70 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was collected, dried, dissolved in 200 ml of dry DMF, and diluted with NMM.
The solution was neutralized to pH 7 to obtain a Boc-free solution. On the other hand, Boc-Lys(Z-Cl)-OH・TBA62.46g
(0.128M) was suspended in 600 ml of ethyl acetate, washed successively with 1N hydrochloric acid and water, and the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. A solution of the obtained oil and 17.30 g (0.128 M) of HOBt dissolved in 100 ml of dry DMF was added to the above-mentioned Boc-removed solution, and 24.42 ml (0.128 M) of WSC was added dropwise to this while cooling to -15°C, followed by cooling to room temperature. The mixture was stirred overnight. After the reaction, the solvent was distilled off under reduced pressure, the residue was added to 3% aqueous sodium bicarbonate solution, and the precipitated crystals were thoroughly washed with water and then dried. This result was dissolved in methanol, and ether was added to precipitate it. The obtained precipitate was suspended in ethyl acetate,
The desired product [25] was obtained by repeating the steps three times. Yield: 114.42g (yield 91.8%) TLC: Rf ₂ = 0.34, Rf ₃ = 0.68 Melting point: 200-202°C [α] ²⁸ _D −26.94° (C = 1.0, DMF) 26) PF (13-17) ;Boc−Lys(Z−Cl)−His−
Leu-Asn-Ser(Bzl)-OH [26] Compound [25] 86.0g (80mM) was dissolved in 500ml of acetic acid, 150g of zinc powder was added thereto, stirred at room temperature for 5 hours, and the reaction solution was filtered. Zinc dust was removed. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were collected to obtain the desired product [26]. Yield: 84.70g (yield 95.2%) TLC: Rf ₂ = 0.47 Melting point: 240-250℃ [α] ³⁰ _D −19.16° (C = 1.0, DMF) Elemental analysis [C ₄₅ H ₅₂ O ₁₂ N ₉ Cl・2CH ₃ COOH・
As 2H ₂ O] C% H% N% Calculated value 53.76 6.63 11.52 Measured value 52.83 6.36 11.35 Amino acid analysis; Asp1.01(1), Ser0.83(1), Leu/
(1), Lys0.93(1), His0.97(1) 27) PF(13−34); Boc−Lys(Z−Cl)−His−
Leu−Asn−Ser(Bzl)−Nle−Glu(OBzl)−
Arg(Tos)−Val−Glu(OBzl)−TrP−Leu−
Arg(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Leu
−Gln−Asp(OBzl)−Val−His−Asn−Tyr
(Bzl-Cl ₂ )-NH ₂ [27] Compound [20] 10.77g (3.2mM) with skatole
Add 0.46g (3.2mM), 25ml dimethyl sulfide, 2.5ml ethanedithiol and 25ml TFA,
After stirring at °C for 10 minutes and at room temperature for 60 minutes, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected, dried, dissolved in 100 ml of dry DMF, and neutralized to pH 7 with NMM. Then this
HOBt0.51g (3.8mM) and compound [26]4.23
g (3.8mM) and cooled to -15°C with 0.70ml of WSC.
was added, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, water was added to the residue, and the resulting precipitate was collected, washed with water, and dried to obtain the target product [27]. Yield: 13.60g (yield 100%) Melting point: 138-160.5℃ [α] ²⁸ _D -19.6゜ (C=0.56, DMF) Amino acid analysis: Asp2.96(3), Ser0.62(1),
Glu3.02(3), Val1.72(2), Leu3(3), Tyr1.06(1),
Lys3.01(3), His1.43(2), Arg1.98(2), Trp0.60(1),
Nle1.06(1), 28) PF (11−12); Boc−Leu−Gly−OBzl [28] Dry 4.99 g (20 mM) of Boc−Leu−OH・H ₂ O
It was dissolved in 30 ml of THF, 50 ml of dry benzene was added thereto, and the solvent was distilled off by cofluorination. The obtained oil was dissolved in 70 ml of dry THF, and H-Gly-
OBzl・TosOH (20mM) and HOBt2.7g
Add WSC5 (20mM) and then cool to -5°C.
ml and then stirred at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure, the residue was dissolved in 100 ml of ethyl acetate, and then dissolved twice with 1N hydrochloric acid and twice with 5% aqueous sodium bicarbonate.
Washed twice with water. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the desired product [28] as an oil. 29) PF (10−12); Boc−Asn−Leu−Gly−
OBzl [29] Add the oil obtained above [28] to -15℃ while cooling.
Add 40 ml of 4.39N hydrogen chloride/dioxane solution,
After stirring for a minute, the mixture was concentrated under reduced pressure. Add ether to the residue, remove the resulting precipitate, and dry.
Dissolved in 30ml of DMF. This was cooled to -5℃.
Add Et ₃ N to adjust pH to 7, then add 0.3 g of HOBt
(2.2mM) and Boc-Asn-ONP7.77g
(22mM) was added and stirred at room temperature for 3 days. Add water to the reaction solution, and collect the precipitate in chloroform.
Extracted with 200ml. Add 1N hydrochloric acid to the chloroform layer, 5
After washing with % sodium bicarbonate solution and water in that order, and drying with anhydrous sodium sulfate,
The solvent was distilled off under reduced pressure, and the residue was crystallized from ethyl acetate-hexane to obtain the desired product [29]. Yield: 8.0g (yield 73.8%) Melting point: 152-156°C [α] ²⁴ _D -36.1° (C = 1.0, DMF) 30) PF (9-12); Boc-His-Asn-Leu-
Gly-OBzl [30] To 7.36 g (15.5 mM) of compound [29], 5 ml of methylene chloride was added, and then 32 ml of TFA was added under ice-cooling, followed by stirring at room temperature for 60 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the precipitate was collected, dried, and dissolved in 40 ml of dry DMF.
The pH was adjusted to 7 with NMM to obtain a Boc-free solution. On the other hand, Boc-His(Tos)-OH・DCHA10.99g
(18.6mM) in 150ml of ethyl acetate and 90ml of 0.5N sulfuric acid
The ethyl acetate layer was washed three times with water, dried over anhydrous sodium sulfate, and then the ethyl acetate was distilled off under reduced pressure to obtain an oil. This oily substance and HOBt2.5g
(18.6mM) was dissolved in 60ml of dry DMF, and the solution was added to the above-mentioned Boc-removed solution. Then, 3.4ml (18.6mM) of WSC was added while cooling to -15°C, and the mixture was stirred at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, washed three times with 5% aqueous sodium bicarbonate and twice with water, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. Ether was added to the residue and the precipitated crystals were collected. This crystal has some Tos removed from His, but in order to remove it completely, we need to remove this crystal.
Dissolve in 100ml of DMF, add 7.05g of HOBt to it,
Stirred at room temperature for 3 days. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, washed twice with 5% sodium bicarbonate water and then with water, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. Ether was added to the precipitated crystals to obtain the desired product [30]. Yield: 7.32g (yield 74.8%) TLC; Rf ₂ = 0.1 31) PF (8-12); Boc-Nle-His-Asn-
Leu-Gly-OBzl [31] To 7.32 g (11.6 mM) of compound [30], 5 ml of methylene chloride was added, and then 30 ml of TFA was added under ice-cooling, followed by stirring at room temperature for 40 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the precipitate was collected, dried, and dissolved in 40 ml of dry DMF.
The pH was adjusted to 7 with NMM. This includes HOBt1.9g
(13.92mM) and Boc-Hle-OH3.23g
Add a solution of (13.92mM) in dry DMF,
After adding 2.5 ml of WSC (13.92 mM) while cooling to -15°C, the mixture was stirred at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure, water was added to the residue, the precipitate was collected and diluted twice with 5% sodium bicarbonate solution, twice with 1N hydrochloric acid, and three times with water.
The product was washed several times and dried to obtain the desired product [31]. Yield: 3.70 g (yield 42.9%) TLC; Rf ₂ = 0.20 32) PF (8-12); Boc-Nle-His-Asn-
Leu-Gly-OH [32] Compound [31] 2.8g (3.8mM) in 100% ethanol
ml, 300 mg of 10% Pd/C was added thereto, and hydrogen gas was passed through it at room temperature for 3 hours. Insoluble matter precipitated in the reaction solution, so after filtering and washing with DMF,
The liquid was concentrated under reduced pressure. Add ethanol-ether to the residue to remove the precipitate and dry it to obtain the desired product [32]
I got it. Yield: 1.76g (yield 71.1%) Melting point: 112.5℃ TLC; Rf ₂ = 0.05 Amino acid analysis: Asp0.96(1), Gly0.98(1), Leu/
(1), His0.95(1), Nle0.94(1) 33) PF(8-34); Boc−Nle−His−Asn−
Leu−Gly−Lys(Z−Cl)−His−Leu−Asn−
Ser(Bzl)−Nle−Glu(OBzl)−Arg(Tos)−
Val−Glu(OBzl)−TrP−Leu−−Arg(Tos)−
Lys(Z−Cl)−Lys(Z−Cl)−Leu−Gln−AsP
(OBzl)−Val−His−Asn−Tyr(Bzl−Cl ₂ )−
NH ₂ [33] Compound [27] 10.60g (2.5mM) of skatole
Add 0.33g (2.5mM), 25ml dimethyl sulfide, 2.5ml ethanedithiol and 25ml TFA,
After stirring at °C for 10 minutes and at room temperature for 50 minutes, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected, dried, and then dissolved in 100 ml of dry DMF, and the pH was adjusted to 7 by adding NMM under ice cooling. To this solution were added 0.36 g (2.7 mM) of HOBt and 1.76 g (2.7 mM) of compound [32], and after adding 0.5 ml of WSC while cooling to -15°C, the mixture was stirred at room temperature overnight. The precipitate was collected, washed with water, dried, and reprecipitated from ethanol-ether to obtain the desired product [33]. Yield: 10.94g (yield 91.7%) Melting point: 140.5-162℃ [α] ²⁸ _D -1.94゜ (C=0.52, DMF) Amino acid analysis: Asp3.87(4), Ser0.76(1),
Glu3.34(3), Gly0.77(1), Val1.84(2), Leu4(4),
Tyr1.04(1), Lys3.28(3), His2.37(3), Ars2.14(2),
Trp0.73(1), Nle2.07(2) 34)) PF(7); Boc−Leu−OPAC[34]Boc−
15.0g (60mM) of Leu-OH・H ₂ O and 17.9g (90mM) of phenacyl bromide were dissolved in 100ml of DMF, and 12.5ml (90mM) of Et ₃ N was added dropwise to this under ice cooling, followed by stirring at 30°C for 2 hours. did. Then, add 4.42g (45mM) of potassium acetate and stir at room temperature for 45mM.
After stirring for a minute, DMF was distilled off under reduced pressure. Dissolve the residue in ethyl acetate, add 5% sodium bicarbonate solution twice,
After washing twice with water and drying the ethyl acetate layer over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was left in an ice chamber and the precipitated crystals were dried to obtain the desired product [34]. Yield: 21.23g (yield 100%) TLC; Rf ₁ = 0.89 35) PF (6-7); Boc-Gln-Leu-OPAC
(35) Add 20ml of methylene chloride to 20.96g (60mM) of compound [34], then add 80ml of TFA under ice cooling,
After stirring at room temperature for 40 minutes, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected; after drying, it was dissolved in 70 ml of dry DMF, and the pH was adjusted to 7 by adding NMM under ice cooling. In this solution
HOBt8.1g (60mM) and Boc-Gln-
Add a solution of 14.78g (60mM) of OH in 90ml of dry DMF, and add 10.9ml of WSC while cooling to -15℃.
(60mM) was added dropwise, and the mixture was stirred at room temperature overnight.
After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, then dissolved twice with 5% sodium bicarbonate solution and twice with 1N hydrochloric acid.
Washed twice with water and three times with water. The ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The precipitated crystals were collected by adding hexane and dried to obtain the desired product (35). Yield: 17.25 g (yield 60.2%) ) TLC; Rf ₁ = 0.38 36) PF (5-7); Boc−Ile−Gln−Leu−
OPAC [36] Add 10 ml of methylene chloride to 17.19 g (36 mM) of compound [35], then add 70 ml of TFA under ice cooling,
After stirring at room temperature for 60 minutes, the reaction solution was concentrated under reduced pressure. After drying the residue under reduced pressure, it was dissolved in 130 ml of dry DMF, and the pH was adjusted to 7 with NMM under ice cooling. In this solution
HOBt5.3g (39.6mM) and Boc-Ile-OH・
Add a solution of 9.5g (39.6mM) of 1/2 H ₂ O in 70ml of dry DMF, and add 7.2ml of WSC while cooling to -15℃.
(39.6mM) was added dropwise, and the mixture was stirred at room temperature overnight.
After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium bicarbonate was added to the residue, and the resulting precipitate was collected, washed with 5% aqueous sodium bicarbonate, twice with 1N hydrochloric acid, and three times with water in that order, and dried. did. This precipitate was reprecipitated from ethanol-ether to obtain the desired product [36]. Yield: 16.35g (yield 76.9%) TLC; Rf ₁ = 0.41, Rf ₂ = 0.68 37) PF (4-7); Boc-Glu(OBzl)-Ile-Gln
-Leu-OPAC [37] 16.24g (27.5mM) of compound [36] was added to 10ml of methylene chloride, and then 70ml of TFA was added under ice-cooling.
Next, 70 ml of TFA was added under ice-cooling, and after stirring at room temperature for 60 minutes, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected, dried, and dissolved in 100 ml of dry DMF. Then, under ice cooling, NMM was added to adjust the pH to 7. 4.09g of HOBt in this solution
(30.25mM) and Boc-Glu(OBzl)-OH10.2g
(30.25mM) dissolved in 50ml of dry DMF was added, and 5.5ml of WSC was added dropwise while cooling to -15℃.
Stir overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, and 5% aqueous sodium bicarbonate was added to the residue to remove the resulting precipitate.
Washed and dried in sequence. The desired product [37] was obtained by reprecipitation from ethanol-ether. Yield: 21.68g (yield 97.1%) TLC; Rf = 0.52 38) PF (3-7); Boc-Ser(Bzl)-Glu
(OBzl)-Ile-Gln-Leu-OPAC [38] Add 10 ml of methylene chloride to 21.46 g (26.5 mM) of compound [37], then add 90 ml of TFA under ice cooling,
After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was taken, dried, and then dissolved in 150 ml of dry DMF.NMM was then added under ice cooling to adjust the pH to 7. Add 3.9g (29.15mM) of HOBt and Boc-Ser to this solution.
(Bzl)-OH 8.6g (29.15mM) dried DMF 50ml
Add the dissolved solution to WSC5.3 under cooling to -15℃.
ml (29.15mM) and stirred overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium bicarbonate was added to the residue, and the precipitate was collected. This was washed twice with 5% sodium bicarbonate solution, 1N hydrochloric acid, and four times with water, and then suspended and taken up in ether to obtain the desired product [38]. Yield: 24.8g (yield 94.7%) TLC; Rf ₁ = 0.53 39) PF (2-7); Boc-Val-Ser(Bzl)-Glu
(OBzl)-Ile-Gln-Leu-OPAC [39] To 24.68 g (25 mM) of compound [38] was added 20 ml of methylene chloride, then 100 ml of ice-cold TFA, and the mixture was stirred at room temperature for 50 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was taken, dried, and dissolved in 120 ml of dry DMF, and then NMM was added under ice cooling to adjust the pH to 7. Add 4.05g of HOBt (30mM) and Boc-Val- to this solution.
Add a solution of 6.5g (30mM) of OH dissolved in 80ml of dry DMF, and add 5.5ml (30mM) of WSC while cooling to -15℃.
was added dropwise, and the mixture was stirred at room temperature overnight. A precipitate was deposited in the reaction solution, so it was collected by adding water, washed twice with 5% sodium bicarbonate, twice with 1N hydrochloric acid, and four times with water, and then suspended in ether and taken. Obtained object [39]. Yield: 26.32g (yield 96.8%) TLC; Rf ₁ = 0.49 40) PF (1-7); Boc-Ser (Bzl)-Val-Ser
(Bzl)−Glu(OBzl)−Ile−Gln−Leu−OPAC
[40] 20 ml of methylene chloride was added to 26.07 g (24 mM) of compound [39], and then 100 ml of TFA was added under ice cooling, followed by stirring at room temperature for 40 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was collected, dried, and dissolved in 100 ml of dry DMF, and then NMM was added under ice cooling to adjust the pH to 7. Add 3.9g (28.8mM) of HOBt and Boc-Ser to this solution.
Add a solution of 8.5g (28.8mM) of (Bzl)-OH in 50ml of dry DMF, and cool to -15°C with 5.3ml of WSC.
(28.8mM) and then stirred at room temperature overnight.
A precipitate was deposited in the reaction solution, so water was added to remove it, and the precipitate was washed in the order of 5% sodium bicarbonate solution, 1N hydrochloric acid, and water.
The desired product [40] was obtained by performing the steps of suspending and removing in ether twice. Yield: 28.0g (yield 92.3%) TLC; Rf ₁ = 0.53 41) PF (1-7); Boc-Ser (Bzl)-Val-Ser
(Bzl)-Glu(OBzl)-Ile-Gln-Leu-OH [41] Compound [40] 12.6g (10mM) was dissolved in 300ml of acetic acid, 15g of zinc powder was added thereto, and the mixture was stirred at 50°C for 4 hours. , zinc powder was separated. Acetic acid was distilled off under reduced pressure, ether was added to the residue, and the precipitated crystals were collected and washed to obtain the desired product [41]. Yield: 11.15g (yield 97.4%) Melting point: 260℃ (decomposition) TLC: Rf ₁ = 0.14, Rf ₂ = 0.64 Amino acid analysis: Ser1.81(2), Glu2.02(2),
Val0.95(1), Leu1(1), Ile0.92(1) 42) Protection-[Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ]-h-PTH
(1-34)NH ₂ ; Boc−Ser(Bzl)−Val−Ser
(Bzl)−Glu(OBzl)−Ile−Gln−Leu−Nls−
His−Asn−Leu−Gly−Lys(Z−Cl)−His−
Leu−Asn−Ser(Bzl)−Nle−Glu(OBzl)−
Arg(Tos)−Val−Glu(OBzl)−Trp−Leu−
Arg(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Leu
−Gln−Asp(OBzl)−Val−His−Asn−Tyr
(Bzl) _-Cl2 ) _-NH2 [42] Compound [33] 10.86g (2.28mM), skatole 0.30g (2.28mM), dimethyl sulfide 25 under ice cooling
ml, ethanedithiol 2.5 ml and TFA 25 ml, and after stirring at room temperature for 60 minutes, the mixture was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was taken and dried, then dissolved in a mixture of 100 ml of dry DMF and 10 ml of DMSO, and the pH was adjusted to 7 by adding NMM under ice cooling. To this solution were added 0.37g (2.74mM) of HOBt and 3.14g (2.74mM) of compound [41], and then 0.50ml (2.74mM) of WSC was added while cooling to -15°C, followed by stirring at room temperature overnight. Add water to the reaction solution,
The resulting precipitate was collected, thoroughly washed with water, and then washed with ethanol-ether to obtain the desired product [42]. Yield: 12.87g (yield 97.3%) Melting point: 139.5-175℃ [α] ²⁸ _D -1.97゜ (C=0.51, DMF) Amino acid analysis: Asp3.72(4), Ser2.76(3),
Glu5.58(5), Gly0.69(1), Val2.86(3), Ile1.11(1),
Leu5(5), Tyr0.99(1), Lys2.87(3), His2.19(3),
Arg2.06(2), Trp0.65(1), Nle1.96(2) 43) [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] −h−PTH(1−
34) 3.5 ml of anisole, 0.35 ml of ethanedithiol, 3.5 ml of dimethyl sulfide and 35 ml of anhydrous HF were added to 2.9 g (0.5 mM) of NH ₂ compound [42] while cooling at 0° C., and the mixture was stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure, ether was added to the residue, the resulting precipitate was collected, and then dissolved in 0.1N acetic acid. Apply this solution to a Dowex x 1 (acetate type) column (3.5 x 12
cm), and only the ninhydrin-positive fraction was collected and lyophilized to obtain 1.87 g of crude product.
Dissolve this in 50ml of 0.1N acetic acid, charge it to a CM-cellulose column (2 x 33cm), and apply a linear concentration gradient of 0.05M ammonium acetate (PH5.1) to 0.4M ammonium acetate (PH6.0)1. Elution was performed. Each fraction is divided into 9.0ml,
74 with a spot near Rf ₆ = 0.30 due to TLC
~84th fraction was collected and freeze-dried. Dissolve this in as little amount of 0.1N hydrochloric acid as possible, and apply this solution to a Sephadex G-25 column (3 x 115
cm) and eluted with 0.1N acetic acid. The absorbance of each fraction was measured at UV 280 nm, and only the fractions with one large peak were collected and freeze-dried [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ].
-h-PTH(1-34) _NH2 was obtained. Yield: 140 mg TLC; Rf ₆ = 0.30 Amino acid analysis (6N containing 3% thioglycolic acid)
Hydrolyzed with hydrochloric acid); Asp3.98(4), Ser2.10(3),
Glu4.93(5), Gly0.97(1), Val2.66(3), Ile0.87(1),
Leu5.00(5), Tyr1.11(1), Lys3.26(3), His2.30(3),
Arg2.03(2), Trp0.62(1), Nle2.22(2) High performance liquid chromatography; Column; Nucleosil ₅ C ₁₈ (4nmID×150mm) Buffer; 0.1% acetic acid-acetonitrile containing 0.1M phosphoric acid (The ratio of acetonitrile is
Flow rate: 1 ml/min Detection: 225 nm Measurement result: Peak detected only at 19.07 min.

Claims (1)

[Claims] 1 Formula H-Ser-Val-Ser-Glu-Ile-Gln-Leu-
Nle−His−Asn−Leu−Gly−Lys−His−Leu−
Asn−Ser−Nle−Glu−Arg−Val−Glu−Trp−
Leu−Arg−Lys−Lys−Leu−Gln−Asp−Val−
A peptide represented by His-Asn-Tyr- _NH2 or a salt thereof.