JPS6034992A - Peptide derivative and determination of enzyme c1-esterase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application Human blood contains an active enzyme known as 01-esterase proenzyme, which is activated by the binding of antibodies and antigens. Activated by C1-esterase. This enzyme is activated by the complement system in a cascade to other active pro-enzymes. These activated enzymes lyse cell membranes of bacteria or dead red blood cells and therefore play an important role in immunological defense. Blood serum is C
It inhibits C1-esterase and also contains an important inhibitor called C1-esterase inhibitor. In the case of inflammation, C1-esterase is activated and, depending on the concentration of esterase inhibitors in the blood, the complement system is activated rapidly or slowly. From a clinical standpoint, in such cases, the degree of CI-esterase in Takumi's blood is also C1.
- It is advisable to also measure the esterase inhibitor concentration.

PRIOR ART Currently, this measurement is carried out by immunological and titrimetric methods, which are complicated and have little accuracy (W, J.).

Canady)b co-authored with Hisonosen, “Immunc.
16, pp. 229-233 (19
76): D, OgetOn and his editor, Thro
mb6eiqResearch'', Volume 9, 217-22
See Nito (1976)].

Problems to be Solved by the Invention An object of the present invention is to carry out the measurement of C1-esterase more quickly and accurately.

By the way, it has been found that the above-mentioned problems can be solved when certain simple peptide derivatives, which are the object of the present invention, are used as substrates.

The present invention relates to a dye having the formula: %Formula %) [wherein R1 is capable of being detached by enzymatic hydrolysis to form a colored or fluorescent compound, and is substituted with an aromatic group or a heterocyclic group. 4 represents an amine group, R2 represents hydrogen or a) a linear or branched alkanoyl group having 2 to 6 carbon atoms, b) a cyclohexylcarbonyl group, C) a carbon atom in the alkanoyl 2 to 4 ω-carboxyl-1ω-methoxycarbonyl- or ω-ethoxycarbonyl-alkanoyl groups, d) 1 to 4 carbon atoms in alkoxy? a straight-chain or branched alkylsulfonyl group having θ) an alkylsulfonyl group having 1 or 2 carbon atoms in the alkyl or phenyl- or hap-tolyl-
a sulfonyl group, f) an unsubstituted or mono-substituted benzoyl group, or g) a benzyloxycarbonyl group whose nucleus is unsubstituted or Hrt-substituted, and R3 represents a benzyloxycarbonyl group whose nucleus is 16
represents an unsubstituted or substituted benzyl group; is a benzyl group, )mI 4 enyl group, cyclohexyl group, cyclohexylmethyl group, 4-hydroxybenzyl group, 4-hydroxycyclohexylmethyl M&, and m is a numerical value of zero, and Y
Amino acids defined by: have the L- or D-configuration or R4 represents hydrogen and m represents the numerical value 0.1 or 2] or their derivatives with mineral or organic acids. Regarding salt.

For example, R1 is a p-nitrophenylamino group, 1- or 2
-naphthylamino group, 4-methoxy-2-naphthylamino group, 4-methyl-7-coumarylamino group or 1.3-
It may be a di(methoxycarbonyl)-5-phenylamino group.

For example, R3 is a benzyl group, 4-methylbenzyl group, 4-
It may be a methoxybenzyl group or a 2-16- or 4-chlorobenzyl group.

R2 represents an alkanoyl group having 2 to 6 carbon atoms or an alkoxycarbonyl group having 1 to 4 carbon atoms, Y represents 4 single bonds, R3 represents a cabenzyl group, and Hl and X represent the above. Peptide derivatives have a particular sensitivity to C-neesterase.

Examples of peptide derivatives of the above general formula include the following compounds: 5oa-Lye(t-Cbo)-Gly-Arg-p
NA, AcOH, 2ACOH.

H-Lys (g-Cbo)-Gly-Arg-pNA
, Ac-Lys(e-Cbo)-Gly-Ar
g-pNA, AcOH, CH50CO-Lyg(t-
Cbo)-Gly-Δrg-pNA, AcOH102
15000-Lye(ε-Cbo)-Gly-Arg
-pNA, AcOH, 1so-Butoo-Lye (
ε-cbo)-Gay-Arg-pNA, AcOH,
CH3CH20O-Lys(ε-Cbo)-017-to rp-pNA, to 〇〇〇, C) 1. , (CH2)2C
O-Lye(ε-Cbo)-Gly-Arg-pNA
, A COH, C:H, cl(20cO-OH2C
o-L7FJ (t-Cbo)-Gly Arg-
pNA, AcOH1BOC-Lye(g-Cbo) A
la-Arg-pNA, AcOH, H-Lye(1-
cbo)-8la-Arg-pNA.

20F3C! OOH, AC-Lye(g-Cbo)-A
la-Arg-'pNA,AC! Oh.

CH,000-Lye(ε-C!bo)-Ala-Ar
g-pNA, Δc01(, soc-oly-Lyg(ε
-Cbo)-()ly-Arg-pNA, AcOH, 2
cv, cooH.

H-Gly-Lye(g-Cbo)-Gly-Arg-
pNA, CH, Oo-Co-G17Lys(C!bo
)-Gly-Arg-pNA, AcOH, CH3(!H
2-Co-G1y(g-CbO)-G17-Arg-p
NA, AcOH0 The peptide derivatives according to the invention can be prepared by methods customary in peptide synthesis, for example by the method described below: 1) Pigment-forming group 1 (11,,: attached to the carboxy group of the O-terminal arginine) In this case, the α-amino group of arginine is stabilized by a protective group, for example a carbobenzoxy group or a t-butoxycarbonyl group, and the δ-guanidyl group of arginine is stabilized by protonation, nitration or tosylation, for example by HCJ. The C-terminal group R1 is also useful as a storage group during stepwise formation of the peptide chain.Other conservative groups are used to attach other amino acid derivatives until the desired peptide chain is fully constructed. Therefore, it can be selectively removed as necessary.

Finally, the remaining protective groups can be completely removed without affecting R1 [see, for example, Miklos and co-authors, "Peptide 5ynthesis"]
s”, pp. 163-165 (1966), Intor
Science Publishers Publishing].

2) Initially constructing the peptide chain (Bodenek 7, supra), the C-terminal carbobexyl group of the aldinino is secured by a customary nisfur group, such as a methoxy, ethoxy or benzyloxy group. The ester group can be removed by alkaline hydrolysis,
However, the t-butoxy group must be selectively removed using trifluorohydrogen An (If the δ-guanidyl group of 0alinine is converted into a Dislocation ((shi, its l(9) racemization does not occur. This is followed by bonding of the dye-forming group R1. The δ-guanidine group of arginine becomes a nitro group or a tosyl group.) N-terminal α of
-Amino group is carbobenzoxy group, p-methyl-1p-
When Hφ is protected by a methoxy- or 1-zp-chloropenzyloxycarbonyl group or a monobutoxy group, these protecting groups are also removed at the same time. Elimination can be carried out by treating the washed peptide derivative with anhydrous chloride in a room temperature, during which all of the above amino- or δ-guar; Elimination can be carried out using 2N-HBr in glacial acetic acid if the bound peptide derivative does not contain nitro- or tosyl binding groups.
It can also be carried out by treating at room temperature using

EXAMPLE The following example details the preparation of peptide derivatives according to the invention. The temperature is Mr. Serra's.

Analysis of the eluates and products obtained in the examples was carried out by thin layer chromatography using glass plates (Merck, F254) coated with silicon dioxide. Thin layer chromatograms were developed with the following solvent system: n-butanol/acetic acid/water (3:1:1).

The following abbreviations were used: Ac -acetyl A (j20 niacetanhydride AcOH diacetic acid Ala = L-alanine β-Ala di β-alanine Arg di L-arginine BO (!: t-butoxycarbonyl r-But =
4-aminobutyric acid B = pen1toyl B220-hydrothermal benzoic acid CHA: L-3-cyclohexylalanine (!H(
) = L-2-cyclohexylglycine D-CHG
: D-2-cyclohexylglycine CRT=
L -3-(' 4-Hydroxycyclohexyl)alanine-nuclear hydrogenated tyrosine Cbo nicarbopenzoxy DMF dimethylformamide DPA: 5-amide-isophthalic acid-dimethyl ester D8C= fll Ir4 chromatodaram or one tuffi Fit - Ethyl gto = ethoxy+! ! t3N Nitriethylamine Gly = Glycine HMPTA = N , N , N', N', N'', N
”-hexylmethyl phosphoric acid triamide 1so-BuO-imbutoxy LMS: solvent system L7. == L-lysine MCA=7-amido-4-methylcoumarin MeO-methoxy MeOH: methanol NA-naphthylamide 0T) NP: p- Nitrophenoxy pNA == p-nitroanilide Ph' Gly = L-2-phenylglycine ph
e = L-phenylalanine D-PhO-D-phenylalanine Suenisuccinyl THF-tetrahydrofuran To θ = p-toluenesulfonyl Unless otherwise specified, amino acids are in the L form.

Example 1 cbo-Arg-OH,H (J 1.6.0 ,!i' in anhydrous) IMPTA 9 Q mA, vacuum dried over P2O5 in a three-necked round-bottomed flask with an internal volume of 250 mA.
(47,0 mmol)? Dissolved at 20°C with exclusion of moisture. Initially, K HMPTA j was added to the resulting solution at room temperature.
"t3N 4.74,!i' (47,
0 mmol) solution? , then Kp-nitrophenyl incyanate (100% excess) 16.49 (100 mmol) was added in small portions. After reaction for 24 hours at 20° C.) most of the IMPTA was distilled off in vacuo. residue 2
The combined AcOH extract was extracted several times with 60 AcOH (Sephadex)-G-15, which was then equilibrated with 30 AcOH.
- applied onto the column and eluted with 0-CH ACOH. Both parts of the ACOH eluate, which could be desorbed with the release of p-nitroaniline by treatment with Trizocin, were lyophilized. An amorphous powder of 12.6 & was obtained, which is L in p8aVC.
It was homogeneous in MS.

Elemental analysis and calculations from the empirical formula C2oH2,N605C1 yielded the following convergence value: C=51.29'2(5
1, (57chi), H = 5.48chi (5,42%), N
=17.92% (18.08chi), CI! = 7.50
% (7,63%). The numbers in parentheses are calculated values.

lb, 2HBr, H-krg-pNA Compound 1a4.65. under moisture exclusion. 2 N -HBr in glacial acetic acid (10 mmol) 40 m/! The mixture was stirred for 2045 minutes. At this time, the amino acid derivative is dissolved in the presence of CO2. 250 ml of anhydrous ether was added dropwise to the reaction solution under vigorous stirring. At that time, 2HBr, H-Ar
g-pNA was precipitated. The ether phase is separated by suction filtration, and then the same phase is filtered at 1QQm/! The benzyl bromide formed as a by-product and the excess HBr and AcOH were removed by washing four times with anhydrous ether. Residue f MeOH
Dissolved in 5Qmi, diluted to -14.5 with Et3N and concentrated to dryness in vacuo at 606C. The product so obtained was dissolved in 75 ml of MeOH.
Flow through a "Sephadex" LH-20 (cross-linked Agistrunk 9L) cuff column equilibrated with .

LMS in one half of the eluate I', , ps
4.18 g of amorphous compound 1b (
A theoretical quantity of 91.6 was obtained. The following v value was obtained from elemental analysis and empirical formula 'It3' from 12H20'60KBr2; C261.15% (51,60
%), +(=4.35% (4,42chi), N218.8
4% (18,43%), Br = 54.81% (35
,03,%) 1c, Obo-Gly-Arg-pN
A,) (Br compound 1b4.5.!? (10 ml J mol) kliL,<dissolved in ml of distilled DMF3Q-io', 9 u<tii'IBt under stirring,
N1.4 (E+l 10 mmol) was added. Formed E t 3 N, ) I B r: filtration j! The yL was washed with 1.1 ml of cold DMF. Cζ1 under stirring (
Cbo-G], y-OpNP 3.65
g (11 mmol) and the mixture was allowed to react under water and ih for 2-3 hours. At that time, r of the reaction solution
l! '2If gradually increased to about 20~7. This melt
Cool to 10'U and cut to 10'U.
; 70ml (5ml) buffered. Reaction solution? -10 for about 2 hours and at room temperature for 6 hours. This treatment was repeated once again at t!INO, 70 rui, and after another 16 hours the reaction solution was evaporated to dryness in vacuo at 50 mF. "Sephadex" has become
FIV was obtained by gel filtration on a G-150 column.

The AcOH eluate, which was separated by trypsinization with the release of p-nitroaniline, was concentrated to dryness in vacuo 40'. The residue was dissolved in 150 ml of MeOH and concentrated again to dryness. P2O5
After drying above at 60 °C, 5.85 fl (theoretical amount of 88.6 fl) of an amorphous compound 1c, which was homogeneous in 1.IAS in DSc, was obtained. Elemental analysis and empirical formula: C
20246.66 section (46.65 Buri, II
= 5.04 (4.98%), N = 17.88%
(17,51%) Sword Br=14.20% (
14,11%) Compound 1c4.56. ! i' (8 mmol) was mixed with 32 ml of 2N-HBr and 62 rn/ml of glacial acetic acid while excluding water.
! The mixture was treated at 20 °C for 40 minutes with stirring. At that time,
The peptide derivative gradually dissolved under CO2 evolution. When the reaction solution was heated to 250 mA with anhydrous ether under vigorous stirring, 2HBr and H-Gly-Arg-pNA were precipitated. The ether phase was filtered off with suction and the stamp phase was then washed four times with 1 ooml per portion of dry ether to remove most of the benzyl bromide formed as a by-product and the excess HBr and AcOH. f MeOH5Q
Dissolved in ml. +; After adjusting the pH to 4.5 with t3N,
The solution was condensed to dryness in vacuo at 60°. The residue thus obtained was dissolved in IMθOH5Qm/ and MeOH
Purification was performed on a "Sephadex" LH-200 column equilibrated with. Fractions of the MθOH eluate, which were separated by trypsinization to liberate p-nitroaniline, were concentrated to dryness in air at 30°. Obtained Fuyu hair vacuum drying box P
After drying at 40° above 20δ, LMS medium-
An amorphous compound that is 1. i 3.78,! i' (theoretical i (92.1% of
77%), 11=4.59chi (4.52%), N=19
．． 47% (19,11% > and BT=30.78% (
31, 14).

iQ, BOC! -Lye(E-ChO)-417-At
-g, -pNA, HBr compound 1a 2.5711 (
5 mmol) was dissolved in freshly capped DMF 20" (cooled and stirred to 7 and -10 FK)'; t3N
O,70i14 (5 mmol) was added. Formed F
=t 3 N, 11B r was collected by filtration and cooled (7t
: Washed 7 times with DMF. BoC-L78 (ε-Cbo)-0pNP 2.7 on the search knife at τ-10°
6, S? (5,5 mmol) addition, 'imZ
. The reaction mixture was reacted at 11t1 for 2 to 3 hours while blocking water, and then the temperature of the reaction solution was gradually raised to 20°C. This solution is re-cooled at -11)K.Et3N r3
．． ．． 35 me +: 2.5 mmol) and 11 ml. The reaction solution was allowed to react for 2 hours at -20°C and for 2 hours at room temperature. IGt3N O, 35"'
After 16:00, the reaction solution was concentrated in air at 50° C. to solidify. 50 Chi ACO)
(5Q !II/! Medium+-1 melt and 50%-ΔcO
7-cephadex "G-15" balanced with H fl'
Lamu 12 was 9, and 1 cup was 2; Torinosindokoro J'l! From 4τ, p -- the free descending fraction of troanilino t°1. Ta. Residue's (MoOH
100: nt Medium (c molten l2, next (・de this molten tr
f was re-evaporated to dryness. Iu et al.
1 dry 4"r: After drying at 6o0 over 9q'+ medium P2O5, DSQK:
106.57g of the compound (106.57g of the 89.8th part of the II debate) was collected.

Elemental analysis and Norohe 7 formula' C,,3H48NaO,B
The following numerical value was obtained by calculation of r 7”+・(41:
C=49. Ro 8 section (49,37 capital), H= 5.00
% (6,0', °chi), 11 = 16.06% (1
5,86 chi) and Br=9.85 yen (10,05 chi).

Amino acid analysis showed that the desired amino acid was obtained with the correct F: 01y 1.00: Lye0.99: A>-6
0,97if, Boo-L7n(i Cbo)-Gl
y-Arg-pNA, Ac0Hie4 (B prepared from
oo-L7F+ ((-0bo)-(11y-Arg
-pNA, RBr 7.95! l (10 mmol)
was dissolved in 75 ml of 60% aqueous MsOH. Add this solution to the acetate of 6 amberite "JRA-40"
10 columns. Column 601-aqueous MeOH
When dissolved using ion exchange, Hsr becomes 8L!
Replaced by OH. The eluate was concentrated to dryness in vacuo at 400 ml. After drying at 400 °C on a vacuum drying box pP205, bromide-free HOC-LY (ε-cbo)-Gly
'rg--pNA, AcOH7,58,9 (, Q theory'
97.9% of J) was obtained.

By this method, organic acids such as zeic acid, propionic acid, phosphoric acid, tartaric acid, citric acid, fL acid, benzoic acid, chlorobenzoic acid, tiL@
n, salicylic acid or phthalic acid? can be manufactured. For example, the hydrochloride type "Amberly 1" JRA-401 is used as the ion exchanger, and the above-mentioned ion exchanger is treated with a sumo wrestler and an
Conversion to M-4 OH form followed by 601-aqueous MoO
It can be converted to the desired acid salt form by treatment with a solution of a 1:1 mixture of the desired organic acid and its sodium salt in H.

Example 2 Commercially available (r) Cbo-Arg-MC! A, H (J 13
．． 0,! i' (25-9 mmol) from Example 1bFc in 104 ml of 2N-HBr solution in glacial acetic acid (208
mmol). Dry residue MeOH4Q
Dissolved in Q7 and “Sephadex” LH-20
Purified on 0 column. MeOH separated with release of 4-methyl-7-amino-coumarin by trypsin treatment
Both portions of the eluate were concentrated to dryness in vacuo at 5-00.

The resulting residue was placed in a vacuum drying box at P20. ．． After drying at 40'G above, homogeneous amorphous compound 2b was obtained in LMS at D8cVc. Elemental analysis and empirical formula: 011'1H2
Calculations from 3N5ONBr2 yield the following numbers:
O=39.40% (38,96%), H-4,61
% (4,70%), N = 14.48% (14,2
0%) and Br = 31.90% (32,40%
) 2C, Obo-Gly-Arg-MOA, HBr Compound 2b and 01) O-()17-OpHP 3.65 g
(11 mmol) in freshly distilled DMF 75 mA
added to. After cooling to -10°, fit3 under +yr stirring
N at first 1.40d (10 mmol) then 0.7
0 ml (5 mmol) was added. The mixture was allowed to react first for 6 hours at -10° and then for 4 hours at room temperature with exclusion of moisture. The reaction bath O was cooled again to -10°, and the Kt3N
The mixture was buffered with 0.70% and stirred overnight at 20°C. The reaction mixture was concentrated to dryness in vacuo at 50 °C, followed by IC residue f5
Q % -Act) (dissolved in 2Q Q mA and purified on a "Sephadex" G-150 column. Fraction of the AcOH eluate separated by trypsinization with the liberation of 4-methyl-7-amino-coumarin. 40 minutes in vacuum
It was concentrated to dryness. The obtained residue was stored in a vacuum dry box using P2O.
After drying at 60° on 5C, amorphous compound 2 (! 4.989' (82.5% of theory), which was homogeneous in LMS in D6C) was obtained. Elemental analysis and empirical formula:
The following numerical values were obtained from C26H31N606Br: c=51.48chi (51.75%), H=
5.24% (5.18%),'=1 6.70%
(13,9?i%) and Br = 13.14%
(13,24%)2(1,2HBr, IT-Gly-
Arg-MOA compound 2c 4.83,! i' (
8 mmol) in glacial acetic acid according to Example 1d.
It was deblocked with 32 ml. The crude product was dissolved in 1 ml of MeCm and purified on a column of "Sephadex" LH-2Q. The MθOII eluate was separated by trypsinization to liberate 4-methyl-7-amino-coumarin and concentrated to dryness in vacuo at 60°C. The resulting residue was stored in a vacuum drying box.
After drying over 2O5 at 40[deg.] 4.05 g (92.0% of theory) of amorphous compound 2 a were obtained which was homogeneous in LMS in DSC. Elemental analysis and empirical formula:
Calculations from cloI (26N604Br2 revealed the following numbers: C = 39.02% (39,29 chi), 1 (= 4.78 width (4,76 coefficient), N = 15.
39% (15,27%) and sr = 28.72%
(29,04chi) 2e, Boc-Log-CbO-G1-8r
-MaA, HBr compound 2 a 2.75,! i'
(5 mmol) was added to BOQ-Lye-(ε-
cbo)-〇T)NP 2.769 (5-5 mmol). The obtained crude product was diluted with 50%-AcO
Dissolve in 75ml of H and “Sephadex” G-15
Purified on 0 column.

Both parts of the AcOH eluate, separated by trypsinization with the release of 4-methyl-7-amino-coumarin, were concentrated to dryness in vacuo at 40°. Residue, P2O5 in 1 raw drying box
After drying at 60° above, the amorphous compound 283.419 was homogeneous in LMS in DSC (82.0 of theoretical
h) was obtained. Elemental analysis and empirical formula: C57H,5
Calculations from 1NBOoBr revealed the following numbers: 20 = 53.13chi (53,43%), H = 6.
24% (6,18 chi), N=13.76% (13,47
) and Br m9.45'! = (9,61%)
Amino acid analysis yielded the desired amino acids in the correct ratio: alyl, 00: Lys 1.02: Arg
O,982f, Boa-Lye(ε-0bo)-Gx
y-Arg-MCA, AcOH compound 20B, 32.
9 (10 mmol) was converted into the corresponding acetate salt according to Example 1f. The raw HL7.95,! i' (98.0% of theory) was obtained.

Example 6 Internal volume 1000+n/! Dry in a three-necked round-bottomed flask with C.
bo-Arg-OH0HCi 4.4 B,'7 (
freshly distilled anhydrous DMF 15 Q
mA, and anhydrous THF 300 m/! 20 in a mixture of
It was dissolved in -10 y2t3N10.2 under stirring and water itching solution cooled,! 7 (0.1 mole)?
Added. This mixture was then added with THF 5 for 20 minutes.
Q chloroformic acid isobutyl ester in ml 13.6
59 (0.1 mol) at a reaction temperature of 15°
I prayed that it wouldn't go overboard. Furthermore, 10 at -10 to -5°
After reacting for 20 minutes, the reaction mixture was 20.5 μl of 5-amino-isophthalic acid-dimethyl ester in 75 μl of KDMF.
92 & (0.1 mol) solution in 60 minutes,
At this time, the reaction temperature was always kept below -5°. The reaction mixture was allowed to react for an additional hour at -5°. After that, 2
Stir overnight at 0 and then cool to -15 with Et3N, H
CJ was crystallized.

Filter the formed 1ct3N,iz and add a small amount of cold DM.
After washing with F. The filtrate together with the washing solution in vacuo for 50 min.
It was concentrated to dryness. 50%-AcOH1000TL of the residue
1 and equilibrated with 50% -AcOH.
The 5-amino-
A separated with the liberation of isophthalic acid-dimethyl ester
Both parts of the cOH eluate were concentrated to dryness in vacuo at 40°. After drying the residue at 50° over P2O3 in a vacuum drying box, homogeneous amorphous compound 3a in LMS in DSC 24.6
fl (45.91 of theory) was obtained. Elemental analysis and calculation from the empirical formula 'C24H3°N 50, %J revealed the following value: CI = 53.211
(53,78%), H=5.71% (5,64chi)
, N=13.20% (13,07chi) and CZ=6.5
Part 2 (6,62%) 3b, 2HBr, H-Arg-DPA Compound 3a21.44. ! ii' (40 mmol) was deblocked according to Example 1b. After work-up, the crude product obtained was purified by dissolving in 250 ml of MeOH and filtering on a "Sephadex" LH-200 column. Both portions of the MeOH eluate, separated by trypsinization to liberate 5-amino-isophthalic acid dimethyl ester, were concentrated to dryness in vacuo. After drying the residue over 40 ml of P2O5 in a vacuum drying box, an amorphous compound 3 bl 9.639 (93.1% of theory 1') homogeneous in DSC and LMS was obtained. Elemental analysis and empirical formula:
C16H25N! Calculations from +058r2 revealed the following numbers: a = 36.82% (36, 45%
), H = 4.67% (4,78 chi), N = 13.45
Chi (13,28%) and Br = 29.85% (30
, 311) Compound 3b 5.27.9 (10 mmol) was prepared as Obo-Gly-OpNP 3.6 according to Example 1C.
517 (11z-remol). The crude product obtained after work-up was diluted with 50% -ACOH20'Om
AcO was dissolved in AcO and purified on a G-150 column. Trypsinization liberated 5-amino-isophthalic acid dimethyl ester.
Both portions of the H eluate were concentrated to dryness in vacuo at 400 °C. Residue?
Amorphous compound 3 Q 5.29 homogeneous in LMS in DSc after drying at 600 min over P2O5 in a vacuum drying box.
P (86.0% of theory) was obtained. The following numerical values were obtained from the elemental analysis and the total nVC from the empirical formula 'c26H3A"6o8"r: c-48.50chi (48,99
h), H = 5.28% (5.22 h), N = 12.9
2% (13,18%) and Br=12.33% (12,
53%) 3t1.2HBr, H-Gly-Arg-D
5.10 g (8 mmol) of PA compound 3c were deblocked with 2N HBr3211V in glacial acetic acid according to Example 1d. After working up the crude product obtained, MeOH 100 m/
and purified on a "Sephadex" LH-200 column. Me separated by trypsinization to liberate 5-amino-isophthalic acid dimethyl ester.
Both portions of the OH eluate were concentrated to dryness in vacuo at 30°. After drying the residue over P2O5 in a vacuum drying box for 40 minutes, the amorphous compound 3d4.25 was homogeneous in LMS in DSC.
# (90.9% of theory) was obtained. Elemental analysis and calculations from the empirical formula "18H2[]N606Br2 revealed the following values: C = 36.85% (37
,00chi), [(= 4.90% (4,83%), N
= 14.72 Chi (14,38%) and Br = 26
．． 95% (27,35%) 3θ, BOC-Lys(ε-
Cbo)-Gly-Arg-DPA, HBr compound 3
a 2.929 (5 mmol) to BO0 according to Example 1θ
-Lys (ε-0bo)-0pNP 2.7611
(5.5 mmol) and the crude product obtained after work-up was dissolved in 50% AcOH 10Q solution and
The fractions of the AcOH eluate separated by trypsinization to liberate 5-aminoisophthalic acid dimethyl ester were concentrated to dryness in vacuo at 40°.The residue was purified on a Sephadex G-150 column. P2O in dry box
After drying over 60 h of the homogeneous amorphous compound 3 e in DEIO in LMEI (theoretical amount of 8
4.1%) was obtained.

Elemental analysis and empirical formula: The following values were obtained by calculation from 037H53"8"□, Br: c=st, os%
(51,33chi), H=6.25% (6,17%),
N2 13.26% (12,94%) and Br-9
, 10chi (9,23chi) From the amino acid branch, the correct ratio of the desired amino acids was found: Gly 1.00 : Lye 1.00 : Arg
o, 973f, Boo-Lys(g-Obo)-Gl
y-Arg-DPA, AcOH compound 688.661
(10 mmol) of Example 1, fK was converted into the corresponding acetate salt.

8.24.9' (97.5% of theory) of product was obtained.

Example 4 Boo-Lys(t-(!bo)-8la-Arg-
2-NA, AcOH4b, 2HBr, H-Arg-2-
NA commercially available cbo-Arg-2-NA,) lci9-4
0 g (20 mmol) in glacial acetic acid according to Example 1b.
It was deblocked with a solution of 8-HBr8Qmi. The immobilized product after work-up was dissolved in 150 m/ml of MθOH and purified on a column of "Sephadex" LH-2Q. Both parts of the MθOH eluate separated by trypsin treatment to liberate 2-naphthylamine? Concentrate to dryness in vacuo at 30°. The residue is a homogeneous amorphous compound in LMS in DSQ after drying at 40° on a vacuum, empty drying box PO5.
b 60 g (93.2% of theory) of B were obtained.

Elemental analysis and empirical formula: 016H23N 50B r 2
The calculations from revealed the following value: c=42.
08% (41,67%), H=5.1296 (5,03
%), N=14.68% (15,19%) and Br=
33.96% (34.65%).

4c, Obo-Ala-Arg-2-NA, HBl-
Compound 4b 4.6 g (10 mmol) was prepared from cbo-Ala-01) NP 3.801/ (1
1 mmol). The crude product obtained after work-up was dissolved in 50%-Acol (1501 nl) and purified on a "Sephadex" G-150 column.

AC separated with release of 2-naphthylamine by trypsin treatment! The fraction of the OH eluate was concentrated to dryness in vacuo at 40°C. After drying the residue at 60° over P2O5 in a vacuum drying box, a homogeneous amorphous compound was obtained in LMS in D8Q Vr.
C4,95,! ? (84.5% of theory) was obtained. Empirical two-element analysis and 027 H33N 604 B
Calculations from r revealed the following numbers: 0=5
5.72 inches (55,39%), H2 6.73 inches (5,6%)
8%), N = 14.68chi (14,35%) and Br
== 13.42% (13,65%).

4d, 2HBr, H-Ala-Arg-2-NA compound 404.6811 (8 mmol) from Example 1dK was deblocked with 28 mA of 2N-HBr in glacial acetic acid. The purified product obtained after work-up was dissolved in 1 ooml of QMeOH and purified on a "Sephadex" LH-200 column. Both aliquots of the MeOH eluate separated by trypsinization to liberate 2-naphthylamine were heated in vacuo for 300 min.
Concentrate to dryness. The residue was dried on P2O5 in a vacuum drying box for 40 minutes.
After drying at °C, an amorphous compound 4d4.08.9 (95.8% of theory) homogeneous in LMS in DSQ Vc was obtained. Elemental analysis and real formula 1"lQ't8N6c2
sr2 Carrano calculations revealed the following numbers: C2 43.9% (42,87%), H=5.32% (5
,30% >, N = 16.02% (15,79%
) and Br==29.68% (60°02chi) 4e, BOC-Lye(ε-Cbo)-Ala-Ar
g-2-NA, HBr compound 4 d 2.66 lI (
Example 1e shows that Boo-Lye (g-Ob
o) -0pNP 2.76 // (5.5 mmol)
I reacted. 50% of the crude product obtained after post-treatment
-AcOH1Q Q mA Medium K Solubility/l'Shikatsu"
The first major fraction of the AcOH eluate was purified by trypsinization to free the 2-naphthylamine. The first major fraction of the AcOH eluate was concentrated to dryness at 40' in air and then dried in vacuo. P2o5 top 60 in dry box
It was dried at 0.

Amorphous compound 4θ homogeneous in LMS in DEIQ
Ro, 45. ? (Theoretical amount of 84.8 h) was obtained.

Elemental analysis and calculations from the empirical formula 38H51N807Br yielded the following sand value: CC505, 88% (
56,08%), H=6.63% (6,56chi), N
= 14.02% (13,77%) and Br=9.80
% (9,82 T) Amino acid analysis revealed the correct ratio of the desired amino acids: Ala 1.00, L71111.02, Arg 0.
974f, Boc-Lys(ε-cbo)-Ala-
Δrp-2NA AcOH compound 408.14.9 (
10 mmol) Two examples were converted from 1fK to the corresponding acetate salts.

7.65.9 (yP theory, best 96.5%) of this product was recovered.

Example 5 Sufficiently dried cbo-krg-OH, HCf 3.4
51 (10 mmol) iiiHMPTA 100rn
It was dissolved under water exclusion during the period of time. After cooling to -10°, Kt
3N1.39+++/! (10 mmol)? Chloroformic acid isobutyl ester 1.35F (10 mmol) dissolved in the solution and then in HMPTA 2 Q ml
The temperature was maintained at -10° to -5° during the 15 minute drop. Thereafter, 71.72 g of 1-naphthi/l/7, (12 mmol) in 15 ml of HMPTA was added to the resulting solution.
was maintained at the above temperature. The reaction mixture was concentrated to dryness at 80 °C in air. The residue was converted to MθOHj[
It was purified by dissolving in 13 ml of water and filtering through a column of "Sephadex" LH-2Q in MeOH. Both parts of the eluate separated by trypsinization with the release of 1-naphthylamine were found to be homogeneous in LMIE in DSQ. Both portions were concentrated to dryness. Amorphous compound 5 a 2.82! ! (60.1% of theory) was obtained.

Elemental analysis and calculation from empirical formula: C24H28N503c1 revealed the following value: CC601,0
7% (61,53%), H=6.101 (6,01 section)
, N=15.05chi (14.9Qchi) and 7.38 to C
H (7,54%) 5b, 2HBr, H-Arg-1-NA compound 5a
9.40.9 (20 mmol) was deblocked with a solution of 2 N-HBr 3 Q u/ in glacial acetic acid according to Example 1b. After treatment), the +1 product was converted to IAeOH.
Dissolve in 150ml and “Sephadex” LH-2
Purified on Q column. Treatment with trypsin liberated 1-naphthylamine. Both portions of the MeOH eluate were concentrated to dryness in vacuo at 60 mL. Place the residue in a vacuum drying box.
,0. After drying at 40° above, the homogeneous amorphous compound 5b8.40 & (theoretical amount of 9
0.8 h) was obtained. Elemental analysis and empirical formula: Calculations from C16■I23N50Br2 revealed the following values: c = 42.2D (41.7S 7
h), H = 5.08 h (5.03%), N = 15
．． 33% (sections 15 and 19) and Bl-=34.10%
(Sections 34 and 65).

Compound 5b 4.6 g (I Q mmol) Example 1
cbo-Ala-OpNP 3.809 (1
1 mmol). The crude product obtained after work-up was dissolved in 50% AcO)l 15 (1 ml) and purified on a Sephadex G-15 column.

Both fractions of ACOH-i, separated by trypsinization to liberate 1-nanothylamine, were concentrated to dryness in vacuo at 40°. After drying at 60° on P2o5 in an air softening box, the residue was homogeneously amorphous in LMS at D8C.
c 4.809 (82.1% of theory) was obtained. By elemental analysis and calculation from the empirical formula = 027H33N604Br, the following values were obtained: 0 = 55.62 coefficient (
55, 9chi), H=6.70% (5,68%), xq
=14.63 coefficient (14,35%) and Bl-=13.3
5% (13,65%).

5a, 2HBr, H-Ala-Arg-4-NA compound 504.68 & (8 mmol) was deblocked with 28 ml of 2N-HBl in glacial acetic acid. Crude product obtained after work-up + 1MeOHIQQ
The 1-naphthylamine was prepared on a column of ``Sephadex'' LI (-20), which was soluble in mA, by trypsin treatment; The residue was dried at 40' on p2o5 in an empty drying box and then dried at L in DSQ.
Amorphous compound 5d was obtained which was homogeneous in MS. Experimental formula:
Elemental analysis and printing of C□, H2RN, 02Bγ2
The following value was obtained: c=43.09% (42-
87%), 5.38% (5,30%) for H, N=
16.10% (15,79%) lit, (J Sr
= 29.80% (30.02%)5e, BO
(!-Lys(t-(!bo)-Ala-Arc(-1
-NA, HBr-1-11 spider. 1-1-2 Compound 5 d2.66,? (5 mmol)e Example 1e
BOQ-Lys (t-0bo)-. pNP
2.769 (5.5 mmol). The post-treated crude product was reduced to 5 Q% -AT! OH
i[]0+++Kon Purified on Kif7ML and "Sephadex" G-150 columns. The first major fraction of the AcOH eluate, separated by trypsinization with the liberation of 1-naphthylamine, was concentrated to dryness at 40' in air and then dried at 600 over P20 in a vacuum drying phase. ,D
homogeneous amorphous compound 5e3 in L+AS at sc(・at
．． 46, P (theoretical cup of 85 chi) was obtained. Elemental analysis and empirical formula: The following numerical value was obtained by calculation using e38H, , N8Q, and Br: c = 55.98% (56
,08ru), I(=6.68φ(6,56%), N=
13.02 (13.77%) and Br=9.80 (
9,82 person in charge.

Amine f'β analysis revealed the correct ratio of theta phase amino acids: Ale 1.00 : Lys 1.01 : Arg
O,975f, BOC-Lye(ε-Oba)-Ala
-Arg-1-NA, +5e8.14g after AcOH combination
10 mmol) was converted into the corresponding a7date column according to Example 1f.

This product7. '77,? (Theory t: 9B, 0%
) is the first one.

14su6 Commercially available Obo-Arg-4-MeO-2-NA, HCj
! i Q,Q ji (20 mmol) was desolocked with 30 TTll of 2N HBr in glacial acetic acid according to Example 1b. The crude product obtained by post-treatment was treated with MeOHi 5
Q rrJ ``#understood and ``Sephadex'' L)
Xrt D" on a 1-200 column. Jl a of 4-methoxy-2-naphthylamine by trypsinization
The main fraction of the M e OH eluate separated below was concentrated to dryness in vacuo at 30°. Place the residue in a vacuum drying box.
After drying at 40 °C on 2O3, an amorphous compound 6b (91.4% of theory) was obtained which was homogeneous in DSC and LMS. Elemental analysis and actual route set: C
By calculating HF from 1 Hpsk"hO2Br2, this change was obtained: 0 = 41.22% (41,57
Palpitation), H-5,19% (5,13chi), II == 1
4.40% (14,26%) and Br = 32.0
1 fr (32,53%) (5c, C!bo-Ala
-Arg-4-MQO-2-NA,! (Dr compound 6
, b 4.91 g (10 mmol) of C according to Example 1C
Reacted with bo-Ala-OpNP 5.809 (11 mmol). After work-up, the crude product obtained was 50
AcOH was purified on a Sephadec® G-150 column dissolved in 50 ml of ΔcOH. The tsushifraction/was concentrated to dryness at 40° in a cellar. After drying at 60° over P2O5 in an empty drying box, the residue was
Uniform bub in MS, 1. (Constant skeletonization 0 substance r6c4.8
6F/C ri'6ii +, 79.0% of i) is 10 ranomata. Elemental analysis and empirical formula: C28H35N60. B
By calculating from r, the following value becomes clear r(became: c
=54. "+8chi C54, 64 section), 14 = 5.8
1% (5,73%), 11=13.93% (16,
65%) and Br2 12.75% < 12.98%)
6d, 21iHr, +(-Ala- to rg-4-MeO
-2-L48 Compound 6e 4.31.9 (7 mmol) was also added to 21 yam-11 in ice 01゛2'' according to 7 Example 1d.
Unblocked with Br2εj ml+. The crude product obtained after work-up was reconstituted in IMeOH 1001 and Sephadex'.

Made F/7 on LH-20 force shims. Trypsinization (formation of 4-methogine-2-naphthylamine) for N14 min.
One contraction was carried out at 0°. The residue was dried on P2O5 in an empty dry phase.
After drying at 0°, 3.74 g (theoretical amount of 95.0 g) of homogeneous amorphous compound 6b in LMB with DSC number τ'd)
did not serve. Elemental analysis and empirical formula: 020H3ON60
The following numerical values became clear from the calculations from 38r2: 0 = 43.01φ (42,72 yen), H = 5.44
%(5,38no), IJ = 15. ．． Section 25 (14,
951) and Br = 28.031 (28-4M>
60, BOC-Lys(ε-○bo)-Ala-Arg
-4-MeO-2-NA, HBI-Compound 6 d 2.
81 g (5 mmol) was added to Boo-Ly according to Example 1θ.
s(ε-Cbo)-0pNP 2.769 (5.5 mmol). The crude product obtained by post-treatment was 50 p-CcW 125 m/! The free 4-methoxy-2-naphthylamine was purified by trizocine treatment on a Sephadex G-15 column. The residue was concentrated to dryness at 40° in a p° air-drying box phase, and after drying at 60° above, L−4 in DSC.
683.319 (78.5 parts of theory) of a homogeneous amorphous compound in S was obtained. Elemental analysis and empirical formula: ['
! A total of 9 from 3gH53NBOBBr gave the following values: c=55.05% (55,51ch), H26
．． 66% (6,57chi), N=1'3.40% (13,
28%) and Br=9.60% (9,47%) 6f, Boo-Lys(g-Cbo)-Ala-Arg
-4-MeO-2-NA, cOH compound 6e, 8.
44. ! i+ (10 mmol) was converted into the corresponding acetate) kM according to Example 1f.

This product 8.05! ! (97.8% of theory bi')
It was done.

The following table lists numerical data regarding the degradability of the peptide derivative according to the present invention by C1-esterase. -T')8
00) Siltyrosine ethyl ester unit (TTEg)
To a mixture of 0.0154 of the solution containing C1-esterase, 2
0.2 ml was added at 67°. Thereafter, the elimination products (e.g., p-nitroaniline, 4-meth□'toxy-2-naphthylamide/or 4-methyl-7-aminocoumarin) generated during the decomposition of the peptide derivative are used for a short period of 5 minutes. The induced optical density ΔOD is narrowed to 405 nm.
◎ Fluorescent elimination products (e.g. 1- or 2-naphthylamine or 1,6-di(methoxycarbonyl)-
In the case of 5-amino-benzene), the optical density addition was determined at the corresponding emission wavelength. From the measured value of the increase in optical density per unit time (2) to the molar extinction coefficient, i7 of the decomposition product formed per unit time is calculated in nanomoles.

Table 1 2.97 2 2.50 3 2.60 4 2.55 5 2.50 6 2.27 7 2.97 85.27 9 7.27 10 6.47 11 5.87 12 5.60 1ro 4.93 14 4.13 15 2,. 57 16 2.67 17 1.00 18 3.67 19 4.13 20 9.50 21 5.90 22 8. tJU 23 9.87 24 7.63 25 6.50 26 2.10 27 2.93 28 2.83 29 3.57 3 [) 4.63 31 '2.67 32 1.87 36 3.67 34 4 .00 35 4.60 36 5.80 37 5.13 38 6.40 39 9.53 40 1.00 41 10.50 42 0.67 43 4.90 44 0.67 45 Ro, 30 46 3.17 47 4.50 48 1. [J0 49 7.27 50 0.83 51 6.70 52 0.67 53 1.00 54 0.50 55 6, Ro0 56 Ro, 10 57 3.17 58 2.83 59 2.33 60 5. 83 61 2.67 62 5.00 63 5.67 The degradability is expressed in nanomoles of degradation products formed by 1 TTic1-esterase per minute.

01-esterase inhibitor concentration in blood plasma 6
111 can be carried out as follows: pH7
, 4. A mixture of 1.6 ml of Trisimida Tor buffer with an ion concentration of 0.2 and 0.1 ml of titrate blood plasma was mixed with 4fJ#c1-esterase o, i y + ε at 67°
Keep at constant temperature for 4 minutes. 2 of the substrate according to the invention to the culture.
X 10-3 moles of water fG 7(+, 0.2 me
was added. When the substrate has a p-nitroanilino group as the chromogen group (R1), the breakdown of the decomposition product p-nitroaniline (R"-H) liberated per minute is 4 []
Measured spectrophotometrically at 5 nm.

The amount of p-nitroaniline liberated per minute is determined as described above in a test system that does not contain blood but has the same composition. Based on the difference between the two measurements, blood plasma is 01
- Measure the esterase inhibitor concentration.

Claims (1)

[Claims] 1. Formula: % Formula %) [In the formula, R1 can be colored by enzymatic hydrolysis or removed under (? a) a linear or branched alkanoyl group having 2 to 6 carbon atoms; b) a cyclohexylcarbonyl group; C) ω-cal having 2 to 4 carbon atoms in the alkanoyl? Xyl-1ω-methoxycarbonyl- or ω-ethoxycar? Nyl-alkanoyl group, d) Straight-chain or branched alkoxycarbonyl group having 1 to 4 carbon atoms in alkoxy, e) 1 to 2 carbon atoms per q in alkyl? f) an alkylsulfonyl group or a phenyl- or p-tolyl-sulfonyl group having an unsubstituted or substituted pen-t
yl group, or g) a benzyloxycarbonyl group whose nucleus is unsubstituted or substituted, R3 represents a benzyl group whose nucleus is monosubstituted or substituted, and X is a glycyl group or represents an alanyl group, and Y is a single bond or has the formula NH-(C!H2)-cH-co- (wherein R4 is a benzyl group, I4 phenyl group, cyclohexyl group, cyclohexylmethyl group, 4- represents a hydroxybenzyl group, a 4-hydroxycyclohexylmethyl group, and m has a value of zero, and the amino acid defined by Y has an L- or D-scratch resistance of 1, or R4 represents hydrogen, and m has a threshold value of 0. .1
or 2) or its salts with mineral acids or organic acids. 2, R1 is p-nitrophenylamino group, 1- or 2-
The derivative according to claim 1, which is a naphthylamino group, a 4-methoxy-2-naphthylamino group, a 4-methyl-7-coumarylamino group, or a 1,3-di(methoxycarbonyl)-5-phenylamino group . 6. The derivative according to claim 1 or 2, wherein R3 is a benzyl group, 4-methylbenzyl group, 4-methoxybenzyl group, or 2-1ro- or 4-chlorobenzyl group. 4. Is R2 an alkanoyl group having 2 to 6 carbon atoms or 1 to 4 carbon atoms in alkoxy? represents an alkoxycarbonyl group, and Y represents a single bond, and R3
represents a benzyl group] Claim 1 or 2
A derivative of JJ Kishu. 5, Boo-Lye (g-cbo)-417-4rg-
pNA, AcO[(. 2AOOH, H-Lye(g-Cbo)-Gly-Ar
(x-pNA, Ac-Lrys(ε-cbo)-Gl
y-Arg-pNA, AcOH, cH3oco-Lys
(ε-Obo)-Gly-Arg-pNA, Ac0HX
C21(5000-Lye(ε-Cbo)-()1y-
Arg-pNA, ocOH, iqo-ButOcO-L
ys(g-Cbo)-Gly-Arg-pNA, AcO
H, cH, cH2co-Lye (g-Obo)-G
ly-Arg-pNA, AcOH, CH3(ljH2)
2CO-Lrys(ε-Cbo)-Gly-Arg-p
NA, AcOH5cH3cH2oco-C)(,,-C
o-Lye (i-cbo)-Gly-Arg-pNA
, Ac0)I. BOC-Lye(ε-Cbo)-Ala-Arg-pN
A, AcOH5H-Lye(ε-Cbo)-Ala-A
rg-pNA, 20F3c! OOHXAc-Lys(g
-Cbo)-Ala-Arg-pNA, AcOH, OH
3000-Lys(ε-Cbo)-Aha-Arg-p
NA, AcOH, 5oe-Gly-Lye (ε-Ob
o)-Gly-Arg-pNA, AcOH, 2CF*C
00H0H-G:ly-Lys(g-cbo)-Gay
-Arg-pNA 10H30-co-Gly-Lye
(ε-Cbo)-Gly-Arg-pNA, AcOH
, CH3-CH2-Co-Gly-Lye (g-Cbo
)-Gly-Arg-pNA, AcOH, according to any one of claims 1 to 4. 6. Enzyme C1-esterase? A medium containing or in which the enzyme is produced or consumed? In the quantitative measurement method, the medium has the formula: % formula %) [wherein R1 is 6U, which is colored by enzymatic hydrolysis or forms a fluorescent compound, and is substituted with an aromatic group or a heterocyclic group. R2 represents hydrogen or a) a straight-chain or branched alkanoyl group having 2 to 6 carbon atoms, b) a cyclohexylcarbonyl group, C) in the alkanoyl group; ω-carboxyl-1ω-methoxycarg with 2 to 4 carbon atoms
-Ethoxycarbonyl-alkanoyl group, d) Straight-chain or branched alkoxycarbonyl group having 1 to 4 carbon atoms in the alkoxy group, θ) 1 or 2 carbon atoms in the argyl group? f) an unsubstituted or 1r1-substituted benzoyl group, or g) a benzyl group whose nucleus is unsubstituted or h′-substituted; represents an oxycarbonyl group, R3 represents a benzyl group whose nucleus is unsubstituted or substituted it, or represents a glycyl group or an alanyl group, Y is a single bond or has the formula NH- (CH2), -C! H-CO- (wherein R4 represents a pencil group, a 4-phenyl group, a cyclohexyl group, a cyclohexylmenal group, a 4-hydroxybenzyl group, a 4-hydroxycyclohexylmethyl group, and m is a numerical value of zero and is defined by Y) Does the amino acid have the L- or D-configuration? Or R4 represents hydrogen and m is the number 0.1
or 2)) and is released per unit time by the catalytic hydrolysis action of the enzyme on the peptide derivative.
p. photometry, spectrophotometry, fluorescence spectrophotometry or i&,
Enzyme C1 characterized by being measured by a vapor chemical method
- Esterase assay.