AP313A

AP313A - New isosteric peptides.

Info

Publication number: AP313A
Application number: APAP/P/1992/000418A
Authority: AP
Inventors: Butrus Atrash; David Michael Jones; Micheal Szelke
Original assignee: Ab Astra
Priority date: 1991-08-28
Filing date: 1992-08-17
Publication date: 1994-02-10
Also published as: BG98583A; EP0605462A1; MX9204767A; HU9400589D0; JPH06510059A; CN1069736A; AU2499092A; SI9200193A; MA22632A1; TNSN92076A1; TW221816B; ATE212643T1; DZ1613A1; FI940945A; AP9200418A0; IL102840A0; CA2116527A1; WO1993005069A1; EP0605462B1; NO940669L

Abstract

This invention relates to new competitive inhibitors of thrombin, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic diseases.\

Description

DESCRIPTION

This invention relates to new competitive inhibitors of thrombin, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic diseases such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, e.g. following angioplasty and coronary bypass operations.

BACKGROUND

Blood coagulation is the key process involved in both haemostasis (i.e. prevention of blood loss from a damaged vessel) and thrombosis (i.e. the pathological occlusion of a blood vessel by a blood clot). Coagulation is the result of a complex series of enzymatic reactions outlined in the Scheme 1 below where the various clotting factors are designated by Roman numerals .

Thrombin plays a central role in coagulation, whether the process is initiated by the intrinsic or extrinsic pathways: it activates platelets, it converts fibrinogen into fibrin monomers, which polymerise spontaneously into filaments, and it activates FXIII, which in turn crosslinks the polymer to insoluble fibrin. Thrombin further activates FV and FVIII in a positive feedback reaction.

BAD ORIGINAL

INTRINSIC

SYSTEM

F XII

FXI

Scheme 1 ho

EXTRINSIC

ProthromDin F II

Thrombin

^r XII/

AP 0 0 0 3 1 3

ART

The first inhibitors of thrombin based on electrophilic ketones were developed as described by M. Szelke and C.M. Tones in ΞΡ-Α1-0,118,280 , G3 priority date 4th March 1983. These earlier compounds were derived from the P-j - ?2' pentapeptide sequence of the fibrinogen Aa chain in which the scissile P^ - Pj_' peptide bond was replaced with the -CO-CH2-moiety, forming a keto isostere to the corresponding peptides.

Other known examples of serine proteinase inhibitors based on electrophilic ketones include the following:

(a) M. Kolb et al. (Merrel-Dow) EP-A2-0,195,212 (Priority date 4.2.86) describing peptidic a-keto esters and amides, (b) B. Imperial! and R.H. Abeles, Biochemistry 1986.

25. 3760 (peptidyl fluoroalkyl ketones), (c) Ueda et al., Biochem. J. 1990. 265. 539 (peptidyl fluoroalkyl ketones).

(d) D. Schirlin et al. (Merrel-Dow) EP-A1-0,362,002 (priority date 1.9.88) describing fluoroalkylamide ketones .

(e) P. Bey et al. (Merrel-Dow) EP-A2-0,364,344 (priority date 7.10.88) describing α,β, δ-triketo compounds .

(f) E.N. Shaw et al. (Research Corporation) US4,318,904 (priority date 25.04.80) describing peptide chloro-methyl ketones e.g. H-DPhe-Pro-Arg-Ci^Cl

Inhibitors of thrombin based on peptide aldehydes have been reported by S. Bajusz et al. in J. Med. Chem.

1990. 33. 1729, and in (Richter Gedeon Vegyeszeti Gyar R T) EP-A2-0,185,390 (priority date 21.12.84).Thrombin

BAD ORIGINAL :

,1 *Ί it:r3 is p-ptides uprising C - --m n a 1 borcnic derivatives ot arginine and isot hi ;nium anal agues there-?: have been reported t .-..2-. Xettner et al. (Du Pont) EP-A2-0,293,831 (priority dates 5.6.87 and 6.4 . 33) .

There are examples of thrombin inhibitory arginine derivatives or analogues not containing an electrophilic ketone, e.g.:

(a) S. Okamoto et al. (Mitsubishi Chemical Industries

Ltd.) ΞΡ-Α1-0,008,746 (priority date 31.08.78) describing ary 1sulphony1 arginine amides e.g. argatroban.

'bi J. Stursebecher et al., Pharraazie 1981. 36. 639 (arylsulphonyl p-amidinophenylalanine amides).

An object of the present invention is to provide novel and potent thrombin inhibitors with competitive inhibitory activity towards their enzyme i.e. causing reversible inhibition. A further object is to obtain inhibitors which are orally bioavailable and selective in inhibiting thrombin over other serine proteases. Stability, duration of action, and low toxicity at cherapeutic dosages are still further objects of the invention .

DISCLOSURE OF THE INVENTION

Compounds

It has been found that compounds of the general formula _1, either as such or in the form of physiologically acceptable salts and including stereoisomers, are potent inhibitors of thrombin:

NH

AP'O Ο Ο 3 1 3

1C

R

,R<

In formula 1., and when occurring below unless specified otherwise, the following applies:

A represents -CH₂-, -CH=CH-, -CH₂-CH₂- or -CH₂-CH₂-CH₂ j ->

and R“ are the same or different and each represents H or X-Β-, where S is a straight or branched alkylene group having 1-3 carbon atoms and X is H, methyl, ethyl, a cycloalkyl group having 3-6 carbon atoms or R'CO-, where R' is OH, a straight or branched alkoxy group having 1-4 carbon atoms, NH₂ or NHR'', where R' ' is a straight or branched alkyl group having 1-4 carbon atoms, or X is a carboxylic acid mimic, known per se, selected from -PO(OR''')₂, -SO-jH and 5-(IH)-tetrazolyl, and R' ' ' is H, methyl or ethyl, or B is -SO₂~ and X is methyl or ethyl;

m is 0, 1 or 2, R^ represents a cyclohexyl group and

R^^a represents H; or

BAD ORIGINAL i ' Hi 0 0 9A

i.od P' repres

sents 0 or S ί 01 , where p is 0, 1 or 2;

R“ represents H; a straight or branched alkyl or a cycloalkyl having 1 to 6 carbon atoms unsubstituted or substituted with one or more fluoro atoms and/or substituted with a phenyl group; a substituted or unsubstituted aromatic ring selected from phenyl,

4-methoxy-phenyl, 4-tertiary-butyl-phenyl,

4-methyl-phenyl, 2-, 3- or 4-tri fluoro-methyl-phenyl, phenyl substituted with 1-5 fluoro atoms; or -CH ' ΙΡβ ) -phenyl .

Compounds of formula 1. relate to the peptide sequence of human fibrinogen Aa chain representing modified subsites P₃ - ^p]_'^:

H-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala10

According to a preferred embodiment the invention relates to compounds of Formula X, wherein

A represents -CH2-CH2- or -CH2-CH2-CH2-;

R·'· is H and R^ represents HOCO(CH₂)_nCH^CH-OCO(CH₂)_n-, and n is 1 or 2;

or

AP0 00 3 1 3

Y 13 Ο η is ϊ, R^J represents a cyclohexyl group and R^Jrtrepresents n;

Particularly advantageous embodiments of the invention are represented by the compounds:

HOOC-CH₂-DCha-Pro-Arg-CH₂ -O-CH₂-CF₃

HOOC-CH₂-DCha-Pic-Arg-CH₂-O-CH₂-CF₃

HOOC-CH₂-DCha-Pic-Arg-CH₂-O-nBu

H00C-CH₂-CH₂-DCha-Pro-Arg-CH₂-0-nBu HOOC-CH₂-CH₂-DCha-Pro-Arg-CH₂-0-CH₂ ~CF₃

HCOC-CH₂-DCha-Pro~Arg-CH₂-0-nBu

Medical and pharmaceutical use

In a further embodiment the invention relates to treatment, in a human or animal organism, of conditions where inhibition of thrombin is required. The compounds of the invention are expected to be useful in particular in animals including man in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disease states in which the compounds have a potential utility, in treatment and/or prophylaxis, include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis. Further, the compounds have expected utility in prophylaxis of atherosclerotic diseases such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds are expected to be useful together with thrombolytics in myocardial infarction. Further, the compounds have expected

bad orig>^nal o

in prophylaxis fcr reccclusicn hr

have expected utility in prevention of rethrombosis after microsurgery. Further, the compounds are expected to be useful m anticoagulant treatment in connection with artificial organs and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in haemodialysis and disseminated intravascular coagulation. The daily dosage will normally be within 0.1 mg to 10 g of active ingredient. Intravenous solutions preferably contain 0.1-100 mg/ml, while a dosage unit preferably contains 1-1000 mg and is preferably administered 1-4 times a day.

A further expected utility is in rinsing of catheters and mechanical devises used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro.

Preparation

A further objective of the invention is the mode of preparation of the compounds of the invention. Thus the invention further relates to a process for preparation of compounds according to formula _1, which process comprises (method I) displacement by R⁴Y (Y=O,S) of the halogen of a halomethylketone of the formula

N(W*)

H

L

O

Halide

AP 0 00 3 1 3

S';cr. as benzyloxy carbonyl, (as illustrated in

Procedures (A), (D) and (F)), reduction of the ketone to alcohol, removal of the amino terminal protecting group, standard peptide coupling, followed by oxidation of the alcohol, giving the protected tripeptide ketone, removal of the amino terminal protecting group, followed by N-alkylation, (as illustrated in Procedures (A), (B) , (G) and (H)), and deprotection, or replacing a protected dipeptide in the coupling reaction referred to above and illustrated (eg. Procedure (A) (iii) ), with an amino-terminal-N-alkylated-N-tri fluoroacyl-protected dipeptide (Procedure K), followed by oxidation and deprotection, or (method II) alkylation, with an R⁴halide, of an α-ketol of the formula

w/w²) (W²)HN

ΌΗ

O

H

L wherein W^x and W are as defined above, (as illustrated in Procedure (E)), and then further reacting as in method I, or (method III) by using the modified DakinWest reaction, Angew. Chem. Int. Ed. Engl. 8. (1969) 981, as applied to tripeptides, J. Org. Chem.,50 (1985)1112: reacting a compound of the formula (or alternatively to directly use an amino-terminal-NBAD ORIGINAL

9 wherein W and W are as defined above, with (TCH₂CO)₂θ wherein T is halogen, R^O or R^S and 4-DMAP, and then further reacting as in method I.

In those cases where the reaction results in a mixture of stereoisomers, these are optionally separated by standard chromatographic or re-crystallisation techniques, and if desired a single stereoisomer is isolated .

DETAILED DESCRIPTION OF THE INVENTION

The following description is illustrative of aspects of the invention.

2 Synthesis and pharmacy

The chemistry used to prepare the inhibitors claimed here is outlined in the Synthesis schemes (Procedures A to H) and Procedures I and K. The necessary alkoxy- or phenoxy-methyl ketones of arginine were mainly prepared

cP

AP 0 0 0 3 1 3 by (Σ'· displacement by R⁴O- of the Br of bromomethylketones , either using preformed NaOR⁴(Procedure A) or by the use of KF (Procedures D and F) or (ΙΣ) by alkylation of arginine α-ketol using Ag2O/R⁴I (see Procedure Ξ).

Standard peptide coupling reactions were used to introduce DCha, Pro and their analogues. The carboxyalkyl group on the N-terminus was introduced either by alkylation using bromo-acetates or by Michael addition to tertiary butyl-acrylate (Procedures A, B) or by using pre-incorporation of carboxy-alkyl in the dipeptide moiety (see Procedure K). All protecting groups were then removed (see Deprotection Procedures a-c below).

General experimental procedures:

Standard work-up refers to ethyl acetate extractions, usually washing with 0.3M KHSO^, 1M KHCO3, H2O and brine followed by filtration through Whatman Phase Separatory paper and drying by toluene azeotroping. TLC was carried out on commercial Merck Silicagel 60F254 coated glass plates. Visualization was by a combination of UV light, heating followed by fluorescamine spray or heating followed by chlorination (CI2 tank) and spraying with 1% starch/KI solution. Flash chromatography was carried out on Merck Silicagel 60 (40-63 μπϋ under pressure of Amino acid analysis was performed using the Beckman Gold System. Peptides were hydrolysed (6N HC1 + phenol at 110°C for 22 h) then injected and the proline peak quantified. MPLC was carried out in glass columns (Anachem) packed with Vydac C18 15-25 gm silica, using gradients of 1% TFAMeCN into 1% TFA-H2O with monitoring at 226 nm.

BAD ORIGINAL 4 ^τ Μ, Π ί: ΜΑ

F Γ ;i c	tiers were an.	a 1 y c e i by H.- L 2 and t	he pure	on
pcc i	ed and Iyophi	11 s e c . t. r L _ v.as c a r r	red out	us	mg a
0 C C z	tra-Physics 3	7 0 C Series chr oma t : g	raphy st	at	1 0 c
3 0 l· ’/	ent system as	for MRLC with detec	tio.n at	21	0 nm .
F1 c>;	1.5 ml/mm.	Column.· Nsvapak CIS,	4 pm (S	x	100 1
cart	ridge, Waters	). All intermediates	were

characterised by NMR (Hitachi-Perkin Elmer R24 60 MHz or Jeol 2^0 MHz instruments). All final peptides were characterised by their EA3 mass spectra (Μ-Scan Ascot, Berks ., U.K.) .

Preparation of starting materials:

3oc-A.rg (Z₂ ) -CH₂ ^3r '· (i) Boc-Arg(Z₂)-OH (10 mmol) in dry TH? (50 ml) and NMM (10 mmol) was cooled to -10°C and iBC (10 mmol) added dropwise keeping the temp. X -10°C. After 10 min at -10°C the mixed anhydride was poured into CH₂N₂-ether (25 mmol in 150 ml) . After 3h excess CH₂N₂ was destroyed with acetic acid and the solution washed with H₂O (3 x) and brine. Drying and evaporation gave the diazoketone as a yellow oil. IR 2100 cm-1 (COCHNo).

(ii) The diazoketone (10 mmol) in dry ethyl acetate (200 ml) was cooled to -15°C and 1 M H3r/ethyl acetate (about 11 ml) added dropwise. When the yellow colour was discharged TLC (ethyl acetate/hexane) showed complete conversion of the diazoketone to bromomethyl ketone. The solution was rapidly transferred to a separating funnel and washed with 1 M KHCO3, brine, dried and evaporated to leave a solid. Dissolution in hot EtOH and cooling gave the bromomethyl ketone as an amorphous white powder.

NMR (CDC1₃): δ 1.3 (5, 9H), 1.5-1.85 (m, 4H) , 3.75-3.95 (m + s, 4H), 4.3 (m, 1H) , 5.05 (s, 2H) , 5.15 (s, 2H),

AP η η 0 3 1 3

5.65 id, 2H), 7.30 (m, 10H), 9.2 (br, s, IH), 9.3 (br, s, IK). Melting point: Softens at 50°C then slowly decomposed >70°C.

3oc-DCha-X-0NSu (X = Pro, Pic or Aze):

(i) Boc-DCha-OH (10 mmol) in CH₂C1₂/DMF (1:5 50 ml) was treated with HONSu (11 mmol), cooled to O°C and WSCDI (13 mmol) added. After 30 min it was warmed to room temperature. TLC after 3h showed complete formation of Boc-DCha-ONSu. Addition of Et₂O (200 ml) and washing with H₂O (3 x) , brine, drying gave the ester as a colourless foam.

(ii) The N-hydroxysuccinimido ester (10 mmol) in

CH₂C1₂ (50 ml) was treated with H-Pro-OBzl.HC1 or HPic-OBzl.HCl or H-Aze-OBzl (11 mmol) and iPr₂NEt (20 mmol). After stirring for 3h, standard ethyl acetate/0.3 M KHSO^ work-up gave the dipeptide ester which was pure enough to be used in the next step.

(iii) The Boc-DCha-X-OBzl in THF was hydrogenated over 5% Pd/C at STP for 4h. Filtration and evaporation gave the acid as a solid or foam. Re-crystallisation (iPr₂O or Et₂O/hexane) gave the pure products.

Boc-DCha-Pro-OH (solid m.p. 163 - 166°C): NMR (CDCl-j) δ 0.8-2.05 (m, + s at 1.4, 26H), 3.4 (m, IH), 3.85 (m,

IH), 4.5 (m, 2H), 5.2 (m, IH).

Boc-DCha-Pic-OH (solid m.p. 121-122°C): NMR (CDCl^) δ 0.8-2.05 (m, + s at 1.45, 28H), 3.35 (m, IH), 3.95 (m, IH), 4.6-4.9 (m, IH) , 5.4 (m, IH) , 5.6 (m, IH), 8.8 (br, s, IH) .

HAD ORIGINAL

* f. ^f’ ^{} MA

Bos-,.Me)DCha- Pro -OMCu:

Boc-.'Met DPhe-CH was hydrogenated over 5¾ Fth-C in ?G% acetic acid-H^O at 0.41 MPa for 3 days giving quantitative yield of Boc-'Me)DCha-CH. Boc-(Me)DCha-CK (10 tncl) and MMM (10 mmol) in Cl^Ci? (50 ml) was cooled to -15^0 and Pl^PO-Cl (10 mmol) added. After 20 min, H-Pro-OBzl.HCl (11 mmol) and NMM (20 mmol) was added. After Ih it was allowed to warm to room temperature. After 2h standard work-up and flash chromatography (40% ethyl acetate/hexar.e) gave pure Boc-(Me)DCha-Pro-OBzl as a colourless oil (80%).

MMR (CDC1₃): δ 0.5-2.2 (m, + s at 1.4, 26H) 2.65 (s, 3H), 3.5 (m, 2H), 4.3-5.0 (m, 2H) , 5.1 (5, 2H) , 7.30 (s, 5H).

This was converted into the N-hydroxysuccinimido ester as described for Boc-DCha-X-ONSu below.

Synthesis schemes

Procedure (A)

8oc-Arg(Z₂)-CH₂Br

Boc-DCha-X-N OR⁴

H OH ;iv)

AP 0 0 0 3 1 3 'OR⁴ (i)

CMP_| •20° (iii)

1) HCl-dioxan

Dess-Martin penodinane

Boc-Arg(Z₂)-CH₂OR⁴e.g. R⁴- CH₂CF₃. CH(Ph)CF₃Aryl

2) Boc-DCha-X-ONSu or (Boc-(Me)DCha-X-ONSuJ (·')

NaBH< MeOH. 0^c

Boc-N^V^OR⁴H OH (v)

1) HCl-dioxan

2) RO₂CCH₂Br

Boc - DC ha - X - Arg (Z₂) - C H₂O R⁴ -► or iPr₂NEl,MeCN. Δ (Boc-(Me)DCha-X-Arg(Z₂)-CH₂OR⁴) or omit or Procedure G or Procedure H

X - Pro. Pic, Aze

R*. (CH₂)₃-NZ-C(NH)-NHZ

Y-DCha-X-Arg(Z₂)-CH₂ORⁱ

Y-H, RO^CHj (RO₂CCH₂)₂(R=Bzl,tBu or Et) procedure .(BI

1)

2)

Boc-DCha-X-Arg(Z₂)-CH₂OR⁴ 3)

HCl-dioxan

ElOAc/

M KHCO₃ .

-tBuO₂CCH₂CH₂-DCha-X-Arg(Z₂)-CH₂OR tBu-acrylate

MeOH, Δ e.g. R = nBu,CH₂CF₃ bad ORIGINAL ft > f I u ν V M/4 16

P'ocedure fC) i Rh_?(OAc)j

Boc-Arg(Z₂)-CHN₂ ♦ HOR⁴ __fe.g. R*= CH₂CF₃.CH(Ph)CF3,CH(CF₃)₂

Boc-Arg(Z₂)-CH₂OR⁴

Then continue as in Procedure (A)

Procedure (D)

Boc-Arg(Z₂)-OH (')

CI₃CCH₂-OH

CH₂C1₂

WSCDI

4-DWAP

Boc-Arg(Z₂)-OTce (ii)

1) HCI-dioxan

2) Boc-DCha-X-ONSu

3) Zn-HOAc (hi)

1) NMM.iBC.THF

2) CH₂N₂-ether

Boc-DCha-X-Arg(Z₂)-OH -► Boc-DCha-X-Arg(Z₂)-CH₂Br

3) HBr-EtOAc.-10° (iv)

CNF

R*YH

KF.RT.24h

Boc-DCha-X-Arg(Z₂)-CH₂YR⁴

Then continue as in Procedure (A)

Y= o.s

R⁴- nBu.Aryl

For Example 1: R⁴YH=HO₂CCOPh

BADOB'^g'^NAL S

APO 00 3 i 3

Procedui&JE) (»)

KF

Boc-Arg(Z₂)-CH₂Br ♦ HO₂CCOPh

Boc-Arg(Z₂)-CH₂O₂CCOPh

CNF

3h (ii)

THF

1M KHCO3

-►

24h

Boc-Arg(Z₂j-CH₂OH (iii) Ag~O ch₂ci₂-►Boc Arg(Z₂)-CH₂OR⁴e g. R⁴«Me.Ei.nPr,nBu

Then continue as in Procec

5 Procedure (Fl

Boc-Arg(Z₂)-CH₂Br * R⁴CH	KF	8oc-Arg(Z₂)-CH₂OR⁴
DMF,24h
e.g R = CH₂CF₃, CH₂(CF₂)₂CF3.		Then continue as in Procedure
Ph(4-OMe)

BAD ORIGINAL ft ί t ξ 0 ii 0 RA

Procedure IG)

1) HCI-dioxan

3oc-DCha-X-Arg(Z₂)-CH₂OR⁴ -►

2) MeSO₂Ci

We - SO₂- DCha- X· Arg (Z ₂) -C H₂C P e.g. R⁴-CH₂CF₃ c „

Procedure rH)

1) HCI-dioxan

Boc-DCha-X-Arg(Z₂)-CH₂OR⁴ -*- ChCH₂-DCha-X-Arg(Z₂)-CH₂OF.^J

2) ChCHO/NaCNBH₃ .

e.g. R - CH₂CF₃

AP 0 0 0 3 1 3 •9

Preparation procedures:

The following preparation procedures illustrate the above methods I-III as well as subsequent steps to final compounds.

Procedure (A) (i) Boc-Arg(Z₂)-CH₂Br (10 mmol) was added as a solid to a preformed solution of the alkoxide or phenoxide (alcohol or phenol 10 mmol and 80% NaH-oil, 10 mmol) in DMF (40 ml) at -20°C under N₂ . After 30 min the solution was warmed to room temperature. 2 hours later 0.3 M KHSO4 was added to neutralize any alkoxide remaining and the DMF removed under vacuum. The crude product was partitioned between ethyl acetate and H₂O, the ethyl acetate layer washed with brine, dried and evaporated. Flash chromatography or crystallisation gave the pure alkoxyketones.

Boc-Arg(Z₂)-CH₂OPh (solid): NMR (CDCI3): δ 1.41 (s,

10H)	, 1.64 -	1.68	(m,	3H) ,	3.92 (dd, 2H)	, 4.	,5 (m, 1H),
4.62	(q, 2H)	, 5.1	(s,	2H) ,	5.2 (s, 2H),	5.5	(d, 1H),
6.8	(d, 2H),	6.95	(t,	1H) ,	7.2-7.45 (m,	12H)	, 9.2 (br,

s, 1H), 9.3 (br, s, 1H). Melting point 115-118°C.

Boc-Arg (Z₂) -CH₂OCH₂CF₃ (solid): NMR (CDCl-j): δ 1.35 (s, 10H), 1.55-1.75 (m, 3H), 3.7 (q, 2H), 3.85 (m, 2H), 4.2 (q + m, 3H), 5.05 (s, 2H), 5.15 (s, 2H), 5.7 (d, 1H), 7.15-7.35 (m, 10H), 9.15 (br, s, 1H), 9.3 (br, s, 1H) . Melting point 87-90°C.

(ii) The alkoxymethyl or phenoxymethyl ketone in MeOH/THF (1:1) at O°C was treated with NaBH^ (1 equiv.). After 10 min, 0.3 M KHSO₄ was added to pH 7

BAD ORIGINAL ft ar.d the mixture evaporated to remove MeOH/THE. Ethyl acetate was added ar.d after standard work-up (ethyl acetate/0.3 M KHSO^) the alcohol was isolated as a di astereorr.er ic mixture.

(iii) The alcohol was treated with 4M HC1 in dioxan for 15 min at room temperature and evaporated. The residue in CH2CI2 (1 mmol in 5 ml) was treated with Boc-DCha-XONSu (1 equiv.) and iPr₂NEt (to pH 9 on wet pH paper). After 3h, standard work-up gave the modified tripeptide which was purified by flash chromatography (ethyl acetate-hexane mixtures containing 1% acetic acid). Yield: 50-85%.

(iv) The tripeptide alcohol in CH2CI2 was treated with Cess-Martin periodinane (3 equiv.) (Dess, D.B. and Martin, J.C. J. Crg. Chem. 1983, 48. 4155-4156). After 2'n stirring at room temperature, standard work-up (Et₂O/l M KHCO3/Na2S₂O2) gave the crude tripeptide ketones which were purified by flash chromatography (ethyl acetate-hexane).

(v) The ketone was treated with 4 M HCl-dioxan for 15 min at room temperature and evaporated. The residue in dry MeCN (1 mmol in 5ml) was treated with benzylbromoacetate or tertiary butyl bromoacetate (1.2 equiv.) and iPr₂NEt (3 equiv.). After reflux for 2h the solution was evaporated and flash columned (ethyl acetate-hexane) giving the benzyloxycarbonylmethyl or tertiary butyloxycarbonylmethyl peptides as oils (40-50%).

In Examples 30 and 31, 2.5 equiv. of the bromoacetate were used to achieve bis-alkylation.

_ba0 o»®**⁴AP 0 0 0 3 1 3

Procedure .'3)

The peptide alkoxymethylketone was treated with excess 4 M HCl-dioxan for 15 min at room temperature. Evaporation gave the HCl salt which was partitioned between ethyl acetate and 1 M KHCO^ · The ethyl acetate was separated, dried and evaporated giving the free amine which was taken up in MeOH and freshly distilled tertiary butylacrylate added (1.5 equiv.). Reflux for 4h gave the tertiary butoxycarbonylethyl peptide which was purified by flash chromatography in ethyl acetate/hexane.

Procedure (C)

Boc-Arg(Z2)-CHN2 (1 mmol? dissolved in the alcohol R^OH (5 ml) was treated with Rh^lCAc)^ (cat.). After several hours at room temperature, TLC analysis showed no diazoketone remaining. The alcohol was removed in vacuo and the product isolated by flash chromatography using ethyl acetate/hexane mixtures.

Procedure (D) (i) Boc-Arg(Z2)-OH (10 mmol) in dry CH2CI2 (50 ml) was treated with 2,2,2-trichloroethanol (11 mmol) and 4DMAP (1 mmol), cooled to O°C and WSCDI (13 mmol) added. After 30 min, it was allowed to warm to room temperature and stirred for 24h. Evaporation and partition between ethyl acetate/0.3 M KHSO₄, followed by 3 x washes with 0.3 M KHSO₄, 1 x H₂O, 1 x brine, drying and evaporation gave the Tee ester which was used as such.

(ii) The Tee ester (10 mmol) was treated with 4 M HClBAD ORIGINAL ft , t * dioxar. (50 ml) for 20 min at room temperature and then evaporated. After drying, the residue in CH2CI2 (50 ml) was treated sequentially with Boc-DCha-X-ONSu (10 mmol) (X = Pro, Pic) and i?r₂NEt (to pH 9 on wet pH paper).

After lh, standard work-up (ethyl acetate/0.3 M KHSO4) gave the tripeptide ester as an oil. The Tee ester (10 mmol) in 90% acetic acid-H2O (50 ml) was treated at 5 min intervals with small portions of freshly activated Zn over lh. After a further lh, the mixture was filtered and the solution evaporated. Standard work-up (ethyl acetate/0.3 M KHSO4) gave the tripeptide acid which was purified by flash chromatography on silica (2% aretre acid-ethyl acetate) giving the tripeptide acid as a colourless foam (80% over 3 steps).

(iii) The tripeptide arid was converted into the bremomethyi ketone using the same procedure as that described for Boc-Arg (Z₂ )-C^Br . The tripeptide bromomethyl ketone was obtained as a colourless oil by

20	flash chromatography mixtures .	using ethyl acetate/hexane
	NMR (CDC1₃): δ 0.9 (m), 1.15 (m) , 1.25 (m) , 1.35 (s), 1.6 (m) , 1.85 (m), 2.1 (m) [total 30H] , 3.3 (m, IH),
25	3.7 (m, IH), 3.95 (m, 4.4 (m, IH) , 4.5 (m, 5.2 (s, 2H), 7.2-7.4 2H) .	2H) , 4.15 (s, 2H), 4.25 (m, IH) , IH), 5.0 (d, IH), 5.1 (dd, 2H), (s, m, 10H), 9.2-9.5 (2 br, s,
30	(iv) The tripeptide bromomethyl ketone (1 mmol) in dry DMF (5 ml) was treated with fluorinated alcohol, phenol

or thiol (1.2 mmol) and anhydrous potassium fluoride (1.5 mmol) and stirred at room temperature for 24h. Evaporation followed by standard work-up and flash chromatography gave the tripeptide ketones.

Sr *

AP 0 0 0 3 1 3

Boc-DCha-Pro-Arg(Z₂)-CH₂O-Ph(4-Me): NMR (CDC1₃) δ 0.9

f.m) ,	1.15 (m), 1.25 (m) , 1.35	(s)	, 1	. 6	(m), 1.	85 (m)	/
2 .1	(m) [total 30H) , 2.38 (s,	3H)	, 3	.4	(m, IH)	, 3.9	(m,
IH) ,	4.1 (br, s, 2H) , 4.4 (m,	IH)	, 4	.6	(m, IH)	, 4.7	(m,
IH) ,	4.9 (q, 2H), 6.9 (d, IH),	7 .	15	(d.	IH) , 7	.4-7.5
(m,	2H), 7.45 (s, 10H), 9.4 (br,	s.	IH)	, 9.6 (	br, s,

IH) .

For Example 37 the protected sulphide was oxidised to the sulphone using m-chloroperbenzoic acid in dichloromethane at room temperature.

Procedure (E) (i) Soc-Arg(Z₂)-CH₂Br (10 mmol) and benzoyl formic acid (12 mmol) in DMF (40 ml) were treated with KF (14 mmol). After stirring for 3h the DMF was evaporated and the product partitioned between ethyl acetate/H₂O. Drying and evaporation gave the crude benzoyl formate ester which was purified by crystallisation (CH₂Cl2~ hexane) giving the product as a white solid (86%).

NMR	(CDC1	₂ ) : δ 1.	4 (s, 9H),	1.65-1.9	(m,	4H) ,	3.95 (m,
2H) ,	4.3	(m, IH),	4.95 (q.	2H), 5.15	(ABq	(, 2H)	, 5.25
(s,	2H) ,	5.9 (d,	IH), 7.35	(m, 10H),	7.5	(t, 2	Η) , 7.65
(t,	IH) ,	8.15 (t,	2H), 9.25	(br, s,	IH) ,	9.45	(br, s,

IH). Melting point 130-132°C.

(ii) The benzoyl formate ester (5 mmol) in THF (200 ml) and 1 M KHCO₃ (200 ml) was stirred vigorously at room temperature for 24h. The THF was separated and evaporated and the aqueous phase extracted with ethyl acetate which was combined with the material from the THF. Crystallisation from C^C^-hexane gave the aketol as a white solid (90%) .

BAD ORIGINAL §1 * Μ ο ο a ί,-MR (CDC1₃) : δ 1.4 (s, 3.95 (m, 2 Η) , 4.25 (m, 5.6 (d, IH), 7.35 (m, s, IH). Melting point

9H)	, 1.7	(m, 4 H) ,	2.95 (t, IH),
2H)	, 5.15	(s, 2H>	, 5.25 (s, 2H)
OH) 01-	, 9.25 (br, s, 103°C .	IH), 9.4 (br,

(iii) The α-ketol (1 mmol) in dry CH2CI2 (5 ml) was treated with alkyl iodide (5 to 10 mmol) and silver oxide (2 mmol). The mixture was refluxed in the dark for 2 to 17 hours (e.g. Mel, EtI, nPrl: 2h; nBuI: 5h). Evaporation followed by flash chromatography (ethyl acetate-hexane) gave the alkoxymethyl ketones as colourless oils (50-85%).

3oc-Arg(Z₂)-CH₂°^Et (oil): NMR (CDCI3) . 4 2H!

2H;

s, 9H) , 1.5-1.8 (m, 4H), 3.4 (q, 4.1 (q, 2H), 4.45 (m, IH), 5.15 5.4 (d, IH), 7.35 (m, 10H), 9.25 : δ 1.15 (t, 3H) ,

2H), 3.95 (t, (3, 2 H), 5.25 (s, (br, s, IH), 9.4 (br, s, IH).

Boc-Arg(Z₂)-CH₂NBu (oil): NMR (CDC1₃): δ 0.9 (t, 3H), 1.25-1.8 (m) + 1.4 (s) [17H], 3.3 (dd, 2H), 3.95 (t,2H), 4.05 (q, 2H), 4.45 (m, IH), 5.1 (s, 2H), 5.2 (s, 2H), 5.35 (d, 1H>, 7.35 (m, 10H), 9.25 (br, s, IH) , 9.4 (br, s, IH).

Procedure (F)

Boc-Arg (Z₂)-CH₂Br was treated with CF^C^OH,

CF₃(CF₂)₂CH₂OH or Ar-OH and KF in DMF using the procedure outlined in Procedure (D) (iv).

Procedure (G)

The Boc protected peptides were treated with 4 M HCldioxan for 15 min at room temperature and evaporated. The residue in CH₂C1₂ was treated with MeSO₂Cl (1.1 equiv.) and iPr₂NEt (2.5 equiv.). After lh standard

AP 0 0 0 3 1 3 work-up and flash-chromatography gave the methylsulphonylpeptides which were deprotected as in □eprotection procedure (a).

Procedure (H)

The peptides were Boc deprotected as above and the HCl washed out using ethyl acetate/1 M KHCO₃ partition. The free amines in MeOH cooled to 0°C were treated with the aldehyde Ch-CHO (1.5 equiv.) and NaCNBH-j (1 equiv.). After 1 hour evaporation in the cold and flash chromatography gave the N-alkylated peptide.

□eprotection using Deprotecticn procedure (a).

Procedure (I)

3oc-(3-trans-phenyl) -D, Lprolir.e was prepared as described in Chung et al. J. Org. Chem. 1990, 55, 270, and coupled to Η-Pro-OBzl as described above for Boc(Me)DCha-Pro-OBzl. The dipeptide was then converted to its -ONSu ester as described below.

Boc-(3-trans-cyclohexyl)-D, Lproline was prepared from the phenyl analogue by hydrogenation over 5% Rh-C in 90% HOAc-H₂0 at 0.41 MPa for 3 days.

Procedure (K)

Synthesis of the intermediate N- (BzlO₂C-CH₂-),N(CF₃CO)-DCha-Pro-ONSu (i) H₂ -Pd/C (ii) BzlO₂C-CHO, NaCNBH

DCha-Pro-ONSu

Z-DCha-Pro-OtBu (iii) (CF₃CO)₂O (iv) TFA (v) HONSu, WSCDI

BAD ORIGINAL fc (ι) Z-DCha-Pro-OtBu (made ty standard peptide coupling reactions) was hydrogenates in THF over 5% Pd-C at standard temperature and pressure for 24 h. Filtration and evaporation provided H-ZCna-Pro-OtBu (oil, 100%).

(ii) The previous product (2 mmol) and benzyl glyoxylate (1 equivalent) in benzene were subjected to three evaporations (fresh benzene added each time) to remove water. The residual imine (2 mmol) in 1% acetic acid/methanol (8 ml) was treated with NaCNBH^ (2 mmol).

After 1 h, evaporation followed by flash chromatography on silica (60% EtOAc-hexane) gave BzlO₂CCH₂-DCha-Pro-C't3u, 385 rr.g (41%) .

(iii) The above product (380 mg) in dry CH₂CL₂ (8 ml) was treated with Et-jN (2 equivalents) and (CF₃CO)₂O '1.2 equivalents). After 40 nm, evaporation and flash chromatography (silica, 30% EtCAC-hexane) gave the N-(BzlO₂C-CH₂-), N-(CFjCO)-BCha-Pro-OtBu as an oil,

390 mg (86%) .

⁴HNmr(COCl₂) - complex due to presence of 4 rotamers eg. tBu group at δ 1,4-1.5 was split four times in the ratio 1:0.25:0.8:0.4. U 0,9(m), l.l(m), 1.65(m),

1.95{m), 2.2(m) [17H); 1.4-1.5 [4xs, 9H]; 3.1 (m),

3.5(m), 3.7(m)[2H]; 4.3-4.6 [m,3H]; 5.05-5.4 (m,3H); 7.35 [.s,5H).

(iv) The above product (335 mg) was treated with

CH₂CL₂-TFA (1:1,8 ml)for 2.5 h at room temperature.

Evaporation followed by three evaporations from toluene gave the free acid, 100%.

(v) The previous acid was converted to its -ONSu ester (100%) using HONSu as described previously for BocOB

AP 0 0 0 3 1 3

DCha-OH .

The intermediate may be coupled to H-Arg(Z)2^_CH2-Y-R⁴using the general methods already outlined.

Deprotection procedure a provides the peptide protected with N-CF-j-CO-. The N-CF^-CO is removed by deprotection procedure d.

Deprotection procedures:

(a) The protected peptide in MeOH / H2O {3:1) containing 1M HCl (2 equivalents) was hydrogenated over 5% Pd/C at STP for 40 min. Filtration (0.2 μπι) and evaporation was followed by lyophilization from water to give the peptides as fluffy white solids. Purification, if required, was carried out by MPLC (see general procedures).

(b) The protected peptide was first treated with TFA/CH2CI2 (1:1) for 1 hour and evaporated, then hydrogenated as above under (a).

(c) The COCOPh group was first hydrolysed as in (E)(ii) then hydrogenated over H2Pd/C as in (a).

(d) Removal of N-CF3-CO (N-trifluoroacetyl):

The N-trifluoroacetyl peptide was dissolved in MeCNH2O-O.88O ammonia (1:1:1) and kept at room temperature for 24 h. Evaporation followed by purification if necessary provided the peptide.

Examples

The following examples illustrates the principles of

BAD ORIGINAL A

Examples of compounds of the invention are listed in

Table 1, Table 2 indicates the procedures used in their preparation and described in the section titled

Preparation procedures, and Table 3 presents characterising data for the compounds listed.

Examples 1-37

he inven

AP 0 0 0 3 1 3

Formula

Η-DCha-Pro-D, LArg-CH₂~OH H-DCha-Pro-D, LArg-CH₂-O-Me

H-DCha-Pro-Arg-CH₂-0-CH₂-CF₃H-DCha-Pro-Arg-CH₂-O-CH(CF₃)₂H-DCha-Pro-Arg-CH₂-O-C*H (Ph) -CF-j

HOOC-CH₂-DCha-Pro-D, LArg-CH₂-O-CH₂-CF-j Et-OOC-CH₂-DCha-Pro-D, LArg-CH₂-O-CH₂-CF₃

HOOC-CH₂-DCha-Pro-D, LArg-CH₂-O-CH₂-CF2-CF2-CF₃H00C-CH₂-DCha-Pro-Arg-CH₂-0-Ph

HOOC-CH₂-DCha-Pro-D, LArg-CH₂-Ο-Ph (4-OMe) HOOC-CE₂-^DCha-Pro-D, LArg-CK₂-Ο-Ph(4-tBu) HOOC-CH₂-DCha-?ro-D, LArg-CH₂-O-Ph {4-Me) HOOC-CH₂-DCha-Pro-D, LArg-CH₂-Ο-Ph (4-F) HOOC-CH₂-DCha-Pro-D, LArg-CH₂ -O-Ph (3-F) HOOC-CH₂-DCha-Pro-D, LArg-CH₂ -Ο-Ph (2-F) H00C-CH₂-DCha-Pro-D,LArg-CH₂-0-Ph(3-CF₃) HOOC-CH₂-DCha-Pro-D, LArg-CH₂-Ο-Ph (4-CF₃) HOOC-CH₂-DCha-Pro-D, LArg-CH₂-Ο-Ph (2-CF₃) HOOC-CH₂-DCha-Pro-D,LArg-CH₂-O-C₆F₅HOOC-CH₂-DCha-Pro-D, LArg-CH₂-O-Et

HOOC-CH₂-DCha-Pro-Arg-CH₂-0-nPr HOOC-CH₂-DCha-Pro-Arg-CH₂-0-nBu HOOC-CH₂-DCha-Pro-Arg-CH₂-0-iBu

HOOC-CH₂-DCha-Aze-D, LArg-CH₂-O-CH₂-CF₃HOOC-CH₂-DCha-Pic-D, LArg-CH₂-O-CH₂-CF₃

HOOC-CH₂-DCha-Pic-Arg-CH₂-0-nBu

Me-DCha-Pro-D, LArg-CH₂-O-CH₂-CF₃

Me-SO2-DCha-Pro-D, LArg-CH₂-O-CH₂-CF₃Ch-CH₂-DCha-Pro-D, LArg-CH₂-O-CH₂-CF₃ (HOOC-CH₂) ₂-DCha-Pro-D, LArg-CH₂-O-CH₂-CF₃

BAD ORIGINAL A

Table 1 (continued)

Example rormula

No .

(KOOC-CH₂)₂-DCha-Pro-Arg-CH₂-O-iBu

HOOC-CH₂-CH₂-DCha-Pro-D,LArg-CH₂-O-nBu

H00C-CH₂-CH₂-DCha-Pro-Arg-CH₂-0-CH₂-CF₃

H-D,LPro (3-trans-Ph) -Pro-D, LArg-CH₂-O-CH₂-CF₃

H-D,LPro(3-trans-Ch) -Pro-D,LArg-CH₂-O-CH₂-CF₃

KOOC-CH₂-DCha-Pro-Arg-CH₂-S-nBu

7 KOOC-CH₂-DCha-Pro-D, LArg-CH₂-SO2-nBu * absolute configuration R or S

DATE RECEIVED dtecwh GENCH-C.	2 9 MAR 199¼ ' —---
action OFFICER
FILE

AP Ο Ο Ο 3 1 3

Table 2

Example Preparation No. procedure

Deprotection procedure

1	D			c
2	E			b
3	A	or	C	b
4	A	or	C	b
5	A	or	C	b
6	A			a
/	A			a
3	F			a
9	A	or	D	a
10	F			a
11	D			a
*	D			a
13	D			a
14	D			a
15	D			a
16	D			a
17	D			a
18	D			a
19	D			a
20	E			a
21	E			a
22	E			a
23	E			a
24	A			a
25	A			a
26	E			a
27	A	and	F	a
28	A	and	G	a
29	A	and	H	a
30	A			a

bad original ' t ί e ο ο «μ

Table 2	' cont ir.uei
Exarcle	Preparat ran	Deprotect
Mo .	procedure	procedure

31	E	a
32	E and 3	b
33	A and 3	b
34	I and A	b
35	I and A	b
36	D	a
37	D	a

AP ο Ο Ο 3 1 3

Table 3

Example Mol . Wt * * retent ion Mo . (min)/system	FAB MS (M + 1)	AAA: Peptide HPLC
Content ( % ) *	t ime
1	438.57	439	56/Pro	10.3	/E
2	452.60	453	65/Pro	9.9	/E
3	520.6	521	59/Pro	8.6	/A
4	588.6	589	60/ Pro	10.7	/A
5	596.7	597	61/Pro	12.2	/A
6	578.64	579	72/Pro	14.8	/C
n !	606.69	607.7	65/Pro	22.0	/B
8	678.65	679.8	76/Pro	18.2	/G
9	572.71	573.3	73/?ro	19.9	/3
13	602.74	603.8	76, Pre	13.4	/G
11	628.82	629.5	77/Pro	22.0	/G
12	586.74	587.2	75/Pro	16.0	/G
13	590.70	591.4	76/Pro	15.2	/G
14	590.70	591.4	63/Pro	15.0	/G
15	590.70	591.4	54/Pro	14.0	/G
16	640.71	641.5	68/Pro	19.0	/G
17	640.71	641.6	70/Pro	20.0	/G
18	640.71	641.4	65/Pro	18.4	/G
19	662.66	663	49/Pro	21.0	/G
20	524.67	525.4	79/Pro	15.5	/B
21	538.69	539	67/Pro	9.2	/F
22	552.72	553	69/Pro	19.3	/B
23	552.72	553.4	56/Pro	9.2	/F
24	564.61	565	67/Aze	13.6	/D
25	592.66	593	90/?ic	8.4	/D
26	566.75	567.4	86/Pic	17.0	/G
27	534.63	535.6	70/Pro	18.8	/B
28	598.69	599	71/Pro	11.2	/D

bad original ο ΜΑ

Table 3 (continued)
Example Mol. Wt	FAB MS	AAA: Peptide	KPLC
» * Γ Θ t G Ω11C Γ. No .	(M + 1)	Content ( % ) *	t ime

(min)/system

29	616.77	617	64/ Pro	14.5 /C
30	636.67	637.6	70/Pro	12.0 /A
31	610.76	611.2	67/ Pro	10.0 /F
32	566.75	567.5	65/Pro	19.7 /3
33	592.66	593	46/Pro	10.6 /F
3 4	540.59	541	56/Fro	14.3 /C
35	546.64	547	49/Pro	15.9 /C
36	568.78	569.4	45/Pro	17.4 /G
3 7	600.78	601 . 3	62/Pro	14.6 /G
'Sased	on amino	acid as	indicated

**See General experimental procedures. Times are given for L-Arg epimers. D-epimers (minor) usually run about 0.5 min earlier .

System A: 20% increased to 80% of 1% TFA-MeCN into 1%

tfa-h₂o	ι over 25 min.	(20-80%
System	B: 10-60%, 30	min
System	C: 10-90%, 30	min
System	D: 30-100%,30	min
System	E: 10-90%, 20	min
System	F: 20-100%,20	min
System	G: 20-70%, 30	min

bad

AP 0 0 0 3 1 3

Example 38

Solution for continuous intravenous administration

A solution is prepared from the following ingredients:

Thrombin inhibitor

Sodium chloride for injection mg 4.5 g

Water for injection up to 500 ml

The active constituent and the sodium chloride are dissolved in the water whereafter the solution is filtered and then sterilised by autoclaving or by filtration through a sterile 0.2 |im filter and aseptically filled into sterile infusion bottles.

Example 39

Solution for injection.

A solution is prepared from the following ingredients:

Thrombin inhibitor 5 g

Sodium chloride for injection 9 g

Water for inj. up to 1000 ml

The active constituent and the sodium chloride are dissolved in the water whereafter the solution is filtered and then sterilised by autoclaving or by filtration through a sterile 0.2 μπι filter and aseptically filled into sterile ampoules (5 ml).

Example 40

Solution for nasal administration

A solution is prepared from the following ingredients:

Thrombin inhibitor 10 g

Glycerol 200 g

Methyl p-hydroxybenzoate 1 g bad original

J)

Propyl p-hydroxybenzoace ¹ ί I ' > ι« f« , ι 'J ’

0.2 g >.‘ater for inj . up to 1 0 3 0 mi

The active constituent and the preservatives were dissolved in glycerol and the mam part of the water. The volume is then adjusted to 1000 ml and the solution is filled into sterile polyethylene containers.

Example 41

Tablets for oral administration

1000 tablets are prepared from the following ingredients;

Thrombin inhibitor 100 g

Lactose 200 g

Polyvinyl pyrrolidone 30 g

Microcrystalline cellulose 30 g

Magnesium stearate 6 g

The active constituent and lactose are mixed with an aqueous solution of polyvinyl pyrrolidone. The mixture is dried and milled to form granules. The microcrystalline cellulose and then the magnesium stearate are then admixed. The mixture is then compressed in a tablet machine giving 1000 tablets, each containing 100 mg of active constituent.

AP 0 0 0 3 1 3

Example 42

Gelatine capsules for oral administration

Gelatine capsules are filled with a mixture of the following ingredients:

Thrombin inhibitor	50	mg
Magnesium stearate	3	mg
Lactose	100	mg

Biology

Determination of thrombin clotting time and ICjqTT:

Human thrombin <T 6769, Sigma Chem Co) in buffer solution, pH 7.4, 100 μΐ, and inhibitor solution, 100 μΐ, are incubated for one min. Pooled normal citrated human plasma, 100 μΐ, is then added and the clotting time measured in an automatic device (KC 10, Amelung).

The clotting time in seconds is plotted against the inhibitor concentration, and the IC^qTT is determined by interpolation .

IC^qTT is the concentration of inhibitor that doubles the thrombin clotting time for human plasma. ρΙΟςθΤΤ is the -log 10 of IC^qTT in mol/1. The results are presented in Table 4.

BAD ORIGINAL Q

Table 4

Example oIC^qTT No .

1	7.71
2	7.81
3	7 .92
4	7.38
5	7.77
6	8.04
7	7.70
3	’“f G. Λ i . Z '-t
9	8.45
10	7.90
11	7.86
12	8.25
13	8.22
14	8.15
15	8.12
16	7.77
17	8.68
18	7.30
19	8.27
20	8.17
21	8.14
22	8.69
23	7.64
24	8.01
25	8.00
26	7.89
27	7.52
28	6.85
29	6.47
30	7.38

AP Ο Ο Ο 3 1 3

Table 4 (continued)

Example pIC₅₀TT

No .

31	6.88
32	7.63
33	7.78
34	7.03
35	7.25
36	7.57
37	7.72

BAD original

’ r	t 0 fc _v q<,
ABBREVIATIONS	4 0
4-DMAP =	4-dimethylamino pyridine
AAA =	amino acid analysis
Arg =	L-argmine
Arg(Z₂) = arginine	UN, N-dibenzyloxycarbonyl-L-
Aze =	L-azetidine-2-carboxylic acid
Boc =	tertiary butoxy carbonyl
Bu =	butyl
Bzl =	benzyl
Ch =	cyclohexyl
Cha =	L-S-cvclohexylalanine
DMF =	dimethyl formamide
Et =	ethyl
EtOAc =	ethyl acetate
FAB =	fast atom bombardment
FI to FXIII =	coagulation factors I to XIII
FII_a to FXIII_a =	activated form of coagulation
factors	II to XIII
Gly =	glycine
HMW-K =	high molecular weight kininogen
HOAC =	acetic acid
HONSu =	N-hydroxysuccinimide
HPLC = chromatography	high performance liquid
iBC =	isobutyl chloroformate
Kail =	kal1ikrein
Me =	methyl
MPLC = chromatography	medium pressure liquid
NMM =	N-methyl morpholine
Nph =	naphthyl
Ph =	phenyl
Pic =	L-pipecolinic acid

AP Ο Ο Ο 3 1 3

PL =	phospholipids
Pr =	propyl
Prekail -	prekallikrein
Pro =	L-proline
STP =	standard temperature and pressure
Tee =	2,2,2-trichloroethyl
TFA =	trifluoracetic acid
THF =	tetrahydrofuran
Val =	L-valine
WSCDI =	water soluble carbodiimide
z =	benzyloxy carbonyl

Prefixes η, i and t have their usual meanings: normal, iso and tertiary.

BAD ORIGINAL

Η. ..

'Ί/*'*

Claims

A compound of the general formula

20 wnerein:

A represents -CH₂-, -CH=CH

-CH₂-CH₂- or -CK₂-CH₂-CH₂25 and are the same or different and each represents H or X-Β-, where B is a straight or branched alkylene group having 1-3 carbon atoms and X is H, methyl, ethyl, a cycloalkyl group having 3-6 carbon atoms or R'CO-, where R' is OH, a straight or branched alkoxy group having 1-4 carbon atoms, NH₂ or NHR'', where R'' is a straight or branched alkyl group having 1-4 carbon atoms, or X is a carboxylic acid mimic, known per se, selected from -PO(OR''')₂, -SO^H and 5-{IH)-tetrazolyl, and R''' is H, methyl or ethyl, or B is -SO₂- and X is methyl or ethyl; and

AP 0 0 0 3 1 3 m is 0, 1 or 2, R³ represents a cyclohexyl group and R^3a represents H; or m is 1 and R³ represents a cyclohexyl or phenyl group and R^3A forms an ethylene bridge together with R¹ ;

Y represents O or S(O)p, where p is 0, 1 or 2; and

R⁴ represents H; a straight or branched alkyl or a cycloalkyl having 1 to 6 carbon atoms unsubstituted or substituted with one or more fluoro atoms and/or substituted with a phenyl group; a substituted or unsubstituted aromatic ring selected from phenyl,

4-methoxy-phenyl, 4-tertiary-butyl-phenyl,

4-methyl-phenyl, 2-, 3- or 4-trifluoro-methyl-phenyl, phenyl substituted with 1-5 fluoro atoms; or -CH(CF₃)-phenyl, either as such or in the form of a physiologically acceptable salt and including stereoisomers .
2 . A compound according to claim 1 wherein

A represents -CH₂-CH₂- or -CH₂-CH₂-CH₂-;

R¹ is H and R² represents HOCO(CH₂)_n- or

CH₃CH₂OCO(CH₂)_n-, and n is 1 or 2;

Y is O ;

m is 1, R³ represents a cyclohexyl group and R^3arepresents H.

BAD ORIGINAL &
3. A compound according to claim 1 selected from

H-DCha-Pro-Arg-CH₂-OH,

H-DCha-Pro-Arg-CH₂-0-Me,

H-DCha-Pro-Arg-CH₂-0-CH₂-C?₃ ,

H-DCha-Pro-Arg-CH₂-0-CH(CF₃)₂,

H-DCha-Pro-Arg-CH₂-0-(R or S)CH( Ph)-CF₃, HOOC-CH₂-DCha-Pro-Arg-CH₂-0-CH₂-CF₃, Et-0OC-CH₂-DCha-Pro-Arg-CH₂-O-CH₂-CF₃ , H00C-CH₂-DCha-Pro-Arg-CH₂-0-CH₂-CF2-CF2-CF₃, HOOC-CH₂-DCha-Pro-Arg-CH₂-O-Ph, HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Ph (4-OMe) , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Ph (4-tBu) , H00C-CH₂-DCha-Pro-Arg-CH₂-0-Ph(4-Me) , HOOC-CH₂-DCha-Pro-Arg-CH₂-G-Ph (4-F) , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Ph (3-F) , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Ph(2-F) , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-?h (3-CF₃ ) , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Ph(4-CF₃ ) , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Ph(2-CF₃ ) , HOOC-CH₂-DCha-Pro-Arg-CH₂-O-C6F5 , HOOC-CH₂-DCha-Pro-Arg-CH₂-0-Et, HOOC-CH₂-DCha-Pro-Arg-CH₂-0-nPr, HOOC-CH₂-DCha-Pro-Arg-CH₂-0-nBu, HOOC-CH₂-DCha-Pro-Arg-CH₂-0-iBu, HOOC-CH₂-DCha-Aze-Arg-CH₂-O-CH₂-CF₃ ,

HOOC-CH₂ -DCha- Pic-Arg-CH₂ -0-CH₂ -CF₃ , HOOC-CH₂-DCha-Pic-Arg-CH₂-0-nBu, Me-DCha-Pro-Arg-CH₂-0-CH₂-CF₃ , Me-S02-DCha-Pro-Arg-CH₂-0-CH₂-CF₃ , Ch-CH₂-E>Cha-Pro-Arg-CH₂-0-CH2-CF₃ , (HOOC-CH₂) ₂-DCha-Pro-Arg-CH₂-0-CH₂-CF₃, , (HOOC -CH₂ ) 2 -DCha - Pro-Arg -CH₂ -O-iBu,

HOOC-CH₂ -CH₂ -DCha - Pro-Arg-CH₂ -O-nBu,

HOOC-CH₂ -CH₂ -DCha - Pro-Arg-CH₂ -O-CH₂ -CF₃ , H-DPro (3-trans-Ph) -Pro-Arg-CH₂-0-CH₂-CF₃,

APO 00 3 1 3

H-DPro (3-trans-Ch) -Pro-Arg-CH₂-0-CH₃-CF₃,

HOOC-CH₂-DCha-Pro-Arg-CK₂~S-nBu and

H00C-CH₂-DCha-Pro-Arg-CH₂-S02-nBu, either as such or in the form of a physiologically acceptable salt and including stereoisomers.
4. A compound according to claim 1 selected from H00C-CH₂-DCha-Pro-Arg-CH₂-0-CH₂-CF₃, HOOC-CH₂-DCha-Pic-Arg-CH₂-O-CH₂-CF₃, HOOC-CH₂-DCha-Pic-Arg-CH₂-0-nBu,

HOOC-CH₂-CH₂-DCha-Pro-Arg-CH₂-0-nBu,

H00C-CH₂-CH₂-DCha-Pro-Arg-CH₂-0-CH₂-CF₃ and

HOOC-CH₂-DCha-Pro-Arg-CH₂-0-nBu, either as such or in the form of a physiologically acceptable salt and including stereoisomers.
5. A compound according to any of claims 1-4 for use in therapy .
6. A process for preparing a compound according to any of claims 1-4 , which process comprises (method I) displacement by R^Y (Y=O,S) of the halogen of a halomethylketone of the formula wherein W² is an amino terminal protecting group and W²is a protecting group , reduction of the ketone to ,£AD OR'G'^nAL

4 6 alcohol, removal of the amino terminal protecting group, standard peptide coupling, followed by oxidation of the alcohol, giving the protected tripeptide ketone, removal of the amino terminal protecting group, followed by N-alkylation, and deprotection, or replacing a protected dipeptide in the coupling reaction referred to above with an amino-terminal-Nalkylated-N-trifluoroacy1-protected dipeptide, followed by oxidation and deprotection, or (method II) alkylation, ketol of the formula halide, of an awith an R - wherein further (method

1 ?

W and W are as defined above, and then reacting as in method I, or

Ί 9 wherein W and W are as defined above, with (TCH2CO)2O wherein T is halogen, R⁴O or R⁴S and 4-DMAP, and then further reacting as in method I, and if

AP 0 0 0 3 1 3

- 47 desired forming a physiologically acceptable salt, and in those cases where the reaction results in a mixture of stereoisomers, these are optionally separated by standard chromatographic or /e-crystallisation techniques, and if desired a single stereoisomer is isolated.
7. A pharmaceutical preparation comprising an effective amount of any of the compounds claimed in claims 1-4, and furthermore comprising one or more pharmaceutical carriers.
8. Use of compound according to any of claims 1-4 as an active ingredient for manufacture of a pharmaceutical preparation for inhibition of thrombin in a human or animal organism.
9. Use of compound according to any of claims 1-4 as an anticoagulant agent.
10. A compound according to any of claims 1-4 for use in obtaining inhibition of thrombin in a human or animal organism in need of such inhibition.
11. A compound according to any of claims 1-4 for use in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues in a human or animal organism.
12. A compound according to claim l substantially as hereinbefore described.
13. A process for preparing a compound according to any of claims 1-4 substantially as hereinbefore described.
14. A compound according to claim 1 prepared by the process claimed in claim 6 or 13.
15. A pharmaceutical preparation according to claim 7

A3STRACT

The invention relates to new competitive inhibitors ci thrombin, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use cf the compounds as anticoagulants for prophylaxis and treatment of thromboembolic diseases, according to the formula

R³

NH

NH (CHR^3A)_m

R wherein :

R¹ and R² are the same or different and each represents

H or X-B-, where B is a straight or branched alkylene group having 1-3 carbon atoms and X is H, methyl, ethyl, a cycloalkyl group having 3-6 carbon atoms or R'CO-, where R' is OH, a straight or branched alkoxy group having 1-4 carbon atoms, NH₂ or NHR'', where R'' is a straight or branched alkyl group having 1-4 carbon

4?

AP Ο Ο Ο 3 1 3 stems, or X is a carbozt/lic acid mimic, known per se, selested frcm - PC- 'OR' ' ' ,₂ , -SO3H and 5-!IH,-tetrazolyl, and ' is H, methyl cr ethyl, or B is -SO₂- and X is met hy1 or ethyl;

m is 0, 1 or 2, R^ represents a cyclohexyl group and R^A represents H; or m is 1 and represents a cyclohexyl or phenyl group

13 and R^A f_{orrns an} ethylene bridge together with ;

Y represents 0 or S(O)_C, where p is 0, 1 or 2;

R^ represents H; a straight or branched alkyl or a

15 cycloalkyl having 1 to 6 carbon atoms unsubstituted or substituted with one or more fluoro atoms and/or substituted with a phenyl group; a substituted or unsubstituted aromatic ring selected from phenyl,

4-methoxy-phenyl, 4-tert iary-butyl-phenyl,

2 3 4 -methyl-phenyl, 2-, 3- or 4-trifluoro-methy1-pheny1, phenyl substituted with 1-5 fluoro atoms; or -CH (CF-j )-pheny 1, either as such or in the form of a physiologically acceptable salt and including stereoisomers .