INHIBITORS AND SUBSTRATES OF THROMBIN

Abstract: Peptides which act as inhibitors or substrates of thrombin are derived from the formula: D-Phe-Pro-Arg or its analogues, wherein Phe is substituted by (a), wherein Ar1 and Ar2 are the same or different and are selected from the group consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphthyl, indolyl and saturated groups corresponding to these, optionally substituted by up to three groups selected from C1-C3 alkyl and C1-C3 alkoxy, L1 and L2 are the same of different and are selected from the group consisting of CH2, CH2-CH2, O-CH2, S-CH2, Ar-L taken together optionally means H, diphenyl-methyl, fluorenyl or saturated groups corresponding to these, but one of the Ar-L cannot be H when the other Ar-L means H or benzyl, and Arg may be substituted by an amino acid of formula (b), wherein Y = [CH2]n-Q, (c), where Q = H, amino, amidino, imidazole, guanidino or isothioureido and n = 1-5, preferably 3-5, or C3-C9 alkyl and C5-C10 aryl or alkylaryl optionally substituted by up to three groups selected from hydroxy and C1-C4 alkoxy; Z = CN, COR1, (d) or (e), where R1 = H, OH, CH2Cl, CH2-CH2-CO-pip, CF2-CF2-CO-pip, (f), (g), CH2-CH2-CO-Pro-NHEt, CF2-CF2-CO-Pro-NHEt or a chromophoric group e.g. pNA, MCA, R2 and R3 are the same or different and are selected from the group consisting of OH, OR6 and NR6R7, or R2 and R3 taken together represent the residue of a diol; where R6 and R7, which are the same or different, are C1-C10 alkyl, phenyl or C6-C10 arylalkyl, R4 and R5 are the same or different and are selected from R2, R3, Gly-pip, Ala-pip or Gly-Pro-NHEt.

Description: INHIBITORS .AND SUBSTRATES OF THROMBIN This invention relates co thrombin inhibitors and substrates. Thrombin, the lasc enzyme in Che coagulacion system, cleaves soluble fibrinogen to fibrin, which is hen crosslinked and forms an insoluble gel forming Che matrix for a thrombus. When a vessel is damaged, che above process is necessary co scop bleeding. Under normal circumstances chere is no measurable amounc of thrombin present in plasma. Increase of che thrombin concentration can result in formation of clots, which can lead co hromboe bolic disease, one of che most common serious medical problems of our time. Thrombin contribuces to haemostatic control by means of several biological reactions. In addition to its primary funccion, che conversion of fibrinogen co fibrin, chrombin accivaces Faccor XIII, which is responsible for Che crosslinking of fibrin. Thrombin also aces by means of a positive feedback mechanism involving che accivacion of Faccors V and VIII, which boch are necessary for its own formation from prothrombin. Thrombin has another essencial role: its binding co platelets initiates platelet release and aggregation which is responsible for primary haemostasia. Fibrinolysis is the process which causes an enzymatic dissolution of fibrinogen and fibrin clocs. Plasma contains a protein, plas inogen, which under Che influence of various accivacors is converted to plasmin, a proteolytic enzyme, the activity of which resembles that of fibrin. Plasmin breaks down fibrin to fibrin degradation products. Under normal conditions, the fibrinolysis system is in balance with the coagulation system. Small thrombi formed in Che blood stream can be dissolved enzymacically and the circulation through che vessels can be restored by the accivacion of che fibrinolycic system in the body. If the fibrinolycic activity is too high, it may cause or prolong bleeding and if it is too low compared to the activity of the coagulation system, there is a risk of thrombosis. The reactions of thrombin are further controlled by natural inhibitors in plasma. The most important of these are ancichrombin III and heparin. These two compounds have been isolated and are therapeutically and prophylactically used in conditions where there is an imbalance in the haemostatic mechanisms with risk for prothrombin activation. Mainly two types of therapeutic agents are used for the prevention of thrombosis. The heparins act by accelerating the inhibition of thrombin by antithrombin III. Coumarin derivatives, the oral anticoagulants, e.g. Warfarin, prevent the generation of thrombin by blocking the post-translational vitamin R-dependent y-carboxylation in the synthesis of prothrombin. Neither Heparin nor Warfarin are ideal. Heparin must be given parenterally and as it functions as a cofactor to antithrombin III it has no effect without this inhibitor. The effect of Warfarin develops very slowly and individual doses must be adjusted by frequent tests. None of these anticoagulants is specific for thrombin, they also inhibit other serine proteases and both of them may cause bleeding if the doses are not correctly balanced. Thus, direct acting, specific thrombin inhibitors, having oral activity would be useful alternatives to the above anticoagulants. Much research in this area has resulted in the synthesis of different kinds of inhibitors of thrombin. By imitating amino acid sequences of fibrinogen, the important natural substrate of thrombin, several good short peptide substrates for thrombin have been synthesized. The very first developed sequence with affinity for the active site of thrombin was Phe-Val-Arg [1] which mimics the fibrinogensequence preceding the bond split by thrombin. This sequence has later been improved co give D-Phe-Pro-Arg and D-Phe-Pip-Arg which have been used i chro ogenic substrates, e.g. D-Phe-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA[l] and i inhibitors of thrombin e.g. che pepcide aldehyde D-Phe-Pro-Arg-H [2], ch irreversible inhibicor D-Phe-Pro-Arg-CH2Cl [3], inhibicors wich kecomechylene bond e.g. D-Phe-Pro-Arg-k-Gly-piperidide [4] and in che recencl synchesized pepcide boronic acid inhibicors e.g. Z-D-Phe-Pro-boroΔrg [5] and che nicrile: Boc-D-Phe-Pro-ArgCN [6]. Thus, D-Phe-Pro-Arg has been considered che besc sequence for about 15 years, and ic has been shown Co have very good affinity for the active site of chrombin, in substrates (Km around 10~^M) as well as in inhibicors (Ri 10" * ~H Co 10-9M) . We have now found chac by exchanging Phe, in he D-Phe-Pro-Arg sequence, for some unnacural, aromatic amino acids, with a specified structure, and by using chese new sequences co construct novel substrates and inhibitors we obtained significantly improved subscrace and inhibicor propercies. The new substrates show better kinetic constants (Km and kcat) and Che inhibitors better inhibition constant (Ki) . Reduction of blood pressure is a side effect observed in many of Che previous hrombin inhibicors concaining Arg or Arg analogues like Gpa and Apa [7]. This side effect which in some compounds can be disturbingly serious is believed to depend on the positively charged guanidino or amidino group of the side chain of Arg or its analogues. Surprisingly, this side effect of inhibitors in the present application is markedly reduced even when the inhibitors have an Arg or Arg analogue. We have also surprisingly found thac by changing Che side chain co a non- basic alkyl or alkylaryl group of a certain size, Che affinity for thrombin is still very good although the affinity for other serine proteases is greatly reduced, i.e. these inhibitors/substrates are more specific for thrombin than corresponding compounds containing Arg. With this non-basic side chain the blood pressure lowering side effecc is greacly reduced. The present invention provides chrombin inhibicors and substrates derived from D-Phe-Pro-Arg or its analogues wherein Phe is substituted by H2N~C-G00H Li L 2 I \ Ari Ar2 and Arg may be substituted by H2N-GH-COOH. I Y Suitably, the inhibitors/substrates are of formula I, in which X = H, CH3 or an N-protecting group, e.g. Ac, Bz,Cbz,Boc; Y = tCH2] n -Q » CH2-^=^-Q where Q = H, amino, amidώo, imidazole, guanidino or isothiour ido and n = 1-5, preferably 3-5, or C^-C- alkyl " and " C^-C.- aιγl or optionally substituted by up to three groups selected frαn hydroxy and C 1 ~C 4 alkoxy; R2 y 0 / Z - CN, CORi, B or «p where R3 ^R5 H. OH, CH 2 C1, CH 2 - H 2 - O-piP, CT j -CB CO-piPf O^-CH-CO-pip, CT^-CF-CO-p CH2-CH2-C0-Pro-NHEt f CF 2 -CF 2 -CO-Pro- HEt or a clircitophoric group e.g. pNA., MCA, 2 and R3 may be the same or different and are selected frαπ. the group consisting of OH, 0R $ and N 67, or R2 and R-3 caken cogecher represenc Che residue of a diol; where Rg and R7, which may be che same or differenC, are c l~_10 alkyl, phenyl or Cg-Cio arylalkyl, R4 and R5 may be the same or different and are selected from I ,, R- Gly-pip, Ala-pip or Gly-Pro-NHEt; Arj-Li Aaj = C(NH2>-COOH where Aτ2~ 2 Arj and Aτ2 may be the same or different and are selected frctn the gro consisting of phenyl, thienyl, pyridyl, naphchyl, Chionaph hyl , indolyl and .. saturated groups corresponding to these, optionally substituted by up to th groups selected frcm C^-C, alkyl and C^-C, a.lkαxy, and 1x2 may be che same or different and are selected frcm the grou consisting of CH2, CH2-CH2, 0-CH2 > S-CH2 > .Ar-L taken together may mean H, diphenyl-methyl f fiuorenyl or saturated groups co._TesponcLi.ncj to these, but one of the Ar-L cannot be H when the other Ar-L means H or benzyl; .A 2 = CΪ-2 CH-COOH or its C1-C3 alkyl substituted NH derivatives, where KQ β CH2, CH2-CH2, S-CH2 > S-C(CH3)2 or CH2-CH2-CH2. Preferably the Phe substitute is Dpa, Nal or Dba and preferable Arg substitute includes Irg, Gpa, Apa and non-basic amino acids such as Pgl, Mbg, Chg. Examples of compounds which may be preferably used in che invention include: Ac-D-βNal-Pro-boroArg pinanediol ester Z-D-Dpa-Pro-boroIrg pinanediol ester Z-D-Dpa-Pro-boroPg1 pinacol ester Ac-D-βNal-Pro-boroMbg pinanediol ester C__3-D-Dpa-Pro-Arg-H Boc-D-Dpa-Pro-Gpa-H CH 3 -D-Dpa-Thi-Mbg-H H-D-Dpa-Pro-Arg-k-Gly-pip Z-D-Dpa-Pro-Arg-CH-Cl Boc-D-Dpa-Pro-ArgCN H-Dpa-Pro-Arg P (0Ph) 2 H-D-BNal-Pro-Pgl P (OPh)-Gly-pip H-D-Dpa-Pip-Arg-pNA H-D-BNal-Pro-Chg-pNA Further examples of compounds which may be-preferably used in the invention are those listed in Examples 10 to 22 below. Inhibition data for some of the new compounds are shown in Tables 1-7. The advantages of- replacing Phe by amino acids according to the invention are clearly shown in the Ki values, which are generally 3 to 10 times better for the new compounds, as well as in the prolongation of the thrombin time. The importance of the D-form of the N-terminal amino acid is also evident from Table 1. The drastic reduction of the blood pressure lowering side-effect with compounds according to the invention is shown in Table 2. TABLE 1 - In-vitroassays D-Phe-Pro-Gpa-k—Gly-Pip 4 D, -Dpa-Pro-Arg—k-Gly-Pip 6 D-Dpa-Pro-Arg—k-Gly-Pip 7 L,Dpa-Pro-Arg—k-Gly-Pip 8 D-Fgl-Pro-Arg—k—Gly-Pip 9 D,L-G(-Nal-Pro-Arg--k-Gly-Pip 14 D,L-β-Nal-Pro-Arg~k-Gly-Plp 54 D-β-Nal-Pro-Arg—k-Gly-Pip TABLE 2 - In-vitroassay Boc-D-Phe-Pro-Arg-H Boc-D,L-Dpa-Pro-Arg-H Boc-D, -Dpa-Pro-Gpa-H 33 Z-D-Phe-Pro-Pgl-H 48 Z-D,L-Dpa-Pro-Pgl-H 53 Boc-D-Phe-Pro-His-H * The concentration needed to double the plasma thrombin time ** 4mg/Kg given i.v. to anaesthetized cats *** lmg/Kg given i.v. bolus to anaesthetized New Zealand white rabbits, 2 per point ND = Not determined. TABLE 3 Ki (u ) Boc-D-Phe-Pro — ArgCN 0.8 Boc-D , L-Dpa - " - 0.05 SUBSTITUTE SHEET TABLE 4 - In-vitroassay Ki(μM) TT(μM) 15 Boc-D,L-Dpa-Pro-Pgl (OH) 2 16 D, -Dpa-Pro-Pgl P (OH) 2 17 Z-D-Dpa-Pro-Pgl P (OPh) 2 18 D-Dpa-Pro-Pgl P (OH) 2 19 Z-D-Phe-Pro-Pgl P (OPh) 2 20 D-Phe-Pro-Pgl P (OPh) 2 21 D-Phe-Pro-Pgl P (OH) 2 23 D-Dpa-Pro-Pgl P (OPh) 2 32 H-D-Dpa-Pro-Mpg P (OPh) 2 9.1 40 H-D-Phe-Pro-Mpg P (OPh) 2 TABLE 5 - In-vitroassays Ki(pM)* TT(μM) APTT(μM) 12 Z-D-Dpa-Pro-BoroIrg-OPin 1.19 26 Z-D-β-Nal-Pro-BoroIrg-OPin 0.216 36 Z-D-Fgl-Pro-BoroIrg-OPin 1.23 37 Ac-D-Dpa-Pro-BoroIrg-OPin 0.156 38 Z-L-Dpa-Pro-BoroIrg-OPin 0.239 46 Z-D-Cha-Pro-BoroIrg-OPin 0.123 *Inhibitor Pre-Incubated for 30 min with Thrombin. TABLE 6 - In-vitroassays 22 Z-D-Phe-Pro-BoroMbg-OPin 41 Z-D-Phe-Pro-BoroPhe-OPinac 10 Z-D-Phe-Pro-BoroAcet-OPinac 11 Z-D-Phe-Pro-BoroPgl-OPinac 43 Z-D-Phe-Pro-BoroOct-OPinac 51 Z-D-Dpa-Pro-BoroMpg-OPin 59 Z-D-Phe-Pro-BoroMbg-OPin TABLE 7 - In-vitroassay Km(μM) H-D-Phe-Pip-Arg-pNA 10.0 34 H-D-β-Nal-Pip-Arg-pNA 6.9 35 H-D, -Dpa-Pip-Arg-pNA 9.1 SUBSTITUTE SHEET Those compounds of che invention which are chrombin inhibicors hav anti-thrombogenic properties and may be employed for indications when a anti-thrombogenic agent is indicated. Generally, these compounds may b administered orally or parenterally to a host to obtain an anti-thrombogeni effect. In the case of larger mammals such as humans, Che compounds may b adminiscered alone or in combinacion wich pharmaceutical carrier or diluent a a dose of from 0.02 to 15 g/Kg of body weight and preferably 1-10 g/Kg t obtain the anti-thrombogenic effect, and may be given as single dose or i divided doses or as a sustained release formulation. When an extracorporea blood loop is to be established for a patient, 0.1-lmg/ g may be adminiscere incravenously. For use wich whole blood from 1-lOmg per litre may be provide to prevent coagulation. Pharmaceutical diluents are well known and include sugars, starches and water which may be used co make tablets, capsules, injectable solutions and che like. The compounds of che inven ion may be added Co blood for che purpose of preventing coagulation of che blood in blood collecting or distribution containers, tubing or implantable apparatus which comes in contact with blood. The advantages of the compounds of the invention include oral activity, rapid onset of activity and low toxicity. In addition, these compounds may have special utility in the treatment of individuals who are hypersensitive to compounds such as heparin. In the following examples, the symbols have the following meanings: Aa ~~ amino acid Ac = acetyl Boc a t-butyloxycarbonyl Bu =■ butyl Bzl = benzyl DCC = dicyclohexylcarbodiimide DIEA = diisopropylethyl-amine boroAa = boronic acid analogue of Aa p Aa = phosphonic acid analogue of Aa -k- = amide bond replaced by CO-CH2 The following non-limicing examples illustrate the preparation of the compound in this invention. The synthesis of some of the different inhibicor Cypes are ou lined in Scheme 1 to 8 and the detailed descriptions are given in the examples below. HPLC The following condicions were adopted for the analysis of most of th synthetic compounds on reversed-phase HPLC (RP-HPLC): column; SuperPac Pep- (4x250mm) , eluant; A = water containing 0.12 TFA, B ~~ acetonitrile containin 0.1% TFA, gradient; 50% to 90% B in A in 25 min, flow rate; l.Oml/min detection; UV absorbance at 210 nm. TLC Thin layer chromacography (TLC) was carried out on the following compounds using precoated silica plates (Merck, F254) in the following systems: A, Chloroform-ethyl acetate (2:1); B, chloroform-methanol-acetic acid (20:4:1). C, n-butanol-acetic acid-ethyl acetate-water (1:1:1:1)', D, chloroform-methanol (9:1); E, pyridin-ethyl acetate-acetic acid-water (5:5:1:3); F, chloroform- methanol-ammonia (1M) (60:35:5). The spots were visualized by ninhydrin and chlorine-dicarboxidine spray reagents (CM. Swahn and J. Gyllander, J. Chromatogr. (1979) 170, 292: NMR spectra Magnetic resonance spectra were recorded at 250 MHz using a Bruker instrument. F-T AMPLE S 1. Synthesis of Dpa, Z-Boc-Dpa-Pro and Z-Dpa-Pro-Arg (Cf. Scheme 1) (a) DL-Dpa.HCl To a solution of potassium tertiary butox de (6.75g, 0.06 mol) in tertiary butanol (350ml) was added, at room tempearature under argon, ethyl acetamido cyanoaetate (lOg, 0.059 mol). When the solution had become clear bromodiphenyl methane (14.55g, 0.059 mol) was added. The mixture was stirred at 20°C for 24h, then evaporated under reduced pressure. The solid residue was treated with ethyl acetate (500ml) and water (175ml). The organic phase was dried (Na2S0 and concentrated to give yellow crystals. The crystals were washed repeatedly with ether and dried to give ethyl 2-diρheπylmethylacetamido cyanoacetate (ll.δlg, 58%, m.p. 181-185°C). The ester (ll.δlg, 34.4 mol) was mixed with hydrochloric acid (20%) and refluxed for 30h. The reaction mixture was allowed to cool and the crystals were collected, washed (ether), and dried to give HC1.D,L-Dpa (7.82g, 81.8%). (b) Z-DL-Dpa To a solution of D,L-Dpa.HCl (0.56g, 0.0021 mol) in NaOH (2N, 5ml), cooled to 0 β C and vigorously stirred was added, dropwise, benzyl chloroformate (0.39g, 0.33ml, 0.0023mol). The reaction mixture was kept at pHIO and at 5°C to 10°C. The solution was warmed to room temperature and stirred vigorously for lh. The solution was washed with ether (4 times) and acidified to pH3 with HC1 (5N) . The mixture was excracced wich dichloromechane and the organic phase dried and coπcentraced co give Z-DL-Dpa (0.73g, 97%, m.p. 214-217°C). (c) Z-D.L-Dpa-ONSu To a scirred solucion of Z-D,L-Dpa (1.88g, 0.005 mol) and N-hydroxy succinimide (0.575g, 0.005 mol) in dry 1,2-dimechoxyechane (30ml) ac 0°C was added dicyclohexyl carbodiimide (l.03g, 0.005 mol). The mixcure was maintained at 0°C for 4h. The suspension was filtered and the filtrate was concentrated to dryness to give an oil which was triturated with ether and filtered to give Z-D,L-Dpa-0NSu (2.15g, 91%, m.p. 139-142°C). (d) Z-D-Dpa-Pro and Z-L-Dpa-Pro To a solution of proline (0.78g, 0.0068mol) and NaHC0 3 (0.57g, 0.0068mol) water (8ml) wa β added a solution of Z-D,L-Dpa-0NSu (2.15g, 0.0045mol) 1,2-dimethoxyethane (15ml). After 2h the solvent was removed under redu pressure and water (5ml) was added. The solution was acidified (cone. HCl) pH2 to give whice cryscals (1.98g, m.p. 113-117°C). Fraccio recryscallisacion from ECOAc gave as he firsc crop one diascereomer as a sol (0.7g, m.p. 180-183°C, FAB MS:M+473; 1H nmr: 7.26 (15H, m, 3xPh), 5.66 (IH, CH), 5.23 (IH, m, CH) , 4.40 (IH, d, CH), 2.03 (2H, s, CH2), 2.20 (4H, 2xCH 2 ); 13 C nmr:172.19 (CO), 156.1 (CO), 139.17 (CO), 127-128 (Ph), 66. (CH2), 59.48 (CH), 55.58 (CH), 24.15 (CH 2 ). Furcher cryscallisacion from c mocher liquor gave as a second crop a mixCure of diascereomers (0.43g, m. 126-130°C). Addicion of pecroleum echer. (b.p. 60-80°C) gave che ocher isom (0.54g, m.p. 128-131°C), FAB MS:M+473; H nmr: 7.29 (15H, m, 3Ph), 5.55 (IH, CH), 5.23 (IH, m, CH), 4.47 (IH, d, CH), 2.04 (2H, s, CH2 , 1.20-2.20 (4H, , CH 2 ); 13C:172.77 (CO), 156.13 (CO), 139.48 (CO), 126.99-128.72 (Ph), 66. (CH2), 59.62 (CH), 55.48 (CH), 53.54 (CH), 47.44 (CH2), 27.94 (CH 2 ), 24.5 (CH 2 ). (e) Z-D-Dpa-Pro-Arg(Mtr)OPh To a solution of Z-D-Dpa-Pro-OH (0.472g, lmmol) and HOSu (0.115g, lmmol) i dimethoxyethane (20ml) was added DCC (0.206g, lmmol) with cooling over an ic water bath, the solution was then stirred at r.t. for 3h, the DCO formed wa filtered off and the solution was concentrated to dryness to give an oi (0.57g). To a solution of H-Arg(Mtr)-0H (0.42g, 1.lmmol) and Et3N (0.12g 1.lmmol) in DMF (25ml) was added a solution of Z-Dpa-Pro-OSu (0.57g) i dimethoxyethane (15ml) with cooling. The solution was stirred at roo temperature for 3h. The solvent was evaporated and the residue was dissolve in H2O (20ml) and MeOH (10ml). The solution was acidified to pH2 and the MeO was removed under reduced pressure. The solid formed was filtered off and dried to give Z-D-Dpa-Pro-Arg(Mtr)OH (0.766g, 91%). The structure of the compound was confirmed by 1H NMR. Fgl and Nal and their corresponding di- and tripeptides were synthesized in a manner analogous to the above procedures. 2. Synthesis of peptide a inophosphonic acid inhibitors (Cf. Schema 3) (a) Diphenyl l-(N-benzyloxycari3onyl)am--τopentanephosphonate A mixture of triphenyl phosphite (9.3g, 30mmol), n-hexanal (4.50g, 45mmol ) benzylcarbamate (4.53g, 30mmol), glacial acetic acid (5ml) was stirred for 45min. The mixture was then heated at 80-85°C for lh and volatile by-products were removed in vacuo with heating on a boiling water bath. The oily residue was dissolved in methanol (40ml) and left for crystallization at -10 β C to give 7.28g, m.p. 70-72°C, 52% yield. The structure was confirmed by proton NMR. (b) Diphenyl 1-aπιinopentanephosphonate Diphenyl-l-(N-benzyloxycarbonyl) aminopentanephosphonate (0.93g, 2.0mmol) was dissolved in ethanol (30ml) and acetic acid (0.2ml) was added. Then 10% palladium on charcoal (lOOmg) was added and the mixture was hydrogenated for 4h. The catalyst was filtered off, washed with ethanol (5x5ml) . After removal of the solvent an oil was obtained. The oil was washed with water to remove acetic acid and dissolved in chloroform, dried (MgSOή.) , concentrated to dryness to give oily product, 0.45g, 68% yield. The structure was confirmed by proton NMR and MS. (c) Z-D-Dpa-Pro-Pgl-P (OPh) _? Z-D-Dpa-Pro-OH (O.llg, 0.25mmol) was dissolved in dry chloroform (2ml) containing EC3N (0.035ml) and cooled to -5°C. Ethyl chloroformate (0.026ml, O.275mmol) was added and the mixture kept at -5°C for 30 min. A solution of diphenyl 1-aminopentanephosphonate (83mg, 0.25mmol) in dry chloroform (2ml) containing Et-N (0.025g, 0.25mmol) was added. The mixture was stirred at r.t. for 12h. Solvent was removed in vacuo. The resulting oil was chromatographed (CHCI3 then 2% MeOH in CHCI3) to give 123mg as crystals, 63% yield. Th structure was confirmed by proton and 31p NMR. (d) H-D-Dpa-Pro-Pgl P (OPh) , •p Z-D-Dpa-Pro-Pgl r (OPh) 2 (50mg, 0.063 mmol) was dissolved in ethanol (5ml) and acetic acid (0.01ml) was added. 10% Pd/C (25mg) was added and the mixture was hydrogenated at r.t. for 3h. The cacalysc was filcered off and echanol removed in vacuo. The resulting oil was treated with water (5ml) and chloroform (20ml). The chloroform phase was dried (MgSO^.) and concentrated to dryness to give crystals, 41mg, 91% yield. The structure was confirmed by H and 31p NM . (e) H-D-Dpa-Pro-Pql P (OH) ? Z-D-Dpa-Pro-Pgl^ (0Ph) 2 (lOOmg, 0.127mmol) was dissolved in ethanol (10ml) and acetic acid (0.1ml) was added. Then 10% Pd/C (50mg) was added and the mixture was hydrogenated at r.t. for 3h. The catalyst was filtered off, P θ2 (lOO g) was added and the mixture was hydrogenated at r.t. for 4h. The catalyst was filtered off, solvent was removed and the residue was treated with 20ml of water and chloroform (60ml). The organic layer was dried (MgSOή.) , concentrated to dryness to give 67mg as cyrstals, 92% as overall yield. The structure was confirmed by 1R and 31p NMR. Z-D-Phe-Pro-PglP (OPh)9 This compound was synthesized by the above procedure in 73% yield. The structure was confirmed by H and 31p NMR. H-D-Phe-Pro-Pgl * (OPh) 2 The compound was synchesized by che above procedure in 90% yield. The structure was confirmed by 1H and 31p NMR. H-D-Phe-Pro-Pgl 7 (OH)7 The compound was synthesized by the above procedure in 89% overall yield. The structure was confirmed by 1H and 31p NMR. (f) Diphenyl 1-(N-allyl)amino-4-Pyridylmethyl-phosphonate To a solution of 4-pyridinecarboxyaldehyde (1.07g,10mmol) and allylamine (0.61g,10mmol) in ether (30ml) was added anhydrous sodium carbonate (2.76g). The solution was stirred at r.t. overnight, then sodium carbonate was filtered off. To the reaction mixture, diphenylphosphite (2.34g 10mmol) and triethylamine (1.01g,10mmol) were added with cooling over an ice-water bath. It was stirred at r.t. overnight. After removal of the solvent an oily residue was obtained which was chromatographed (1:1 Petroleum ether/ethyl acetate) to give 2.85g (65%) as an oil. (g) Diphenyl 1-(N-allyl)amino-4-(tert- butyloxycarbonyl)butyl-phospohonate 4-(tert-butyloxycarbonyl)amino-butylaldehyde diethyl acetal (2.91g,lOmmol) was dissolved in acetone (20ml) in the presence of IN hydrogen chloride (1ml) and PPTS (150mg). The reaction mixture was refluxed for 3h. The solvent was removed and the residue was dissolved in chloroform and dried (MgSO.). After removal of MgSO. the solution was stirred and allylamine (0.61g,lOmmol) and anhydrous sodium carbonate (2.76g) were added. The reaction suspension was stirred at r.t. overnight. Then sodium carbonate was filtered off. To the solution obtained, diphenylphosphite (2.34g,lOmmol) and the triethylamine (l.Olg,lOmmol) were added. It was stirred at r.t. for 2 days. The residue obtained after evaporation was chromatographed on silica gel (1:1 Petroleum/ethyl acetate) to give 200mg as a yellow waxy SUBSTITUTE SHEET solid ( 5% ) . (h) Diphenyl 1-amino-4-pyridyl-methyl-phosphonate The N-allyl protected compound (1.0g,2.3mmol) was dissolved in ethanol (25ml). To the solution was added 10% Pd/C (300mg) and it was refluxed for 20 hours. The reaction was followed by HPLC, retention time: lO.Omin for the product and 12.0min for the starting material. After removal of the solvent the product was obtained by chromatography, 0.51g (56%) as a yellow oil. The following tripeptides were synthesized according to Scheme 5: Z-D-Phe-Pro-Cpg P (0Ph) 2 :0.36g(67%) Z-D-Phe-Pro-Epg P (0Ph) 2 :0.31g(87%) Z-D-Phe-Pro-Pyg P (0Ph) 2 :0.41g(35%) Z-D-Phe-Pro-Dmg P (0Ph) 2 :0.25g(70%) Z-D-β-Nal-Pro-Mpg P (OPh) 2 :54mg(32%) H-D-Phe-Pro-Epg P (OPh) 2 :126mg(71%) H-D-Phe-Pro-Dmg P (0Ph) 2 :85mg(52%). Abbreviations: Mpg = methoxypropylglycine Cpg ■= 4-cyanophenylglycine Epg = 2-ethylpropylglycine Pyg - 4-pyridylglycine Dmg = 3,3-dimethylpropylglycine Nal = naphtylalanine PPTS = Pyridinium p-toluenesulfonate SUBSTITUTE SHEET 3. Synthesis of peptide aminoboronic acid inhibitors (a) (+) -Pinanediol 4-brcπιo-R-l-aminobutane boronate hydrochloride The cicle compound was prepared as described by D . S. Matteson et al (1984) in Organometallics , 3_ > 1284-1288 and in European patent appl . 293881A2. (b) Z-D-Dpa-Pro-Irq-OPin.HCl To a solution of Z-D-Dpa-Pro-OH (236mg, 0.5πmol) in THF (5ml) in che presence of criechylamine (7C|ul, 0.5mmol) was added isobucyl chloroformace (65 l, 0.5mmol) ac -15°C and he solucion was scirred ac -13 β C for 13 min. Afcer che addicion of (+) - pinanediol 4-bromo-R-l-aminobucaneboronace hydrochloride (183mg, 0.5mmol) in CHCI3 (3ml) followed by chac of Et3 (70 / ul, 0.5mmol), he reaccion ixcure was scirred ac che same cemperacure for 2h, and Chen below 10°C for 2h. THF was removed under reduced pressure and che residue was dissovled in ethyl acetate (50ml), which was washed with 1% NaHC03, water, 0.2N HCl and water, and then dried over Na2Sθ - Removal of solvent gave an oily product quantitatively. HPLC analysis showed one major peak at the retention time of 22.8 min along with several minor components. To a solution of the above compound (2/5th of total amount synthesized, 0.2mmol) in ethanol (1ml) was added thiourea (61mg, 0.8mmol) under an atmosphere of argon at room temperature. After stirring for 4 days, ethyl acetate (70ml) was added to the reaction mixture, which was washed with 1% NaHC03, water, 0.2N HCl and then water, and dried over Na2S04< The. residue obtained by removing the solvent was treated with n-hexane to get the product as a powder. Reprecipitation from ethyl acetate wich 2:1 mixcure of eChyl echer and n-hexane gave a product (98.9mg, 60.6%, two step overall). Retencion time on RP-HPLC analysis was 13.5 min under the conditions described at the general procedure. H NMR analysis in deuceraced chloroform gave a complex paccerrt because of the existence of proline residue in the molecule, however, the typical signals corresponding to pinanediol were observed as proper racios. _ _ 4. Z-D-P e-Pro-boroMbq-OPin (Cf. Scheme 4) A solution of pinanediol (dichloromethyl)boronate (1ml, 1.2g, 4.6mmol) i THF (7ml) was placed in a septum fitted flask (100ml), and 1,1-dimethylpropan magnesium chloride (4.6ml, 4.6mmol) added dropwise from a dry syringe at 0°C. The reaction mixture was left stirring under nitrogen at room temperature After 7 hours TLC showed mainly one spot [Rf = 0.82, chloroform:pet.ethe (1:1)]. The solvent was removed and the residue dissolved in ether (50ml) washed with water (2x10ml), dried (MgSOή.) and filtered. The ether was removed and the crude product purified on a column of silic gel, eluted with hexane and 10% of chloroform to give the -»-chloroboroni ester as a pale yellow oil (0.55g, 40% yield). The above compound (0.55g, l.δmmol) in THF (5ml) was added via a doubl ended needle at -78°C to a solution of lithiumbis(trimethyl-silyl) amid (1.8ml, 1.8mmol) in THF (5ml) under nitrogen. The reaction mixture was kept overnight at 20°C then the solvent was removed. The crude product was dissolved in petroleum ether (40-60°C) (25ml) to precipiate out the inorganic salt (LiCl). The reaction mixture was filtered, cooled to -78°C and dry ethereal HCl, 1M (3 equiv, 5.4ml, 5.4mmol) added. The flask was kept in a fridge overnight. Next morning, the reaction mixture was filtered to isolate the hydrochloride (0.41g, 1.29mmol, 72% yield) as a white solid. Z-D-Phe-Pro-OH (0.45g, 1.lmmol) was dissovled in THF (7ml) and the equivalent of N-me hylmorpholine (O.llg, 1.lmmol) added. The solution was cooled to -20°C and one equivalent of isobutylchloroformate (0.149g, 1.lmmol) added dropwise. After 10 min., a solution of the above aminohydrochloride (0.348g, 1.lmmol) dissolved in THF (7ml) was transferred under nitrogen, and triethylamine (O.llg, 1.lmmol) added to the reaction mixture. The reaction mixture was stirred for one hour at -20°C, followed by 2h at room temperature. Insoluble material was removed by filtration, then the solvent removed by evaporation, and che residue dissolved in echyl acetate (30ml). The organic layer was washed wich 0.2N hydrochloric acid (10ml), 5% aqueous sodium bicarbonate, saturated solution of sodium chloride and water. The organic phase was then dried over anhydrous MgSOή,, filtered and the solvent evaporated to give a white solid which was purified on a column of silica gel eluted with light petroleum to give the desired product (0.59g, 81%). The structure was confirmed by *H NMR and MS. 5. PREPARATION OF 0 -BROMO BORONIC ESTERS (Cf. Scheme 6) All the reactions involving boron used purified anhydrous reagents. Reactions were carried out under argon or nitrogen used directly from the cylinder through a glass line. In a 250 ml reaction flask fitted with a reflux condenser was placed 1-bromo-l-propene (3.63g, 30mmol). Dibromo borane -methyl sulfide complex in dichloromethane (60ml, 60mmol) was then added to the reaction flask dropwise and the mixture was refluxed under nitrogen for 5h. The solvent was removed and the reaction mixture washed with water and dried (MgSO.). A dry round-bottomed flask (lOOml) was charged with the bromo boronic acid (0.5g, 3mmol) and pinanediol (0.52g, 3mmol) a magnetic follower and dry ether (20ml), fitted with a septum and flashed with nitrogen. The reaction mixture was left stirring for two hours until the solid dissolved, the organic phase was washed with water (10ml), separated, dried (MgSO.) and filtered. The crude product was purified on a column of silica gel (230-400 mesh), eluted with chloroform (the product was eluted before the pale red ring). The first fraction (100ml) was collected and the solvent evaporated to give -K-bromo boronic ester (0.8g, 88.6%) as a colourless liquid. SUBSTITUTE SHEET 6. Synthesis of isosteric etcmethylene inhibitors (Cf. Scheme 2) (a) R ^D-Dpa-P.ro-Arg(Mtr)-k-Gly{-Me (modified Parkin-West reaction) Boc-D-Dpa-Pro-Δrg(Mtr)-OH (0.126g, 0.15mmol) was added to monomethylsuccinyl anhydride (0.259g, l.Ommol). Et3 (0.042ml, 0.30πmol), DMAP (1.8mg, 0.015mmol) and pyridine (0.1.2ml) were added, the reaction flask was fitted with a reflux condenser, and the reaction was heated at 45-50 β C. The reaction mixture was stirred for lh, then NaHCθ3 (5%, 5ml) was added and the stirring was continued for an additional 30min. The product was extracted into ethyl acetate and washed wich AcOH (0.1N) and brine. The organic layer was dried (MgSOή.), filcered and concencraced co dryness Co give an oily residue which was chromacographed on silica gel (grade 9385, 50g). Elucion wich CHCl3:CH3θH,98:2 gave the product after removal of che solvenc as a brown oil (0.134g, 98%). The scruccure was confirmed as Boc-Dpa-Pro-Arg(Mtr)-k-GlyOMe by 1H NMR (250MHz), and by FAB mass spectrometry. (b) Z-D-Dpa-Pro-Arg(Mtr)-k-Cly-pip A solution of Z-D-Dpa-Pro-Arg(Mtr)-k-Gly-OMe (O.lg, 0.lmmol) in MeOH (10ml) was cooled to 0°C and NaOH (IN, 0.22ml, 0.22mmol) was added with stirring for 2.5h at room temperature. The solution was neutralized to ρH7 and the MeOH was removed under reduced pressure. The aqueous solution was acidified (pH2) and extracted by ethyl acetate and dried (Na2S0 ^ • ae solvent was removed under reduced pressure to give an oil. To a solution of the oil and HOSu (12mg, 0.lmmol) in dimethoxy ethane (20ml) was added OCC (21mg, 0.lmmol), with cooling. The solution was stirred at room temperature for 20h, and piperidi (17mg, 0.2mmol) was added to the solution with cooling and the solution w stirred at room temperature for a further 3h. The solution was concentrated t dryness and the product was purified by chromatography on silica ge (MeOH:CHCl3, 92:2) to give Z-D-Dpa-Pro-Arg(Mtr)-k-Gly-pip (78mg, 81%). Th structure of the product was confirmed by 1H NMR and FAB mass spectrometry. In a separate experiment the L-isomer Z-L-Dpa-Pro-Arg(Mtr)-k-Gly-pip, wa synthesized by the above procedure in 55% yield, (c) H-D-Dpa-PrO-Arg-k-Gly-pip.2TEA Z-D-Dpa-Pro-ArgMtr-k-Gly-pip (52mg, 0.053mmol) was dissolved in 0.9ml of TF and 0.1ml of thioanisole at room temperature. After stirring for 4h. TFA wa removed at reduced pressure,and the residue was triturated with ether. Th crystals were collected and washed with ether (51mg of Mtr deprotected product) . The crystals were then dissolved in 5ml of methanol and 21mg of 10% Pd/C was added. After 20h. hydrogenation at room temperature the catalyst was removed by filtration and the solvent evaporated. The residue was triturated with ether to give white crystals. 34mg (75%), m.p. 146-151 (dec). HPLC showed two equally, big peaks, from the two forms containing D and L Arg. The structure was confirmed by 1H NMR and FAB mass spectrometry. In a separate experiment the L-isomer of Z-Dpa-Pro-Arg(Mtr)-k-Gly-pip was deprotected to give H-L-Dpa-Pro-Arg-k-Gly-piρ.2TFA in 43% yield. 7. Synthesis of peptide aldehydes (Cf. Scheme 8) (a) Z(N02).Arg N Q .3(0CH3) N,0-dimethylhydroxylamine hydrochloride (1.45g, 14.9mmol) was dissolved in 10ml of DMF, and the solution kept at 0°C. Diisopropylethyla ine (1.92g, 14.9mmol) was first added and then Z-(N02)Arg in 10ml of DMF, HOBT (1.92g, 14.2mmol) and WSC.HCL (2.99g, 15.6mmol). After 5h reaction at 0°C the solution was left at room temperature overnight. The solvent was removed at reduced pressure and 100ml of EtOAc and 25ml of H2O were added. The organic phase wa diluted with ether and washed with Na 2 C0 3 (0.5M), H2O, H2SO4 (0.1M) and H2O then dried and the solvents removed giving 3.66g of product. The wate solutions were combined, extracted and treated in the same way as above givin further 1.69g. Totally 5.35g (95%) which was chromatographed on Sephadex LH-2 *ith 95% ethanol. Yield 4.60g (82%) of homogenous product (TLC in S^ and S2). NMR confirmed the structure. (b) Arg(NO2)-N H3(OCH3) .HBr The above compound was deprotected in HBr/HOAc at room temperature for 45min in the usual manner. The product was homogenous according to TLC in S3, S4 and S 5 . (c) BθC-D,L-Dpa-Pro-Arg(N02)-NCH3(OCH3) Arg(N02)-NCH3(0CH3).HBr (4.9g, 13mmol) was dissolved in DMF, the solution cooled co -5°C and EC3N added co alkaline reaccion. Boc-DL-Dpa-Pro OH (5.5g, 12.5mmol), HOBT (1.7g, 12.5mmol) and WSC (2.8g, 14.5mmol) were added and che reaccion mixcure scirred for 2h. ac -5°C and Chen scirred ac room cemperacure overnight. The solvent was evaporated at reduced pressure, EtOAc and H2O were added, the organic phase separated and extracted with 0.5M NaHCθ3 (3x30ml), NaCl solution (4x20ml), dried ( aSθ4 and the solvent removed. Yield 8.3g (97%) TLC (S2) shows one spot. The expected structure of the compound was confirmed with NMR. (d) £_c-D,L-Dpa-Pro-Arg-NCH^(OCH3) .HCl The above compound (2.33g, 3.4mmol) was dissolved in 240ml of MeOH and 3.6ml HCl (1M). The catalyst, 10% Pd/C (0.6g) was added and hydrogenation performed at room temperature for 20h. The catalyst was filtered and the solvent removed at reduced pressure. Remaining 2.3g solid contained some starting material which was removed by ion exchange chromatography on Sephadex QAE + Cl ~ with 50% EtOH. Yield 1.74g (76%). TLC ( 'S Δo a,„na,, sh.owed. a singl, e spot. (e) Boc-D , L-Dpa-Pro-Arg-H . HCl The above compound (0.5g, 0.74mmol) was dissolved in 40ml of dri (molecular sieve, 4A) THF and che solucion was cooled co -40°C and 3.2ml DIBAH (IM toluene solution, 3.2mmol) was added dropwise during stirring argon atmosphere. After 3h. 12.8ml of 0.25M citric acid was added. T aluminium salts were centrifuged and washed several times with THF H2O (4:1) From the combined liquid phases THF was removed at reduced pressure and th product extracted with EtOAc. The solvent was removed, the product dissolve in 15ml of 20% HOAc and chromatographed on Sephadex G15 with 20% HOAc a eluenc. Yield 172mg (38%). The compound shows a double spoc in TLC (S2 an S3) probably showing che cwo isomeres wich D- and L-Dpa, respeccively. NMR an MS were in agreemenc wich che expecCed scruccure. 8. Synthesis of Boc-D,L- a-P-X>-ArgCN.HCl (a) Z-D,L-Dpa-PTO-Arq-NH?.HCL Arg-NH2.2HC1 and Boc-D,L-Dpa-ProOH were coupled in che normal way wich HOB and DCC in DMF. Yield 53%. (b) Boc-D,L-ppa-Pro-ArgCN.HCl The above cripepcide amide (0.50g, 0.79mmol) and cosylchloride (0.50g, 2.55mmol) were dissolved in 2ml of pyridine ac room ce peracure and scirred for 24h. The pyridine was evaporaced ac reduced pressure, 5ml of pyridine and 0.5ml of water were Chen added and scirring concinued for 2h. Afcer evaporation at reduced pressure the residue was triturated with a small amount of water, dissolved in EtOAc, dried ( a2S0 and chromatographed on Sephadex QA∑Φsi®. The fractions containing the product were evaporated, dissolved in 10% HOAc and freeze dried. Yield:0.33g (68%). [ σUD 2 2°=. -144° (C-0.5, 50% HOAc). The structure was confirmed with 1H NMR and mass spectroscopy. 9. Synthesis of Dba ( a) Preparation of N-formyl-N,°(,β-tribenzylalanine cyclohexylamide Bzl-NH 2 288 μl (2.64 mmol) and benzyl phenethylketone 592 mg (2.64 mmol) were dissolved in MeOH (5 ml) at rt and the solution was stirred overnight. To the mixture were added formic acid 99.6 μl (2.64 mmol) and cyclohexylisocyanide 298 μl (2.4 mmol) at rt, which was allowed to react at rt for 2 weeks. Insoluble material in MeOH was collected by filtration and washed with MeOH, ether and then n-hexane. The crude product was recrystallized from CHC1 3 with the 2 to 1 mixture of ether and n-hexane. Yield 540 mg (48.2%), NMR in CDClg? =0.8-1.8 cyclohexyl (11H), =2.1-4.75 CH 2 (8H), $ =5.45-5.55 NH (IH), 2=7.0-7.4 phenyl (15H), =8.25 (main) and 8.4 (minor) formyl (IH). (b) Preparation of Bzl-Dba The fully protected Dba 400 mg (0.85 mmol) was dissolved in 2.5 ml of TFA and 3 ml of 11 N HCl and the solution was kept at 145 C C with water-cooled condenser and stirred for 20 h. After removal of TFA, the pH of the solution was adjusted around 7 by addition of 10 N NaOH. Further addition of ether gave a powder which was collected and washed with ether. Yield 124 mg (40.4%). SUBSTITUTE S eHπc[ T 10. Synthesis of Z-D-Phe-Pro-Pgl-H (a) Pgl Pentylglycine was obtained by the Strecker* synthesis, using hexanal, in a yield of 31.6% (*Vogel, Textbook of Practical Organic Chemistry). (b) Z-Pgl To a solution of pentylglycine (0.5g, 3.5mmol) in a mixture of water (4ml) and THF (4ml) to give a 0.5M solution, in the presence of triethylamine (0.64ml, 1.22eg.) was added Z-OSu (0.963g, 3.86mmol.) at room temperature, solution became clear after 15min. TLC after 2h indicated some starting material and so a further portion of triethylamine (0.2ml) and Z-OSu (200mg) was added. TLC after a further 2h indicated no starting material and so the solution was poured onto water (50ml) and was extracted by CHCl-(50ml). The organic phase was washed by HCl (IM, 20ml) and dried (MgSO.) and concentrated to give Z-Pgl (1.18g) as a gummy solid, which was recrystallised (DCM/Petroleum ether bp60- 80°C) to a white crystalline solid (0.8g). Structure was confirmed by H Nmr. (c) Z-Pql-NMe(OMe) To a solution of hydroxylamine (184mg, 1.05eq.) was added DIPEA (0.33ml, 1.05eq.), and after 5min a solution of Z-Pgl (0.5g, l.δ mol) then HOBT (0.242mg, 1 eq.). The solution was cooled to -15°C and a solution of WSCI.HC1 (0.378mg,l.leq. ) was added. The solution was maintained at -15°C for 30min. then allowed to warm to room temperature and pH adjusted to r- * >pH4 by addition of acetic acid. TLC after 16h shows little SUBSTITUTE SHEET starting material and so the solution was poured onto NaHCO-. (100ml) and extracted by Et 2 0 (50ml). The Et-,0 phase was washed by NaCl (20ml). The aqueous phase was washed by Et 2 0, the organic phases were combined and then concentrated to give Z-Pgl.NMe(OMe) (324mg). Structure confirmed by H Nmr and mass spec. (d) PglNMe(OMe) To a solution of Z-PglNMe(OMe) (324mg) in MeOH (10ml) was applied a vacuum, then argon. Purging was repeated twice, but on the third time Pd/C ("O.5g) was added. Purging was repeated twice more and on the third time the vacuum was quenched by bubbling Hydrogen through the solution. AcOH (0.5ml) was then added. After 90min TLC indicated no starting material and so the solution was filtered (celite), washing with a large volume of MeOH, and concentrated to give PglNMe(OMe)(380mg), as a gum. Structure was confirmed by 1 H Nmr. (e) Z-D-PheProPqlNMe(OMe) To a solution of Z-D-PhePro (0.187mg, leq. ) in DMF (2ml) was added DIPEA (0.084ml, leq.) and PyBOP (0.25mg, leq.), with stirring under argon. After lOmin a solution of PglNMe(OMe) (0.09mg, 0.479mmol), in DMF (1ml), was added. After 90min TLC indicated no starting material and so the solution was poured onto HCl (IN, 50ml) and extracted by Et 2 0 (50ml). The Et 2 0 layer was washed by NaHCOg (50ml, 1.2N) and NaCl (sat'd, 20ml) and dried (MgSO.). Repeated chromatography on silica gel (Merck 9385), eluting with CHClg/MeOH gave Z-D- Phe-Pro-PglNMe(OMe) (200mg). Fab Ms shows 567 (10%, M+H), SUBSTITUTE SHEET and 589 (100%, M+Na) as required, the structure was also confirmed by H Nmr. (f) Z-D-Phe-Pro-Pgl-H To a solution of Z-D-PheProPglNMe(OMe) (33mg) in THF (2ml) at - 40°C was added Di-isobutylaluminium hydride (IN, 0.155ml, 2.5eq.), under argon. The solution was allowed to warm to room temperature and stirred for 18h. TLC showed no starting material and so was quenched by H 2 S0. (IN, 0.5ml) and stirred for lOmin. The aqueous phase was then extracted by EtOAc (20ml). the organic phase was dried (MgSO. ) and concentrated to give Z-D-PheProPgl-H (31mg). The structure was confirmed by H Nmr and mass spectrum, and the compound was sent for biological testing, as compound number 33. SUBSTITUTE SHEET 11. Z-L-Val-pNa 4-nitroaniline (67.5g,0.48mol) was dissolved in pyridine (dried over 4A sieves,750ml), and the solution was cooled down with ice. PC1 3 (34.3g, 0.25mol) was dissolved in pyridine (350ml) and added dropwise to the nitroaniline solution. The solution was allowed to stand for 30 min. at room temperature. To the solution was added a solution of ZZVal-OH (112g,0.44mol) in pyridine (250ml). The reaction mixture was stirred at room temperature for 1 week, then the pyridine was removed on a rotavapor. The residue was treated with NaHC0 3 (2%). The product crystallized and was filtered and washed with water. The product was dissolved in boiling EtOH (2000ml, 95%) and the hot solution was filtered and left to stand overnight at room temperature. The crystals were filtered giving Z-L-Val-pNA (116.9g, 71.6%). 12. (a) Boc-Dpa Boc-Dpa was obtained in an analogous manner to the procedure of Examples 1(a) and (b). ( b ) Boc-Dpa-Pip-Arg-pNA (Cf . Scheme 7 ) To a solution of Boc-Dpa.HCl (150mg, 0.396mmol) in DMF(5ml) was added TBTU (133.5mg, 0.416mmol, 1.05eq.) and DIPEA (0.069ml, 0.396mmol, leq.), to give a solution of basic pH. PipArgpNA.TFA (238mg, 0.396mmol, leq.), was then added. pH of the solution was acidic so a further portion of DIPEA (0.05ml) was added. After stirring for 16h, TLC showed a little starting material and so a further portion of DIPEA was added (0.034ml, 0.5eq. ). TLC after a further 3h showed no starting material and so the solution was poured onto EtOAc (50ml) and washed by HCl(0.5N, 200ml), dried (MgSO. ) and concentrated to give 430mg of gum. Trituration with Et~ and petroleum ether (b.p.60-80 β C), over 5 minutes, gave a powder which was filtered, avoiding drying in the air, to give a BocDpaPipArgpNA.TFA as a powder, 207mg, 62% yield, Rf 20min on Rp HPLC (pep-S, 35-70% MeCN+0.1%TFA, 25min, lml/min. ). (c) Dpa-Pip-Arg-pNA To solid BocDpaPipArgpNA (lOOmg, 0.119mmol), was added TFA (2ml), with cooling in an ice bath for lOmin. The ice bath was then removed and the solution stirred for a further 10 min. TLC indicated no starting material, so the solution was concentrated under vacuum (oil pump). Washing with Et 2 0(150ml), until the filtrate was neutral pH, gave a powder dried under vacuum to DpaPipArgpNA.2TFA, 92mg, (90%), as a powder, of retention time (15.5min on Rp HPLC pep-s, 4x250mm, 35-70% MeCN +0.1% TFA). SUBSTITUTE SHEET ( d) Boc-D-Nal-Pip-Arg-pNA To a solution of Boc- -D-Nal(150mg, 0.474mmol) in DMF (5ml) was added TBTU (164mg, 0.51mmol, 1.05eq.) and DIPEA (0.083ml, 0.474mmol, 1 eq.). PipArgpNA.TFA (285mg, 0.474mmol, 1 eq.) was then added. After 30 min TLC, using the Sakaguchi reagent(8-hydroxyquinoline, Br 2 , NaOH), indicated mainly starting material, and so DIPEA (0.083ml, leq. ) was added. After 30min. TLC indicated mainly starting material and so the reaction was diluted by EtOAc (50ml) and washed by HCl (0.5N, 100ml). The organic phase was dried (MgSO.), and concentrated to give an oil (490mg). Recrystallisation from CHC1 3 /petroleum ether bp 60- 80°C/Et 2 0 gave 90mg, 20.1% yield, of Boc-Dpa-PipArgpNA.TFA as a powder. (e) H-D-NalPipArgpNA.2TFA To solid Boc- -D-NalPipArgpNA.TFA (50mg, 0.055mmol), cooled in an ice bath, was added TFA (2ml), with stirring. After lOmin the ice bath was removed, TLC after 30 min still showed some starting material and so solution was stirred for a further 10 min, then concentrated (oil pump). Trituration with Et 2 0 have a powder. The powder was washed with Et^O, until the eluant was neutral, dried overnight at room temperature to give H-D-NalPipArgpNA as a powder, 42mg, 92% yield. Retention time 11.5min on Rp HPLC. SUBSTITUTE SHEET Example 13 The following compounds were prepared by the route substantially as outlined in Examples 2a to 2e: Yield Physical data to confirm structure 15 Boc-D,L-Dpa-Pro-Pgl (OH) 2 96% Nmr,Ms. 16 D,L-Dpa-Pro-Pgl P (OH) 2 17 Z-D-Dpa-Pro-Pgl P (OPh) 2 18 D-Dpa-Pro-Pgl P (0H) 2 92% Nmr, FABMs[M+H]502,m.p.130-133. 19 Z-D-Phe-Pro-Pgl P (OPh) 2 20 D-Phe-Pro-Pgl P (0Ph) 35% Nmr, FABMs. p Δ 21 D-Phe-Pro-Pgl (OH) 2 23 D-Dpa-Pro-Pgl P (0Ph) 2 32 H-D-Dpa-Pro-Mpg P (OPh) 2 H, 31 PNmr,HPLC. 40 H-D-Phe-Pro-Mpg P (OPh) 2 HPLC. 55 H-D-Phe-Pro-Apg P (OPh) 2 HPLC. 56 H-D-Phe-Pro-Epg P (0Ph) 2 71% 57 H-D-Phe-Pro-DPg P (OPh) 2 52% Nmr. Example 14 The following compounds were synthesised according substantially to Examples 2a to 2d: Z-D-Phe-Pro-Epgl(OPh) 87% Z-D-Phe-Pro-CpgL(OPh)^ 67% Z-D-Phe-Pro-Pyg (QPh)^ 35% Z-D-β-Nal-Pro-Mpg (OPfi). 32% Example 15 The following compound was synthesised according to Examples 2a and 2b: MTy (0Ph) 2 Example 16 The following compounds were synthesised according substantially to Example 2f: p D-Phe-Pro-Pyg p (OPh) ? D-Phe-Pro-AegL(Boc) θPh) 9 D-Phe-Pro-Npg (0Ph) 2 Example 17 The following compounds were synthesised according substantially to Examples 6a to 6c: 4 D,L-Dpa-Pro-Arg—k—Gly-Pip 5 D-Phe-Pro-Gpa—k—Gly-Pip 81.2% Nmr,FABMs,m.p.110-114. 6 D-Dpa-Pro-Arg—k—Gly-Pip 74.5% Nmr,FABMs,m.p.146-151. 7 L-Dpa-Pro-Arg—k—Gly-Pip 42.7% Nmr,FABMs,m.p.136-140. 8 D-Fgl-Pro-Arg—k—Gly-Pip 81.3% Nmr,FABMs. 9 D,L-K-Nal-Pro-Arg~k—Gly-Pip 81% Nmr,FABMs,m.p.123-127. 14 D,L-β-Nal-Pro-Arg—k—Gly-Pip 55% FABMs. fceηriTUTE SKCHT 54 D-β-Nal-Pro-Arg— k — Gly-Pip 41% FABMs . Example 18 The following compounds were synthesised according substantially to Example 10: 33 Z-D-Phe-Pro-Pgl-H 99.0% Nmr, FabMs[M+H]508,7% 48 Z-D-Dpa-Pro-Pgl-H quantitative Nmr, FabMs[M+H]584,35% 53 Boc-D-Phe-Pro-His-H 90% Nmr, FabMs[M+H]484 Z-N-Me-Phe-Pro-Pgl-NMe(OMe) Example 19 The following compounds were synthesised according substantially to Example 11: 34 H-D-β-Nal-Pip-Arg-pNA 90.6% Nmr 35 H-D,L-Dpa-Pip-Arg-pNA 81% Nmr H-D-Phe-Pro-Phe-pNA Example 20 The following compounds were synthesised according substantially to Example 4: 22 Z-D-Phe-Pro-BoroMbg-OPin 89% Nmr 41 Z-D-Phe-Pro-BoroPhe-OPinac 68.6% Nmr 59 Z-D-Phe-Pro-BoroMbg-OPin 90% Nmr Example 21 The following compounds were synthesised according substantially to Example 3a: 10 Z-D-Phe-Pro-BoroAcet-OPinac 42% Nmr 11 Z-D-Phe-Pro-BoroPgl-OPinac 41.5% Nmr 39 Z-D-Dpa-Pro-BoroMpg-OPin 43 Z-D-Phe-Pro-BoroOct-OPinac 77% Nmr 51 Z-D-Dpa-Pro-BoroMpg-OPin quantitative Nmr Example 22 The following compounds were synthesised according substantially to Examples 3a and 3b: 12 Z-D-Dpa-Pro-BoroIrg-OPin 61% Nmr, FABMs[M]780,13%. 26 Z-D-β-Nal-Pro-BoroIrg-OPin 41.8% Nmr, FABMs[M+H]755,10%. 36 Z-D-Fgl-Pro-BoroIrg-OPin 49% Nmr, FABMs[M+H]778,11%. 37 Ac-D-Dpa-Pro-BoroIrg-OPin 8.1% Nmr, FABMs[M+H]689,14%. 38 Z-L-Dpa-Pro-BoroIrg-OPin 39% Nmr, FABMs[M+H]781,12%. 46 Z-D-Cha-Pro-BoroIrg-OPin 41% Nmr, FABMs[M+H]711,10%. SUBS' 1" ? ' -35- n- oμ 2 - CθQ/ • 1-fCl o C4. - α - >«-CN - oαH I MH, • R ΛiΛil FΛ( O - Λ C I + ti- cLi - αwλ t i -* ■ «-• * ι*ιH VG. 0-.-A « cHo + CtHs-CHz-o -κι« 2 + C fe W_rC>) Kc lA C ς H f - -P (o " P^ 2 _ 1 I0 η ' d/c κ 2 co 3 0 II P /c 0 II H 2 NCHP(OPh) 2 SCHEME 5 Scheme 6 SUBSTSTUTE SHEET, D,L-Aa BocPipArgpNA BOC-D, -Aa H-PipArgpNA.2TFA TBTU Boc-D,L-Aa-Pip-Arg-p.NA.2TFA TFA H-D, -Aa-Pip-Arg-pNA.2TFA Scheme 7 SUBSTITUTE SH CH 3 ( CH 2 ) 4 CH0 CH 3 (CH 2 ) 4 CH -C0 2 H \ Pentylglycine(Pgl) NH 2 .HCl (1,1) Z-Pgl (3,1) Z-Pgl-NMe(OMe) (4,1) H-Pgl-NMe(OMe) (5,1) X-Pep-Pgl-NMe(OMe) (6,1) DIBAL-H Scheme 8 SUBSTITUTE SHEET Pla-sπa thr πbin rime (TT) A volume of 150μl of citrated normal human plasma and 20μl of buffer or sample were warmed at 37°C for 1 min. Coagulation was started by adding ISOul of freshly prepared bovine thrombin (5NIHu/ml saline) and the coagulation time was recorded on a coagulometer. A phosphate buffer, pH7.8, containing 0.1Z bovine serum albumine and 0.02% sodium azide was used. The samples were dissolved in DMSO and diluted with the buffer. When no inhibitor was used DMSO was added to the buffer to the same concentration as that used in the samples. The inhibitor concentrations were plotted against the thrombin times in a semilogarithmic graph from which the inhibitor concentration that caused a doubling (40 sec) of the thrombin time was determined. Determination of Ki The inhibition of human ot-thrombin was determined by the inhibition of the enzyme catalyzed hydrolysis of three different concentrations of the chromogenic susbtrate S-2238. 200ul of sample or buffer and 50jul of S-2238 were incubated at 37°C for 1 min and 50ul of human <X.-thrombin (0.25 NIHu/ l) was added. The initial rate of inhibited and uninhibited reactions were recorded at 405π . The increase in optical density was plotted according to the method of Lineweaver and Burlce. The Km and apparent Km were determined and Ki was calculated using the relationshi : Ki = Km app __ - Cardiovascular effects Cats weighing 2-3kg were anaesthetized with Mebumal, given as an intraperitnoeal injection. The central blood pressure and heart rate were recorded on a Grass Polygraph by means of a catheter inserted in the femoral DETERMINATION OF km The km of substrates with human <X-thrombin were determined by measuring the absorbance at a series of dilutions of substrate, (page 753, Longman Scientific & Technical, 5th edition, 1989.) DETERMINATION OF ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) FOR IN-VITRO SAMPLES A volume of 150μl of citrated (3.2%) normal human plasma was incubated at 37°C with sample (20^ιl) or buffer (20μl, control) for 1 min. To the solution was added reconstituted "AUTOMATED APTT" (available from Organon Teknika,0.1ml). Each solution was activated at 37°C for 5 min. After activation, calcium chloride (0.1ml, 0.025M, prewarmed at 37°C) was added and clot detection was timed using a "semiautomated coagulometer" (Nach, Schnicter und Gross). IN-VIVO TOXICITY DATA Deposition of 11 - indium-labelled platelets was monitored after lmg/kg intravenous doses, via a cannula in the marginal ear vein of New Zealand white rabbits, as outlined in G.R.May, CM.Hero, K.D.BUTLER, C.P.PAGE, JOURNAL OF FHARIYIACOLOGICAL METHODS, 24,ppl-35, 1990. Peripheral blood pressure was monitored by a pressure sensor in the carotid artery. EX-VIVO APTT, TT Data was obtained as for in-vitro tests, but using plasma samples obtained at 0,1,10,30 and 60 minutes after dosing of lmg/kg, i.v. bolus injection in a marginal ear vein in New Zealand white rabbits, which were anaesthetized from a canula in the carotid artery. Mty XNH-CH-Z Pyg XNH-CH-Z ■ •! OMe 
 Dpg XNH-CH-Z AM A COMPOUND NO. O O — mm --» Cxi 3 Sis 8 S:S& ε s F. I* -50- References 1. Claeson, G, and Aurell, L. Annals of New York. Academy of Sciences , 370, 79-811 ( 1981 ) . 2. Bajusz, S., Barabas, E., Tolnay, P. et al. Inc. J. Pepcide Protein Res. J , 217-221 (1978). 3. Kettner, C. and Shaw, E. Thrombosis Research JΛ, 969-973 (1979). 4. Szelke, H. and Jones, D.M. DS patent 4,638,047-A. 5. Kettner, C. and Shenvi, A.B. European patent appl. 293881 (1988). 6. Stuber, ., Kosina, H. and Heimburger, N. Int. J. Peptide Protein Res. 31_, 63-70 (1988). 7. Kaiser, B,, Hauptmann, J. and Harkwardt, F. Die Pharmacie 42, 119-121 (1987).

Claims: CIAIMS 1. A peptide derived from the formula D-Phe-Pro-Arg or its analogues, wherein Phe is substituted by wherein Ari and Δτ2 *ι*6 tbe ^aae or different and are selected frαn the gro consistinz of phenyl , thienyl, pyridyl, naphthyl, thionaphthyl , indolyl and .saturated groups corresponding to these, optionally substituted by up to th groups selected frαn C 1 - 3 alkyl and ^-C * alkoxy, Lj .and 2 are the s-ame or different and .are selected f.rcra the grou consisting of CH2, C&2- ~ CE-2, O" 0 ^* S-CH2 , op4-ionαi ) Δr-L taken together j^ meansH, diphenyl-methyl, fluo.renyl or saturated groups corresponding to these, but one of the .Ar-L cannot be H when the other Ar-L means H or benzyl , 2. A peptide according to claim 1, wherein Phe is substituted by Dpa, Nal or Dba. 3. A peptide according to claim 1, wherein Arg is substituted by H, N-CH-COOH, 2 I Y wherein Y ■ CH2] n -Q ι CH2-^≤^*Q where Q » H r amino, amidino, imidazole, guanidino or isothioureido and n = 1-5, prafe.rahly 3-5, or C--C 8 alkyl ancf C--C. Q aryl or a . lkylaryl optionally .substituted by up to three groups selected fixm hydroxy and C.-C, alkoxy . 4. A peptide according to claim 3, wherein Arg is substituted by Irg, Gpa, Apa or a non-basic amino acid. 5. A peptide according to claim 4, wherein the non- basic amino acid is selected from Pgl, Mbg and Chg. 6. A peptide of the formula X-Aa. -Aa,-NH-CH-Z, 1 2 I Y wherein X - H, CH3 or an N-protecting group, e.g. Ac, Bz,Cbz,Boc; Y ■ [CH2Jn~Q > CH 2 -^^^"Q where Q » H, amino, .amidino, άmidazole, gu___idino or i≤othiourεido and n = 1-5, preferably 3-5, or C.-C- alkyl andC ς - 10 ary l or alkylaryl optionally substituted by up to three groups selected frαn hydroxy and C.-C. alkoxy; R 2 R / 0 / Z ■ CN, CORi, B or ηp where *1 - H, CE, Ca-Cl, CH--CH 2 -CO-pip, CF 2 -CF 2 -COpip, CH∑-CH∑-CO-Pro-NHEt, CF 2 -CF 2 -CD-P.ro-NHEt or a chrαnophoric group e.g. pNA, WC , R2 and R3 are the same or different and are selected frαn the group consisting of OH, 0R $ and NR. 7, or R2 and R3 taken together represent the residue of a diol; where Rg and R7, which are the same or different, are c l" c 10 alky 1 * phenyl or Cg-Cio arylalkyl , B- and R5 xe the same or different and are selected frαn R j , R^, Gly-pip, Ala-pip or Gly-Pro-NHEt; Δrι- ι ai = « C NH2 -C00H where ΔT2- 2 Arj and τ2 α.τ the s.ame or different and are selected frαn the grou consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphthyl , indolyl and •saturated groups corresponding to these, optionally substituted by up to thr groups selected frαn C--C3 alkyl and C--C, alkoxy, - * l and L2 °-~-e the same or different and are selected from the gr consisting of CH2, CH2-CH2, 0-CH2* S-CH2 > otionally Ar-L taken together / meanSH, diphenyl-methyl, fluorenyl or saturated groups corresponding to these, but one of the Ar-L cannot be H when the other Ar-L means H or benzyl; A&2 -* or its Cj-C3 alkyl substituted derivatives, where R » CH2, CH2-CH2, S-CH2, S-C(CH3>2 or CH2-CH2-CH2. 7. A peptide according to claim 6 which is selected from the following: Ac-D-0Nal-Pro-boroΔrg pinanediol ester Z-D-Dpa-Pro-boroIrg pinanediol ester 2-D-Dpa-Pro-boroPgl pinacol ester Ac-D-BNal-Pro-boroMbg pinanediol ester CH3-D-Dpa-Pro-Arg-H Boc-D-Dpa-Pro-Gpa-H CH3-D-Dpa-Thi-Mbg-H H-D-Dpa-Pro-Arg-k-Gly-pip Z-D-Dpa-Pro-Arg-CH 2 Cl Boc-D-Dpa-Pro-.ArgCN H-Dpa-Pro-Arg P (0Ph) 2 H-D-BNal-Pro-Pgl P ( OPh ) -Gly-pip H-D-Dpa-Pip-.Arg-pNA H-D-BNal-Pro-Chg-pNA and the compounds listed in E χ .amples 10 to 22 herein. 8. A substrate of thrombin, which is or includes a peptide according to any one of claims 1 to 7. 9. .An inhibitor of thrombin, which is or includes a peptide according to any one of claims 1 to 7. 10. A method of inhibiting thrombin in a mammalian host, comprising administering an effective amount of a peptide according to any one of claims 1 to 7. 11. A method according to claim 10, wherein the peptide is administered in combination with a pharmaceutically acceptable carrier or diluent. 12. A method according to claim 10, wherein the peptide is administered at a dose of from 0.02 to 15mg/kg of body weight. 13. A method according to claim 12, wherein the dose is from 1 to lO g/kg of body weight. 14. A method according to claim 10, wherein the peptide is administered as a single dose or in divided doses or as a sustained release formulation. 15. A method according to claim 10, wherein the host is a human host. 16. A method of preventing coagulation of mammalian blood, comprising adding thereto an effective amount of a peptide according to any one of claims 1 to 7. 17. A method according to claim 16, wherein the amount of peptide added of from 1 to lOmg/litre. 18. A method of establishing an extracorporeal blood loop for a patient, comprising administering intravenously from 0.1 to lmg/kg of body weight of a peptide according to any one of claims 1 to 7. 19. Use of a peptide according to any one of claims 1 to 7 as an inhibitor or substrate of thrombin. 20. Use of a peptide according to any one of claims 1 to 7 in the preparation of a medicament for the treatment or prevention of thrombosis. 21. A pharmaceutical composition for inhibiting thrombin in a mammalian host, comprising a peptide according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or diluent.

Inventors: CHENG LEIFENG, CHINO NAOYASHI, CLAESON GORAN KARL, DEADMAN JOHN JOSEPH, ELGENDY SAID MOHAMED ANWAR, KAKKAR VIJAY VIR, SCULLY MICHAEL FINBARR, CHENG, LEIFENG, CHINO, NAOYASHI, CLAESON, GORAN, KARL, DEADMAN, JOHN, JOSEPH, ELGENDY, SAID, MOHAMED, ANWAR, KAKKAR, VIJAY, VIR, SCULLY, MICHAEL, FINBARR
Applicants: THROMBOSIS RES INST, THROMBOSIS RESEARCH INSTITUTE
Application year: 1991
Main classification: C07K5
IPC codes: A61K38, A61P7, A61P43, C07K5, C07K7, C12N9