CA1161432A - Tripeptide derivatives and their application in assaying enzymes - Google Patents

Abstract

ABSTRACT
Tripeptide derivatives having the formula I

wherein H5 is a chromogenic substituted amino group which is capable of being split off by enzymatic hydrolysis to form a coloured or fluorescent split product H - R5. The tripeptide derivatives of formula I are used for assay-ing certain enzymes, more particularly factor Xa. Enzyme-bearing materials are reacted with the said tripeptide deriv?ives. The quantity of split product H - R5 released by th? enzymatic action of the tripeptide derivative is determined photometrically, spectrophotometrically. fluor-escence-spectrophotometrically or electrochemically. The quantity of released split product H - H is proportional to the quantity of enzyme present in the starting materials.

Description

Description TRIPEPTIDE DERIVATIVES AND THEI~ hPPLICATION
IN ASSAYING ENZYMES
.
Technical Field ~he present invention relates to tripeptide de-rivatives which are very easily split by certain enzymes of the enzyrne class 3.4.21., more particularly factor Xa.
Therefore, -the new tripeptide derivatives are intended to be used as substrates ior quantitatively assayin~ the 10 said enzyrnes, more particularly factor Xa.
Back~_ound Art Factor Xa is a proteolytic enzyme which is form-ed in the blood coagulation cascade by activation of the proenzyme f`actor X and which, to~ether with phospholipid 15 and calcium ions, proteolytically splits factor II (pro-thrombin) at two points of the peptide chain and converts the said factor into factor IIa (thrombin) which finally causes coagulation. In certain pathological disorders, e.g. liver diseases, vitamin deficiency, etc., and in the 20 dicoumarol therapy, the formation of factor X is reduced.
In hereditary disturbances in the synthesis of factor X
the formation of factor Xa is, of course, also reduced correspondingly. Therefore, it is important to have at one's disposal a direct enzymatic assay method which al-25 lows factor Xa to be assayed photometrically in bloodplasma in a simple and accurate manner.

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The main methods for assaying factor Xa are the followin~:
a) Biological assay method [cf. "Thrombosis and Bleeding Disorders", Nils U. Bang, Georg Thieme Verlag, p. 196 (1971)]: Factor X is activated to factor Xa by means of venom of Russel viper and calcium ions. In a one-step operation prothrombin is activated to -thrombin by factor Xa in the presence of factor V and phospholipids, and thrombin converts in~icator fibrino~en into fihrin.
10 The clotting time is measured. The required factors II and V and fibrinogen are supplied by a substrate which is free from factor X. The clotting time is influenced by the de-gree of activation of factor X. The activation degree, under otherwise constant conditions, is a function of the ; 15 concentration of factor X in the sample. This biological method allows no more than a rough assay to be carried out since the clotting time is read off subjectively by the ex-perimentator. Furthermore, manipulated plasma is required durin~ the preparation of which mistalces can occur. More-20 over, the fibrinogen which is acting as an indicator is not formed directly, but via activated thrombin (indirect method).
b) Biochemical method [cf. "Thrombosis and Bleed-ing Disorders", N.U. Bang, Georg 'l'hieme Verlag, p. 196/7 25 (1971)]: If the factor X preparations to be tested are sufficiently pure, a more accurate assay method can be ayplied. Esnouf and Williams (1962) have shown ~hat factor Xa has an esterase activity and hence splits a~ino acid esters. However, 50 to 100 ~g of factor X are required for 30 this assay. On the other hand, lower concentrations of fac-tor Xa can be determined by using carboxybenzoxy-phenyl-alaninep-nitrophenyl ester and measuring the quantity of p-nitrophenol released per time unit. This assaying method has the following disadvantages: The said ester undergoes 35 an autohydrolysis at the applied pH of 8 and, moreover, is not specific for factor Xa since it responds also to many other enzymes. The ester is not solu~le in water so that acetone is required. As a result Gf all these disadvant-ages this assaying method is inaccurate and costly.
c) The published German patent application OS No.
25 52 570 discloses tetrapeptide derivatives which are in-tended to be used as substrates for assaying factor Xa.Bz-Ile-Glu-Gly-Arg-pNA.HCl is disclosed as an example of a tetrapeptide derivative which is split by factor Xa with formation of p-nitroaniline. The formation of p-nitroani-line can be followed spectrophotometrically. This method 10 of assayin~ factor Xa is somewhat more accurate than the above-described biological and biochemical assaying methods.
However,the tetrapeptide derivatives described in German patent application DE-OS No. 25 52 570 are not suf-15 ficiently soluble in water to allow -the assay of factor Xa to be carried out at substrate saturation. In the case where extremely low concentrations of factor Xa have to be determined, e g. in pathological plasma, the said tetra-peptide derivatives are not sufficiently sensitive to al-20 low reasonab y accurate measuring values to be obtained.If the quantity of factor Xa to be measured were increased by adding a ~urther quantity of plasma, a precipitation of the tetrapeptide derivative would take place under the in-fluence of plasmaproteins, and as a result it would be im-25 possible to perform the enzyme assay.
Disclosure ot lnvention Now, new tripeptide derivative~ were found which are very easily soluble in aqueous media and which have a surprisingly high sensitivity to factor Xa.
The tripeptide derivatives of the invention have the following general formula O O
Rl _ D - NH - CH - C - N - CH - C - Arg - R5 wherein Rl represents an alkanoyl group which has 2 to 8 carbon 35 atoms and which may Garry an amino group in the ~-posi-~ 2 tion, a phenylalkanoyl group which has 2 to 4 carbon atomsin the alkanoyl radical and the phenyl radical of which may carry an amino group in t~le p-position, a cyclohexyl-c~rbonyl group which may be substituked with an aminome-thyl radical in the 4-position, a benzoyl group which may be substituted with a methyl radical, an amino group or halo~en, e.~. Cl or Br, in the o- or ~-position, an alk-oxycarbonyl group having l to 8 carbon atorns in the alk-oxy group, a benzyloxycarbonyl Irroup which m~y be substi-10 tu~d with methoxy, methyl or chlorine in the p-position, an alkanesulfonyl group having l to 4 carbon atoms, a phe-nylsulfonyl group which may be substituted with methyl in the p-position or an ~- or ~-naphthylsulfonyl group, R2 represents a straight-chained or branched alkyl radical 15 having l to 6 carbon atoms, a hydroxyalkyl radical havin~
l to 2 carbon atoms, an alkoxyalkyl radical ha~ing l to 2 carbon atoms in the alkyl and l to 4 carbon atoms in the alkoxy, a benzoxyalkyl radical having l to 2 carbon atoms in the alkyl, an ~-carboxyalkyl or ~ -alkoxycarbonylalkyl 20 radical which has l to 3 carbon ato~ns in the alkyl and -the alkoxy group of which is straight-chained or branched and has l to 4 carbon atoms, an ~ -benzyloxycarbonyl-alkyl radical having l to 3 carbon atoms in ttle alkyl, or a cyclo-hexyl-, cyclohexylmethyl-, 4-hydroxycyclohexylmethyl-, ~5 phenyl-, benæyl-, 4-hydroxybenzyl- or imidazol-4-yl-methyl radical, R3 represents a straight-chained or branched alkyl radical having l to 4 carbon atoms, R4 represents hydrogen or a methyl or ethyl radical, and 30 R5 represents an amino group which is substituted with aromatic or heterocyclic radicals and which is capable of being split off hydrolytically with formation of a colour-ed or fluorescent compound H - R5.
Preferably, the strongly basic guanidino group of 3~ arginine is stabilized, e.g. by protonation with a mineral acid such as HCl, HBr, H2S04 or H3P04, or an organic acid such as formic, acetic, propionic, lactic, citric, oxalic, Z

tartaric, benzoic, phthalic, tricnloroacetic or trifluoro acetic acid. The nature of protonation has no influence whatsoever on the sensitivity (susceptibility) of the tri~
peptide derivativesto the enzymes.
The removable chromogenic group represented by R5 in formula I can be, e.g., a p-nitroph~nylamino, 1-carb~
oxy-2-nitrophen-5-yl-amino, 1-sulfo-2-nitro-phen- 5-yl-amino, ~-naphthylamino, 4-methoxy-~-naphthylamino, 5-nitro--naphthylamino, quinon-5-yl-amino, 8-nitro-quinon-5-yl-10 amino, 4-methyl-coumar-7-yl-amino or 1,3-di(methoxycarb-onyl)-phen-5-yl-amino group (d~rived from 5-amino~iso-phthalic acid dimethyl ester).
The tripeptide derivatives of formula I can be prepared according to known methods which are briefly 15 described hereinafter:
1) The chromogenic group E~5 is attached to the carboxy group of the C-terminal arginine, whilst its ~-amino group is protected by a protecting group, e.g. a carbobenzoxy or tert.-butoxycarbonyl group, and the ~-20 guanidyl group of arginine is ~rotected by protonation,e.g. with ~C1, or nitration or tosylation. The C-te~minal group R5 serves also as a protective group during the step-wise building up of the peptide chain. The remaining pro-tective ~roups can be removed selectively as needed in 25 order to attach the next ~lino acid derivatives until the desired peptide chain is completely built up. Finally, the remaining protective groups can be entirely removed with-out group R5 bein~ affected (cf. e.g. Miklos Bo~lansky et al., "Peptide Synthesis") Interscience Publishers, p.
30 163-165, 1966).

2) First of all, the peptide chain is built up (according to Bodansky, loc. cit.) whilst the C-terminal carboxyl group of arginine is protected by a usual ester group, e.g. a methoxy, ethoxy or benzyloxy group. The 35 ester groups can be removed by alkaline hydrolysis, except for the tert.-butoxy group which has to be removed select-ively by means of trifluoroacetic acid. If the ~-guanidyl ~ ~i4~
-- 6 ~
group of arginine is protonated, the ester group is re-moved by trypsin, no racemization takin~, place in this case. Thereafter, the chromogenic group R is introduce~.
If the ~-guanidino group of arginine is protected by a nitro or tosyl group and the N-terrninal a-amino group of the tripeptide derivative is protected by a carbobenzoxy group or a p-methyl, p-methoxy or p-chloro-benzyloxycarb-onyl group, or a tert.-butoxy ~roup, all these protective groups are removed simultaneously. The removal can be 10 achieved by treating the protected tripeptide derivative with anhydrous HF at room temperature, and as a result all the above-mentioned amino and ~-guanidino protective groups are removed. The removal can also be carried out by treatment with 2N HBr in glacial acetic acid at room 15 temperature if the protected tripeptide derivative does not contain any nitro or tosyl groups as protective groups.
The tripeptide derivatives of f`ormula I are sub-stantially more readily soluble in water than the tetra-peptide derivatives described in German patent applica-20 tion DE-OS No. 25 52 570 and thus allow the enzyme assays to be carried out at substrate saturation required for ob-taining reliable measuring results. The tripeptide deriva-tives of for~nula I are significantly more sen~,itive than the tetrapeptide derivatives disclosecl in German patent 25 appl;cation No. 25 52 570 and c~n, therefore, also be used ror a3saying extremely low concerltrations of ~actor Xa.
The published Dutch patent apl)lication OS No.
76 07 433 discloses tripeptide derivat;ives having the 30 general formula H - D - Al - A2 ~ A3 N 1' 2 3 amino acid radicals) which have a hlgh sensitivity to certain enzymes of enzyme class ~.C. 3.4.21., e.g. throm-bin and plasmin. This high sensitivity is attributed to the fact that the N-terminal D-amino acid has an unsub-stituted a-anlino group. Indeed, if the a-amino group of the N-terminal D-amino acid is substitilted, i.e. if the hydrogen in the formula is replacesl b~ a protective or blocking group, e.g. by benzo-Jl or carbobenzoxy, the sensitivity of the tri~,eptide deriva~ive to enzymes of enzyme class 3.4.21. is drastical~y reduced. On the grounds of these findings one could have expected that the new tripeptide derivatives of formula I wherein the ~-amino grollp of the N-terminal D-amino clcid carries a pro-tective or blocking group woulcl not or only slightly be split by enzymes of enzyme class 3.4.21. ~owever, contrary 10 to all expectations, the new tripeptide substrates sur~
prisingly have a very high sensl-tivity to factor Xa.
A further advantage of the tripeptide substrates of the invention as compared to the tetrapeptide deriva-tives disclosed in German patent a~)plication D~-OS No.
15 25 52 570 resides in the fact that their synthesis is simpler and less costly.
The tripeptide substrates of the invention can be used f`or ~luantitatively assaying-r certain enzymes of en-zynle class E.C. 3.4.21. (cf. "Enzyrne Nomenclature", El_ 20 sevier Scientific Publishing Company, Amsterdam, 1973, ~. 238 ~f.), e.g. factor Xa in blood plasma, and via fac--tor Xa also for the quantitatlve assay of o~ler biologi-cally im~ortant factors, e.g. factor X (proenzyme o~ fac-tor Xa), antiI`actor Xa ~= antit~lromblrl I:l:l or heparin co-25 factor) or heparin in blood plasma.
~ These various assays can be carried out in themanner described hereinafter:
1) Assay of factor Xa and factor X:
Citrated human plasma is used and first activated 30 with Russel viper venom (RVV = Russel viper venom) in or-der to convert factor X present in the plasma into factor Xa. The following reàgents are used:
Buffer TRIS-imidazole buffer, pH 8.4, ionic strength 0.3 RVV Freeze-dried Russel viper venom preparc,tion (fur-nished by Laboratoire Stago, 92500 Asnières s/Sei-ne, France), dissolved in 2 m~ of` bufrer contain Z

ing CaC12 at a concentration of 0.015 M
Substrate - Cbo-D-Leu-Sar-Arg-pNA.AcO~, dissolved in distilled H20, concentration: 2 x 10 3 M.
~ -test cuvette is charge(l wi-th 0.~ ml of` RVV re-agent heated to 37C and then with 0.02 ml of` human ci-trated plasma. The two components are immediately mixed and then incubated for 60 seconds at 37C. The resulting activaticn mixture is diluted witn 1.6 ml of buffer heat-ed to 37C and mixed with 0.2 ml of 2 x 10 3 M substrate 10 solution. Then~ the change in the optical density of the mixture caused by the released ~)-nitroaniline is rneasured spectrophotometrically at a wave length of 405 nm. The measured increase in the optical density per mirlute is di-rectly proportional to the quantity of factor Xa present 15 in the activated plasma. The measured increase in the op-tical density per minute is also a measure for the quan-tity of factor X present in the starting plasma since as a result of the activation a ~iven quantity of f~ctor X
is converte(l stoichiometrically into the corresponding 20 ~luantity of` factor Xa.
2) Assay of antifactor ~a:
A test sample is prepared by diluting citrated human plasma with TRIS-imidazole buffer at a ratio of l:lO.
10~ ~1 o~ a heparin solutiorl in Ti~IS-imida~ole bufrer (con-25 tainin~ 3 USP units of heparin per ml of buffer) are addedto 100 ~1 of the diluted plastna, an~l thc mixture is in-cubclted f~r 2 minutes at 37C. 100 ~11 of a solution of factor Xa ~reparation "Diagen" (furrlished by Diagnostic ~ea~ents, 'l`hame, Great Britain) containing ~.5 units of 30 factor Xa ~)er ml are added to the incubate. The mixture is incubated for 3 minutes at 37C. The incubate is di-luted with 0.6 ml of TRIS-imida201e buffer heated to 37C
and immediately mixed with 100 ~1 of a 2 x lO 3 M solu-tion of Cbo-D-Leu-Sar-Arg-pNA.AcOH (substrate) in distil-35 led water heated to 37C.

11~14~

In a parallel experiment a blank test sample isprepared in the same manner but us;nr the same volumetric quantity of buffer instead of diluted plasma.
The increase in the optical density at 405 nm is measured spectrophotometrically f`or both samples (test sample and hlank sample). The difference between the in-crease in the optical denslty of the blank sa~.ple per mlnute (~ODblank/min.) and the increase in the optical density of the test sample (AODtest/min.) is a measure 10 for the quantity of factor Xa inhibited by the antifactor Xa-heparin complex and hence a measure for the quantity of antifactor Xa ~- antithrombin III) initially present in the p1.asma.

3) Assay of heparin in blood plasma:
A test sample is prepared by dilutin~ plasma of a heparin-treated patient with TRIS-imidazo]..e buf.fer at a ratio of` 1:10. The diluted plasma is incubated for 1 to 2 minutes at 37C. 100 ~1 of` a so1.ut:ion Or factor Xa pre-parati.on "Diagen" (furnished by Dia~nostic Reagents, Thame, 20 Great Britain) containing 8.5 units of factor Xa per ml are added to 200 ~1 of the incubate, and the mixture is lncubated for 3 minutes at 37C. The lncub~te is diluted with ().~, ml of TRIS-imi.da%ole buf~`er heated to 37C and imnledlately mixed with 100 ~1 o a 2 x 10 3 M solution of 25 Cbo-D-Leu-Sar-Arg-pNA.AcOH (substrate) in distilled water.
In a parallel experiment I ~:Lank test sample is ~)repared in ~he same manner, but usinrr the same (luantity of correspondingly diluted normal plasma ~heparin-free) instead of the diluted hepàrinized plasma.
The increase in the optical density per minute at 405 nm is measured spectrophotometrically for both samples.
The difference between the increase in the optical density of the blank sample per minute (~ODblank/min.) and the in-crease in the optical density of the test sample per min-35 ute (~ODtes~min.) is a measure for the inhibitory activity of the heparin bound to heparin-cofactor (antithrombin III).
J

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-- 10 _ Qntifactor Xa ~= heparin-cofactor or antithromb-in III) alone inhibits factor Xa but slowly so that only small quantities of factor Xa are inhibited within a short incubation period (e.g. 3 minutes). How~er, if antifactor 5 Xa enters into contact with heparin, the two components form a complex which is a rapid inhibitor of factor Xa and which will completely combine with factor Xa within a short incubation time (e.g. 3 minutes).
Since the inhibition of factor Xa is proportional 10 to the quantity of heparin present as lon~ as sufficient antifactor Xa is present the quant;.ty of heparin initial-ly present in the patient's plasma can be determined by means of a calibration curve establi.shed with normal plasma and increasing quantities of added heparin on the erounds 15 of the potentiating action of the added heparin.
In the measurement of the quantity of the coloured split product H - R5 (p-nitroaniline) released in the re-action between factor Xa and the tripeptide derivatives (substrates) of the invention advantage is taken of the 20 fact that the split product has an UV spectrum which dif-fers from that of the substrate and is shifted towards higher wave lengths. The substrates of the invention have an absorption maximum at about 310 nm an(i a molar extinc-tion coefficient of about 13,000. The absorption of the substrates at 405 nm is virtually nil. The split product H ~ ~5, i.e. p-nitroaniline, formed by enzymatic hydro-lysis of the substrate has an absorption maximum at 380 nm and h molar exti.nction coeffici.ent of a~)out 13 ~200. At 405 nm the extinction coefficient is only moderately reduced, 30i.e. to about 10,400.
In the case of substrates wn.ich contain a ~-raph-thylamino, 4-methoxy-~-naphthylamino coumar--7-yl-amino or isophthalylamino group as the chromo~enic rroup the quantity of split produc-t H - ~5 released by factor Xa is 35measured by fluorescence-spectrophotometry. In a test sys-tem comprisin~ factor Xa buffer and substra-te the energy-poorer emitted light is continuously measured at 400 to470 nm af-ter the fluorescent split product has been con-tinuously excited by energy-richer light at 300 to 400 nm.
The quantity of split product formed per time unil; is a measure for the existing factor Xa activity. According to definition 1 ~mole of split product formed per minute cor-responds to 1 enzyme unit of factor Xa, as based on a gi-ven substrate.
The sensitivity of the above-described assayin~
10 methods can be further increased by converting the split product H - R5, before measurement of its quantity, into a more intensely coloured compound by coupling with a dia-zo compolmd in the case where R5 is a p-nitrophenylamino, l-carboxy-2-nitro-phen-5-yl-amino, 1-sulfo-2-nitro-phen-15 5-yl-amino, 5-nitro-~-naphthylamino or 8-nitro-quinon-5-yl-amino group.
Best Mode For Carryin~Out The Inv ntion In the following working exam~les the preparation of -the tri~eptide derivatives of the inven-tion is descri-2~ bed in a detailed manner. Temperatures are indicated in centigrades.
The analyses of the eluates and ~roducts obtained according to the examples were carried out by thin layer chromatography using glass plates coated with silicon di-oxide ~el (Merck, F 254). The thirl layer chromato~rams were develope(1 by means of the solvent system n-butanol/acetic acid/water ~3;1:1).
The following abbreviations are used:

D-l~adi = D-~-amino-adipic acid Ac = acetyl Ac20 = acetic anhydride AcOH = acetic acid Ala = L-alanine D-Ala = ~-alanine AOA = D-~-amino-octanoic acid Arg = L-arginine D-Asp = D-aspartic acid BOC = tert.-butoxycarbonyl Bu - butyl But = L-2-aminobutyric acid D-But = D-2-aminobutyric acid Bz = benzoyl ~zl = benzyl Bz~0 = benzoic anhydride ChA = quinonyl amide D-CHA = D-3-cyclohexylalanine D-CHG = D-2-cyclohexylglycine D-CHT - D-3-(4-hydroxycyclohexyl)-alanine = tyrosine substituted in the nucleus Cbo = carbobenzoxy DMF = dimethylformamide DPA = dimethyl ester of 5-amido-iso-phthalic acid Et = ethyl Et3N - triethylamine Gly = glycine D-Glu ~ D-glutamic acid D-His = D-histidine HMPTA = N,N,N',N',N",N"-hexamethyl-phosphoric acid triamide D-Ile ~ D-isoleucine D-Leu = D-leucine MCh = 7-amido-4-methylcoumarin MeO = methoxy MeOH = methanol NA = naphthylamide D-Nleu = D-norlellcine D-Nval = D-norvaline OtBu = tert.-butoxy OpNP = p-nitrophenoxy pNA = p-nitroanilide l~ Pr = propyl D-Ph'Gly= D-~_phenylglycine ~-Phe = D-phenylalanine Sar = sarkosine = N-methylglycine D-Ser = D-serine SS = solvent system TFA = tri.fluoroacetic acid THF = tetrahydrofurall TLC = thin layer chrorrl.lto~r.l~)hy (or thin layer chrom~.togram) D-Thr = D-threonine Tos ~ p-toluenesulfonyl D-Tyr = D-tyrosine D-Val = D-valine If not ot.herwise stated, the arnino aci.ds in the peptide chains have the L-form.

Examyle 1 Cbo-D-Leu-Gly-Ar~-pNA.H~r la. Cbo-Arg-pNA.HCl In a 250 ml three-necked flask 16.0 g ~47 mM) of 5 Cbo-Arg-OH.HCl, which had been dried in vacuo over P205, were dissolved in 90 ml of abs. HMPTA at 20 in the ab-sence of humidity. To the resulting solution there was added at room temperature first a solution of 4.74 g (47 mM) of Et3N in 10 ml of HMPTA and then portionwise 16.4 g 10 (100 mM) of p-nitrophenyl isocyanate (100% excess). After a reaction time of 24 hours at 20 the major portion of HM*TA was removed by distillation in vacuo. The residue was extracted several times with 30yo AcOH. The residue was discarded. The combined AcOH extracts were further 15 purified by passing them through a column of "Sephadex G-15" (Trade Mark) equilibrated with 30~lo AcOH and eluted wi~h 305~ AcOH. The fraction of the AcOH eluate which was split by treatment with trypsin with release of p-ni-troariline was freeze-dried. There were thus obtained 12.6 20 g of an amorphous powder which was homogeneous in the SS
as shown by TLC. Elementary analysis and calculation from the empirical formula C20H25N605Cl gave the following va-lues: C = 51.2g% t51-67%). H = 5.48% (5.42~), N = 17.92%
(18.08~7), Cl = 7.5~ (7.63~). The values within brackets 25 have been calculated.
lb. 2HBr.H~Ar~-pNA

4.65 g (10 mM) of compound la were treated, while stirring, with 40 ml of 2N HBr in glacial acetic acid for 45 min. at 20 in the absence of moisture. The amino acid 30 derivative dissolved with C02 evolution. The reaction so-lution was added dropwise wlth vigorous stlrring to 250 ml of absolute ether. This resulted in the precipitation of` 2HBr.H-Arg~pNA. The ethereal phase was sucked off, whereupon the solid phase was washed 4 times with portions 35 of 100 ml of abs. ether in order to remove benzyl bromide which had formed as a by~product as well as excess HBr and AcOH. The residue was dissolved in 50 ml of MeOH, the pH

Z

was adjusted to 4.5 by the addition of Et3N, and the solu-tion was concentrated to dryness in vacuo at 3C. The re-sulting product was dissolved in 75 rnl oî MeOH and passed through a column of "Sephadex" Ll~-20 (cross-linked dextran

5 gel) equilibrated with MeOH. From a fraction of the eluate there were obtained 4.18 g (91.6qo of` the theory) of amor-phous compound lb which was homogeneous in the SS as shown by TLC. Elementary analysls and calculation from the em-pirical formula C12H25~N603Br2 gave the following values:
10 C = 31.15% (31.60%), H = 4.35~ (4.42a,b) ~ N = 18.84~ (18.43 and Br = 34.81% (35.03~).
lc. Cbo-Gly-A~-pNA.HBr 4.56 g (10 mM) of compound lb were dissolved in 30 ml of freshly distilled DMF, and the solution was cooled 15 to -10. 1.40 ml tlO rr~) of Et3N were added to the solu-tion, while stirring. The formed Et3N.HBr was removed by filtration and washed with a small ~uantity of cold DMF.
3.65 g (11 n)M) of Cbo-Gly-OpNP werc added at -10 to the filtrate, while stirring, and the reaction was allowed to 2~ proceed for 2-3 hours in the absence of moisture, whereby the temperature of the reaction solution gradually reached about 20. The solution was again cooled -to -10 and buf-fered with 0.70 ml (5 mM) of Et3N. The reaction solution was allowed to react for about 2 hours at -10 and for 25 about 3 hours at room temperature. This procedure was re-peated with O.70 ml of Et3N, and aFter 16 hours the reac-tion solution was concentrated to dryness in vacuo at 50.
The residue was dissolved in 75 ml of 50% AcOH and puri-fied by gel filtration on a column of "Sephadex" G-15 3~ equilibrated with 50% AcOH. The fraction of the AcOH elu-ate which was split by treatment with trypsin with re-lease of p-nitroaniline was concentrated to dryness in vacuo at 40. The residue was dissolved in 150 ml of MeOH
and agaiil concentrated to dryness. The resulting residue 35 was dried in a vacuum desiccator at 60 over P205 to ob-tain 5.85 g (88.3% of the theory) of amorphous compound lc which was homogeneous in the SS as shown by TLC. Element--- 16 --ary analysis and calculation from the empirical formula C22H28N706Br gave the following values: C - 46.33~ (46.65 %), H = 5.04% (4.98;~), N = 17.88% (17.31%) and Br = 14.20%
(14.11%).
5 ld. 2HBr.H-GlY-Ar~:-pNA
4.56 g (8 n~) of compound lc were treated, while stirring, with 32 ml of 2N HBr in glacial acetic acid for 40 min. at 20. The dipeptide derivative gradually dis-solved with C02 evolution. The reaction solution was added 10 dropwise with vigorous stirring to 250 ml of abs. ether, and this resulted in the precipitation of 2HBr.H-Gly-Arg-pNA. The ethereal phase was sucked off, whereupon the so-lid phase was washed 4 times with portions of 100 ml of abs. ether in order to remove benzyl bromide which had 15 formed as a by-product as well as excess HBr and AcOH. The residue w~s dissolved in 50 ml of MeOH. The pH was adjust-ed to 4.5 by means of Et3N, and the solution was concen-trated to dryness in vacuo at 30. The resulting residue was dissolved in 50 ml of MeOH and purified on a column of 20 "Sephadex" LH-20 equilibrated with MeOH. The fraction of the MeOH eluate which was split by treatment with trypsin wit h release of p-nitroaniline was concentrated to dry-ness in vacuo at 30. The resulting residue was dried in a vacuum desiccator at 40 over P205 to obtain 3.78 g (92.1%
25 of the theory) of amorphous compound ld which was homo-geneous in the SS as shown by TLC. Elementary analysis and calculation from the empirical formula C14H23N704Br2 gave the following values: C = 32.31% (32.77~), H = 4.59%
(4.52U~o), N = 19.47% (19.11%) and Br = 30.78% (31.14%).
30 le. Cbo-D-Leu_Gly_Ar~-pNA.HBr 2.57 g (5 n~q) of compound ld were dissolved in 20 ml of freshly distilled DMF, and the solution was cooled to -10. 0.70 ml (5 n~l) of Et3N were added to the solution, while stirring. The formed E;t3N.HBr was removed by filtra-35 tion and washed with a small quantity of cold DMF. 2.13 g(5.5 rr~) of Cbo-D-Leu-OpNP were added at -10 to the fil-trate, while stirring. The reaction mixture was allowed 3~

to react for 2-3 hours in the absence of moisture, whereby the temperature of the reaction solution gradually reached about 20. The solution was again cooled to -10, buffered with 0.35 ml (2.5 mM) of Et3N and allowed to react for 5 about 2 hours at -20 and for a further 3 hours at room temperature. This procedure was repeated with 0.35 ml of Et3N, and after 16 hours the reaction solution was concen-trated to dryness in vacuo at 50. The residue was dis-solved in 50 ml of 50% AcOH and purified by gel filtration 10 on a column of "Sephadex" G-15 equilibrated with 50% AcOH.
The fraction of the AcOH eluate which was split by treat-ment with trypsin with release of p-nitroaniline was concentrated to dryness in vacuo at 40. The residue was dissolved in 100 ml of MeOH, and the solution was again 15 concentrated to dryness. The resulting residue was dried in a vacuum desiccator at 60 over P205 to obtain 3.08 g (90.6% of the theory) of amorphous compound le which was homogeneous in the SS as shown by TLC. Elementary analysis and calculation from the empirical formula C28H39N807Br 20 gave the following values: C = 49.06% t49.49%), H 3 5.82%
(5.78~), N = 16.85% (16.49%) and Br = 11.59% (11.76%).
The amino acid analysis confirmed the presence of the expected amino acids in the correct proportions:
Gly : 1.00 - D-Leu : 0.99 - A g : 0.97.
~xample 2 Cbo-D-Leu-Gly-Ar~-MCA.HBr 2b. 2HBr.H-Ar~-MCA
13.0 g (25.9 mM) of commercial Cbo-Arg-MCA.HCl were deblocked according to Example lb by means of 104 ml (208 30 mM) of a solution of 2N HBr in glacial acetic acid. The dry residue was dissolved in 400 ml of MeOH and purified on a column of "Sephadex" LH-20. The fraction of the MeOH
eluate which was split by treatment with trypsin with release of 4-methyl-7-amino-coumarin was concentrated 35 to dryness in vacuo at 30. The resulting residue was dried in a vacuum desiccator at 40 over P205 to obtain L4~

11.2 g (87.7~ of the theory) of amorphous compound 2b which was homogeneous in the SS as shown by TLC. Elemen-tary analysis and calculation from the empirical formula C16H23N503Br2 gave the following values: C = 3~.40%
5 ( 38 . 96~o), H = 4.61~ (4.70~), N = 14.48% (14.20~o) and Br =
31.90% (32.40~).
2c. Cbo-Gly-Ar~ MCA.HBr 4.93 g (10 mM~ of compound 2b and 3.65 g (11 mM) of Cbo-Gly-OpNP were added to 75 ml of freshly distilled DMF.
10 After cooling to -10, there were added, while stirring, first 1.40 ml (10 mM) and then 0.70 ml (5 mM) of ~t3N. The mixture was allowed to react, in the absence of humidity, first for 3 hours at -10 and then for 4 hours at room temperature. The reaction solution was again cooled to 15 -10, buffered with 0.70 ml of Et3N and stirred overnight at 20. The reaction mixture was concentrated to dryness in vacuo at 50, and the residue was dissolved in 200 ml of 50% AcOH and purified on a column of "Sephadex" G-15.
The fraction of the AcOH eluate which was split by treat-2n ment with trypsin with ~elF~ase of 4-methyl-7-amino-cow~rin was concentrated to dryness in vacuo at 40. The thus obtained residue was dried in a vacuum desiccator at 60~ over P205 to yield 4.98 g (82.5% of the theory) of amorphous compound 2c which was homogeneous in the SS as shown by TLC. Elementary analysis and calcul~tion from the empirical formula C26H31N606~r gave the following values: C = 51.48% (51.75~), H = 5.24~ (5.18%), N = 13.70~o ( 13 . 93%) and Br - 13.14% (13.24%).
2d. 2HBr.H-Gl~-Ar~-MCA
4.83 g (8 mM) of compound 2c were deblocked accord-ing to Example ld by means of 32 ml of 2N HBr in glacial acetic acid. The resulting crude product was dissolved in 100 ml of MeOH and purified on a column of "Sephadex" LH-20. The fraction of the MeOH eluate which was split by 35 treatment with trypsin with release of 4-methyl-7-ami-no-coumarin was concentrated to dryness in vacuo at 30.
The resulting residue was dried in a vacuum desiccator at L4~Z

40~ over P205 to give 4.05 g (92.05~ of the theory) of amor-phous compound 2d which was homogeneous in the SS accord-ing to TLC. Elementary analysis and calculation from the empirical formula C18H26N604Br2 gave the followin~; values:
5 C = 39.02~ (39.29~o), H = 4.78% (4.76%), N = 15.39%
(15.27~) and Br = 28.72% (29.04%).
2e. Cbo-D-Leu-Gly-Ar~-MCA.HBr 2.75 g (5 ~) of compound 2d were reacted with 2.13 g (5.5 n~) of Cbo-D-Leu-OpNP in accordanse with Ex-10 ample le. The resulting crude product was dissolved in 75ml of 50% AcOH and purified on a column of "Sephadex"
G-15. The fraction of the AcOH eluate which was split by treatment with trypsin with release of 4-methyl-7-ami-no-coumarin was concentrated to dryness in vacuo at 40.
15 The residue was dried in a vacuum de5iccator at 60 over P205 to obtain 2.91 g (81.2% of the theory) of amorphous compound 2e which was homo~eneous in the SS as shown by TLC. E;lementary analysis and calculation from the empir~
ical formula C32H42N707Rr gave the following values: C =
20 53.13% (53.63%), H = 6.01C1o (5.91%), N = 13.91% (13.689~) and Br = 10.88~ (11.15~).
The amino acid analysis confirmed the preser~ce of the expected amino acids in the correct proportions:
Gly : 1.00 - Leu : 1.02 - Arg : 0.98.
Example 3 Cbo-D-Leu-Gly-Ar~-DPA HBr 3a. Cbo-Ar~-DPA.HCl 34.48 g (0.1 mole) of dried Cbo-Arg-OH.HC1 were dissolved in a 1000 ml three-necked flask in a mixture of 150 ml of freshly distilled anhydrous DMF and 300 ml of abs. THF at 20. To the solution, cooled to -1(), there were added, while stirring, 1().2 g (().1 mole) of Et3N in the absence of humidity. Then a solution of 13.65 g (0.1 mole) of isobutyl chloroformate in 50 ml of THF
35 was added dropwise within 20 minutes, whereby the reaction temperature was never allowed to exceed -5. After an ad-ditional reaction time of 10 minutes at a temperature of -10 t;o -5 a solution of 20.92 g (0.1 mole) of dimethyl 5-amino-isophthalate in 75 ml of D~iF was added dropwise within 30 minutes, whereby the reaction temperature was never a,llowed to exceed -5. The reaction mixture was al-5 lowed to react for another hour a-t -5. Then it was stir-red overni,~;ht at 20 and subsequently cooled to -15 in order to let the Et3N.HCl crystallize. The formed Et3N.
HCl was filtered off and washed with a small amount of cold Dr~F. The filtrate and the washing solution were con~
10 centrated to dryness in vacuo at 50. The residue was dis-solved in 1000 ml of 50% AcOH and puri.fied by gel filtra-tion on a column of "Sephadex" G-15 equilibrated with 50%
AcOH. The fraction of the AcOH eluate which was split by treatment with trypsin with release of dimethyl 5~ami-15 no-isophthalate was concentrated to dryness in vacuo at 40. The residue was dried in a vacuum desiccator at 50 over P205 to obtain 24.6 g (45.9~o of the theory) of amor-phous compound 3a which was homogeneous in the SS as sho~
by TLC. Elementary analysis and calculation from the em-20 pirical formula C24H30N507Cl ~ave the following values:
C = 53.21% (53.78%), H = 5.71% ( 5.64'yo)~ N = 13.20%
(13.07%) and Cl = 6.5~,~ ~6.62%). , 3b. 2HBr.H-Ar,~-DPA
21.44 g (40 n~l) of compound 3a we~re deblocked ac-25 cording to ~xample lb. ~fter the usual treatrnent the re-sulting crude product was dissolved in 250 ml of MeOH and puri~ied by gel filtration on a column of "Sephadex" LH-20. The fraction of the MeOH eluate which was split by treatment with trypsin with release of dimethyl 5-ami-30 no-isophthalate was concentrated to dryness in vacuo. The residue was dried in a ~acuum desiccatvr at 40 over P205 to obtain 19.63 g (93.1% of the theory) of amorphous com-pound 3b which was homo~;eneous in the SS as shown by TLC.
l~lementary analysis and calculation from the empirical 35 ~`ormula C16H25N505Br2 gave the following values: C =
36.825~ (36.45C~o)~ H = 4.67% (4.78'Jlo)J N = 13.45% (13.28%) and Br = 29.85% ( 30.31%).

3c. Cbo-Gly~Ar~-DPA.HBr - 21 -5.27 g (10 mM) of compound 3b were reacted accord-ing to Example lc with 3.65 g (11 mM) of Cbo-Gly-OpNP.
The crude product obtained after the usual treatment was dissolved in 200 ml of 50% AcOH and purified on a column of "Sephadex" G-15. The fraction of the AcOH eluate which was split by treatment with trypsin with release of dimethyl 5-amino-isophthalate was concentrated to dryness in vacuo at 40. The residue was dried in a vacuum desic-10 cator at 60 over P205 to give 5.29 g (83.0qo of the the- ~
ory) of amorphous compound 3c which was homogeneous in the SS as shown by TLC. Elementary analysis and calculation from the empirical formula C26H33N608Br gave the follow-in~ values: C = 48.50% (48.99%), H = 5.28% (5.22~0), N =
15 12.92~0 (13.18%) and Br = 12.33% (12.53~b).
3d. 2HBr.H-Gly-Arg-DPA
5.10 g (8 mM) of compound 3c were deblocked accord-ing to Example ld by means of 32 ml of 2N HBr in glacial ~cetic acid. The crude product obtained after the usual 20 treatment was dissolved in 100 ml of` MeOH and purified on a column of "Sephadex" LH-20. The fraction of the MeOH
eluate which was split by treatment with trypsin with release of dimethyl 5-amino-isophthalate was concentrated to dryness in vacuo at 30. The residue was dried in a va-25 cuum desiccator at 40 over P205 to ~ive 4.25 g (90.9% ofthe theory) of amorphous compound 3d which was homogeneous in the SS as shown by TLC . Elementary analysis and calcu-lation from the empirical formula C18H28N606Br2 gave the following values: C - 36.85% (37.00qv), I-l = 4.90% (4.83%), 30 N = 14.72% (14.385~) and Br = 26.95% (27.35~).
3e. Cbo-D-Leu-Gly-Ar~-DPA-HBr 2.92 g (5 mM) of compound 3d were reacted accord-ing to Example le with 2 .13 g ( 5 . 5 mM) of Cbo-D-Leu-OpNP.
The crude product obtained after the usual treatment was 35 dissolved in 100 ml of 50/J AcOH and purified on a column of "Sephadex" G-15. The fraction of the AcOH eluate which was split by treatment with trypsin with ~elease of di-methyl 5-amino-isophthalate was concentrated to dryness in vacuo at 40. The residue was dried in a vacuurn desiccator at 60 over P20S to obtain 3.11 g ( 82 .9% of the theory) of amorphous compound 3e which was homogeneous in the SS as 5 shown by TLC. Elementary analysis and calculation from the empirical formula C32H44N709Br gave the following values:
C = 50.88% (51.20~o), H = 5.99% (5.91"j~o), N = 13.26a~ (13.0~o) and Br = 10. 48~o ( 10 . 64010) .
The amino acid analysis confirmed the presence of 10 the expected amino acids in the correct proportions:
Gly: 1.00 - Leu: 1.00 - Arg: 0.97.
Example 4 Cbo-D-Leu-Sar-Ar~-2-N.4.HBr 4b. 2HBr.H-Ar~:-2-NA
9.40 g (20 n~) of commercial Cbo-Arg-2-NA.HCl were deblocked according to Example lb with a solution of 80 ml Or 2N HBr in glacial acetic acid. The product obtained af-ter the usual treatment was dissolved in 150 ml of MeOH
and purii`ied on a column of "Sephadex" LH-20. The fraction 20 of the MeOH eluate which was split by treatment with tryp-sin with release of 2-naphthylamine was concentrated to dryness in vacuo at 30. The residue was dried in a vacuum desiccator at 40 over P205 to ob~ain ~.60 g (93.2% of the theory) of amorphous compound 4b which was homogeneous in 25 the SS as shown by TLC. Elementary analysis and calcula-tion from the empirical formula C16~l23N50Br2 gave the fol-lowing values: C = 42.08% (41.67,~), I{ = 5.12% ~5.03%), N =
14.68% (15.19%) and Br = 33.96% (34.65%).
4c. Cbo-Sar-Ar~;-2-NA.HBr 4.6 g (10 n~) of compound 4 b were reacted accord-ing to Example lc with 3.80 g (11 rrM) of Cbo-Sar-OpNP. The crude product obtained after the usual treatment was dis-solved in 150 ml of 50~ AcOH and purified on a column of "Sephadex" G-15. The fraction of the AcOH eluate which was 35 split by treatment with trypsin with release of 2-naph-thylamine was concentrated to dryness in vacuo at 40. The residue was dried in a vacuum desiccator at 60 over P2~5 _ 23 -t~ obtain 4.95 g (84-~ of the theory) of amorphous com-pound 4c which was homogeneous in the SS as shown by TLC.
Elementary analy~is and calculation from the emp~cal for-mula C27H33N604Br gave the following values: C = 55.72%
(55.39%), H = 6.73% (5.68~o), N = 14.68% (14.35~o) and Br =
13.42% (13.65%).
4d 2HBr.H-Sar-Ar~-2-NA
.

4.68 g (8 mM) of compound 4c were deblocked accord-ing to ~xample ld by means of 28 ml of 2N HBr in glacial 10 acetic acid. The crude product obtained after the usual treatment was dissolved in 100 ml of MeOH and purified on a column of "Sephadex" LH-20. The fraction of the MeOH
eluate which was split by treatment with trypsin with release of 2-naphthylamine was concentrated to dryness in 15 vacuo at 30. The residue was dried in a vacuum desiccator at 40 over P205 to give 4.08 g (95.810 of the theory) of amorphous compound 4d which was homo~eneous in the SS as shown by TLC. Elementary analysis and calculation from the empirical formula ClgH28N602Br2 gave the following 20 values: C = 43.9% (42.87%), H = 5.32% (5.30%), N = 16.02~o (15.79%) and Br = 29.68% (30.02%).
4e. Cbo-D-Leu-Sar-Ar~-2-NA.HBr 2.66 g (5 mM) of compound 4d were reacted accord-ing to Example le with 2.13 g (5.5 ~) of Cbo-D-Leu-OpNP.
25 The crude product obtained after the usual treatrnent was dissolved in 100 ml of 5~% AcOH and purified on a column of "Sephadex G-15". The first main fraction of the AcOH
eluate which was split by treatment with trypsin with r~lease of 2-naphthylamine was concentrated to dryness in 30 vacuo at 40 and dried in a vacuum desiccator at 60~ over P205. There were thus obtained 3.01 g (86.2~o of the theo-ry) of amorphous compound 4e which was homogeneous in the SS as shown by TLC. ~lementary analysis and calculation from the empirical formula C33H44N705Br gave the follow-35 ing values: C = 57.05qo (56.73%), H = 6.40% (6.35~o) ~ N =14 . 30% ( 14 . 03%) and Br = 11.12% (11.44%).

z -- 2~ --The amino acid analysis confirmed the presence of the expected amino acids in the correct proportions:
Sar: 1.00 - Leu: 1.02 - Arg: 0.97.
E;xample _5 Cbo-D-Leu-Sar Ar~-4-MeO-2-NA.HBr 5b. 2HBr.H-Ar~-4-MeO-2-NA
10.0 ~ (20 ~) of commercial Cbo-Arg-4-MeO-2-NA.HCl ;~ere deblocked according to Example lb by means of 80 ml of 2N HBr in glacial acetic acid. The crude product obtain-10 ed af`ter the usual treatment was dissolved in 150 ml of MeOH and purified on a column of "Sephadex" LH-20. The main fraction of the MeOH eluate which was split by treat-ment with trypsin with release of 4-methoxy-2-naphthyl-amine was concentrated to àryness in vacuo at 30. The re-15 sidue was dried in a vacuum desiccator at 40 over P205 togive 8.98 g (91.4~ of the theory) of amorphous compound 5b which was homogeneous in the SS as shown by TLC. Element-ary analysis and calculation from the empirical formula C17H25N502Br2 gave the following values: C = 41.22%
20 (41.57~), H = 5.19% (5.13yo), N = 14.40% (14.26%) and Br =
32. 01~o (32.53~o).
5c. Cbo-Sar-Ar~-4-MeO-2-NA.HBr -4.91 g (10 n~) of compound 5b were reacted accord-ing to Example lc with 3.80 g (11 mM) of Cbo-Sar-OpNP. 1'he 25 crude product obtained after the usual treatment was dis-solved in 150 ml of 50% AcOH and purified on a column of "Sephadex" G-lS. The first main fraction of the AcOH elu-ate which was split by treatment with trypsin with re-lease of 4-methoxy-2-naphthylamine was concentrat;ed to 30 dryness in vacuo at 40. The residue was dried in a vacuum desiccator at 60 over P205 to obtain 4.86 g (79.0% of the theory) of amorphous compound 5c which was hornogeneous in the SS as shown by TLC. Elementary analysis and calcula-tion from the empirical formula C28H35N605Br gave the fol-35 10wing val les: C 2 54 . 38% ( 54.64%), H = 5.81% (5.730,10), N =13.93~ (13.65%) and Br = 12.75% (12.985~).

3'~

5d. _Br.H-Sar-Ar~-4-MeO-2-NA
4.31 g (7 mM) of compound 5c were deblocked accord-ing to Example ld with 28 ml of 2N HBr in glacial acetic acid. The crude product obtained after the usual treat-ment was dissolved in 100 ml of MeOH and purified on a 5 column of "Sephadex" LH-20. The main fraction of the MeOH
eluate which was split by treatment with trypsin with formation of 4-methoxy-2-naphthylamine was concentrated to dryness in vacuo at 80. The residue was dried in a vacuum desiccator at 40 o~er P205 to obtain 3.74 g 10 (95.0% of the theoryJ of amorphous compound 5d which was homogeneous in the SS as shown by TLC. Elementary ana-lysis and calculation from the empirical formula C20H30N603Br2 gave the following values: C = 43.01%
(42.72%), H = 5.44~ (5.38%), N = 15.25~o (14.95~o') and Br =
28.03,~ (28.42%).
The amino acid analysis confirmed the presence of the expected amino acids in the correct proportions:
Sar : 1.00 - Leu ; 1.01 - Arg : 0.98.
5e. Cbo-D-Leu-Sar-Ar~-4-MeO-2-NA.HBr 2.81 g (5 nM) of compound 5d were reacted accord-ing to Example le with 2.13 g (5.5 n~) of Cbo-D-Leu-OpNP.
The crude product obtained after the usual treatment was dis-sciLvedinl~5nld`50% AcOH and purified on a column of "Sepha-dex" G-15. The first main ~`raction of the AcOH eluate 25 which wa~ split by treatment with trypsin with re].ease of 4-methoxy-2-naphthylamine was concentrated to dryness in vacuo at 40. The residue was dried in a vacuum de-siccator at 60 over P205 to give 2.98 g (81.8~o of the theory) of amorphous compound 5e which was homogeneous in 30 the SS as shown by TLC. Elementary analysis and calcula-tion from the empirical ~ormula C34H46N706Br gave the fol-lowing values: C = 56.28% (56.04%), H = 6.34% (6.36C~o) ~ N
= 13.75% (13.46j~o) and Br = 10.68% (10.97%).
EKample 6 Cbo-D-Leu-Gly-Ar~;-pNA.AcOH

1~ki14~

6.80 g (10 mM) of Cbo-D-Leu-Gly-Arg-pNA.HBr (pre-pared according to Example 1) were dissolved in 75 ml of 60% aqueous Me~H. The solution was poured on a column of "Amberlite" JRA-401 in the acetate form. The column was eluted by means of 60yo aqueous MeOH, the HBr being replaced by AcOH by ion exchange. The eluate was con-centrated to dryness in vacuo at 40. After drying in the vacuum desiccator at 40 over P205 there were obtain-ed 6.62 g of bromide-free Cbo-D-Leu-Gly-Arg-pNA.AcOH
10 (gg.010 of the theory). Other salts with organic acids, e.g. formic acid, propionic acid, oxalic acid, tartaric acid, citric acid, lactic acid, benzoic acid, chloroben-zoic acid, salicylic acid or phthalic acid~ can also be prepared from the above named tripeptide deri~ative ac-15 cording to the same method. The ion exchanger can be e.g."Amberlite" JRA-401 in i~ hydrochloride form, and the des~red acid salt form can be obtained by converting the said ion exchanger into the basic OH-form by treatment with caustic soda solution and then 20 with a solution of a 1:1 mixture of the desired organic acid and its sodium salt in 60qo aqueous MeOH.
~xample_7 Tos-D-Leu-Glv-Ar~r~-pNA.HBr 6.26 g (10 mM) of 2HBr.H-D-L,eu-Gly-Arg-p~A (pre-25 pared according to ~xample 7f, see Table 3) were dissolved in 30 ml of distilled DMF. The solution was cooled to -15, and first 2.80 ml (20 mM) of ~t3N and immediately after-wards 1.91 g (10 mM) of tosyl chloride were added, while stirring, in the absence of humidity. After a reaction 30 time of about 2 to 3 hours at -15, the reaction mixture was a~owedtofurth~r react overnight at room temperature. The reaction mixture was again cooled to -15. The precipitated mixture of Et3N.HBr and Et3N.HCl was removed by filtra-tion and washed with a small amount of cold DMF. The 35 filtrate and the washing solution were combined and con-centrated to dryness in vacuo at 50. The residue was dissolved in 75 ml of 50~0 AcOH. The solution was purified ~R~J?7~

_ 27 -on a column of "Sephadex" G-15 equilibrated with 50~0 AcOH.
The main fraction of the AcOH eluate which was split by treatment with trypsin with r~e~eof p-nitroaniline was concentrated to dryness in vacuo at 40~. The residue was 5 dissolved in 100 ml of MeOH, and the solution was again concentrated to dryness. The residue was dried in a vacu-um desiccator at 60 over P205 to obtain 6.65 g (95.0~o of the theory) of amorphous compound ros-D-Leu-Gly-Arg-pNA.
HBr which was homogeneous in the SS as shown by TLC. ~le-10 mentary analysis and calculation from the empirical form-ula C27H39N8S07Br ga~e the following values: C = 46.85 (46.35a/~ 1 = 5.66% (5.62%) ~ N = 16.28~o (16.02%) ~ S =
4.43~0 (4.58'~o) and Br = 11.22~ (11.42%).
Instead of using tosyl chloride as the acylation a~ent corresponding amounts of acetyl chloride, butyrY1 chloride, octanoyl chloride, benzoic acid chloride, p-methylbenzoic acid chlo~ de, 2-chlorobenæoic acid chlor-ide, methanesulfonyl chloride, n-butanesulfonyl chloride, benzenesulfonyl chloride, a-naphthalenesulfonyl chloride, methyl chloroformate, isobutyl chloroformate, octyl chloro-forrnate, benzoic acid anhydride, acetic acid anhydride, phenylacetic acid-OpNP, phenylpropionic acid-OpNP,.cyclo-hexylcarboxylic acid-OpNP, N-Cbo-4-aminomethylcyclohexyl-carboxylic acid-OpNP, N-Cbo-~ -amino-n-butyric acid-OpNP, ~,N-Cbo- ~ -aminooctanoic acid-OpNP or N-Cbo-4-aminomethyl-benzoic acicl-OpNP can also be used.

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- 52 _ The susceptibility of the tripeptide derivatives ~f.the invention to factor Xa is illustrated numerically in the following Table 4 and compared with the suscept-ibility cf the known prior art substrate Bz-Ile-Glu(Y-OH)~Cly-Arg-pNA.HCl.
`
Table_4 hctivity of 1 ml of aqueous sol1ltion of bovine factor Xa and o~ factor Xa as obtained by activation of factor X in 1 ml of nor~al human blood plasma by n-eans of RVV, ex-10 pressed in nanomoles of split product H - R5 released per minute _________________ substratebovi.ne factorhuman factor concentrati.on Xa Xa 2 x 10 ~
______________ __________________________________________ 15 ~ubstrates according to exalrlples 1~.40 815 6 25~) 1520

7 950 705

8 27~0 1568

9 2010 1105 1~ l3~2 825 lL 245 100 12 196~ 1528 1436 146~

13 1385 12~5 4~3Z

Table 4 ( cont ' d) 23 2~14 1000 12~5 495 3S 21~ 1055 37 2 ~0 11 8~' 5 570 6~ 4~0 1~5 75 lB0 130 Table 4 (cont'd) 53 68~ 510 54 '740 505 5~3 2540 1430 12~0 1025 ~i6 1370 965 67 141~ 1005 ~8 1490 1010 7~ 12~5 795 73 1~65 875 78 79~ 440 ~ 940 635 82 ~3~ 1380 84 2~10 1690 . Table 4 (cont~l) ~7 330 146 ~9 42~ 170 92 610 41~
93 7~ 630 94 102U ~45 96 ~30 5~5 97 18~0 1420 ~3~ 1230 1()8 99 199~ 1515

10~ 12~0 1055 101 ~080 1290 103 2~.60 1460 Comparison substrats* 490 820 ____________________________~____________________________ * Bz-Ile-Glu(Y-OH)-Gly-Arg-pNA.HCl (disclosed in Germ~n patent application Nc.. DE-OS 25 52 570) ________________________________________________________ The values of the factor Xa activity r~iven in Table 4 were determined experimentally in the manner described hereinafter:
For determining the aetivity of' bovine factor Xa 1.8 ml of T~IS-imidazole buf`fer having a pH of a . 4 and an ionic stren~th of 0.3 was well mixed at 37C with 0.025 ml of an aqueous solution of factor Xa preparation "Diagen"
(furnished by Diagnostic Reagents Ltd., Thame, Great 3ri-tain) obtained by dissolvin~g the contents of one vial of 10 "Diagen" in 0.5 ml of water. To tne mixture there was added 0.2 rnl of a 2 x 10 3 M agueolls solution of a sub-- 5c, -str~t~ ~f the invention. Thereafter, the quantity of split product H - R5 in nanomoles released per minute was de-termined~ and from the obtained value the corresponding value for 1 ml of factor Xa solution was calculated.
For determining the activity of human f`actor Xa 0.01 ml of normal citrated plasma was mixed with 0.20 ml of a solution of Russel viper venom in TRIS-imida~ole buf-fer having a pH of 8.4 and an ionic strength of 0.3 and containing 15 mmoles of CaC12 per ml. The mixture was in-i.0 cubated for 75 seconds at 37C in order to activate ~ac-tor X present in the plasma and convert it completely in-to factor Xa. To the incubate there was added first 1.40 ml of TRIS-imidazole buffer (pH 8.4, ionic strength 0.3) heated to 37C and then 0.40 ml of a 2 ~ 10 3 M aqueous 15 solution o. a substrate of the invention. Thereafter, the ~uantlty of s~)lit product H - R5 in nanomoles released per ~ninute ~as determined, and fronl the obtained value the corresponding value for 1 ml of citrated plasma was cal-culated.

Claims (11)

C l a i m s

1. Tripeptide derivatives having the formula I

wherein R1 represents an alkanoyl group which has 2 to 8 carbon atoms and which may carry an amino group in the .omega.-position, a phenylalkanoyl group which has 2 to 4 carbon atoms in the alkanoyl radical and the phenyl radical of which may carry an amino group in the p-position, a cyclohexylcarb-onyl group which may be substituted with an aminomethyl radical in the 4-position, a benzoyl group which may be substituted with a methyl radical, an-amino group or halo-gen in the o- or p-position, an alkoxycarbonyl group hav-ing 1 to 8 carbon atoms in the alkoxy ,group, a benzyloxy-carbonyl group which may be substituted with methoxy, methyl or chlorine in the p-position, an alkanesulfonyl group having 1 to 4 carbon atoms, a phenylsulfonyl group which may be substituted with methyl in the p-position or an .alpha.- or .beta.-naphthylsulfonyl group, R2 represents a straight-chained or branched alkyl radical having 1 to 6 carbon atoms, a hydroxyalkyl radical having 1 to 2 carbon atoms, an alkoxyalkyl radical having 1 to 2 carbon atoms in the alkyl and 1 to 4 carbon atoms in the alkoxy, a benzoxyalkyl radical having 1 to 2 carbon atoms in the alkyl, an .omega.-carboxyalkyl or .omega.-alkoxycarbonylalkyl radical which has 1 to 3 carbon atoms in the alkyl and the alkoxy group of which is straight-chained or branched and has 1 to 4 carbon atoms, an .omega.-benzyloxycarbonyl-alkyl ra-dical having 1 to 3 carbon atoms in the alkyl, or a cyclo-hexyl-, cyclohexylmethyl-, 4-hydroxycyclohexylmethyl-, phenyl-, benzyl-, 4-hydroxybenzyl- or imidazol-4-yl-methyl radical, R3 represents hydrogen or a straight-chained or branched alkyl radical having 1 to 4 carbon atoms, R represents hydrogen or a methyl or ethyl radical and R represents an amino group which is substituted with aromatic or heterocyclic radicals and which is capable of being split off hydrolytically with formation of a coloured or fluorescent compound H - R5, with the exception of tripeptides in which R1 is benzyloxycarbonyl and R is isopropyl or cyclohexyl, and salts thereof with acids.

2. Tripeptide derivatives according to claim 1 wherein R5 is a p-nitrophe-pylamino, l-carboxy-2-nitrophen-5-yl-amino, 1-sulfo-2-nitrophen-5-yl-amino, .beta.-naphthylamino, 4 methoxy-~-naphthylamino, 5-nitro-a-naphthylamino, quinon-5-yl-amino, 8-nitro-quinon-5-yl-amino, 4-methyl-coumar-7-yl-amino or 1,3-di(methoxy-earbonyl)-phen-5-yl-amino group (derived from 5-amino-isophthalic acid dimethyl ester).

3. Tripeptide derivatives according to claims 1 wherein the strongly basic guanidino group of arginine is stabilized by protonation with a mineral or organic acid.

4. Tripeptide derivatives according to claims 1 and 3 wherein the guani-dino group of arginine is stabilized by protonation with acetic acid.

5. Tripeptide derivatives according to claim 1: Cbo-D-Leu-Gly-Arg-pNA.-AcOH, Cbo-D-Leu-Sar-Arg-pNA.AeOH, Cbo-D-Ph'Gly-Gly-Arg-pNA.AeOH, Cbo-D-Nleu-Gly-Arg-pNA.AeOH, Cbo-D-Nleu-Sar-Arg-pNA.AeOH, Cbo-D-Nval-Gly-Arg-pNA.AcOH, Cbo-D-CHA-Gly-Arg-pNA.AeOH, Cbo-D-CHA-Sar-Arg-pNA.AeOH, Cbo-D-CHT-Gly-Arg-pNA.AeOH, Cbo-D-CHT-Sar-Arg-pNA.AeOH, Cbo-D-CHG-Gly-Arg-pNA.AcOH, CH3SO2-D-Nleu-Gly-Arg-pNA.AcOH, isobutoxy-CO-D-Nleu-Gly-Arg-pNA.AcOH, BOC-D-Leu-Gly-Arg-pNA.HBr, 4-MeO-Cbo-D-Leu-Gly-Arg-pNA.HBr, CH30-CO-D-CHA-Gly-Arg-pNA.AcOH, C2H50-CO-D-CHA-Gly-Arg-pNA.AcoH, CH3SO2-D-CHA-Gly-Arg-pNA.AcOH, 4-Me-Cbo-D-Leu-Gly-Arg-pNA.AcOH, 4-Cl-cbo-D-Leu-Gly-Arg-pNA.AcoH, BOC-D-(.alpha.)-AOA-Gly-Arg-pNA.AcOH.

6. Process for quantitatively assaying factor Xa in a medium which con-tains factor Xa or in which factor Xa is formed or consumed, which comprises reac-ting the said me-dium with a tripeptide derivative according to claim 1 and measuring, by photometric, spectrophotometric, fluorescen-ce-spectrophotometric or electrochemical methods the quantity of the coloured or fluorescent split product H -R5 released per time unit by the catalytic hydrolytic ac-tion of factor Xa on the tripeptide derivative.

7. Process according to claim 6 wherein blood plas-ma of human beings or warm-blooded animals is treated with an activator in order to convert quantitatively the in-active factor X present in the plasma into the active fac-tor Xa, the activated plasma is reacted with the said tri-peptide derivative in a buffer system, and the quantity of split product H - R5 released by factor Xa from the tri-peptide derivative per time unit is determined.

8. Process according to claims 6 and 7 wherein Russel viper venom (RVV) or a thromboplastin reagent is used as the activator.

9. Process for the quantititives assay of heparin in heparinized blood plasma via factor Xa which comprises incubating a predetermined quantity of factor Xa with blood plasma. in a buffer system, measuring the quantity of non-neutralized factor Xa by reacting the latter with a tripeptide derivative according to claim 1 and determin-ing the quantity of split product H - R5 released per time unit and determining the heparin level from the difference between the quantity of factor Xa initially used and the residual quantity of factor Xa.

10. Process for the quantitative assay of anti-factor Xa in human blood plasma via factor Xa which com-prises, on the one hand, preparing a test sample by di-luting citrated human plasma with a buffer solution, add-ing heparin and then factor Xa to the dilute solution and addiing to the mixture a tripeptide derivative according to claim 1, on the other hand, preparing a blank test sample in the same manner, but using the same volumetric amount of buffer solution instead of dilute plasma, determining for both samples the increase in the optical density per minute caused by the formation of split product H - R5, and determining the quantity of factor Xa inhibited by the antifactor Xa-heparin complex and therefrom the quantity of antifactor Xa initially present in the plasma from the difference of the increase in the optical density of the blank test sample per minute and the increase in the op-tical density of the plasma test sample per minute.

11. Process according to claims 6, 9, or 10 wherein, if R5 is a p-nitrophenylamino, 1-carboxy-2-nitro-phen-5-yl-amino, 1-sulfo-2-nitro-phen-5-yl-amino, 5-nitro-.alpha.-naphthyl-amino or 8-nitro-quinon-5-yl-amino group, the split product H - R5, before the measurement of its quan-tity, is converted into a more intensely coloured compound by coupling, with a diazo compound.