C-Terminal peptide alcohol, acid and amide analogs of desulfato hirudin54–65 as antithrombin agents

THROMBOSIS RESEARCH 54; 319-325, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

C-TERRINAL PEPTIDE ALCOEOL,ACID AND ANIDB ANALOGS OF DESULPATO HIRUDINs,_6s AS ANTITRRORRINAGRNTS

John L. Krstenansky*, Marguerite H. Payne, Thomas J. Owen, Hark T. Yates and Simon J.T. Mao Merrell Dow Research Institute, 2110 E. Galbraith Road Cincinnati, Ohio 45215 USA (Received 14.12.1988; accepted in revised form 28.2.1989 by Editor N.U. Bang)

ABSTRACT

with C-terminal Analogs of the antithrombin peptide hirudin oHI?&? to examine the modifications have been synthesized in a-thrombin inhibition. The C-terminal residue, reqyirements for could be replaced with L-amino acids or amino alcohols with Gln side chains without greatly affecting neutrH1 or charged hydrophilic by inhibition of the peptide’s antithrombin potency as determined formation in in vitro. thrombin-induced clot human plasma Derivatives with D- or L-amino carboxamides at position Gd retained activity. Deletion significantly reduced poteircy, but still residue 64 to the amide or alcohol of residue 65 with conversion of derivative resulted in a three-fold loss of potency. In addition to synthesis of peptide alcohols via these results the solid-phase p-nitrobenzhydrylideneisonitroso resin direct displacement of attached peptides with the desired C-terminal amino alcohol is reported.

INTRODUCTION Most inhibitors of a-thrombin either chemically react with the residues of the catalytic site or sterically block the access of substrate (1). Notable exceptions to this are the endogenous endothelial-bound protein, thrombomodulin (2), C-terminal and fragment analogs of the leech anticoagulant protein, hirudin (3). Thrombomodulin binds to a non-catalytic domain of a-thrombin preventing the cleavage of fibrinogen and facilitating the activation of protein C, presumably through induction of a conformational change in a-thrombin (2). Similarly, peptide analogs based on desulfato hirudin inhibit fibrinogen cleavage and subsequent fibrin-clot formation by bind%g6’to a non-catalytic site on a-thrombin (3). A conformational change of a-thrombin is induced upon binding of these peptides as evidenced by the circular dichroic (CD) spectra (4), but cleavage of the tripeptide chromogenic substrate, H-D-Phe-Pip-Arg-pNA, is not blocked at concentrations Key words:

hirudin,

thrombin,

synthetic

peptides, 319

peptide

alcohols

C-TERMINAL PEPTIDE ALCOHOL...

320

Vol. 54, No. 4

that inhibit fibrinogen cleavage (3). Native hirudin has been reported to induce conformational changes in a-thrombin (5), but it also blocks the cleavage of small peptide substrates (6). The minimal functional domain of hirudin’s C-terminus has been determined to lie between residues 56-64, Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu (4). Numerous analogs based on this region have been synthesized in an attempt to increase the potency of these anticoagulants, stabilize the peptides to degradation potential enzymatic and their thrombin-bound determine conformation (7-10). This paper reports the effect of a number of modifications to the C-terminal residue of this class of anticoagulant on their ability to inhibit fibrin-clot formation in vitro. Modifications of the C-terminal functionality of pharmacologically active peptides often result in dramatic alterations in receptor potency, selectivity, activation or pharmacokinetic profile of the molecule. The amide C-terminus of the tachykinin (11) and the neuropeptide Y (12) families is required for full receptor potency and activation. Peptide alcohol modifications in the enkephalins (13) have produced orally active analogs. Modification of the Cterminus also lends stability towards carboxypeptidases. Therefore a thorough investigation into the structure activity relationships of a potential peptide drug requires a delineation of the C-terminal modifications that lead to enhanced potency and stability.

METHODS Synthesis of Peptide C-Terminal Acids and Amides. The peptides were techniaues on svnthesized bv solid-nhase the aoorooriate PAM or D. methylbenzhydrylamine resin on a 0.5 mmol scale with’an’bpplied Biosystems Model 430A peptide synthesizer using the protocols supplied by the manufacturer. The NE-Boc amino acids were double coupled as their preformed symmetrical anhydrides using dimethylformamide (DMF) as the solvent for the first coupling and dichloromethane (DCM) as the solvent during the second The side chain protection was as follows: coupling. Asp(Chx), Glu(Bzl), of the N-terminal succinyl derivatives Lys(t-ClZ), Tyr(2-BrZ). In the case the final Boc protecting group was removed, the resin neutralized with 10% diisopropylethylamine in DCM, washed with DCM and acylated with succinic anhydride in DCM/DMF(1:l) for 30 min. The peptides were removed from the with liquid HF containing 5% anisole at resin and deprotected by treatment -5’C for 40 min. Upon removal of the HF in vacua the peptides were extracted with 30% acetic acid and the extracts werexphilized. The crude peptides were purified by preparative reverse-phase high performance liquid and 0.1% aqueous trifluoroacetic chromatography (HPLC) using an acetonitrile x 250-mm Cl8 column. Identity acid (TFA) eluant system and a Dynamax 21.4 were assessed by analytical HPLC, amino and purity of the materials obtained amino acid analyzer) and fast-atom acid analysis (AAA; Beckman 6300 The bombardment mass spectrometry (FAB-MS; Finnegan TSQ-4C instrument). results are listed in Table I. I

The p-nitrobenzhydrylideneSynthesis of Peptide C-Terminal Alcohols. was svnthesized from unsubstituted 1% resin (oxime resin) isonitroso crosslinked polystyrene beads (2QO-400 mesh; Bio-Rad) as previously reported (15). The substitution level of the resin was 0.97 mmol/g resin. Syntheses either manually in a scintillation vial and sintered glass were performed funnel or in an Applied Biosystems Model 430A peptide synthesizer (ABI) using symmetrical anhydrides of the Boc amino single couplings of the preformed The protocols for the cycles were taken from acids for the coupling step. for use in the automated synthesizer. Nakagawa et al. (16,17) and adapted 1) 25% trifluoroacetic acid (TFA) in The steps of the cycle are as follows: dichloromethane (DCM) for 1.5 min; 2) 25% TFA in DCMfor 16 min; 3) DCMwash

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C-TERMINAL PEPTIDE ALCOHOL...

321

3 times for 0.5 min; 4) isopropanol (i-PrOH) wash for 4 min; 5) DCN wash 3 times for 0.5 min; 6) i-PrOH wash for 4 min; 7) DCHwash 3 times for 0.5 min; 8) I-PrOH wash for 4 min; 9) DCMwash 3 times for 0.5 min; 10) addition of 2 equivalents of the preformed symmetrical anhydride of the Boc amino acid followed by the addition of diisopropylethylamine to make a 5% solution in DCM(approx. 8 equivalents) then stirring for 1 h; 11) DCMwash 5 times for 1 as a per cent of the theoretical min. The weight gain of the resin amount was typically in the range of 38-54X reflecting the loss of peptide from the resin during synthesis due to the labile nature of the linkage. The peptides were constructed on the resin beginning with the penultimate C-terminal residue. Displacement of the protected fragment from the resin was accomplished using 1.5 to 4 equivalents of either alaninol or leucinol(Advanced Chemtech). The reaction took place in DMFover 48 h or in DCH containing 0.8 equiv. acetic acid over 16 h. After filtration of the resin and evaporation in vacua of the filtrate, deprotection was performed with liquid HF contair&g 5Xisole at -5°C for 30 min. Purification of the peptide alcohols was by reverse-phase HPLC and the products were analyzed by analytical HPLC, AAA and FAB-MS. The peptides gave satisfactory analysis for the desired product (Table I). Fibrin-clot Inhibition Assay. Inhibition of plasma clot formation was Human plasma from a healthy female determined as previously described (3). (fasting for 12 h) was collected in a final EDTA concentration of 0.1%. The plasma was immediately sterilized by filtration through a 0.2~urn filter disk It was then aliquoted 1 mL/vial and stored at -2O’C. All peptide (Gelman). samples were assessed using the same plasma preparation. N’-Acetyl desulfato Bovine thrombin (50 uL; 0.2 (3) vas included as a standard. of a 96-well microtiter plate (Falcon) ;::Y;i;1&-~3 ma was added to the wells containing 50 uL of a solution of the synthetic peptide to be tested. After 1 min of agitation and additional incubation for 10 min at 24’C, 100 uL of diluted human plasma (1:lO) in 0.1 M sodium chloride and 0.012 M sodium phosphate (PBS buffer; Sigma) was added and vortexed for 20 sec. The optical density at 405 nm (O.D. ) of the solutions was monitored by an autoreader (EL 309, Bio-Tek Instrd&hts) at 5 min intervals. Typically, the O.D. at 30 min for various concentrations of the peptide was used to construct dose:~~~“~~?uF~~‘f~ ~~ra$~ylCg~f zl”yf (OT?bZle M I ‘,odi,“,“,h$r\~ phosphate, 0.01% sodium azide and 0.1% bovine serum albumin,

,atz pH 7.4.

‘M”tz1:“m

RESULTS AND DISCUSSION Peptide Synthesis. The C-terminal amide and acid peptides were synthesized by standard solid-phase techniques. Double couplings of the preformed symmetrical anhydrides of the N”-Boc amino acid derivatives were performed using first DMF then DCM as a solvent. The peptides were cleaved from the resin and deprotected with liquid HF and purified by preparative HPLC. Analysis of the products was by analytical HPLC, AAA and FAB-MS (Table I). The method for the synthesis of the C-terminal peptide alcohols arose from the need for a solid-phase synthetic method for peptides having unusual amino secondary alcohols 3-substituted 3-amino-Z-hydroxy(e.g., l,l,l-trifluoropropanes) as a C-terminal residue. These types of analogs are intermediates that upon oxidation become trifluoromethyl ketones, which are serine protease mechanism-based enzyme inhibitors. In general, a solid-phase technique using Boc-N” protected amino acids for the synthesis of C-terminal peptide alcohols would be useful for the following reasons: 1) All desired analogs would be accessible without the potential problem of the solution characteristics of the intermediates. 2) The time to perform a synthesis

ASX

21x

Pro

Ala

DFBBIPBBYL#

Leu

Phe

0.94(l) --_ 0.93(l) --

1322 1322

1.00(l) 0.99(l) 1.00(l) 0.99(l) 0.99(l) 0.99(l)

1323

1321

1321

1322

1323

1321 1324

1322 __a

1322

1322 -me

1269

1270

1282

1340

1354

1355 1340

1411

1412

1282

0.99(l) 1.00(l) 0.99(l)

0.99(l) 0.98(l)

calcd.

%I

C-terminalalcohol functionalityand lower case indicatinga g-amino acid.

2.9

2.0

0.7

0.4

1.9

23 0.3;

20

16

18

7.1

(H+E)' Dol. vt. (lw

PAB-Mb

m-e

0.99(l) --_ 0.98(l) --_

l-

'Singleletteramino acid code is used with Sue indicatingN"-succinyl,# indicatinga C-terminalamide, -01 indicatinga

bPast-atoabombardmentmass spectroaetry: (l4+E)* * 1 m.u.

LYS

1.00(l) ___ 0.98(l) ___

Tyr

0.96(l) 1.02(l) 1.00(l) 1.02(l) 0.95(l) --_ 0.99(l) 0.98(l) --0.94(l) 0.97(l) 0.96(l)

3.08(3) 2.00(2) 0.98(l) 0.95(l) --3.10(3) 2.00(2) 0.99(l) 0.94(l) m-0

'Aminoacid analysis (6 N BCl; 24 hr; 106°C)

LC SucYEPIPEEAPB# --11 SucYBPIPEBAPk# ---

5

Ile

MAa

0.97(l) 1.02(l) 0.98(l) 1.00(l) 5.01(5) 1.01(l) DFBBIPEEYLA 1.01(l) 4.02(4) 1.04(l) 1.01(l) 0.97(l) 0.98(l) 0.99(l) 0.95(l) 1.05(l) 0.99(l) DPBBIPEBYLA-o:1.00(l) 4.08(4) 0.95(l) _--_0.95(l) --_ 0.99(l) DFEBIPEEYL-01 0.99(l) 4.05(4) 1.04(l)

DPEEIPBEYLO

Sequence’

T

1.00(l) 4.02(4) 0.99(l) --_ 4.12(4) 2.00(2) 0.97(l) 6 SucYEPIPEBAPO --_ ___ 4.18(4) 1.96(2) 0.98(l) 7 SucYBPIPBEAPO# 4.11(4) 2.01(2) 0.97(l) 0 SucYEPIPEEAPE --3.09(3) 2.03(2) 0.97(l) 9 SucYEPIPBEAPB _-_

4

3

2

1

#

-

TABLE I ANALYYICALANDPIlDtIH-Cl#Y~ITIDNMTA

Vol. 54, No. 4

would be decreased. peptide synthesizers. available.

C-TERMINAL PEPTIDE ALCOHOL...

3) Much of 4) All of

323

the process could be automated in existing the protected amino acids are commercially

Stewart and Morris have reported the reductive cleavage of peptides linked by benzyl esters to solid-phase supports to produce primary peptide alcohols (14). However, this method cannot be used in peptides containing benzyl protected acidic residues. It also is not applicable to the synthesis of secondary or tertiary peptide alcohols. Initially we examined the direct attachment of the hydroxyl group of the C-terminal amino alcohol to the resin as a benzyl ether. While this method may be useful for most amino alcohols, the presence of the trifluoromethyl group caused the alkoxides of these amino alcohols to poorly substitute a Merrifield resin. Peptide alcohol products were obtained from these types of resins but only in very limited amounts (unpublished results). Therefore a method that did not rely on the direct attachment of the C-terminal amino alcohol to the resin was needed. Kaiser an coworkers reported on the use of p-nitrobenzhydrylideneisonitroso resin (oxime resin) for the synthesis of protected peptide fragments to be used for a fragment coupling approach to large peptides (15-17). Since the active ester linkage of the peptide to the resin enables displacement of the protected peptide with nucleophfles (15), we sought to extend the use of these resins as a method for the solid-phase synthesis of peptide alcohols. Indeed, it was possible to obtain Ac-Pro-PheAla-Vfa and Ac-Ile-Phe-Ala-Vfa in 18% and 16% isolated yields after HPLC 3-amino-2-hydroxy-4-methylpreparative purification (Vfa l,l,l-trifluoropentane). These results were Extended to the synthesis of Cterminal peptide alcohol derivatives of hirudin and the syntheses were performed on an automated peptide synthesizer wR&v programming was adapted for the use with these resins. The best displacement conditions were using 1.5 to 2 equivalents (based on resin weight gain) of the amino alcohol in DCM that contains 0.8 equivalents acetic acid as a catalyst for 16 h at room temperature. As had been previously noted by Kaiser and coworkers (15-17), one limitation of the use of these resins is that the length of the peptides produced is limited by the loss of peptide from the resin during synthesis due to lability of the resin linkage. With the hirudin peptides there was approximately a 45% loss of peptide during the synthesis based on resin weight gain. Fibrin-clot Inhibition. Compounds l-5 represent a series of analogs based on the native desulfato hirudin fr&Gent 54-65 (Comnound 1). This Cterminal fragment of hirudin represents oniyone binding domain or-the native protein and therefore has only a fraction of the antithrombin potency of hirudin (K = 50 PM, ref. 6). Compounds 6-11 are a series of C-terminally modified pkptides based on a more optimize1 sequence, 4, that resulted from previous structure-activity relationship studies (7-10). Table I gives the IC values for the analogs in an in vitro a-thrombin-induced fibrin&ot i&!bition assay. Removal of the -hydrophilic side chain of Gln by replacement with Ala (Compound 2) resulted in a 2.5-fold loss ofsurtithrombin potency. Replacement of this neutral hydrophilic residue (Gln of 6) with an anionic side chain (Glu, Compound g) resulted in virtually no chinge in potency. However, replacement with a cationic side chain (Lys, Compound 9) gave a 2-fold loss of potency. The optimal functionality of the C-terminus for antithrombin potency was found to be either an acid or alcohol (2 vs 3). Peptides amides were active, but had reduced potency (6 vs L; 9 vs IO). -It was found that a D-amino acid would be tolerated at position 65, butwith a slight loss of poTency (10 vs 11). Truncation of the peptide by removal of the residue at position s andTermination of the sequence of the peptide as an alcohol or amide gave analogs, 2 and 2, with potency similar to that of

324

C-TERMINAL PEPTIDE ALCOHOL...

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the Ala65 analog, 2, again pointing to the advantage of having a hydrophilic residue at the C-t&-minus. Initial ex vivo studies on analogs of this series and analogs related to them indicates&?-effective anticoagulant levels can be achieved (Dr. Robert J. Broersma, unpublished results). For example, compounds 10 and 11 after intravenous administration at 10 mg/kg into rats produce a zgree 3 anticoagulation and duration profile equivalent to 0.1 mg/kg of hirudin. The effectiveness of these materials in models of disseminated intravascular coagulation and venous thrombosis is currently being studied. In summary, neutral or negatively charged amino acids or amino alcohols were tolerated at position 65 of these hirudin C-terminal fragment analogs. No modification that dramatically increased potency was found. The relative ex vivo and in vivo effectiveness of these compounds is currently being -assessed in airattempt to judge the potential pharmacodynamic benefits of unnatural modifications at the C-terminus. In view of the ability of the Cterminus to tolerate modification, it is interesting that the C-terminal carboxamide analogs had significantly reduced pot$qcy. Fragment analogs of another hirudin variant, hirudin PA and desAsp -hirudin PA , which are equipotent show that C-termi&?6 extension is permissi&66 in these peptides and that the C-terminus itself is not likely to be involved in an interaction with a-thrombin (10). Therefore, the reason for the lessened potency that results from the amide modification is unclear. It is clear that a neutral or negatively charged hydrophilic side chain at position 65 contributes significantly to the potency-of these peptides. ACKUO-S

We thank Drs. John Coutant and Brad Ackerman for Sue Treadway for assistance with the manuscript.

the FAB-MS analyses

and

REFERENCES

l.MARKWARDT,F. Approaches for designing synthetic, low molecular weight inhibitors of clotting enzymes. Folia Raematol. (Leipzig)109, 7-15, 1982. 2.ESMON, N. Thrombomodulin. 454-463, 1987.

Seminars

3.KRSTENANSKY,J.L. and MAO, S.J.T. hirudin using synthetic unsulfated 10-16, 1987.

in

Thrombosis

and

Antithrombin properties N”-acetyl-hirudinls_65.

Bemostasis

13,

of C-terminus

of

PEBS Lett. 211,

and KRSTENANSKY, J.L. Interaction of 4.MA0, S.J.T., YATES, M.T., OWEN, T.J. hirudin with thrombin: Identification of a minimal binding domain of hirudin that inhibits clotting activity. Biochemistry27, 8170-8173, 1988. 5.KONN0, S., FENTONII, J.W. and VILLANUEVA,G.B. Analysis of the secondary structure of hirudin and the mechanism of its action with thrombin. Arch.

Biochem.Biophys. 267, 158-166, 1988. 6.MARKWARDT,F. Biochemistry

and pharmacology Markland,

Animal Venoms H. Pirkle and P.S. Dekker, 1988, pp. 255-269.

of hirudin. In: Bemostasisand (Eds.) New York: Marcel Jr.

7.KRSTENANSKY,J.L., OWEN,T.J., YATES, M.T. and MAO, S.J.T. Anticoagulant peptides: Nature of the interaction of the C-terminal region of hirudin with 1987. a non-catalytic binding site on thrombin. J. Med. Chew 30, 1688-1691,

Vol.

54, No. 4

C-TERMINALPEPTIDEALCOHOL...

B.KRSTENANSKY,J.L., MAO, S.J.T., of the interaction of thrombin anticoagulant peptide hirudin.

Proceedingsof

Leiden:

325

OWEN, T.J. and YATES, M.T. Characterization

with In:

the

C-terminal

region

of

the leech

Peptides: Chemistry and Biology: the Tenth American Peptide Symposium G.R. Marshall (Ed.)

Escom, 1988,

pp. 447-448.

9.0WEN, T.J., KRSTENANSKY, J.L., YATES, M.T. and MAO, S.J.T. N-Terminal requirements of small peptide anticoagulants based on hirudin54_65. J. Hed. Chem. 31, 1009-1011, 1988. lO.KRSTENANSKY,J.L., OWEN,T.J., YATES, M.T. and MAO, S.J.T. Comparison of analogs as hirudin and hirudin PA C-terminal fragments and related antithrombin agents. Throa. Res. 52, 137-141,1988. ll.DUTTA,

A.S.

Agonists

and antagonists

of

substance

P. Drugs of the Future

12, 781-792, 1987. lZ.CHANG,R.S.L., LOTTI, V.J., CHEN, T.-B., CERINO, D.J. and KLING, P.J. Neuropeptide Y (NPY) binding sites in rat brain labeled with 1251-BoltonHunter NPY: Comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens. Life Sci. 37, 2111-2122,1985. 13.ROEMER, D. and PLESS, J. Structure activity relationship of orally enkephalin analogues as analgesics. Life Sci. 24, 621-624, 1979. 14.STEWART, J.M. and MORRIS, D.H. Synthesis phase method. U.S. Patent 4,254,023,1981.

of peptide

alcohols

active

by the solid

lS.DEGRADO,W.F.and KAISER, E.T.

Polymer-bound oxime esters as supports for peptide synthesis. Preparation of protected peptide fragments. J. Org. Chem. 45, 1295-1300, 1980. solid-phase

16.NAKAGAWA,S.H. and KAISER, E.T. Synthesis of protected peptide segments and their assembly on a polymer-bound oxime: Application to the synthesis of a peptide model for plasma apolipoprotein A-I. J. Org. Cher. 48, 678-685, 1983. 17.NAKAGAWA,S.H., LAU, H.S.H., KEZDY, F.J. and KAISER, E.T. The use of polymer-bound oximes for the synthesis of large peptides usable in segment condensation: Synthesis of a 44 amino acid amphiphilic peptide model of apolipoprotein A-l. J. Am. Chem. Sot. 107, 7087-7092, 1985.