JPS6034996A - (nle8,nle18,tyr34)-h-pth(1-34)nh2 - Google Patents

Abstract

NEW MATERIAL:The peptide of formula (Ser is serine; Val is valine; Glu is glutamic acid; Ile is isoleucine; Gln is glutamine; Leu is leucine; Nle is norleucine; His is histidine; Asn is asparagine; Lys is lysine; Arg is arginine; Trp is tryptophane; Asp is aspartic acid; Tyr is tyrosine; all of which are L-isomers; Gly is glycine), its salt or its redioactive derivative labeled with I<125>. USE:Remedy for diseases caused by the lowering of the function of accessory thyroid. A remedy for osteopathy relating to PTH (human accessory thyroid hormone) or a labeling compound for the examination of the function of accessory thyroid. PREPARATION:The carboxyl group of the C-terminal thyrosyl group is converted to amide group, and the protected amino acids and/or lower peptides are condensed by a liquid-phase synthesis process according to the amino acid order of formula. The protecting group of each N-terminal amino group and side-chain functional group is removed at the final stage by acid decomposition process to obtain the peptide of formula.

Description

[Detailed description of the invention]

The present invention provides a novel human parathyroid hormone (th-PTH)
Regarding derivatives. More specifically, the present invention is useful as a therapeutic agent for hypoparathyroidism, as a therapeutic agent involving P T H, or as a labeling compound for testing parathyroid function [i.e., the formula (in the formula: Ser is L-serine, Val is L-valine, G
lu is L-glutamic acid, ■le is L-inleumene,
Gin is L-glutamine, Leu is L-IJin, N
l e is L-norleucine, His is L-histidine, Asn is L-ameparagine, Gay is glycine, Ly
s is L-lysine, Arg is L-arginine, Trp i
, t, L-tryptophan, Asp is L-amepartic acid, Tyr is I, -tyrosine), its salt, or its 1125-labeled radioactive substance. h-PTH is a peptide hormone consisting of gtt amino acids, and its biological activity is at amino acid position 11f1/-3.
/l N-terminal fragment 1-1 or h-PTHNat
, Acad, Sci, U, S, A, + 51+
Otsu 3 to Otsu 7 (/ri7/): ], However, h-P
TH is unstable due to the presence of L-methionine (Met), and when labeled with Met-125, the hormone activity is lost [Recent Prog, Hormone Re+
q,,/g, 2 Otori ~ 295 to 7962)]. In the conventional method for quantifying h-PTH, only the portion that has h-PTH activity is quantified.
-34Z) was used as an antigen to prepare an erectile antibody. However, h-PTH (/-3g) is L-
It is unstable due to the presence of methionine + Met), and when labeled with 125, Met present at the 5-position and 1-g position is oxidized, resulting in inactivation of the hormone activity. Therefore, h that not only has PTH activity and immunoactivity against PTH antibodies, but also has stable hormonal activity even when labeled with h-125, and has stable radioactivity.
-PTH derivatives include CN1e, Nle, and Ty in which Met at positions 5 and 1g are replaced with L-norleucine, and phenylalanine at position 3 is replaced with L-tyrosine.
r)-h-PTH(/-31
5-//No. 3753]. However, this CN1e, Nle, Tyr )-h
-PTf((/-,,?4'l has Me in its molecule)
Since t does not exist, labeling with 125 does not inactivate the hormone, but its hormonal activity is at most similar to that of natural h-PTH (/-
It was not too strong, having the same level of activity as 3g). The wood compound [■] acts as a receptor for PTH.
L known h-PTHll-311) and (Nle.
It not only has TH activity, but also has immunological activity against PTH antibodies, and even when it is tested with 1125, the hormone activity does not decrease, and CN1e, Nle, Tyr) h-PTI
-1(Ty'r')-h-PTH (/-3≠) exhibits an extremely superior effect. For this reason, the present target compound (1
) is an extremely useful peptide as a therapeutic agent for hypoparathyroidism, a therapeutic agent for bones involving PTH, and a labeling compound for testing parathyroid function. The peptide CI) of the present invention can be obtained by converting the carboxyl group of the C-terminal tyrosyl group into an amide group, and retaining the individual amino acids and/or protected lower peptides in the amino acid order represented by the formula CI). is condensed using a liquid phase synthesis method, and in the final stage of the condensation reaction, the protecting group for the amino group at the N-terminus and the protecting group for the functional group on the side chain are removed by acid decomposition. The condensation reaction itself is carried out by repeating the attachment and detachment of a protecting group and the condensation reaction in accordance with the algorithm for peptide synthesis. That is, the various protecting groups used in the preparation of the raw materials for the present peptide 1 [I] and all of these intermediates are those known in peptide synthesis, and therefore include hydrolysis, acid hydrolysis, reduction, amino acid sequence, or hydrazitolysis. Protecting groups which can be easily removed by known means are therefore used - such protecting groups are described in the literature and reference books in the field of peptide synthetic chemistry. For example, protective groups used for ami/groups include formyl group, p-toluenenulfonyl group, o-21-lofenylnulfenyl group, acyl group, ben\roxycarbonyl group, o(or p)-bromobenozyloxycarbonyl group. 0 (or p)-r10benzyloxycarbonyl group, p-21-ropenoxycarbonyl group, penzylmexylcarbonyl group, trichloroethyloxycarbonyl,! old, t-butyloxycarbonyl group,
aliphatic oxycarbonyl groups such as t-amyloxycarbonyl group and diisopropylmethyloxycarbonyl group,
-1 phenyl-isoproboxycarbonyl group, 2-1
E, J: Reacting the 2-p-amine groups with /,3-diamines such as benzoylacetone and acetoneacetone E, J: The resulting enamines It can be protected by formation. Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the abimid group is 3. t-Ditkinbenzi group, Hesu +
p-methoxyphenyl) substituted with a methyl group, etc. The hydratide group is a benzyloxycarbonyl group, a trichloroethyloxycarbonyl group, a trifluoroacetyp group, a t-butyloxycarbonyl group, a trityl group,
2-P-cyphenyl is substituted with an isopropoxycarbonyl group, etc. Entel group includes alkanols such as methanol, ethanol, t-butanol, cyanomethyl alcohol, pencil alcohol, p-bromobenzyl alcohol, p-
Chloro-11 benzyl alcohol-7, 2. A-dichloropencyl alcohol--1-dichloropencyl alcohol, p
-21-benzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol, p-110 benzoylmethyl alcohol
Aralkat of real alcohol nato, 2,4,,4-
Phenols such as trichlorophenol, dichlorophenol, pentachlorophenol, p-210phenol, 2,'I-dinitrophenol, thiophenol, p-nido-cothiophenol thiophenols such as

[Replaced inconveniently.] 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, benzyl remexycarbonyl group, etc.
l/4, ethyloxycarl (7I), etc.Also, as a group suitable for etherification (if
Hensyl group, 2,2-dichlorobenzyl group, tera-hyropyranyl group, t-phytyl group, (many).To protect these hydroxyl groups, 2,2,2-to-1J fluoro-1-t- Butyloxycar 71 mini-l rare aminoethyl group, 2,2.2-) reflux and A-/-benzyloxycarbonylamino group. However, it is not necessary to protect these hydroxyl groups. Examples of groups used to protect the amino group in the guanidino group of arginine include a nitro group, a tosyl group, a benzyloxycarbonyl group, and a mesitylene-non-nulfonyl group. There is no need to protect it. Groups used to retain the imino group of the hinucicin include, for example, pencil group, trityl group, benzyloxycarbonyl group, tosyl group, 2+, :2.2-trifluoro-/-t-)゛thyl group. leoxycarbonylaminoethyl group, 2, 2. 2-) Riflumello/-benzyloxycarbonylaminoethyl group, etc., but this imino group does not necessarily need to be protected. In the present invention, the protection of the α-amino group is carried out using a levonyl group, an L-amyl A oxycarbonyl group, and the amino group of the side chain (i.e., the protection of the ξ-amino group of lysine). , ; 0-IJ lI pencyloxycarboxyl group is used for Φ, α-carboxy/l/ group L))
The pencil ester group is used to protect the carboxyl group of the chain (1 μm), i.e., the side chain carboxyl group of glutamic acid and aspartic acid.
A pencil group is used to protect the hydroxyl group of tyrosine, a 2,o-cyclopencyl group is used to protect the hydroxyl group of tyrosine, and a 1-yl group or a mesitylene-2-fluoronyl group is used to protect the amino group in the guanidino group of arginine. It is preferable to use Completed target compound [IJ plywood? Here, the condensation of 4 amino acids and/or lower peptides is carried out, for example, with an amino acid with a protected α-amino group and an activated terminal carboxyl group or with a free α-amine. or by reacting an amino acid or compound with a terminal carboxyl group and a protected terminal carboxyl group or This can be carried out by reacting the amino acid or peptide 1 with a free terminal carboxyl group and a retained α-amino group. In this case, the carboxyl group Ha. Lillie is acidic anhydride 1
1. Acid anhydride, acid imidazo'JP"II: or active ester, e.g. evacyanomethyl ester, thiophenyl ester, p-210thiophenyl ester, p-21-lophenyl ester, 2.Gouche 1-mouth phenyl ester, 2. 4. 5-1 dichlorophenyl ester,
2. /1. , O-trichlorophenyl ester,
It can be activated by converting it into penkchlorophenyl ether, N-1 hydroxysuccinimide ester, N-hydroxyphthalic acid imide ester, etc. Also, carbonimides, e.g., N, N'-cyclohegin, N-ethyl-N'-3,
-dimethylamitub+1 leech-carbosimil", N,
It can be activated by reacting the N'-carbonyl-ciimigazole with a condensing agent. Preferred condensation methods in the present invention are the acid method, active ester method and carbodiimide method. At each stage of compatibility, it is desirable to use a method that does not cause racemization or a method that minimizes racemization, preferably the AND method, activated ethyl method, Bunosh method (Z.N.
aturforsch, 21 b, 172 O (
/9 Otsu)〕or〕1'Iger method Chem Ber
Particularly suitable is a modified method using N-ethyl-N-3-dimethylaminopropylpropylene (WSC) as condensing agent. As long as the condensation order is the amino acid jl shown by the formula [Eng. For example, consider a C-terminal fragment consisting of the amino acid order 29-3 and a peptide fragment consisting of the amino acid 23-2.
The condensation of 3-2g is suitably carried out by a modified Geiger method using WSC. The resulting C-terminal fragment +-23-3 is linked with a peptide fragment consisting of amino acids in the order of t/g-2.2, which is best carried out by the modified Geiger method using WSC. . The resulting C-terminal fragment l-/g-3
It is preferable to connect a peptide fragment consisting of the amino acid sequence /3 to /7 in the order before 'l, a peptide fragment consisting of the amino acid sequence g to 72, and a peptide fragment consisting of the amino acid sequence 7 to 7. . The protecting group for the α-amino group, such as the t-butyloxycarbonyl group and the t-amyloxycarbonyl group, in condensation reaction 1 of ``2'' is removed with trifluoroacetic acid. A protecting group for the α-carpogycyl group, such as ethyl ester, can be decomposed in a dilute hydroxylation solution or
The phenacyl ester group is decomposed with Zn powder in acetic acid, and the benyl ether group is converted to anhydrous hydrogen fluoride wC, hydrogen Additive IIJ can be decomposed or converted into hiratide. Thus protected N-terminal α-amino group, ξ-amino group, side chain carboxyl group, quanicino group and/or
A tetratriacontapeptide containing a hydroxyl group is obtained. These retained dI# groups are preferably eliminated in one step by acid decomposition, e.g. with anhydrous hydrogen fluoride or trifluoromethanonulponic acid, to give the target compound of formula CI) (9). The peptide L (II is Pef.
It can be made from t. Gel 1 such as Sephadex G-+25, Sephadex Y.Q-50, Sephadex 7, L)(-20)
The peptide of the present invention can be carried out by gel filtration using (61 1'+lI), column chromatography using carboxymethyl cellulone, ion exchange resin, high performance liquid chromatography, etc. CD is produced in the form of a base or its salt (1). The salts include 1, inorganic salts, formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, phenolic acid such as 1i. This peptide 1 [I] is Some kind of inorganic substance 'l' 4 years old or 41 years old
A material can be added to form a 1C button body. This complex refers to an as-yet-unknown compound that is formed upon addition and gives Pepti 1 a sustained effect. Such substances include
For example, inorganic compounds derived from metals such as calcium, magnesium, aluminum, cobalt or zinc, especially slightly soluble salts of these metals such as phosphates, pyrophosphates or phosphates, as well as water Mention may be made of acid compounds or alkali metal borates. Furthermore, Vepti 1 [■] of the present invention is preferably 1tIA
It is used as a labeling reagent. For example, in order to increase the radioactivity of a certain amount of h1, add 125-1 J phosphate buffer (1 bottle H7,/) to Pep-1-1 [1] of the invention and
Add 125I/minT and stir, then add 125I + sulfuric acid, add a small amount of potassium iodide and serum albumin, perform chromatography, and collect the 125I-labeled fraction. 125 by collecting
The radioactive compound labeled with I is prepared. Next, the parathyroid hormone (PTH) activity of the peptide [I] of the present invention and the radioactive substance labeled with 125I will be described. <Preparation of 125I-labeled compound> 7, . 10.5 M'J acid buffer (pH 7,
/ ) S (Blth knee each h -PTI-I (
/-3gl, h-PTIl(/-3glNH2, (N
le, Nle, Tyr, :Ih-PI'
ll (/8' 18 34 -3g) and (Nle, Nle, Tyr "JJ
-h-PTl-I (/-31Ni12)!μf included
W/mt4) containing H,
The reaction was stopped by adding 50 μe of the solution containing 57 ng/me). This contains 0.5 human serum albumin. /
After adding N acetic acid bath 't+K o, s me, charge the column (/xsO port) of Sefadenokutsu G-10, cure with the above acetic acid solution, and calculate the content of each cedar specimen labeled with '25■. I got the picture. <P T I-I activity measurement method> (1) Preparation of P T H receptor 1 liter of SD male and female rats (body weight 200-.25 OS')
After exsanguination of the head and laparotomy, the kidney was removed, its surface membrane was removed, and the renal cortex was cut out and cooled on ice. The following operations are performed at as low a temperature as possible (0-1°C). The above kidney cortex area was treated with 0.25 M Sucron and/or
The renal cortex was soaked in 10mM Tris-HCl buffer containing MEDTA (pH 7.5) (hereinafter referred to as Solution A), and the wet weight (7) of the renal cortex was placed in a glass jacket tube using Teflon Perasle. Add 3 times the volume of solution A (i+i', (l1le)) and homogenize. This homosyne-1.15
Centrifuge at 0X for 70 min, then further centrifuge at 2200X for 15 min. Discard 10 M%.
Suspend the upper layer of the sediment, which is cloudy in color, in Solution A, and c)
Centrifuge the yH suspension at 2.200X for 9.75 minutes.
Rinse from t, open 411 and dispense into t containers, -
Freeze at 70°C and store at -20°C. (2) P T II,! : P T HV septano reaction test product 2μ9/m14 and 70μ? /
A T PMg, 2m
MS MgCl2/o mM, KCl 60 m
M, G T P 20 p M, Ia 7'
ftJ-7-Luxanthine/mM, Creatinohonufe-1-gmM and Bovine Serum Albumin (BSA
)oi% Containing Content 1 River 0 (Hereafter referred to as BM)
This is also done for TH (/-g/1.). Dispense 50 μe each of these q bath solutions into a glass tube, and prepare g bottles of each bath solution. Test sample 1 is kept in ice water to allow other substances such as ATP to decompose. The P T II resator preparation stored at -20°C was heated to Iq' (
Freeze, add creatine kinase previously dissolved in Solution A, and add creatine kinase i O. / mg/me, l) T H l/-1!
Pig-kun (product++r white matter / /I-my / mI
Adjust the temperature to V and keep it in water cooling. After soaking the dispensed sample solution in 2 for 29 minutes in a 111 temperature bath at 37°C, n engineering, 37
℃-C/O/. ) Incubate for I. Then 0. / M acetic acid! ．． e: ? :Iii /iri (pH
After adding 100 μe of Hiroshi0) and immediately placing the tube in ice water, immediately heat the test tube in boiling water for 7 minutes to stop the reaction. (3) Measurement of produced C-AMP The above reaction-stopped sample is diluted 70 to 30 times with distilled water, and protein is removed by centrifugation at 20° OX G for 75 minutes. The supernatant (AMP-jf) was
- (manufactured by Yamasa Kaedeyu Co., Ltd.). (4) Measurement of PTH titer The measured value of C-AMP was converted into the unit of PM/~1) TH receptor protein/min, and this was used as the reaction value, compared to that obtained with the standard product. Parallel line test 2X, 2 for the test item
Test 5i using the point method. (s) PTH activity results (U/〃Nog) h-PTH (/-311-) 300t〉700 Abbreviations described in this specification have the following meanings. Ser + L-serine Val; L-valine Glu; L-glucumic acid 11e + L-isoleucine Qln, L-glucumin Leu; L-leucine Nle; L-norleucine 1iis; L-hifu-tidine Asn: L-asparagine Qly; Glycine Lys: L-lysine Arg; L-arginine Trp; L-tryptophan Asp; L-amebalagic acid Tyr; L-tyrosine Bocit-butyloxycarbonyl Aoc;
o-Chlorobenzyl Aquincarbonyl Bzli Pencil Bzl-Ce2: 2. 6-)9 rt IJ he/
SirvTO5; tosyl friend OE'l; ethyl ester 0Bzl; benzyl enether 0NPip-nitrophenyl ester 0PAC; phenacyl ester TFA i) lifluoroacetic acid Tos OH; p-1' luenesulfuric acid'Et3
N; triethylamine NMM; N-methylmorpholine TBA; t-butylamine DCHA; dicyclohexylamine NaOHi sodium hydroxide THF i tetrahydrofuran DMF i dimethylformamide DMSO; dimethyl nulphoxyto ether; diethyl ether DCC; N,N'-dicyclohexylcarbosiimide WSC; N-ethyl, N'-3-dimethylaminopropyl-1-HOBt;/-hydroxybenzotriazole PF (
) iPF means a protected amino acid or peptide fragment, and the numbers in parentheses indicate the order of the amino acids in formula [E]. Next, production examples of the present invention will be specifically explained with reference to the following examples. In addition, thin layer chromatography (TLC) used in the examples
), the carrier and developing solvent system, and the conditions for amino acid analysis are as follows. <TLC>Support; silicage) vG; developing solvent system; /, chloroform-methanol-acetic acid (9J':5:3
), λ chloroform-methanol-acetic acid (g 3 : /
! ;:5), 3 Chloroform-methanol-vinegar mAg0:2S=2
), Hiroshi Chloroform-echnol-acid ethyl (5:2:
S), left hexane-ethyl acetate (/: /) carrier; cellulose (manufactured by Merck & Co., DC-Alufolien) developing solvent system; B butanol-pyridine-acid-resistant-water (!; : 3
:0, /://) Upper layer <Amino acid analysis> Unless otherwise specified, samples were prepared with N-hydrochloric acid at 710°C, 2 glut.
Hydrolyzed in a sealed tube for g hours. Example / (Nlc, Nla, Tyr)-b-PTH(/
-3g) Production of NH2 1) P F (34'); Boc-Tyr
(Bzl-CI2)-N)I2[1] Boa-Tyr(Bzl-C12) OH32,g'
Dissolve l t (0,/2M) and P-nitrophenol/Otori tcO, 72M) in dry THF, and add DCC21a, 7Otr ((1)
, /JM) was added dropwise, and the mixture was stirred overnight. After the reaction, the precipitate was removed, the furnace solution was saturated with NH3 gas, and the mixture was stirred for S hours. A precipitate was formed, which was dissolved by adding DMF and then concentrated under reduced pressure. The residue was crystallized from ether, taken in a furnace and dried to obtain the desired product [1]. Yield i4'4', 77F (yield IIA 9%) Melting point, 2/
'l~2/Otsu℃ TLC; Rf1=0. Otsu 2 Elemental analysis [as C21H2404N2C12] 6%
■(% N% Calculated value 374I!/Yu S/Otsu 3g measured value!;7
．． lI-, 2! ;, 37 Otsu S/[α expression-yo 3/1°
CC=/, DMF)2) PF (333'l);
Boa-Asn-Tyr (Bzl-C12)
-NH2(2) Compound (1), l! 4j Otsu? (Q m M ) was dissolved in t methylene chloride, and to this was added TFA/QQ ml under water cooling, followed by stirring at room temperature for 30 minutes. After the reaction, methylene chloride and TFA are removed under reduced pressure! The residue was crystallized with ether, then taken and dried. Obtained crystal, B
oa-Ash -OH/ 3.93 V (OtsuQ o+
M ) and HOBvg/s' (Otsu Q m M ) were dissolved in DMF+, and W SC was cooled at -/3°C.
/0.9 g ml (Q+uM) was added and stirred overnight. After the reaction, the precipitate was collected by PF, washed with S% sodium bicarbonate solution (7 times), water (2 times), and methanol (7 times), and dried to obtain crystals of the target product [2]. Obtained. 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, 2 and Og V (Yield g O2S%) Melting point: 2≠0~2t℃ [α] Ren -2≠066 (C=ha DMF) Elemental analysis (as C25H3006N4CI2) C% N%
N% Calculated value S≠25'J: 4Z Otsu 10/3j Kawasada f direct
3 g, 1% Otsuta 3 g 10.3/3) PF
(323'l); Boa-His(Tos)-A
sn-Tyr(Bzl-CI2)-NH2(a) Compound (2)22. /lit (40mM) in a small amount of methylene chloride, and add 100ml of TFk to this under water cooling.
After stirring at room temperature for 30 minutes, T F'A was distilled off under reduced pressure to obtain a BoC-free product. On the other hand, Boc-His(Tos)-OH-DCHA,
2g3f'(4'g +n M) with ethyl acetate
The mixture was made cloudy, washed twice with /N sulfuric acid and twice with water, dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off under reduced pressure. The residue was dissolved in dry DMF and added with the previously mentioned dry DMF solution of the deboa compound and HOBL4,4.
g″i (Vg m M ), then 1/3-
'Cold Prostitution) Lower W S Cg, 7 g ml ('l
g m M ) was added, and the whole solution was stirred at room temperature. After the reaction, FDMF was distilled off under reduced pressure and the washing residue was washed with S% sodium bicarbonate water 7 times and with water twice, and then
After drying, a crude product was obtained. 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 = 2g, g/f/<yield g5/%) Melting point: 770~
773℃ TLC, Ytf3=0 Otsuza, 0. /l 2 part Tos
was obtained by desorption. Elemental analysis (C3B) (as 4409N7C128)C
% N% N% Calculated value 33.9 jλll //, 3F measured value 3
; 3.03 Yuoto S /ノ0≠4) PF (3/
34I HBLIG-Vat-His-Asn-T
yr(Bzl-C12)-NH2(4) Compound C3'J
2g, g/liter C311,0tm M) was dissolved in a small amount of methylene chloride, and 4.20m of TFA was added to this under water cooling.
After stirring at room temperature for 30 minutes, '1'FA was distilled off under reduced pressure. Ether was added to the residue, and the crystals taken out by JNOT were dried on a furnace plate and then dissolved in DMF/GEQmt. This solution was neutralized with NMM and added with BOC-Val-OH.
do/17y<37g7' m M) and HOBt
Dry 30 otr (37, Gua m M)
Add the solution dissolved in Otsu Qml, and then cool at -15°C wscg, g Otsume (37,4' 7 Ill
M) was added and stirred at room temperature overnight. After reaction, D under reduced pressure
MF was distilled off, and the residue was washed 7 times with 5% sodium bicarbonate solution and 3 times with water, and dried to obtain the target product [4]. Yield: 2774g (yield/03..2%) Melting point: TLC; Rf'3 = 0! ; εα piece - 2g, 3g' (C=7.DMF) elemental analysis [
C36■ (as 4608C12) C% N% N% / Calculated value!;'A7!; , 5:g7 /≠ Pulling measurement value 5337 Left 7g /3.1tt35) PF (3
03'l); Boc-Asp(OBzl)-Va
l-Hl a-Aan-Tyr (Bz l -C12
) -NJ(2(5)/Qml) was added, and after stirring at room temperature for 30 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, the precipitated crystals were dried on a furnace plate, and then dissolved in DMF/20ml. This solution was neutralized by adding NMM/Qml, and then the Boa-
Asp(OBz I) -0H/2.3;? (
3 g, A 7 m M ) and I (OBt 5.;
a.2? Dry (3 g Otsu 7 m M) D M F
Add the solution dissolved in gQ ml, then -7,5'℃
After cooling, 70 gml (3 g, 7 mmol) of WSC was added thereto, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was diluted with S% sodium bicarbonate solution.
After washing twice with water, it was dissolved in methanol. Yield: J/, +, 2S' (yield: 2%) Melting point: 2/4
1-co/3°C TLC; Rf3 -0. Otsu [Formula 6-23,2gEC=/, DMF) Elemental analysis C
As C47H57011N9C12]C% N% N
% Calculated value 3t, 711 yo7g /Noot7 Measured value 5ot/7 Tough9 /Su07 6) PF (,2ter311); Boa-Gln-A
sp(OBzl)-Val-His-Asn-Tyr(
Bzl-C12)-NH2[6] Compound C5]3/, //g (3/, 27 mM) was suspended in methylene chloride with S and added with TFA/20mM under water cooling.
After stirring at room temperature for 30 minutes, TFA and methylene chloride were distilled off under reduced pressure. After adding residual ether and drying the precipitated crystals on a furnace plate, dry DMF /QQml
It was dissolved. Neutralize this solution with N M M g me and add Boc-Gin-ONP/, 2 Otl/-y to this.
(3g 17m M) and HOBL 01ll J
? (3,/3 m M) in DMF 100 m
Add the solution dissolved in 1 ml of N M M 3 and then add the solution dissolved in N M
．． After adding 7 g rd - night stir 41! did. After the reaction, DMF was distilled off under reduced pressure, and the residue was washed seven times with S% sodium bicarbonate water and twice with water, suspended in Meknol, and recrystallized from ether to obtain the desired product [6]. Yield j33. /91 (yield 9', ', 3%) melting point,
g/~g3°C TLC, Rr3=0. l17 [αZoD14-r3. qgo< C-/,, DMF
) Elemental analysis CC52H65013Nl As 1c12] 0% I (% N% Calculated value SS Otsu/Left g3 /3.7261 Fixed value 3
-≠g/5? Otsu /307 amino acid analysis; As p
2. / 9 (2), G l 11 /, 0-!
; ('), Val/(+), Tyr O, 73 (11
, His Q, g 3 (1)y) PF (,272
g); Bobney Lys(Z-CI)-Leu-O
Et (7) Boc-Lys (Z-CI) -0I-I・TBA9'
7 Otsu 7 (0,2M') as ethyl acid! The mixture was washed with /N hydrochloric acid and water in that order, dried over anhydrous nitric oxide for 1 year, and then concentrated under reduced pressure to obtain an oil. Dry this
r1(F3oome+: dissolve, add H-Leu −
OEl, ・HCI 3L? , l11”/ (0,,2
M) and 11. OB+, ,,! 70f<0.2M
> and then cooled to -73-℃.
After adding (Q, 2M), the mixture was stirred overnight in a vacuum chamber. After the reaction, TI (F) was distilled off under reduced pressure. The residue was dissolved in ethyl acetate, washed with S% sodium bicarbonate solution, water/N hydrochloric acid, and water in this order, and dried over anhydrous nitrate. After that, it was concentrated under reduced pressure. The residue was left in a cool place to crystallize. Hexane was added and the mixture was evaporated to obtain a roasted product [7]. Yield: /10 Otsu22 (Yield 79S%) ) Melting point, 77~go℃ TLC, R15=0.l1g [α]X-/90g"CC=/, DMF)e) PF
(,2 Otsu-2g); Boa-Lys (Z-CI)-
Lys(Z-CI)-Leu-OEt [8] Compound [
7]/10 Otsu29(0,/)9M) in methylene chloride30
After adding TF A 2 SQ ml to this under water cooling, the mixture was stirred at room temperature for 7 hours. After reaction, TF under reduced pressure
A, methylene chloride was distilled off to obtain an oily de-Boc compound. On the other hand, Boc-Lyg(Z-CI)-0HSTBA'i
i'7/l<0.799M) acid resistance) v300 m
The suspension was washed with 300 ml of /N hydrochloric acid and then water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oil. This was dissolved in dry THF/3Qml, and added to the above-mentioned eliminated Boc compound and HOBv2 < 0.799M).
was dissolved in 23 Qml of dry THF, and then, while cooling to 1/3°C, 2 pml of WSe (pml<o/qqM) was added dropwise, and the mixture was stirred overnight at room temperature for 4jL. After reaction, THF
When the residue was distilled off under reduced pressure, agar-like crystals were precipitated. This was dissolved in acid-resistant ethyl, washed with S% heavy water, water, /N hydrochloric acid, and water in this order, dried with anhydrous nitrification, and concentrated under reduced pressure. The resulting precipitate was treated with hexane and then collected. This was recrystallized from acid-resistant ethyl, ether, and hexane to obtain the desired product [8]. yield,/! ; Otsu, Lpa (yield 9.22%) melting point; //q
~//Otsu℃ TLCi Rr2=0.7g [α] Next day -20.7.2″<C,=7.DMF )9
)PF(2j”-,2g); Aoc-Arg(To
s)-Lys(Z-CI)-Lys(Z'-CI
)-Lcu-OEl.

[9] Compound (e) / 3; (/gqmM) was added to 30ml of methylene chloride, and added with TFA and 230Q under ice cooling.
ml was added, and the mixture was stirred at room temperature for 7 hours. After concentrating the reaction solution under reduced pressure and dissolving the residue in 300 ml of dry DMF,
Neutralized with NMM. Aoc-Arg(Tos)
-0HJ Or' (-20,2mM): I)
MF10Qm16 solution and HOB+1.
273r (,202mM) and then cooled to -/3-°C. WSC37, Oml (,202mM)
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 Jv/l of acid-resistant ethylene. This solution (dipped in 5% sodium bicarbonate solution twice, saturated saline solution, /N hydrochloric acid solution)
The mixture was washed twice with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Add ether to the residue, heat it in the furnace and turn it into the desired object [
9] was obtained. Yield i 2 / 7.9 / S' (Yield 100%
)7 TLC, Rf, = 0.07, Rf2-0 Surprising melting point i7j
~7g℃ [α] Knee/IAo 2'''(C=/,DMF)+(
1) PF (2'l 2g)HBoa-Leu-Ar
g(Tos)-Lys(Z-CI)-Lys(Z-C
I)-Leu-OEt (+o) Compound (9) 2/7. the law of nature? 100ml of methylene chloride and 13Qml of TFA were added to <0.1g3M), and after stirring at room temperature for several minutes, methylene chloride and TFA were added under reduced pressure.
FA was distilled off. The obtained oil was dried with DMF3QQm.
1 and neutralized by adding NMM. Kono solution Ni Bo
c-Leu-OH-l-I2030. riVCo, 20
M) and HOBt 27? <0.20 g M) dissolved in 100 ml of dry DMF was added and then -/j
WS C37,3 ml (0,2011,
After adding M) dropwise, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was added to water, and the precipitated product was poured into a furnace. The product was recrystallized twice using methanol-ether and hexane to obtain the desired product [10]. Collection kj2/3.1.3? C yield 90.3; %) melting point;
157~/Otsu O℃ TLC, Rf, = 0.2g, Rf2 = 0.77 [α)
2D7 y g Ot g'' (C-HaDMF)++)P F
(23-2g); Boc-Trp-Leu-Arg
(Tos)-Lys(Z-CI)-Lys(Z-C
I)-Leu-OEl (++) Compound (10)/33. / 7gl), /,? M)
methylene chloride 100++Il and TFA23;O
After stirring for several minutes at room temperature, methylene chloride and TFA were distilled off under reduced pressure. Dry the residue in DMF, 2
5 and neutralized to pH 7f with NMM. Add HOB to this solution. gll? (0,132M> and Boc-TrP-OH'IO, /7? (0, /J2M
) and then cooled at -73°C, wsc r 4
4.2ml<0. /32M) was added dropwise, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residual S% weight was poured into 11!1 water St, and the precipitated product was poured into a furnace plate. After suspending this in water and heating it, it was recrystallized twice from mechnol ether to obtain the desired product [11]. Yield: / Guco, J7r (yield g/, 2%) Melting point: 765
~770°C TLC, Rf = 0.3/, Rf2 = 0. I2 [α)%
[l y g, otog' (C=/, DMF) + 2) P
F (23-2g); Boa-Trp-Leu
-Arg(Tos)-Lys(Z-CI)-Lys(Z
-CI)-Leu-OH[12) Compound (If)/'IO,A'It (9A,/
4+11M) Dissolved in hot ethanol w'/200 ml, cooled, and after removing a small amount of precipitate in the furnace, /N-
2 ggm/(3xM) of Na OH aqueous solution was added and stirred at room temperature for 7 hours. To the reaction solution/N-TosOH aqueous solution/9
After adding 2 ml < 2 times M), the mixture was separated from the furnace and ethanol was distilled off under reduced pressure. Condensation liquid KIN-TosOH ml (
After adding M) and then 2 t of water, the resulting precipitate was drained. After washing twice with water and drying, the target object [1]
2] was obtained. Yield;/'12.9g? (Yield/O/, /%)TL
Ci'Rf2 =0.7/ Melting point; 725-730℃ [α)%' -37.2g (C-/, DMF) Elemental analysis (as C69H940111N12SCI2°2H20) 0% H% N% Calculated value S Otsu 33 Otsu, 72 //, 113 measurement value S
Otsu, 03 1. Otsu2 //, g3 amino acid analysis; Leu
j (21, Lys-, 2,0g (21, Arg/, /
lf+1, TrpO,g 31)+s) P F (
233'A); Boc-Trp-Leu-Ar
g(Tos)-Lye(Z-CI)-Lys(Z-C
I)-Leu-Gin-Asp(OBzl)-Val
-His-Asn-Tyr(Bzl-C12)-NH2
[13) Compound (6)/, otsuzaf(/,!; rl1M)
was suspended in a small amount of methylene chloride and then cooled with water.
After adding 7 ml of A, the mixture was stirred at room temperature for 30 minutes. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, and the resulting 477 crystals were dried on a furnace plate. These crystals were dissolved in 30 ml of dry DMF and neutralized with a small amount of NMM. Konosoru W c compound (12), 2.4' 3 V (
/, Otsu3111M), ■(OBt O,22? (
/, ojmM) and 20 ml of dry DMF, then -/! After adding 3 ml of WS000 (1.1 times M) while cooling at i.degree. C., the mixture was stirred at room temperature overnight. After the reaction, DMF under reduced pressure
was distilled off, and the residue was washed 7 times with aqueous sodium bicarbonate solution and 2 times with water, suspended in methanol, added with ether, and dried on a furnace plate to obtain the desired product (3). Yield: 3 Otsu 2r (yield 99.7%) Melting point: 2 Otsu 0-270℃ [σ) 2D5 p Otsu Otsu 0 (C=0.3. DMF
) Elemental analysis (as C++6HueO25N2sSCI2) 6% 8% N% 31 Calculated value j 7. / g OtsuO1l /3.2.2
Measured values st, oo Otsuko Otsu /ノ57 Otsu amino acid analysis; As p /, ri11. (2), GIIIO Riotsu (
1), Va I O, 7/ +11, Leu 2.00
(21, Tyr O, 9g (1), Lys 2.0
? (21, His O, 5g (ll, Ar g
Q.? / (11, Trp O,7g (1) +4) P F (2,2); Boa-Glu(O
Bzl) -0PAC(+4) Boc-Glu(OBzl)-0I(/2g, 29 (
Dissolve 0.33M) in DMF and add phenacyl bromide//3. ! ? (0,57M)
After adding 773ml of Et3N (0.37M
> was dropped. After dropping, it was stirred at 30°C for 1 hour, and then potassium acetate was added at 30°C. After stirring for 45 minutes, DMF was distilled off under reduced pressure. QQ ml of acid-resistant ethyl Otsu was added to the residue, washed once with S% sodium bicarbonate solution and twice with water, and the ethyl acetate layer was dried over anhydrous nitric oxide, and the solvent was distilled off under reduced pressure to precipitate crystals. Add hexane to this? I counted the number of houses and got the objective [+4]. Yield j/36/ri2 (yield 702%) TLC, Rf5 = 0.73 +a) PF (+2/J, 2); Boa-Val-Gl
u(OBzl)-0PAC[15] Compound (14)/Ge7. gg? <0.32. ! ;M)
30 ml of methylene chloride was added to the solution, and the mixture was cooled with water.
After adding 30 Qml of A and stirring at room temperature for 7 hours, methylene chloride and TFA were distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were dried several times. The crystals were dissolved in 300 ml of dry DMF and neutralized to pH 7 with NMM. Add this solution to ll0BE3/'I
f<0.26M) and Boa-Veil-OH3A,
'19 r (0,24M) Processing, -/,, t℃
After cooling, 6 ml of WSC guar (Q)6M) was added dropwise thereto, and the mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in chloroform and dissolved in S%
Washing was performed in the following order: sodium bicarbonate solution, water, /N hydrochloric acid, and water. The chloroform layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and hexane was added to the resulting crystals, heated, and then ethyl acetate was added.
The desired product [15] was obtained by recrystallization from ether. Yield, 10 liters 79 (yield 7 lA2%) TLC, Rf
3 = 0 Otsu 3 Melting point; 739-1117°C [α) %9y, 72° (C = /, DMF) + 6)
PF'(20-+2.2); Ape-Arg(To
s)-Val-Glu(OBzl)-0PAC(+e)
Compound [+5] 9. g3 f (0.1 gM) was processed into 50 ml of methane chloride, and added with TFA2 under ice cooling.
00ml methylene and TFA were distilled off. The residue was treated with hexane, the hexane was removed by a decanting method, and ether was added and treated, and the ether was distilled off under reduced pressure. The obtained oil was dissolved in 20Qml of dry DMF and neutralized with NMM. Add HOBt 21 to this solution
A33t ((7,/gM), Aoc-Arg(Tos
)-0H7Otsu0fCO,7g'M) and dry DM1
Add 200ml and add WSC3 under cooling at -/S℃.
After dropping 2.9 g+nl (Q,/g M), the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure and the remaining residue was dissolved in ethyl iv1 acid/l. This solution is
% sodium bicarbonate solution, water, /N hydrochloric acid, and water in this order, and dried over anhydrous sodium sulfate, acid-resistant ethyl ester was distilled off under reduced pressure. The obtained oil was crystallized from acid-resistant ethyl ether, and the obtained crystals were dissolved in ether! The process of calculating the number of objects in the cloud was repeated three times to obtain the target item 6]. Yield 1/≠975f (yield 9≠6%) Ti, c; Rrt = 0.7'l, RI'4 = (7
/Melting point; /10~//II℃ [α)2k//,! ;°(C=/,DMF)'7)
P F (/9 22); Boc-Glu (OB
zl)-Arg(Tos)-Val-Glu(OBz
l)-0P AC [17] Compound (+6)/&7110? <0.170M)? Add 3Qml of methylene chloride, and add TFk30Q under ice cooling.
After stirring at room temperature for 7 hours, methylene chloride and TEA were distilled off under reduced pressure. After working up the residue with ether and distilling off the ether under reduced pressure, the oil obtained was dissolved in 200 ml of dry DMF. This is HOBv 2 left 27? Co/g7M) and Boc-C
1u (OBzl)-OH Otsu 3092 (0, / g 7
M), add 10Qml of dry DMF, -/3
While cooling at °C, WSC3p, 22ml CO, ig7M) was added, and the mixture was stirred at room temperature overnight. After the reaction, the solvent was distilled off, and the residue was poured into a glass of water.
1] was obtained. The solvent was distilled off from the crystal mother surface and crystallized from mechnol-ether to obtain the desired product (2s, OJr. Yield: /ge/llgugu (yield 7o7%) TLC, Rr,
=0. ! ;t, R "4 = 0.g, 2 melting point; //ri~/
2/℃〔α〕ゎ −/kota″′ (C=ha DMF)18)
PF (/g-22); Boc-Nle-Glu (
OBzl)-Arg(Tt+5)-Va 1-Glu(
OBzl )-OPAC[+8] Compound (17) 6. 217 ml of methylene chloride and TFA were added to the mixture under water cooling, and the mixture was stirred at room temperature for 110 minutes, and then methylene chloride and TFA were distilled off under reduced pressure. The residue was crystallized by adding ether and dried. The crystals were soaked in dry DMF and neutralized to 11 H7 with NMM under ice cooling. Add B++c to this solution.
-Nl e -OH/, Otsu 7? (7,,7+nM
) and HOB tO 7′ (7,,,2mM)
Dry DMF 110 ml +: Add the dissolved solution and cool to -15°C WS C/, 3 ml (7,2 + n
After adding M), the mixture was stirred overnight. After reaction, D under reduced pressure
MF was distilled off, water was added to the residue, and the resulting precipitate was filtered and washed with S% sodium bicarbonate solution, water (3 times), /N hydrochloric acid solution (3 times), and methanol in this order. Next, reprecipitation was performed from methanol-ether to obtain the desired product 08]. Yield; j otsu/f (yield 7 g%) TLCi Rr, -0,3t +9) PF (/za-, 22); Boc-NJ e
-Gln (OBzl)-Arg(Tos)-Val-
Glu(OBzl)-0H (193 compound ha) 503t (1A2mM) in acetic acid 30ml
Melt t, add 1g of zinc powder to it, and stir at room temperature.
The mixture was stirred for a certain period of time. After the reaction, the zinc powder was removed from the furnace, and the acetic acid was distilled off under reduced pressure. Add ether to the precipitated crystals? F' was removed to obtain the target product [I9]. Yield: 1Al12f TLCiRf+ =0.1g, Rr2 =07Arg
Q, Ta 4♂3° (wealth), Vnl/(l120) P
F (/g34') HBoc-Nle-Gl
u(OBzl)-Arg(Tos)-val-Glu(
OBzl))-Trp-Leu-Arg(Tos)-
Lys(Z-CI)-Lys(Z-CI)-L
e u-Gl n-A3 P (OBz 1)-Va
1-His-Asn-Tyr(Bzl-Cl2)-N
H2 (20) Compound [13) f, Rir (J, 5mM) with 0 Nucatol
3f (3.3mM), dimethy/l'7. /L/
phyto, l 3 ml. Ethanedithiol +2. ! ; ml and TFA,!
, and stirred at 0°C for 70 minutes and at room temperature for 175 minutes. After the reaction, the reaction mixture was concentrated under reduced pressure. Add ether to the residue, remove the resulting precipitate, dry it, and add 100 ml of dry DMF.
=Dissolved and neutralized to 1) H7 with NMM. Add HOB L O, 3'l f (II m
M ) and compound [+5)tA3 S' (II m
M ) and -/! ;Ck cooling WSC0.73m
After adding 1, the mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, S% sodium bicarbonate solution was added to the residue, and the resulting precipitate was filtered and thoroughly washed with water. This product was dissolved in ethanol, and ether was added to precipitate it twice to obtain the desired product [20]. Yield, //, /2fC yield 9t%) TLC, Rf3=o, 7"s Melting, temperature 230°C (decomposition) [α]ゎ-g73"<C=0.3; 3, DMF) Amino acids Analysis; Asp/, 9 g (21, Glu
3. O'l (3), Val/, Otori (2), Leu
,2(2),Tyr/,07(11,Lys/ri3(2),His0.37(1),Arg/ri7(2)
, TrpO, Jj (1), Nl e /, 07 (l
121) PF (/7); Boc-8er (Bz
l)-0PAC [21] BoC-3er (Bzl)-01 (glj otr(o, J
Dissolve M) in 17 ml of phenacyl chloride (01!5M), add 1.3N ml of phenacyl chloride (01!5M), and cool with water. ;M)
was added dropwise, and the mixture was stirred at 30°C for 3.3 hours. Next, add potassium acetate to this reaction solution 2.2. / ? (0,22
! ;M) was added, and the mixture was stirred at room temperature for 7 hours. After the reaction, TDMF was distilled off under reduced pressure, and the residue was dissolved in 500 ml of ethyl acetate, and washed successively with S% sodium bicarbonate water and water. After drying the ethyl acetate layer with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The mixture was left in a refrigerator to crystallize, and hexane was added thereto to obtain the desired product [21]. Yield j/22.979<yield 997%) TLC, Rf5
=0. g2 [α) 1−//, gg” (, C=/, 0.DMF)
Melting point; Gu! ;-17℃ 22) PF (/Otsu-/7); Boa-As
n-3cr(Bzl)-0PAC1:22) Compound (21)//9. Add 30 ml of methylene chloride to the solution (f<0.29M), and add 2 ml of TFA under ice-cooling. ;O
ml was added, and the mixture was stirred at room temperature for 7 hours. After the reaction, it was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were collected on a furnace plate.
Dry. The crystals were dissolved in 41 OOml of dry DMF and neutralized to pH 7 with NMM. There are also 3 HOBs in this solution.
/3Sri<o,, :z32M) and Boc-Asn-
OH33, g g? (023,jM) was added to this and WSC Genol ml (0,23
2M') was added dropwise, and the mixture was stirred overnight at room temperature. After the reaction,
DMF was distilled off under reduced pressure, and the residue was acid-resistant f/l/50
0 ml E (ii) and j% sodium bicarbonate water were added. During liquid separation, crystals were precipitated, so the crystals were collected with tP and washed with water, and then with ether to obtain crystals of the target product [22]. 1+/, 7% V was obtained.The ethyl acetate layer of the filtrate was concentrated under reduced pressure, and the oily residue was crystallized from ethyl acetate-ether to obtain crystals of the target product [22].
I got it. Yield: Gudo 071 (Yield: 3-2%) TLC〼Rf2=otsuy, Rf4=t), t,;z Melting point: /79~/7℃ [α), -1311@cc=7. 0. DMF)7 Mi/
Acid analysis; Asp /, ;t, :a (+1,
Scr /, Oo (1123) PF (15-/7)
; Old +c-Lcu-Asn-3er (Bzl)-OP
AC [23] Compound [22) g o / l (0153M) k: 4
Methylene chloride j; Qmi was added, and then TFA/! was added under water cooling.
After adding Oml, the mixture was stirred at room temperature for 7 hours. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and the resulting oil was separated by a decanting method, followed by dry DMF/FF. ml and neutralized to 1) H7 with NMM. Add HOBtJ, 2.72 (o/Otsuza M), Boc-Le to this solution.
u -OH-T-T, Oμ/ 99/ /l / /
Add P RJ 'I J+ 1- and DMFlooml and cool to -/3℃ w 5C30,7ml (0,
/AKM) was dropped into the chamber/Mte.The stirred reaction solution turned into a gel, so it was left to stand in an ice chamber for 30 minutes, and then water was added.
The resulting precipitate was plated in a furnace, washed in the order of S% sodium bicarbonate solution and water, and dried to obtain the desired product [23]. Yield', gg, scoV (yield. 3%) TLC, Rf2
=0. gOSRf,=0. gg melting point i/92~/93
°C Elemental analysis (C33H4409N4 L, T: IC%
N% N% Calculated value Otsu/g Otsu 692 g, 73 Measured value Otsu i, gi 'y, os g, s Otsu24) P
F (/'l-/7); Boa-His(
Tos) -Leu-Asn-8er(Bzl)-0
PAC [2 compound [2a) g7,! ;! ;ri(0
,/37M) t,=100 ml of methylene tetrachloride was added, and then 2°Q ml of TFA was added under water 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, and the resulting precipitate was poured onto a furnace plate. The solution was dissolved in MTi'p-l and neutralized to pH 7 with NMM to obtain a deboaming solution. On the other hand, Boa-His(Tos)-OH-DCHAg, 2V (0, /';/M> to 1 acid concentration f) v/AIC suspension 7'15L, /N sulfuric acid! ; Washed with OOml, and the precipitated crystals were separated by furnace. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained oil was dried with DMF/! ; Solution dissolved in Oml and HOBL20
,ll? CO, 131M) was removed from the Boc-depleted solution r(i-
Add ttr- to this and add WSC27 m under cooling to -75℃.
1 (Q/3/M) was added dropwise, and the mixture was stirred at room temperature for 3 days. After the reaction, the solution was distilled off under reduced pressure, the residue was added to water, and the resulting precipitate was filtered, washed with S% sodium bicarbonate solution and water in that order, and dried to obtain the target product [24]. Ta. Collection speed; 10g Otsu Jr (llmataon (2g Yu/%) T LC
, RI'2 =0.20.075TR+3=0. ! ;J
, 0. I7 was obtained from which some TO8 was removed. Melting point -, 1 sll~/S ℃ [α] 275-1g, sg" (C=10.DMF) 25
) PF (/3/7) ; Boc-Ly
s(Z-CI)-Hi5-Lcu-Asn-8cr(
Bz I)-0P AC [25] Compound (24) / 07.9 Otsu? <0. //Otsu M), add 10Qml of methylene chloride, then add TFA2 under water cooling.
After adding 00 ml, the mixture was stirred at room temperature for 70 minutes. The stock solution was then concentrated under reduced pressure, ether was added to the residue, the resulting precipitate was filtered, dried, and then dissolved in 200 ml of dry DMF.
A pH 7K neutralized and de-BoC solution was obtained with MM. On the other hand, Boc-Lys(Z-CI)-OH-TBA
H1 acid x 4-) v Otsu00
The suspension was washed with /N hydrochloric acid and then water, and the ethyl K1 acid layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting oil and HOBt I7.30f? <0.7
2 gM) was dissolved in 7 ml of dry
In addition to the BoC-free solution described in i+, WSC211-11,2mi (0.72 gM) was added dropwise thereto while cooling at -73°C, and the mixture was stirred at room temperature overnight. After the reaction, the iW medium was distilled off under reduced pressure, and the residue was added to 3% sodium bicarbonate solution St. The precipitated crystals were thoroughly washed with water and then dried. The crystals were dissolved in methanol Q and ether was added to precipitate them. The obtained precipitate was suspended in ethyl acetate and the steps of removing P were repeated three times to obtain the desired product [25]. Yield, //I1. G21 (yield 7g%) TLC;
R[2=0.34', R+3=0. Agf'JIB
Point; 200--20-2℃ [α]', 8-2g9
p"<c'=/, o, DMF)26) PF (I3
/7); Boc-Lys(Z-CI)-Hi5
-Lcu-As++-3cr (Bz I)-0H [
26] Compound (25) g Om, OW (g Om M)
acetic acid so. Melt ml, is this Q-zinc powder/30? After stirring at room temperature for S hours, the reaction ak was filtered to remove zinc dust. The reaction p solution was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were heated in a furnace to obtain the target product (26). Yield: g≠702 (yield 9j2%) TLC: Rf2=□17 Melting point i 2110~230℃ [a]%0-i 9 i O<c= /, o, DMF
) Elemental analysis (C45H52012N9CI・, ICH3
As COOH・2H20] 0% N% N% Calculated value I3.7 Otsu to Otsu3 //, 3.2 measured value,
52 and 3 63 Otsu //3s amino acid analysis; As'p
/, 0 / (ll, Ser O, g 3 ('l
, Lcu / (+1, Lys 0.93 (ll, H
isori7(1)27) P F (/ 3 3 'l
) 逼Boc-Lys(Z-CI)-His-Lcu
-Ash-8er (Bzl) -Nle -Glu (OB
ZI)-Arg(Tos)-Val-Glu(OBzl
)-Trl+-Lcu-Arg(Tos)-Lys
(Z-CI)-Lys(Z-CI)-Leu-Gl++
-Asp(OBz l) -Va l-Hl 5-A
sh-Tyr (Bz I -CI2)-NH2[27
) Compound (zoo/ 0.77 f (3,2111M
) and stirred at 0° C. for 70 minutes and at room temperature for 40 minutes, and then the reaction solution was concentrated under reduced pressure. Add ether to the residue, dry the resulting precipitate several times, and then add dry DMF/
QQmlp: After dissolving and neutralizing to pH 7 with NMM,
Stir overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure.
Water was added to the residue, and the resulting precipitate was separated, washed with water, and dried to obtain the desired product [27]. Yield: /3Of (yield 100%) Melting point: /3g~/Otsu0. j3; ℃ [α), -/, 9 Otsu0<c=o, s Otsu, DMF
) Amino acid analysis HAsp, 2.9 Otsu (3), 5erO Otsu 2 (1), Glu 3.02 (31, Val /,
72 (21, Le u 3 (3), Tyr Otsu 0 Otsu (1), Lys 3.0 / (31, His /4
1!3 (21, Arg/, 9 g (2), Trp
Q Otsu 0 (1), l'JIe10 Otsu (1) 28) P F
(/ / −/ 2 ); Boa-Lcu-Gl
y -0Bzl (2B) Boc-Leu-OH・H2O4499? (20mM
) was dissolved in 30 ml of dry THF, 3 Q ml of dry benzene was added thereto, and the solvent was azeotropically distilled off. The resulting oil was dissolved in 70 ml of dry THF and added with H-Gly-OBzl-TosOH (+20mM
) and HOBt. After the reaction, the solvent was distilled off under reduced pressure,
After dissolving the residue with ff1 acid ethyl)vloOmlf, /
It was washed twice with N-hydrochloric acid, twice with 5% sodium bicarbonate solution, and once with water. The ethyl acetate/I layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain an oily target product [2B]. 29) PF (/0-/2); Boa-
Asn-Leu -Gly = OBzl [29] The oily substance [28] obtained in nIS was cooled to 1/3°C≠3
9N hydrogen chloride/dioxane solution 'l (f, 'l Oml
was added, stirred for 0 minutes, and then concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was dried several times and then dissolved in 30 ml of dry DMF. This is Q-t
Under cooling, add Ev 3N to adjust pH to 7, then HOBtO, 3? (,2,2mM) and Boa-Asn-ONP 777S' (22mM
) and stirred at room temperature for 3 days. Water was added to the reaction solution, and the precipitate was extracted with 200% chloroform. The chloroform paste was washed with /N hydrochloric acid, S% sodium bicarbonate solution, and water in this order, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The residue was crystallized from ethyl acetate-hexane to obtain the desired product [29]
Yield: J', Or (yield 730g%) Melting point: /! ;2 /33 Otsu [α) 2; 3 Otsu, /″CC=/, 0.DMF)3o)
P F (9/2) HBoa-Hi 5-As
n-Leu-Gly-OBzl (ao) Compound [29) 7J (/J: 5mM) 3ml of methylene chloride was added, then 32ml of TFA was added under water cooling, and the mixture was stirred at room temperature for 60 minutes. Concentrate the reaction solution under reduced pressure,
Ether was added to the residue, the precipitate was dried on a furnace plate, dissolved in dry DMF/10 ml, and dissolved in N.
A Boa-free solution was obtained by adjusting the pH to 7 with MM.
77 tc/g (M) was added with 1 acid w/30 ml and 0.3 N sulfuric acid Qml and shaken, and the ethyl 41-ate layer was washed three times with water and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain an oil. This oil and HO
Dissolve B L2.3''/ (/J', AmM) in dry DMF, add the solution to the BoC-free solution described in nIJ, and then add WSC3/1.ml while cooling to -/J°C.
/gmM) was added, and the mixture was stirred overnight at room temperature. After the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, washed three times with S% sodium bicarbonate solution 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 filtered. This crystal is His To
Although s has been partially eliminated, in order to completely eliminate it, dissolve this crystal in DMF/QQml and add 1(OBt
705r was added and 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% deuterium chloride water and then with water, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. Add ether to the precipitated crystals?
The number of houses and the object [30] are 1 fathom. Yield near, 32? C yield 7≠g%) TLC collection R12,=0. / 3+) PF (g-/2); Boa-Nle-Hi
s-Asn-Lau-Gly-OBz l (31)
3 ml of methylene chloride was added to Compound (so)7JJ t (//mM), and then 3 Q ml of TFA was added under ice cooling, and the mixture was stirred at room temperature for several minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and the precipitate was collected.
After drying, the solution was diluted with 9 DMF/lQ ml and adjusted to pH 7 with NMM. This is HOB/. 9' (/3.92 mM) and Boc-
Add a solution of Nl e -OH3゜23 (I3゜2 + 11M) in dry DMF, and cool to -75°C with WS.
After adding 3 ml (73,92 mM) of C,1, 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, and the precipitate was collected and washed twice with S% water, twice with /N hydrochloric acid, and three times with water. , (I2 dried target [31]
I got it. Yield: 3.70'/(yield'l-29e6
) TLCHRr2=o2゜32) P F (g −/ 2 ); Boc-N
le-Hi 5-Asn-Leu-Gly-OH [3
2) Compound (a+)+2g? (3, g m M) in 100 ml of ethanol, 1 m/0% Pd
/C300mf/ was added, and hydrogen gas was passed through at room temperature for 3 hours. Insoluble matter precipitated in the reaction solution, so it was filtered and DMF
After washing, the furnace solution was concentrated under reduced pressure. Add ethanol ether to the residue and dry the precipitate for several times to obtain the desired product [3].
2] was obtained. Yield: /7? (Yield 7/, 7%) Melting point, //2.3; °C TLC, Rf2-0.03 Amino acid analysis; As p Q, 9 Otsu (1), Gl,
y 0.9 g (11, Leu / (+), His
Ori 5 (1), Nle O, 94t (+1aa) pp
'(g-3'l); Boc -Nl e-Hi
5-Asn-Le u -GI y-Lys (Z
-CI)-His-Lcu-Asn-8e
r(Bzl)-Nle-Glu(OBzl)-Arg(
Tos)-Val-Glu(OBzl)-TrII-L
cu-Arg(Tos)-Lys(Z-CI)-Lys
(Z-CI)-Leu-Gln-Asp(OBzl
) -Va 1-His-Asn-Tyr(Bzl-
CI2)-NH2[33] Compound (27) / 0 Oy (2,3-mM) with 1--tv0.33? (+2.!;mM)
, Jime 4-ni '71V7 light 23m1. xli7shif
Add OJv, 2.3 ml and TF, ljml,
After stirring at 0° C. for 70 minutes and at room temperature for 50 minutes, the reaction surface was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was dried several times, then dissolved in 100 ml of dry DMF, and the pH was adjusted to 7t by adding NMM under water cooling. Is this solution HOBto3? (,2,7+nM)
Oyobi compound [32)/, 71. t (271TI
M) and cooled to −/3°C.
After adding 3 ml, the mixture was stirred at room temperature overnight. The precipitate was collected in a furnace, washed with water, dried, and reprecipitated from ethanol-ether to obtain the desired product [33]. Yield: 10 1 lr (yield 7.7%) 1 t contact point: /g O5~/otsuno ℃ [α] -/, 9〆 (C=0.32.DMF) Amino acid analysis; Asp3. g 7 (4), 5ar0,7 Otsu (1), Gl u 3.3 'l (3), GIyO,
77 (11, Va I /, g'l (21, Le
u 'l (4), Tyr /, 04' (ll,
Ly s 3.2 g (3), His 2.37 (
al, Arg, 2. / 'l (21, Trp O
, 73 (1), N I e2, 07 (21 Boa-Lcu-OH-H2O/ 310? (4o
+nM) and phenacyl bromide/7ri7 (woQ m
Melt M) in 100mlkC of DMF and add 26ml of ice to this.
T' E t 3 N72.3m1CriQ m M)
was added dropwise, and the mixture was stirred at 30°C for 2 hours. Next, acid-resistant potassium≠112f/C113; mM) was added, and after stirring at room temperature for 13 minutes, DMF was distilled off under reduced pressure. Dissolve the residue in ethyl ffI acid, wash twice with 5% aqueous sodium bicarbonate and twice with water, and dry the ethyl acid layer with anhydrous sodium sulfate.
Reduced pressure] - The solvent was distilled off. The residue was left in an ice chamber and the precipitated crystals were dried to obtain the desired product [34]. Yield j, 2/, 23tC yield 100%) TLC, R1'
,=0. g9 35) PF (Otsu-7); Boa-Gln-L
eu -OP AC[35) Compound (34),! 0.9 A f/ (1 GlnM
) (C chloride) f-ren, :1Qml was added, and then, under ice-cooling, TFAgQml was added, and 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 dried several times, then dissolved in 70 ml of dry DMF, and adjusted to 11 H7 by adding NMM while cooling with water. This solution tr-HOBtdo/2 (OtQ m M ) and Boc-Gln-OH/lA7gf' (Ot Om M
) dissolved in 90 ml of dry iDMF, add -/
, cooled to ℃ in the evening WSC/ 0.9 ml
After adding m M ) dropwise, 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, and then washed twice with S% sodium bicarbonate solution, twice with /N hydrochloric acid, and three times with water. The acid-resistant ethyl layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Hexane was added to the precipitated crystals, and the mixture was dried in a furnace to obtain the desired product [35]. Yield j/723? <Yield 60%) TLCiRf, -0,3g 3s) P F (37); Boa-I 1c-G
ln-Lcu-OP AC(36) Compound 1j5) / 7. /7flc36m M)
Add methylene chloride/Qml to the solution, then add TFA7 under ice cooling.
After adding Qml and stirring at room temperature for 60 minutes, the reaction solution was concentrated under reduced pressure. After drying the residue under reduced pressure, dry DMF/30
1 ml of the solution was dissolved, and the pH was adjusted to 7 with NMM while cooling with water. Add HOB L to this solution.
and Boc-11cmOH-%H20ta3f/C39
Add a solution of 1 ml M) dissolved in 7 ml of dry DMF, and cool to -75°C with WS C7.2 ml (3 ml).
After adding M) dropwise, the mixture was stirred at room temperature overnight. After the reaction,
DMF was distilled off under reduced pressure, t S% sodium bicarbonate solution was added to the residue, the resulting precipitate was filtered, and then diluted twice with S% sodium bicarbonate solution and /N hydrochloric acid.
It was washed three times with water and dried. This precipitate was reprecipitated from ethanol-ether to obtain the desired product [36]. Yield, / Otsu 3S2 (Yield 7 Otsuri%) TLC; Rr, = 0. 'l/, Rf'2=0otza37)
P F (4'-7); Boc-Glu(OBz
l) -11cmGln-Leu-OPACC37)
Compound (36)/Otsu24r (27.5mM) was added to methylene chloride/Qml, and then TFA 7 was added under ice cooling.
Add Q ml, then add 7 Q ml of TFA under ice cooling,
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 removed and dried, then dissolved in ml of dry DMF/QQ, and then NMM was added under water cooling to adjust the pH to 7. Is this solution IkoH, OBtgO? (30,25n+M) OJ:HiBoa-Glu(OBzl) -OH/ 0.2
A solution of f (30,2,3mM) dissolved in 3Qml of dry DMF was added, and 3ml of WSC was added dropwise to the -/S'C while cooling, followed by stirring at room temperature overnight. After reaction, D
MF was distilled off under reduced pressure, S% aqueous sodium bicarbonate was added to the residue, and the resulting precipitate was collected, washed twice with 5% aqueous sodium bicarbonate, /N hydrochloric acid, and nine times with water, and dried. The desired product [37] was obtained by reprecipitation from ehnol-ether. Collection fi! , 2/, AgS'C yield 97/%) TLC;
Rfl = 0.3-2 s8) PF (3-7); Bo c-8er (
Bz I )-Glu(OBzl)-11e-Gln-
Leu -0PAC [38] Compound (37) 2 /, 4'Otsu VC263+n M
) Methylene chloride/Qml was added, and then 90ml of TFA was added under water cooling, and after stirring at room temperature for 7 o'clock, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was oven dried, then dissolved in dry DMF/30 ml, and then adjusted to pH 7 by adding NMM while cooling with water. HOB t 3.9 in this solution? (,2L?/3
Add a solution of m M/jmM) dissolved in 30 ml of 1% MF, -/! ; Cf under cooling WSC3.3ml<2
9/! ;mM) and then stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, 3% aqueous sodium bicarbonate was added to the residue, and the precipitate was collected. This was washed twice with S% sodium bicarbonate solution, /N salt, and acid, and nine times with water, and then suspended in ether and plated in an oven to obtain the desired product [38]. Yield (jk, 2≠gy<yield 911-.7%) TLC;
Rr, = 0.33 39) P'F (2-7); Boa - Val
-8er(Bzl)-Glu(OBzl)-11e
-Gln-Lcu-OPAC[39) Compound (as) 2'1. A g S'(2!;
+nM) 20M of methylene dichloride was added, then TFA/QQml was added under ice cooling, 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,
After drying the resulting precipitate several times, drying D M F
Dissolve in 820 ml, then add NMM under water cooling to 1
Adjusted to 1 H7. This solution is HOBclAO! ;
? (30mM) Oyobi Boa-Val-OH1y, 3
A solution of f (J(1) mM) dissolved in dry DMFgOml was added, and WSC was performed while cooling to -15°C! After dropping 3ml (3Q+M), the mixture was stirred at room temperature overnight. A precipitate was deposited in the reaction solution, so water was added thereto, washed twice with S% sodium bicarbonate solution, twice with /N hydrochloric acid, and once with water, suspended in ether, and washed on the furnace plate. The target product [89] was obtained. Yield; Boc-8cr (Bzl 4g%)
)-Val-3er(Bzl)-Glu(OBzl)-
11e-Gln-Leu-OPAC (40) Compound (39)-7A,07? (211 mM) was added with 2Q ml of methychloride, and then treated with TF under ice-cooling.
After adding 100ml of A, 4! The mixture was stirred for 0 minutes. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and the resulting precipitate was dried on a furnace plate, then dissolved in 10 ml of dry DMF, and the pH was adjusted to 7 by adding NMM under water cooling. This solution KHOB t 3.9 ft' (2g, g
) and Boc-8er(Bzl)-OHg,
! ;? (2 g, gm M) in dry DMF! ;Om
Add the solution dissolved in l and -/! ; ℃c玲tdl下W
After adding 3 ml (21, g m M) of SC&, it was stirred overnight at room temperature. A precipitate was deposited in the reaction solution, so why not add water? After removing F' and washing in the order of S% sodium bicarbonate solution, /N hydrochloric acid, and water, the steps of suspending in ether and heating were performed twice to obtain the target product [4o]. yield;,? Boa-8er (B
zl )-Val-8er(Bzl)-Glu(OBz
l )-11e-Gln-Leu-OH (41) Compound (40) / 2 O' (/On+M) was dissolved in 300 ml of acetic acid, zinc powder/S2 was added thereto, and the mixture was stirred at 50°C for 7 hours. The zinc powder was separated by furnace. Acetic acid was distilled off under reduced pressure, ether was added to the residue, and the precipitated crystals were washed on a furnace plate to obtain the desired product [4]. Yield i//, 7! ; f (yield 7.4t%
) Melting point; 2g0°C (decomposed) TLC, Rf, , = 0.7'I% Rf2 = 0. t'1
Amino acid analysis HSer Otsuza/(2), Glu, 2. oj
(21, Val O, 93; (+l, Leu/(11
, IIeo, 9,2 (l142) protection f1-(Nle, N
je, Tyr) -h-PTH(/-3'l)
NH2; Boa-'Ser(Bzl)-Va
1-8er(Bzl)-Glu(OBzl)-Ile
-Gin-Leu-Nl e-Hi 5-Asn-Le
u-Gly-Lys(Z-CI)-I(is-Leu
-Asn-8er(Bzl)-Nlc-Glu(OBz
l)-Arg(Tos)-Val-Glu(OBzl)
-Trp-Leu-Arg(Tos) -Lys(Z
-CI) -Ly5(Z-CI)-Leu-Gl
n-Asp(OBzl)-Val-His-Asn-T
yr(Bz l-CI2) NH2(42) compound-
(3g) / 0, Zao' (2,2g m M) with 7 Kato tvO, 30? <2.2 gmM), dimethyl soufflefide 2j; m11 ethanedithio-tv
, :l, j; ml and T F A 2 ! ;Ill
was added, stirred at room temperature for 40 minutes, and then concentrated under reduced pressure. Needle powder was added to the residue, and the resulting precipitate was dried on a furnace plate. A mixed solution of 100 ml of dry DMF + 10 ml of DMSO was dissolved therein, and then NMM was added under water cooling to adjust the pH to 7k. Add HOB t O, 37t (
2,7′I m M ) and compound [41] 3.7
'iZs' (J, 74'mM) was added, then −/3
W S C0,30 m' (2,7 II m
After adding M), the mixture was stirred at room temperature overnight. Reaction, reaction solution t
This water was added, and the resulting precipitate was poured into a furnace, thoroughly washed with water, and then washed with Fnofrey ether to obtain the desired product [42]. Yield, I2. g7f<Yield 973%) Melting point: I39.3-/7. ! i℃ [α) r t, free (C=0.!;/, DMF) Amino acid analysis i Asp 3.72 (4), 5erJ7
Otsu (3), Glu 35 g (51, GI7Q, Otsu 7
(1), Va I 2. g l (3), 11e /
, / / (11,Leu,!; (5),Tyr
Q cotton (1), Lysλza7 (3), Hisu, /ri (
3), Ar g , 2. Q Otsu (2), TrpO1 Otsu 5
(1), Nle/Riot (2) 43) (Nle, Nle, Tyr)-h-P
TH (/-, 71%) NH2 Compound C42], 2.9? (0.3mM) to K”
ClC7?7 Rejected 7: 7--Jv3. ! ;m1. :
r-Cundithio-/L 10.35 ml. 3.3 ml dimethylnulfite and 35 ml water HF
1 and borrowed for O minutes. After the reaction, HF was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected. /N Dissolved in acid-resistant. The force of this solution (3,3X/2ty)
n) and only the ninhydrin-positive fraction was collected and lyophilized to obtain a crude product/g7v. This was dissolved in 30 ml of O/N1II'l acid and charged onto a CM-cellulose column (:l x 33 cnI), 0.0,! ; M acid resistant ammonium (pH/)
Elution was performed using a linear concentration gradient of 1/L-0≠M ammonium citrate (pH 60). Fractionate Qml of each fraction, TLC & Co. R16) Ko, 30
('l) The 7th group fraction with the 1st spot was collected and freeze-dried.
．． /N hydrochloric acid, and add this solution to Cef7 Desox G-2.
! ; column (JX/15cm) and charge it to 0. /
It was eluted with Nm acid. Each fraction was UV, 2g□nm
CN1e, CN1e,
Nle, Tyr'] -1+ -P T H(
/ 3≠) NH2 was obtained. Yield, /'lomV TLC, Rrs = 0.30 Amino acid analysis (hydrolyzed with N-hydrochloric acid containing 3% thioglycolic acid) Asp3g (4), Set,! , /
0 (3), GIu4473 (5), Gly O,97
(1), Val,! 6Og(3), 11eOg7(ll,
Lcu j OO(51, Tyl' /, / / (
1+, Ly83.2 Otsu (3), His2.30 (3),
Arg, :l, 03 (21, Trp□Otsu, 2 (
+), N1e2.22 (2) High-speed hedral chroma 1-graphy; Column; Nuclcosil 5 CH (11 mm
IDX/50 m m) Buffer io, /M phosphoric acid containing O acid containing 07 acids, 1 acid nitrile (the ratio of acetonitrile is 20% for the first S minute)
, for the next 20 minutes there is a linear concentration gradient from 20% to 0%) Flow rate: /ml/min detection r22! ;nm Measurement result; Yakonomi peak detected at /9.07 minutes. Patent Applicant Toyo Jozo Co., Ltd. Representative Takashi 1) Tetsuo Procedural Amendment 1992/D 22nd January Manabu Shiga, Commissioner of the Patent Office/, Case Indication/-311) NH2 3. Address of the person making the amendment 632 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture / ≠ "Dianomethyl" specification cylinder 35, page 1, line 2 of the statement of interest for the amendment order
"Methanol-Etherhexane" in Shinyu lines 1-20
is corrected to "methanol-ether-hexane". "Elemental analysis..." on page 39, lines 70 to 3 of the specification
・Delete "7.27 Otsu". Correct GlnJ to j'-Glu in the specification No. GS Shinyuuto. ``Ethanol ether'' in Specification No. 60 Shinyu 4'-, S I''f is corrected to ``ethanol-ether.''

Claims (1)

[Scope of Claims] /), a peptide represented by the formula %, a salt thereof, or an I-labeled radioactive substance thereof.