AU715378B2 - Low molecular weight bicyclic thrombin inhibitors - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): BIOCHEM PHARMA INC.

Invention Title: LOW MOLECULAR WEIGHT BICYCLIC THROMBIN

INHIBITORS

The following statement is a full description of this invention, including the best method of performing it known to me/us: rn LOW MOLECULAR WEIGHT BICYCLIC THROMBIN INHIBITORS FIELD OF THE INVENTION This invention relates to compounds useful for the treatment of thrombotic disorders, and more particularly to novel heterocyclic inhibitors of the enzyme thrombin.

BACKGROUND

Inordinate thrombus formation on blood vessel walls precipitates acute cardiovascular disease states that are the chief cause of death in economically developed societies. Plasma proteins such as fibrinogen, proteases and cellular receptors participating in hemostasis have emerged as important factors that play a role in acute and chronic coronary disease as well as cerebral artery disease by contributing to the formation of thrombus or blood clots that effectively diminish normal blood flow and supply. Vascular aberrations stemming from primary pathologic states such as hypertension, rupture of atherosclerotic plaques or denuded endothelium, activate biochemical cascades that serve to respond and repair the injury site. Thrombin is a key regulatory enzyme in the coagulation cascade; it serves a pluralistic role as both a positive and negative feedback regulator. However, in pathologic conditions the former is-amplified through catalytic activation of cofactors required for thrombin generation as well as activation of factor XIII necessary for fibrin cross-linking and stabilization.

eeoc In addition to its direct effect on hemostasis, thrombin 35 exerts direct effects on diverse cell types that support and amplify pathogenesis of arterial thrombus disease.

The enzyme is the strongest activator of platelets causing 000 them to aggregate and release substances (eg. ADP TXANE) that further propagate the thrombotic cycle. Platelets in a fibrin mesh comprise the principal framework of a white thrombus. Thrombin also exerts direct effects on endothelial cells causing release of vasoconstrictor substances and translocation of adhesion molecules that become sites for attachment of immune cells. In addition, the enzyme causes mitogenesis of smooth muscle cells and proliferation of fibroblasts. From this analysis, it is apparent that inhibition of thrombin activity constitutes a viable therapeutic approach towards the attenuation of proliferative events associated with thrombosis.

The principal endogenous neutralizing factor for thrombin activity in mammals is antithrombin III (ATIII), a circulating plasma macroglobulin having low affinity for the enzyme. Heparin exerts clinical efficacy in venous thrombosis by enhancing ATIII/thrombin binding through catalysis. However, heparin also catalyzes inhibition of other proteases in the coagulation cascade and its efficacy in platelet-dependent thrombosis is largely reduced or abrogated due to inaccessibility of thrombusbound enzyme. Adverse side effects such as thrombocytopenia, osteoporosis and triglyceridemia have been observed following prolonged treatment with heparin.

Hirudin, derived from the glandular secretions of the leech hirido medicinalis is one of the high molecular weight natural anticoagulant protein inhibitors of 30 thrombin activity (Markwardt F. Cardiovascular Drug SReviews, 10, 211, 1992). It is a biopharmaceutical that has demonstrated efficacy in experimental and clinical thrombosis. A potential drawback to the use of Hirudin as a therapeutic agent is likely antigenicity and lack of an S' 35 effective method of neutralization, especially in view of its extremely tight binding characteristics toward thrombin. The exceedingly high affinity for thrombin is unique and is attributed to a simultaneous interaction with the catalytic site as well as a distal "anion binding exosite" on the enzyme.

Thrombin activity can also be abrogated by Hirudin-like molecules such as hirulog (Maraganore, J.M. et al., Biochemistry, 29, 7095, 1990) or hirutonin peptides (DiMaio, J. et al., J. Med. Chem., 15, 3331, 1992).

Thrombin activity can also be inhibited by low molecular weight compounds that compete with fibrinogen for thrombin's catalytic site, thereby inhibiting proteolysis of that protein or other protein substrates such as the thrombin receptor. A common strategy for designing enzyme inhibitory compounds relies on mimicking the specificity inherent in the primary and secondary structure of the enzyme's natural substrate. Thus, Blomback et al. first designed a thrombin inhibitor that was modeled upon the partial sequence of the fibrinogen A(LsB)a chain comprising its proteolytically susceptible region (Blomback, et al., J. Clin. Lab. Invest., 24, 59, 1969). This region of fibrinogen minimally includes the residues commencing with phenylalanine: Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly -Gly-Gly-Val-Arg-Gly-Pro-Arg T scissile bond Systematic replacement of amino acids within this region 30 has led to optimization of the tripeptidyl inhibitory sequence exemplified by the peptide (D)-Phe-Pro-Arg which corresponds to interactions within the P,-P 2 local binding sites on thrombin (Bajusz S. et al. in Peptides: Chemistry 35 Structure and Biology: Proceedings of the Fourth American Peptide Symposium, Walter Meienhofer J. Eds. Ann Arbor Science Publishers Inc., Ann Arbor MI, 1975, pp 603).

Bajusz et al. have also reported related compounds such as (D)Phe-Pro-Arg-(CO)H (GYKI-14166) and (D)MePhe-Pro-Arg- (CO)H (GYKI-14766) (Peptides-Synthesis, Structure and Function: Proceedings of the Seventh American Peptide Symposium, Rich, D.H. Gross, E. eds., Pierce Chemical Company 1981, pp. 417). These tripeptidyl aldehydes are effective thrombin inhibitors both in vitro and in vivo.

In the case of both GYKI-14166 and GYKI-14766, the aldehyde group is presumed to contribute strongly to inhibitory activity in view of its chemical reactivity toward thrombin's catalytic Ser 19 5 residue, generating a hemiacetal intermediate.

Related work in the area of thrombin inhibitory activity has exploited the basic recognition binding motif engendered by the tripeptide (D)Phe-Pro-Arg while incorporating various functional or reactive groups in the locus corresponding to the putative scissile bond (i.e.

P

1 In U.S. Patent 4,318,904, Shaw reports chloromethyl- S 25 ketones (PPACK) that are reactive towards Ser 19 and HisS.

i These two residues comprise part of thrombin's catalytic triad (Bode, W. et al., EMBO Journal 8, 3467, 1989).

0 Other examples of thrombin inhibitors bearing the (D)Phe- 30 Pro-Arg general motif are those incorporating COOHterminal boroarginine variants such as boronic acids or boronates (Kettner, C. et al., J. Biol. Chem., 268, 4734, 1993).

Still other congeners of this motif are those bearing phosphonates (Wang, C-L Tetrahedron Letters, 33, 7667, 00 1992) and a-Keto esters (Iwanowicz, E.J. et al.,Bioorganic and Medicinal Chemistry Letters, 12, 1607, 1992).

Neises, B. et al. have described a trichloromethyl ketone thrombin inhibitor (MDL-73756) and Attenburger, J.M. et al. have revealed a related difluoro alkyl amide ketone (Tetrahedron Letters, 32, 7255, 1991).

Maraganore et al. (European 0,333,356; WO 91/02750; U.S.

5,196,404) disclose a series of thrombin inhibitors that incorporate the D-Phe-Pro- moiety and hypothesize that this preferred structure fits well within the groove adjacent to the active site of thrombin. Variations on these inhibitors are essentially linear or cyclic peptides built upon the D-Phe-Pro moiety.

Another series of patents and patent applications have described attempts to develop effective inhibitors against thrombosis by using alpha-ketoamides and peptide aldehyde analogs (EP 0333356;WO 93/15756; WO 93/22344; WO 94/08941; WO 94/17817).

Still others have focused their attention on peptides, peptide derivatives, peptidic alcohols, or cyclic peptides as anti-thrombotic agents (WO 93/22344, EP 0276014; EP 0341607; EP 0291982). Others have examined amidine sulfonic acid moieties to achieve this same end (U.S.

4,781,866), while yet others have examined para or meta substituted phenlyalanine derivatives (WO 92/08709; WO 30 92/6549).

A series of Mitsubishi patents and patent applications have disclosed apparently effective argininamide compounds for use as antithrombotic agents. The chemical structures 35 described in these documents represent variations of side groups on the argininamide compound 4,173,630; U.S.

o 6 4,097,591; CA 1,131,621; US 4,096,255; US 4,046,876; US 4,097,472; CA 2,114,153).

Canadian patent applications 2,076,311 and 2,055,850 disclose cyclic imino derivatives that exhibit inhibitory effects on cellular aggregation.

Many of the examples cited above are convergent by maintaining at least a linear acyclic tripeptidyl motif consisting of an arginyl unit whose basic side chain is required for interaction with a carboxylate group located at the base of the P 1 specificity cleft in thrombin. Two adjacent hydrophobic groups provide additional binding through favourable Van der Waals interactions within a contiguous hydrophobic cleft on the enzyme surface S. designated the P 3

-P

2 site.

15 The present invention provides thrombin inhibitors that display inhibitory activity towards the target enzyme, thrombin.

The present invention further provides thrombin inhibitors that display inhibitory activity towards the 20 target enzyme thrombin and are provided for in a pharmacologically acceptable state.

Still a further aspect of the present invention is to provide for the use of heterocyclic thrombin inhibitors and formulations thereof as anticoagulant and 25 thrombin inhibitory agents.

Yet a further aspect of the present invention is to provide for the use of heterocyclic thrombin inhibitors and formulations thereof for therapeutic treatment of various thrombotic maladies.

The present invention further provides a process for the synthesis of these low molecular weight thrombin inhibitors. The enzyme inhibitors of the present invention are encompassed by the structure of general Formula I.

SUMMARY OF THE INVENTION The present invention provides for novel compounds that display thrombin inhibitory activity as Sreflected in formula \\melb01\home$\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 7 Y A R Y R1 2 Rl 412

(I)

wherein R12 is attached to either D or E, in the alternative, and is represented by the structure:

Z*RI

0 wherein: A is selected from 0 S, SO, SO 2 0 and NR 8 wherein R, is hydrogen, C1_ alkyl optionally interupted with 1 or 2 heteroatoms; C- 16 aryl, cycloalkyl or heterocyclic ring 15 or a hydrophobic group; B is selected from S, SO,, O, NH, -CH= and CRR 7 wherein R, and R, are independently selected from hydrogen :id: and C16 alkyl provided that when A is S, SO, SO 2 O, or NR,, then B is CRR,; D is selected from (CH-R,) 02 wherein R, is hydrogen,

C,_

alkyl or and CH with a double bond to B when B is or -CH=; E is selected from CH2 and CH substituted with the -C(O)R, provided that only one of D and E is substituted with

-C(O)R;

X is selected from O, N-Rs, or CH-Rs; Y is selected from O, S, SO, SO,, N-R 5 and CH-R, provided that when X is N-R, then Y is CH-R 8 or 0, and when X is 0 then Y is CH-R,; Z is selected from O, S and H,; iR is one of formula VIa to VId: \\melbOl\home$\PClare\eep\specis\4062 8-95 biochem cm.doc 29/03/99 -8- R,N, (Jp R 1 Via VIb )0-8 K v0-7 G G

G

N P- Vic N P T N (P, (J)n VdU U u wherein:

R,,

11 is hydrogen or C -6 alkyl; K is a bond or -NH-; G is C1-4 alkoxy; cyano; -C (NH) -NH- C

-CH

2

-NH-C(NH)-NH

2 a C 6 cycloalkyl or aryl substituted with cyano, -NH 2

-CH

2

-NH

2

-C(NH)-NH

2

-NH-

C(NH)-NH, or -CH,-NH-C(NH)-NH2; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -NH 2

-CH

2

-NH

2

-C(NH)-NH

2

-NH-C(NH)-NH

2 or -CH,-NH-C (NH) -NH U is cyano, -C(NH)-NH, or -NH-C(NH)-NH.; P is a bond, or a bivalent group: ,OH N O CH or 3s 25 s or J is C 6 alkylene optionally substituted with OH, NH 2 and

C

16 alkyl and optionally interrupted by a heteroatom selected from O, S and N; n is 0 or 1; and T is a heterocycle selected from the group consisting of: x x )f R'

R

3 5 X' x o xN \\melbOl\homeS\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 9 wherein

X

5

X

10

X

1 1 and X, are each independently selected from the group consisting of N, or C-X, where X 7 is hydrogen, C 4 alkyl, or C 5 aryl; X, and X 13 are each independently selected from the group consisting of O, S, or CH-X,; and R' is hydrogen, alkyl optionally carboxyl substituted, carboxyl, -C, 0 1 6 alkyl-CO,-C-_, alkyl, C 620 aralkyl, C 3 cycloalkyl, aryl or an aromatic heterocycle.

R, is selected from H and C alkyl optionally substituted with C, aryl, a 6 member heterocycle or a C3_, cycloalkyl ring;

R

3 is selected from H, NR 6 ,R and C16_ alkyl; and R, and R, are independently selected from H; NRR,; C6-, aryl 15 or C37 cycloalkyl optionally substituted with C6_ alkyl; :f C:1-16 alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRR, or a C 6 6 aryl, heterocycle or C,3 cycloalkyl group optionally substituted with halogen, hydroxyl, C_6 alkyl; an amino acid side chain; and a hydrophobic group, and wherein an alkyl is a straight or branched, saturated or unsaturated chain of carbon atoms.

As will be appreciated from the disclosure to follow, the molecules, compositions and methods of this 25 invention are useful as anti-coagulants, or in the treatment and prevention of various diseases attributed to the undesirable effects of thrombin, as well as for diagnostic purposes.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to molecules which inhibit the enzyme, thrombin. These molecules are characterized by a heterobicyclic moiety as illustrated in Formula I: \\melb01\homeS\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 10 R2 Y A Rl 12

(I)

wherein R12 is attached to either D or E, in the alternative, and is represented by the structure: 10 wherein X, Y, Z, A, B, D, E and R1 to R4 are as previously defined.

~Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to" and is not intended to exclude other additives, components, integers or steps.

The term "hydrophobic group" (HG) as used hereinafter, refers to any group which lacks affinity for, or displaces water. Hydrophobic groups include but are not 20 limited to Cl-20 alkyl, C2-20 alkenyl vinyl, allyl) or C2-20 alkynyl propargyl) optionally interrupted by a carbonyl group, forming an acyl group); C6-16 aryl, C3-7 cycloalkyl, C6-20 aralkyl, C6-20 cycloalkyl substituted C1-20 alkyl, wherein the aliphatic portion is optionally interrupted by a carbonyl group forming an acyl group) and the ring portion is optionally substituted with Cl-6 alkyl such as methyl ethyl or t-butyl; or a hydrophobic amino acid side chain. Preferred hydrophobic groups include cyclohexyl, benzyl, benzoyl, phenylmethyl, phenethyl and para-t-butyl-phenylmethyl.

The term "arginyl moiety" represents an arginine amino acid residue or an analogue or derivative thereof.

\\melbOl\home$\PClarke\Keep\speis\40628-95 biochem cn.doc 29/03/99 lOa For example, an analogue or derivative of the natural residue may incorporate a longer or shorter methylene chain from the alpha carbon ethylene or butylene chain); replacement of the guanidino group with a hydrogen bond donating or accepting group amino, amidino or methoxy); replacement of the methylene chain with a constrained group an aryl, cycloalkyl or heterocyclic ring); elimination of the terminal carboxyl descarboxy) or hydroxyl an aldehyde); or a combination thereof.

The term "alkyl" represents a straight or branched, saturated or unsaturated chain having a specified total number of carbon atoms.

S: The term "aromatic" or "aryl" represents an unsaturated carbocyclic ring(s) of 6 to 16 carbon atoms which is optionally mono- or di-substituted with OH, SH, amino NR6R7) halogen or Cl-6 alkyl. Aromatic rings include

S

SO

0e *55 9 \\melbOl\home$\Pclarke\Keep\specis\40628-95 bioche cm.doc 29/03/99 benzene, napththalene, phenanthrene and anthracene.

Preferred aromatic rings are benzene and naphthalene.

The term "cycloalkyl" represents a saturated carbocyclic ring of 3 to 7 carbon atoms which is optionally mono- or di-substituted with OH, SH, amino NRR,) halogen or

CI_

6 alkyl. Cycloalkyl groups include cyclo- propyl, butyl, pentyl, hexyl and heptyl. A preferred cycloalkyl group is cyclohexyl.

The term "aralkyl" represents a substituent comprising an aryl moiety attached via an alkyl chain benzyl, phenethyl) wherein the sum total of carbon atoms for the aryl moiety and the alkyl chain is as specified. The aryl or chain portion of the group is optionally mono- or disubstituted with OH, SH, amino NR 6

R

7 halogen or C,_ 6 alkyl The term "heteroatom" as used herein represents oxygen, nitrogen or sulfur N or S) as well as sulfoxyl or sulfonyl (SO or SO,) unless otherwise indicated. It is understood that alkyl chains interrupted by one or more heteroatoms means that a carbon atom of the chain is .replaced with a heteroatom having the appropriate valency.

25 Preferrably, an alkyl chain is interrupted by 0 to 4 heteroatoms and that two adjacent carbon atoms are not both replaced.

*5SS The term "heterocycle" represents a saturated or 30 unsaturated mono- or polycyclic bicyclic) ring incorporating 1 or more 1-4) heteroatoms selected from N, 0 and S. It is understood that a heterocycle is optionally mono- or di-substituted with OH, SH, amino

NR

6 halogen, CF,, oxo or C,_ 6 alkyl. Examples of suitable monocyclic heterocycles include but are not limited to pyridine, piperidine, pyrazine, piperazine, pyrimidine, imidazole, thiazole, oxazole, furan, pyran and I L.

thiophene. Examples of suitable bicyclic heterocycles include but are not limited to indole, quinoline, isoquinoline, purine, and carbazole.

The term "hydrophobic amino acid" represents an amino acid residue that bears an alkyl or aryl group attached to the a-carbon atom. Thus glycine, which has no such group attached to the a-carbon atom is not a hydrophobic amino acid. The alkyl or aryl group can be substituted, provided that the substituent or substituents do not detract from the overall hydrophobic character of the amino acid.

Examples of hydrophobic amino acids include natural amino acid residues such as alanine; isoleucine; leucine; phenylalanine; and non-naturally ocurring amino acids such as those described in "The Peptides", vol. 5, 1983, Academic Press, Chapter 6 by D.C. Roberts and F.

Vellaccio. Suitable non-naturally ocurring amino acids include cyclohexylalanine and 1-aminocyclohexanecarboxylic.

By "amino acid side chain" is meant the substituent attached to the carbon which is a to the amino group. For a example, the side chain of the amino acid alanine is a methyl group and while benzyl is the side chain for 25 phenylalanine.

Preferably R, is H or alkyl. More preferably R, is H, methyl or ethyl and most preferably R 2 is H.

30 Preferably, R, is H or CI,_ alkyl. More preferably, R, is H, methyl or ethyl, and most preferably R 3 is H.

a Preferably, one of R 4 or RS is a hydrophobic group such as a saturated or unsaturated carbocycle of 5 or 6 members optionally fused to another carbocyclic group while the other is H, C 1 1 alkyl optionally substituted by NR 6 R or I K carboxy. The hydrophobic moiety may be linked via a spacer such as a alkyl chain optionally interrupted with 1 or more 1-4) heteroatoms, carbonyl or sulfonyl (SO,) groups. More preferably, one of R 4 and R s is phenyl, cyclohexyl, indole, thienyl, quinoline, tetrahydroisoquinoline, naphthyl or benzodioxolane linked via Ci 16 alkyl optionally interupted with a heteroatom or a carbonyl while the other is H, carboxymethyl or carboxyethyl.

Preferably, A Preferably, B Preferably, D Preferably, E as previously Preferably, X Preferably, Y Preferably, Z is absent or CH 2 is S or CH,.

is C

H

is CH substituted with -C(0)R I wherein R, is defined.

is CH-R 5 or N-R.

is CH-R. or S.

is O.

In a preferred embodiment, R, is represented by one of formula VIa to VId: U. U a

S

S.r

S.

U..

R

11 P /T (J)n Via K )7

SG

Vie p

"T

U uCX)1-3

R

1 1

N

VIb >)0-8

G

N P T wherein:

R

11 is hydrogen or C,- 6 alkyl; K is a bond or -NH-; G is C,-4 alkoxy; cyano; -CH2-NH 2 -C(NH)-NH; -NH- C(NH)-NH,; -CH2-NH-C(NH)-NH 2 a C 6 cycloalkyl or aryl substituted with cyano, -NH 2

-CH

2

-C(NH)-NH

2

-NH-

C(NH)-NH, or -CH,-NH-C(NH)-NH,; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -NH 2

-CH

2

-NH

2

-C(NH)-NH

2

-NH-

C(NH)-NH, or -CH,-NH-C (NH) -NH 2 U is cyano, -NHL, -C(NH)-NH 2 or -NH-C (NH)-NH 2 P is a bond, or a bivalent group:

OH

OH CH 4 H CCH 3 or S J is CI- alkylene optionally substituted with OH, NH 2 and C1i 6 alkyl and optionally interrupted by a heteroatom selected from 0, S and N; n is 0 or 1; and T is H, OH, amino, a peptide chain, C 1 16 alkyl, C- 1 6 alkoxy,

C,-

20 aralkyl, or heterocycle optionally substituted.

Preferably R 1 is H or methyl and most preferably H.

Preferably K is a bond.

Preferably G is -NH-C(NH)-NH2 attached via a methylene chain of 3-7 carbons or phenyl substituted with -C(NH)-NH 2 attached via a methylene chain of 0 to 3 carbons. More 20 preferably G -NH-C(NH)-NH, attached via a methylene chain of 3 atoms.

Preferably P is Preferably J is selected from: -CH2-S-CH 2 -CH2-; -CH 2

-O-CH,-

-CH,-NH-C1H-CH2-; and a bond when n is 0. More 25 preferably, J is a bond while n is 0.

In particular embodiments of the invention, R, is selected from the following amino acid derivatives prepared according to the procedures described in Bioorg. Med.

30 Chem., 1995, 3:1145 0 0 0 T T n NH n NH nNH NH H2 HN- N)INN e N

NH

2

H

2 N NS-N (4n1i 0

H

~NH

NN

H

2

N

0

H

nNH

H

2

N

0 H H 0-n,

NH

5H 2

N

H

a a.

a. a p a a a a a.

a a a a a.

a. a a.

0

H

N N

NH

2 0

H

T

H

3 C N'NH 2 0o T 0

TT

~N n2 NH 2 n2NH HN NH 2

NH,

T 0T 0

T

TN N H1 2 N NH z, N NH2

NH

2 H H H

NH

2

H

2

N

H

2 N NHHNH :0 0 0 T T

NN

NH

2

H

2

N

H

2

N

16 7 0 IN H T T TI N NH 2 n2 N HrNH 2

(%NH

HH

0 0 0 A2( H2NN 0 0 0

NH

2

N

1N7 1n2 NH2 NH NH 2 NH NH wherein n=1-6, nl=l-2, n2=0-7 and T is as previously defined.

In a preferred embodiment, T is a peptide of 1 to 4 amino acid residues in length and preferably fibrinogen's A or B chain or fragment or derivative thereof. In another preferred embodiment, T is a heterocycle selected from the group consisting of: R• "IX :9 4- XX,, X XR

R.

15 x-x---x *wherein

X

10

X

1 1 and X 12 are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, C_4 alkyl, or C 6 1 6 aryl; X. and X 13 are each independently selected from the group consisting of C, 0, N, S, N-X 7 1 or R' is hydrogen, C 1 16 alkyl optionally carboxyl substituted, carboxyl, alkyl-C0 2

-C

1 16 alkyl, C 6 -2 aralkyl, C 3 7 aryl or an aromatic heterocycle.

Preferably T is selected from the group consisting of:

N

N

/N

N-N

N N

N-

0

N

N

7

S

0 0

U

0* 0* 9*

U

wherein R' is as defined above.

more preferably T is selected from the group consisting of: R'

R'

I I A' 0 wherein R' is as defined above.

More preferably T is selected from the group consisting of: R N R O wherein R' is as defined above.

Most preferably T is

R-

wherein R' is H or C_ 4 alkyl such as methyl, ethyl, propyl or butyl and most preferably wherein R' is hydrogen,. In another embodiment, T is a 1,2 thiazole optionally substituted with R' and\or is attached to J at the 2, 3, 4 or 5 position of the ring.

In particular embodiments, compounds of the invention represented by formulas II, III, IV and V, wherein X, B, R i to R 4 and R are as previously defined.

are

Y,

0*

C

p. C

C.

C.

C

C

C.

C

C C C 00 C. C

C.

-C

CS

C..

(II) (III)

(IV)

R B

R

0

S

H

N

NH O In a particularly preferred embodiment, compounds of the invention are represented by one of formulas VII, VIII, IX and X: R R R2 R2 5 (VII) R (VIII) R Rx R, o 33 3 o 0 R

R

(IX) R Y f 4 4 N (CH 2 )n R N R 3 R,

R

3 0 R1 O 0 wherein B is O, S, -CH 2 or -NH-; Y is selected from O, S, SO, SO2, N-R s and CH-R,; R. is an arginyl moiety or an analog or derivative thereof optionally substituted with an amino acid, a peptide or a heterocycle; R, is H or Ci_ alkyl; R, is selected from H, NRR, and C_ 6 alkyl; and R. and R, are independently selected from H; NRR,; C 6 1 aryl or C 3 cycloalkyl optionally substituted with C.

6 alkyl; 15 C 1 alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRR, or a C 6 16 aryl, heterocycle or C3 cycloalkyl group optionally substituted with halogen, hydroxyl, C, 6 alkyl; an amino acid side chain; and a 20 hydrophobic group; R, is hydrogen, C 1 6 alkyl optionally interupted with 1 or 2 S* heteroatoms; C,, 6 aryl, C, 7 cycloalkyl or heterocyclic ring or a hydrophobic group; and n is 1 or 2.

Preferred compounds according to formula VII include: 0005 6S-benzylhexahydro-5-oxo-5Hthiazolo 2-a] pyridine-3Rcarboxamido (propyl ketoarginine) 0010 6S-benzylhexahydro-5-oxo-5Hthiazolo 2-a] pyridine-3Rcarboxamido (butyl ketoarginine) 0015 thiazolo pyridine-3Rcarboxamido (propylcarbmethoxyketoar ginine) 0020 6S-cyclohexylmethyl oxo-Sil-thiazolo 2-a]pyridine-3Rcarboxamido (benzylketo arginine) 0025 6S-cyclohexyl methyl hexahydro-Soxo-SH-thiazolo[3, 2-alpyridine -3Rcarboxamido (carbmethoxy propyl cyclodithioketalarginine) 0030 6S-cyclohexylmethyl oxo-5H-thiazolo 2-a]pyridine-3Rcarboxamido -Arg- -pipecolilic acid)

S

Ph o

NH

HN NH 2 0 00 0 a 5O

S*

0000 000 0 0 0 00 0: :0.

so 0 0

NH

HN lNH 2 Or 0

NH

HN NH 2 003S 6S-benzylhexa thiazolo 2-alpyridine-3Rcarboxamido (carboxamidopropyl cyclodithioketal arginine) 0040 2-a] pyridine-3Rcarboxarnido -Arg nipecotamide) 0045 6S-cyclohexylmethylhexahydro-S-oxopyridine-3Rcarboxamido( (S)Arg isonipecotamide) 0050 6S-benzylhexahydro-5-oxo-5Hthiazolo[3, 2-alpyridine-3Rcarboxamido (carbxamidopentyl cyclodithioketal arginine) 0055 6S-benzylhexahydro-5-oxo-5Hthiazolo[3, 2-alpyridine-3Rcarboxamido (carbmethoxy propyl cyclodithioketal arginine) 0060 2-alpyridine-3Rcarboxaxnido (l-carboxy-3-thiobutyl ketoarginine) 0NH r0

NH

i-N JNH, 0 NH0

CNH,

HN JNH2 NH NH, 4 -S 0-HN

NH

2 0

NH

HN I r 0~ HN JNH.

9 00 00.: .0 19-4 0065 6S-cyclohexylrnethylhexahydro-5-oxo- 2-alpyridine-3Rcarboxainido (l-carboxy-3-thiobutyl ketoarginine) 0070 2-alpyridine-3Rcarboxainido (l-carboxy-2-methyl-3thiobutyl ketoarginine) 0075 6S-cyclohexylmethylhexahydro-5-oxo- 5H-thiazolo[3, 2-alpyridine-3Rcarboxamido( (3-thiobutyl sulfonicacid) ketoarginine)

S

Q 0 0 0

NH

HN zz< NH, OH

S

NH

HNzzz 0 NH, OH s Os

NH

H 2zz 00 ,00:.

toS too 0080 SH-thiazolo 2-a~pyridine-3Rcarboxamido (iso-quinolinium methyl ketoarginine) 0085 5H-thiazolo t3, 2-alpyridine-3Rcarboxamido (propyl carbmethoxy ketoarginine) 0090 pyridine-3Rcarboxamido ((propylketo)Arg-Phe- Arg-NH.) 0095 6s-benzylhexahydro-5-oxo-5Hthiazolo pyridine-3Rcarboxantido( (propanoic acid) ketoarginine) 0100 6S-benzylhexahydro-5-oxo-5Hthiazolo 2-alpyridine-3Rcarboxamido (propyl carbmethoxy ketoarginine) 0105 2-alpyridine-3Rcarboxamido (ax-benzothiazolo keto arginine); and 0110 2-a]pyridine-3Rcarboxamido (propyl carbmethoxy ketoarginine) Os NH N

HN<-

NH

0N~ 00H

NH

S.

C

C C *9 C. C C C

C

C C 99 9

C

C

9*9*

C.

C C

C

CCC*

9CCC 9C 99 C. 9 C C

CC

C

C.

CCC

C

0205 6-Benzyl-5-oxo-hexahydrothiazolo[3 2 -a]pyridine-3 carboxylic acid [1- (benzothiazole-2-carbonyl) -4guanidino-butyl] -amide 0210 thiazoloj3, 2 -a]pyridine-3 carboxylic acid [1- (benzothiazole-2-carbonyl) -4guanidino-butyl]-amide 0215 6-Benzyl-5-oxo-hexahydrothiazolo[3, 2 -a]pyridine-3 carboxylic acid [1- (benzothiazole-2-carbonyl) -4guanidino-butyl]-amide

H

o

NH

-NH2 HNA-J-NH2

H

o NH-I H N -L-NH 2 a a aa a. 0*

S.

a..

a a a a a a.

a. a a a a.

a *.a 0220 6-Benzyl-8a-methyl-5-oxohexahydro-thiazolo[3, 2a]pyridine-3-carboxylic acid [1- (benzothiazole-2-carbonyl) -4guanidino-butyl]-amnide 0225 8a-Methyl-5-oxo-6-phenethylhexahydro-thiazolo[3 ,2a]pyridine-3-carboxylic acid [1- (benzothiazole-2-carbonyl) -4guanidino-butyji-amide 0230 8a-Methyl-S-oxo-6-phenethylhexahydro-thiazoloj3, 2a]pyridine-3-carboxylic acid [1- (benzothiazole-2-carbonyl) -4guanidino-butyl] -amide &4N~ 0 NH~s o I NH NH 2 me 0

NHS

NH -HNH 2 0240 8a-Methyl-5--oxo- 6 (2-trifluoro methyl-quinolifl-6-ylmethyl) hexahydro-thiazolo[3, 2-a] pyridine-3-carboxylic acid Iii- (benzothiazole-2-carbonyl) -4guanidino-butyll-amide 0245 6-Benzyl-5-oxo-hexahydrothiazolo[3 ,2-ajpyridine-3carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) butyllamide 0250 6-Benzyl-5-oxo-hexahydrothiazolo[3, 2-alpyridine-3carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) butyl]ainide 0255 6-Benzyl-5-oxo-hexahydrothiazolo[3, 2-a]pyridine-3carboxylic acid [4-guanidino-l- (1-methyl-ill imidazole-2carbonyl) butyl]-amide 0260 6-Benzyl-8a-methyl-5-oxohexahydro-thiazolo[3 ,2a]pyridine-3-carbo xylic acid [4guanidino-1- (thiazole-2carbonyl) -butyl]-axnide 0265 5-Oxo-6- (3-cyclohexyl-propyl) hexahydro-thiazolo[ 3 ,2a]pyridine-3-carboxylic acid [4guanidino-1- (thiazole-2carbonyl) butyl]-amide CF3 NN 0 N H NH NH 2

H

o NHS HNA NH 2

H

a.

S

a a a a S a a a

S.

a..

H

0 N o NH me HN L-NH2 me o 'NH HN- AJNH 2

H

N

o 0 NH

S

HN~ NH 2 0275 8a-Methyl-5-oxo-6-(3-phenylpropyl) -hexahydro-thiazolo[3 ,2alpyridine-3-carboxylic acid [4guanidino-l-(thiazole-2carbonyl) -butyll-amide 0280 8a-Methyl-5-oxo-6-(3-phenylpropyl) -hexahydro-thiazolo[3, 2a]pyridine-3-carboxylic acid [4guanidino--(thiazole-2carbonyl) -butyl]-amide 0285 8a-Methyl-5--oxo-6- (2tri fluoromethyl-quinolin- 6-' ylmethyl) -hexahydrothiazolo[3, 2-a]pyridine-3carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyl]amide 0295 6- (1,3-Dioxo-1,3-dihydroisoindol-2-yl) thiazolo[3, 2-a]pyridine-3carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]amide 0305 5-Oxo-6- (3-phenyl-propionyl amino) -hexahydro thiazoloi3, 2a]pyridine-3-carboxylic acid [4guanidino-l- (thiazole-2carbonyl) -butyl]-amide 0315 5-Oxo-6-(3-phenyl-propionyl amino) -hexahydro thiazolo[3, 2a]pyridine-3-carboxylic acid [4guanidino-l- (thiazole-2carbonyl) -butyl]-amide 0 H N H S HN LNH2 N H 0 HN LN2

CF

3 N w 0

NH

NH NH 2 9 S 9 9* 9 9p a a 99 *99a a a 9 9 *59a *9*9 9* a 9 a.

C..

NH

N 0

N

0 0NHrNH 2 More preferred compounds according to formula (VII) include: 0085 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazoloE3, 2a] pyridine-3R-carboxamido (propylcarbo methoxyketoarginine); 0090 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazolo (3,2alpyridine-3R-carboxamido ((propylketo)Arg-Phe-Arg-NH 2 0095 6S-benzylhexahydro-5-oxo-5H-thiazolofj,2-alpyridine-3Rcarboxamido ((propanoic acid) ketoarginine); 0105 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazolo (3,2alpyridine-3R-carboxamido (ax-benzothiozolo keto arginine); 0210 6 -Benzyl- 5-oxo-hexahydro-thiazolo[3, 2-a]pyridine-3 carboxylic acid (benzothiazole-2-carbonyl) -4-guanidinobutyl]-amide; 0220 6-Benzyl-8a-methyl-5-oxo-hexahydro-thiazolo[3 ,2-alpyridine- 3-carboxylic acid (benzothiazole-2-carbonyl) -4guanidino-butylj-amide; 0240 8a-Methyl-5-oxo-6- (2trifluoromethyl-quinolin-6-ylmethyl) -hexahydrothiazolo[3, 2-alpyridine-3-carboxylic acid [1- (benzothiazole-2-carbonyl) -4-guanidino-butyl]-amide; 0245 6-Benzyl-5-oxo-hexahydro-thiazolo[3 ,2-alpyridine-3carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl,)butyl]amide; 0260 6-Benzyl-8a-methyl-5-oxo-hexahydro-thiazolo[3, 2-alpyridine- 25 3-carboxylic acid [4-guanidino-1-(thiazole-2-carbonyl)butyll-ainide; 0265 5-Oxo-6- (3-cyclohexyl-propyl) -hexahydro-thiazolo[3,2alpyridine-3-carboxylic acid [4-guanidino-1- (thiazole-2- P. carbonyl )butyll-amide; 0285 8a-Methyl-5-oxo-6- (2-trifluoromethyl-quinolin-6-ylmethyl) hexahydro-thiazolo[3 ,2-a]pyridine-3-carboxylic acid 14guanidino-1- (thiazole-2-carbonyl) -butyli-amide; and 0315 5 -Oxo -6 (3 -phenyl -prop ionyl amino) -hexahydro thiazolo[3 ,2a]pyridine-3-carboxylic acid [4-guanidino--(thiazole-2carbonyl) -butyl]-amide.

Most preferred compounds according to formula VII include: 0085 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazolo [3,2a] pyridine-3R-carboxamido (propylcarbo methoxy ketoarginine); and 50105 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazolo [3,2alpyridine-3R-carboxamido (c-benzothiozolo keto arginine).

09* Preferred compounds according to formula VIII include: 0325 3-Aminomethyl-2-benzoyl-4-oxooctahydro-pyrrolo[1, 2-a]pyridine-6carboxylic acid (benzothiazole-2carbonyl) -4-guanidino-butyl]-amide 0330 3-Aminomethyl-4-oxo-2-phenylacetyloctahydro-pyrrolo[1, 2-alpyrazine-6carboxylic acid (benzothiazole-2carbonyl) -4-guanidino-butyl]-amide 0335 2-Benzoyl-4-oxo-octahydropyrroloti, 2-a]pyrazine-6-carboxy-ic acid [4-guanidino-1- (thiazole-2carbonyl) -butyl]-amide 0340 4-Oxo-2- (3-phenyl-propiolyl) octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyl]-amide 0345 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid [4-guanidino-1- methyl-thiazole-2-carbolYl) -butyl]amide 0 N H H N

HN

HNHI

a a S

S

a.

a

S

S S** a S S a a a a a.

*5 a a

S..

0350 2- (3-cyclohexyl-propionyl) -4-oxooctahydro-pyrrolofi, 2-a]pyrazine-6carboxylic acid [4-guanidino-l-( 2 thiazole-carbonyl) -butyll-ainide 0355 5-Oxo-7- (3-phenyl-propionyl) octahydro-2-thia-4a, 7-diazanaphthalene-4-carboxylic acid [4guanidino-1- (thiazole-2-carbonyl) butyl]-amide 0365 4-Oxo-2- (4-phenyl-butyryl) octahydro-pyrrolo[1, 2-alpyrazine-6carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyl]-amide 0370 4-Oxo-2-phenylacetyl-octahydropyrrolo[1, 2-ajpyrazine-6-carboxylic acid [4-guanidino-1- (thiazole-2carbonyl) -butyl]-amide 0375 2- (2-Amino-3-phenyl-propionyl) -4oxo-octahydro-pyrroloji, 2-a] pyrazine-6-carboxylic acid [4guanidino-l- (thiazole-2-carbonyl) butyl] -amide 0380 2-[2-Amino-3- (4-hydroxy-phenyl) propionyl] -4-oxo-octahydropyrrolo[1, 2-a] pyrazine-6-carboxylic acid [4-guanidino-l- (thiazole-2carbonyl) -butyl]-antide 0

H

0 NH S' NH-L

NH,

0N

NH

0 H NH 0

HN~

NH

S

*5

SO

S

S

S S S S SS S 5e

S.

S**

0385 2 -Amino-3 fluoro-phenyl) propionyl] -4-oxo-octahydropyrrolol, 2-al pyrazine-6-carboxylic acid [4-guanidino-1- (thiazole-2carbonyl) -butyl]-amide 0 0 NN NH ;)0

NH

0390 4-Oxo-2- (3-phenyl-propyl) -octahydropyrrolo[1,2-a] pyrazine-6-carboxylic acid [4-guanidino-l- (thiazole-2carbonyl) -butyl]-amide 0395 2-[2-Amino-3- (lH-indol-3-yl) propionyll -4-oxo-octahydropyrrololl, 2-a]pyrazine-6-carboxylic acid [4-guanidino-l- (thiazole-2carbonyl) -butyl]-amide 0400 4-Oxo-2- (3-thiophen-3-yl-propionyl)octahydro-pyrrolo[1, 2-a] pyrazine-6carboxylic acid [4-guanidino-1- (thiazole-2 -carbonyl) -butyl]-amide 0405 4-Oxo-2- (3-thiophen-2-yl-propionyl)octahydro-pyrrolo[1, 2-a] pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-amide 0410 2-0(-1 H-Imidazol-4-yl-propionyl) -4oxo-octahydro-pyrrolo[1, 2-a]l pyrazine-6-carboxylic acid [4guanidino-l- (thiazole-2-carbonyl) butyll-amide N ?HN s% 0 NN

NH

0 0 NN NHP

N

NH

0 0 NN NH, o

N

H

2

N

NH

0 N14, 0 N NHD o

N

NH

9* 9 9 *9*9 9 9*9* 9 S 9 9**9 5* 9* 9* 90 *9 9 *9* 0415 2- (2-Axino-3-thiophen-3-ylpropionyl) -4-oxo-octahydropyrrolo[1, 2-a] pyrazine-6-carboxylic acid [4-guanidino-1- (thiazole-2carbonyl) -butyl]-amide 3~f 0420 4-Oxo-2-(1,2,3..4-tetrahydroisoquinoline-3-carbonyl) -actahydropyrrolo[1, 2-a] pyrazine-6-carboxylic acid [4-guanidino-1- (thiazole-2carbonyl) -butyl]-aiaide 0425 2- (Hydroxy-phenyl-acetyl) -4-oxooctahydro-pyrrolo[1, 2-a] pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-amide 0430 2- (2-H-ydroxy-3-phenyl-propionyl) -4oxo-octahydro-pyrroloi, 2 -a] pyrazine-6-carboxylic acid [4guanidino-1- (thiazole-2-carbonyl) butyl]-amide 0435 4-Oxo-2-phenoxyacetyl-octahydropyrrolo[1, 2-al pyrazine-6-carboxylic acid [4-guanidino-1- (thiazole-2carbonyl) -butylil-amide OH 0 Nr NH-P

N

H2N\(

NH

0 2HN s2 0 NN NH~j

N

NH

0*

S

S S S

S

S

SSSS

SO

SO

OS

S..

0440 4-Oxo-2- (3-phenoxy-propionyl) ocatahydro-pyrrololi, 2-alpyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-amide 0445 4-Oxo-2- (2-phenyl-ethanesulfonyl) octahydro-pyrrolofl, 2-a] pyrazine-6carboxylic acid [4-guanidino-l- (thiazle-2-carbonyl) -butyl]-ainide 0450 2- (Naphthalene-2-sulfonyl) -4-oxaoctahydro-pyrrolo[l, 2-a] pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-amide 0455 4- (6-[4-Guanidino-l- (thiazole-2carbonyl) -butylcarbamoyl]-4-oxohexahydro-pyrrolo[l, 2-a] pyrazin-2y1) 4-oxo-3-(2 propyl -pentanoylamino)butyric acid methyl ester 0460 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[l, 2-a] pyrazine-6carboxylic acid f4-guanidino-1)butyl] -amnide 0465 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[l. 2-a] pyrazine-6carboxylic acid [3-guanidino-propyl) amide

NHN

H N. N

NH

0 NN

NHN

NH

00 '41 0 NN I_NH;: 0

NH

S

S S

S*

*5 S S S

S.

S

S p 5 5

S

S

S

SO S *5*S

S.

S

S.

*5 55

S.

S

0 0 H

NH;-/

H

2 N

NH

0

NRH

0H N

H

2

N

0470 4- (6-(4-Guanidino-1- (thiazole-2carbonyl) -butylcarbamoyl]-4-oxohexahydro-pyrrolo[1,2-a]pyrazin 2 -yl) 4-oxo-butyric acid 0475 4-Oxo-2- (3-phenyl-propionyl)octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid (5 -ethyl -thiazole- 2 -carbonyl) -4-guanidino-butyll-amide 0 0 N

NI

0 ?HN§ 0. 0 0* 0 0 0* 0 0 SO 0* S 00 S0.

000 0480 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo1, 2-a]pyrazine-6carboxylic acid [4-guanidino-1- methyl-thiazole-2-carbonyl) -butyllamide 0485 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (4methyl-thiazole-2-carbonyl) -butyllamide 0490 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolojl, 2-alpyrazine-6carboxylic acid[1- ethyl -thiazole-2 carbonyl) -4-guanidino-butyl]-amide 0495 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrroloil, 2-a]pyrazine-6carboxylic acid (4-carbainimidoylpheny) -ainide 00

NH

N

tNNH. 0

NH

NHl -7 0500 4-Oxo-2- (3-phenyl-propianyl)octahydro-pyrrolo(l, 2-aJpyrazine-6carboxylic acid [4-guanidino-1-(5phenyl-thiazole-2-carbonyl) -butyl]amide 0505 4-Oxo-2- (3-phenyl-propionyl)octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid (S-benzyl-thiazole- 2-carbonyl) -4-guanidino-butyl]-amide 0510 4-Oxo-2- (3-phenyl-propionyl)octahydro-pyrrololl, 2-ajpyrazine-6carboxylic acid (4-carbarnimidoylbenzyl) -2-oxo-2-thiazol-2-yl-ethyllamide 0515 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[1, 2 -a]pyrazine-6carboxylic acid [1-(3-carbamimidoylbenzyl) -2-oxo-2-thiazol-2-y1-ethyl]amide 0520 4-Oxo-2- (3-phenyl-propionyl)octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid (1-carbainimidoylpiperidin-4-ylmethyl) -2-oxo-2thiazol-2-yl-ethyl]-amide 0525 4-Oxo-2- (3-phenyl-propioflYl) octahydro-pyrroloti, 2-a]pyrazine-6carboxylic acid (1-carbamimidoylpiperidin-3-ylmethyl) -2-oxo-2thiazol-2-yl-ethyl]-amide 0 'F

NN

N0

NH

0

N

2 o NN

N~

t o f to 0 ft f 0

N

0

N

0NH 0530 4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [1-(1-carbamimidoylpiperidin-2-ylmethyl) -2-oxo-2thiazol-2 -yl -ethyl]-amide 0535 [6-[4-Guanidino-1- (thiazole-2carbonyl) -butylcarbamoyl]-4-oxo-2- (3phenyl-propionyl) -octahydropyrroloti, 2 -a]pyrazine- 3-yl] -acetic acid 0540 3-[6-[4-Guanidino-1- (thiazole-2carbonyl) -butylcarbainoyl]-4-oxo- 2 (3phenyl-propionyl) -octahydropyrrolo[1, 2 -a~pyrazin-3 -yIJ-propioflic acid 0

NN

0 H HO0 0 T,-N 00 00

NH

HNH

a a a a 0545 (1-Carbamimidoyl-piperin-4ylmethyl) -2-oxo-2-thiazol-2-ylethylcarbamoyl]-4-oxo-2- (3-phenylpropionyl) -octahydro-pyrrolo[1, 2a]pyrazin-3-yl) -acetic acid 0550 (1-Carbamimidoyl-piperidin-4ylmethyl) -2-oxo-2-thiazol-2-yle thyl carbamoyl] -4 oxo- 2 (3 -phenyl propionyl) -octahydro-pyrrolo[1, 2alpyrazin-3-yl) -acetic acid 0555 (1-Carbamimidoyl-piperidil- 3 ylmethyl) -2-oxo-2-thiazol-2-ylethylcarbamoyl]- 4 -oxo- 2 (3-phenylpropionyl) -octahydro-pyrrolo[1, 2a]pyrazin-3-yl) -acetic acid 0

N

HO 0 0 HN %N

N~N

M'

2 a.

a a a a a C, a.

a a., 0560 (3-Guanidino-propylcarbamoyl) -4oxo-2- (3-phenyl-propionyl) -octahydropyrroloil, 2 -a]pyrazin-3 -yl) -acetic acid 0565 (3-Guanidino-propylcarbamoyl) -4oxo-2- (3-phenyl-propionYl) -octahydropyrroloil, 2-a]pyrazin-3-yl) -propionic acid 0570 4-Oxo-2- (3-phenyl-propiolyl) octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (thiazolc-2-carboflyl) -butylil-methylamide 0575 4-Oxo-2- (3-phenyl-propiolyl) octahydro-pyrrololl, 2-a]pyrazine-6carboxylic acid (1-carbamimidoylpiperidin-4-ylmethyl)-2 -oxo- 2- 058 0 ([l-Carbamimidoyl-piperidifl- 4 ylmethyl) -2-oxo-2-thiazol-2-Yl-ethylImethyl-carbamoyl) -4-oxo-2- (3-phenylpropionyl) -octahydro-pyrrolo[l, 2a]pyrazin-3-ylI-acetic-acid 0585 4-Oxo-2- (3-phenyl-prOpiolyl)octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid (1-carbamimidoylpiperidin-3 -ylmethyl) -2-oxo-2thiazol 2 -yl -ethyl] -methyl -amide 0 CI_ N/,N

H

_NH

HNN

0 HNj

HN

rH o N

N'H

N

NH

0 0 0 0*0* 0 00** 0 *000 0

N

0 Nt 0 'ft 0 0590 4-Oxo-2- (3-phenyl--propionyl) octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid (3-guanidino-propyl) methyl -amide 0595 2- (Naphthalene-2-carbonyl) -4-oxooctahydro-pyrrolo[l, 2 -alpyrazine- 6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-amide 0600 2- (Naphthalene-l-carbonyl)-4-oxooctahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-ainide 0605 2- (3-Naphthalen-1-yl-proplonyl) 4-oxooctahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carboriyl) -butyl]-ainide 0 CI iiN)

I-

0 'N

HN

tNrZ 0

NH

NH

9 9* 9 9 9 9 9 9*9* 9 9999 99.9 .9.9 0 9 9 9 9.

9 9 99 99 .9.

9 0610 2- (4-tert-Butyl-benzoyl) -4-oxooctahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid f4-guanidino-l- (thiazole-2-carbonyl) -butyl]-ainide 0615 2- (Benzo~l, 3]dioxole-5-carbonyl) -4oxo-octahydro-pyrrolo[l, 2-a]pyrazine- 6-carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyl]-axnide 0

NH

NH

2 0620 2- (3-Benzo[1, 3]dioxol-5-yl-propionyl) 4-oxo-octahydro-pyrrolo[1, 2a]pyrazine-6-carboxylic acid [4guanidino-1- (thiazole-2-carbonyl) butyl]-amide 0625 (2-Methyl-benzylidene) -but-3enoylj-4-oxo-octahydro-pyrrololl, 2a]pyrazine-6-carboxylic acid (1carbamrimidoyl-piperidin-3-ylmethyl) 2-oxo-2-thiazol-2-yl-ethyl]-amide 0630 2-[2-(2-Methyl-benzyliderle)-but-3enoyl]-4-oxo-octahydro-pyrrolo[1, 2ajpyrazine-6-carboxylic acid f1- (1carbamimidoyl-piperidin-4-ylmethyl) 2-oxo-2-thiazol-2-yl-ethyll-amide 0635 2- (2-Benzylidene-pent-3-enoyl) -4-oxooctahydro-pyrrolo[1, 2-alpyrazine-6carboxylic acid (3-guanidino-propyl) amide 0 0 0 HN S.3I 0 N 0 NtNH NNt 0 0

S

*6*S

S

S S

S

*S

4* S

SS

S.

S

SSS

0640 4-Oxo-2- (3-phenyl-propionyl)-octahydropyrrolo[1, 2-a]pyrazine-6-carboxylic acid 4carbamimidayl -benzylaxnide 0645 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-alpyrazine-6-carboxylic acid [4imidazol-1-yl-l- (thiazole-2-carbonyl) butyl]-arnide 0650 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-a]pyrazine-6-carboxylic acid [4- (2-amino-imidazol-1-yl) -1-(thiazole-2carbonyl) -butyl]-amide 0655 4-Qxo-2- (3-phenyl-propionyl) -octahydropyrrolo[i, 2-a]pyrazine-6-carboxylic acid [3- (2-amino-6-methyl-pyrimidin-4-yl) -1- (thiazole-2-carbonyl) -propyl]-ainide 0670 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolofi, 2-a]pyrazine-6-carboxylic acid [3- (2-amino-6-chloro-pyrimidin-4-yl) -1- (thiazole-2-carbonyl) -propyl]-amide 0675 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-a]pyrazine--6-carboxylic acid [3- (6-amino-pyridin-2-yl) -1-(thiazole-2carbonyl) -propyll-amide 0~~Y 0 0

NH

0

N

0 0 s 00

N

N3

N

00 N3 0

NH

0680 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[i, 2-aJpyrazine-6-carboxyiic acid [3- (2-amino-pyridin-4-y1) -2.-(thiazole-2carbonyl) -propyl]-amide 0685 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-a]pyrazine-6-carboxylic acid [2- (2-amino-pyridin-4-yl) -1-(thiazole-2carbonyl) -ethyl]-amide 0690 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-ajpyrazine-6-carboxylic acid [2- (6-amino-pyridin-2-yl) -1-(thiazole-2carbonyl) -ethyl]-amide 0695 2-[4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[l, 2-alpyrazine-6-carbonyl]-3- (thiazole-2-carbonyl) 3, 4-tetrahydroisoquinoline- 6-carboxamidine 0700 2-[4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-a]pyrazine-6-carbonyl]-3- (thiazole-2-carbonyl) 2,3, 4-tetrahydroisoquinol ine-7-carboxamidine 0705 N-[1-[4-Oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo1, 2-a]pyrazine-6-carbonyl]- 5- (thiazole-2-carbonyl) -pyrrolidin-3-ylIguanidi ne 0 N3

N

0 H 00

N

0 ~Nk a a S S

S

a S

S..

0710 4-Oxo-2- (3-phenyl-propionyl)-octahydropyrrolojll,2-apyrazine-6-carboxylic acid [1- (4-arino-cyclohexyl) -2-oxo-2-thiazol-2-ylethyl]-amide 0715 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrroloil, 2-a]pyrazine-6-carboxylic acid [Il- (4-amino-cyclohexylmethyl) -2-oxo-2-thiazol- 2 -yl -ethyl] -amide 0 0720 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolol, 2-ajpyrazine-6-carboxylic acid [1- (4-amino-benzyl) -2-oxo-2-thiazol-2-ylethyl]-amide 0725 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[l, 2-a]pyrazine--6-carboxylic acid [1- (4-aminomethyl-benzyl) -2-oxo-2-thiazol-2yl-ethyl]-ainide 073 0 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-alpyrazine-6-carboxylic acid [1- (3-aminomethyl-benzyl) -2-oxo-2-thiazol-2yl-ethyl]-amide 0735 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-a~pyrazine-6-carboxylic acid (2oxo- l-piperidin-4-ylmethyl-2 -thiazol-2 -ylethyl) -amide 0 Nii 0 HN) o s 0

NH

0

N

00 o 2 S 0 o s a a a..

a a a. a S a *aa* a S a.

aa.

*.S

a 0740 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-alpyrazine-E--carboxylic acid (2oxo-1-piperidin-3-yl-2-thiazol-2-yl-ethyl) ani de 0745 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1,2-ajpyrazine-6-carboxylic acid [1- (3-guanidino-cyclohexylmethyl) -2-oxo-2thiazol-2-yl-ethyl]-aiide 0750 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrroloi, 2-ajpyrazine-6-carboxylic acid (4-guanidino-cyclohexylmethyl) -2-oxo-2thiazol-2-yl-ethyl]-amide 0755 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-ajpyrazine-6-carboxylic acid Iii- (2-guanidino-cyclohexylmethyl) -2-oxo-2thiazol-2 -yl-ethyl]-amide 0760 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-ajpyrazine-6-carboxylic acid [1- (5-benzyl-thiazole-2-carbonyl) -4-guanidinobutyl]-amide 0765 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-ajpyrazine-6-carboxylic acid [4guanidino-l- (5-phenyl-thiazole-2-carbonyl) butyll-amide 0

N~H

HN

HN

NH

2 0 0 s 0

NH

NH

4* 4.

4 4 4 4 9 U 4 9 9**e 9 4 0770 4-Oxo-2-(3-phenyl-propioflyl)-octahydropyridoti, 2-a]pyrazine-6-carboxylic acid [4guanidino-1- (thiazole-2-carbonyl) -butyl]ami de 0775 5-Oxo-7- (3-phenyl-propiolyl) -octahydro-2thia-4a, 7-diaza-naphthalene-4-carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) butyl]-amide 0780 5-Qxo-7- (3-phenyl-propiofyl)-octahydro-2 thia-4a, 7-diaza-naphthalene-4-carboxylic acid (4-carbamimidoyl-benzyl) -2-oxo-2thiazol -2 -yl -ethyl] -amide 0785 5-Oxo-7- (3-phenyl-propionyl) -octahydro-2thia-4a, 7-diaza-naphthalene-4-carboxylic acid (3-carbamimidoyl-benzyl) -2-oxo-2thiazol-2 -yl -ethyl] -amide 0790 S-Oxo--7- (3-phenyl-propiolyl) -octahydro-2thia-4a, 7-diaza-naphthalene-4-carboxylic acid (1-carbamimidoyl-piperidil- 3 ylmethyl) -2-oxo-2-thiazol-2-y-ethyl]-afide 0795 5-Oxo-7- (3-phenyl-propiolyl) -octahydro-2thia-4a, 7-diaza-naphthalefle-4-carboxylic acid (1-carbamimidoyl-piperidil- 4 f 2 N w~ 0__

NH

S IN 0 r- NS 0

KNH

.5 S 5

S

CCC.

S

S S C C, C

C.

5C

C

S..

S

LL 7 0800 (4-[4-Guanidino-l- (thiazole-2-carbonyl) 7 (3-phenylpropionyl) -octahydro-2-thia-4a, 7-diazanaphthalen-6-yl]--acetic acid 0805 5-Oxo-7- (3-phenyl-propionyl) -octahydro-2thia-4a, 7-diaza-naphthalene-4-carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) butyl]-amide 0810 3-[4-[4-Guanidino-1- (thiazole-2-carbonyl- 7 (3-phenylpropionyl) -octahydro-2-thia-4a, 7-diazanaphthalen-6-yl]-propionic acid 0815 5-Oxo-7- (3-phenyl-propionyl) -octahydro-2thia-4a, 7-diaza-naphthalene-4-carboxylic acid [3-guanidino-propyll-amide 0820 5-Oxo-7- (3-phenyl--propionyl) -octahydro-2thia-4a, 7-diaza-naphthalene-4-carboxylic acid (1-carbamimidoyl-piperidin-3ylmethyl) -2-oxo-2-thiazol-2-yl-ethylI-amide 0825 4-Oxo-2- (3-phenyl-propioflyl)-octahYdropyrrolo[1, 2-ajpyrazine-6-carboxylic acid [4guanidino-1- (hydroxy-thiazol-2-y--methyl) butyl]-axnide 0 HO 0 0 NH iH 2 N wNH__0 S N 0 1 N NH 0s

N

NH

S N 0 00N

NHN

0 a a a a as a.

a a *5t* a a 5 a a a C a a a..

a 0 o~ -HN I-N; 0 H

OH

NH

HN=

NH,

it- 0830 4-Oxo-2- (3-phenyl-propionyl)-octahydropyrrolo[i, 2-alpyrazine-6-carboxyiic acid (4-guanidino-l-thiazol-2-ylmethyl-butyl) ainide 0835 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1, 2-ajpyrazine-6-carboxylic acid [4-guanidino-l-thiazoi-2-yl-butyl) -amide 0840 4-Oxo--2- (3-Phenyl-propionyl) -octahydropyrroloi, 2-a]pyrazine-6-carboxylic acid [4methoxy- 1- (thiazole-2 -carbonyl) -butyil-amide 0845 [6-[4-Methoxy-1- (thiazole-2--carbonyl) butylcarbamoyl]-4-oxo-2- (3-phenylpropionyl) -octahydro-pyrroloji, 2-alpyrazin- 3-yiII-acetic acid 0850 (5-Methoxy-2- ([4-oxo-2- (3-phenylpropionyl) -octahydro-pyrrolo[1, 2-a]pyrazine- 6-carbony]-anino) -pentanoyl) -thiazol-S-yl]acetic acid 0855 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrroloi, 2-a]pyrazine-6-carboxylic acid [4amino-i- (thiazole-2-carbonyl) -butyl]-amide 0 3N

N

NH

HN:

NH,

0 0 ?HN

NH

HNt 0 0 SN 0 o0- 0

N'

HO 0 0s 0- 9

S

*5

S

S S S S

S

S

C*S

S

S S *5*S S S

S

555 0860 4-Oxo--(3-phenyl-propionyl) -octahydropyrrolo[1, 2-a]pyrazine-6-carboxylic acid amino-i- (thiazole-2-carbonyi) -pentylil-amide 0865 4-Oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[i, 2-a]pyrazine-6-carboxyiic acid [5guanidino-1- (thiazole-2-carbonyl) -pentyl]amide 0870 2- (3-Naphthalen-2-yl-propiofyl) -4-oxooctahydro-pyrroio[l, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2carbonyl) -butylI-axnide 0875 4-Oxo-2- (3-phenyl-propiolyl) -octahydropyrroio[1, 2-alpyrazine-6-carboxyiic acid [4guanidino-1- (1-methyl-iH-imidazole-2carbonyl) -butyil]-arride 0880 4-Oxo-2- (3-phenyl-propiolyl) -octahydropyrrolo[1,2-a~pyrazine-6-carboxylic acid [4guanidino-1- (thiazoie-2-carbonyi) -butyl]amide 0885 8,8-Dimethyl-4-oxo-2-(3-phelyl-propionyl)octahydro-pyrrolo[i, 2-alpyrazine-6carboxylic acid [4-guanidino--(thiazole-2carbonyl) -butyil-amide 0 H tNH

HN

0 &NH2 0 HH

NH

2

N

0 H 0 1-t S 0

NH

HNH2 0 0

NH

HN:(

S

S

*9

S

S S S. S *5S*

*SS*

S

S

S S S S

S

00

NH

HN

Preferred compounds according to formula (VIII) include: 0325 3-Aminomethyl-2-benzoyl-4-oxo-octahydro-pyrrolo[l, 2-a]pyridine-6carboxylic acid ll- (benzothiazole-2-carbonyl) -4-guanidino-butyl]amide 0330 3-Aminomethyl-4-oxo-2-phenylacetyl-octahydro-pyrrolo[1, 2a]pyrazine-6-carboxylic acid (benzothiazole-2-carbonyl) -4guanidino-butyl]-amide 0515 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid (3-carbamimidoyl-benzyl) -2-oxo-2-thiazol-2yl-ethyl]-amide 0530 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-alpyrazine-6carboxylic acid (1 -carbamimi doyl -piperi din- 2-ylme thyl) -2-oxo- 2-thiazol-2 -yl-ethyl]-amide 0545 (1 -Carbamini doyl -piper in- 4-ylme thyl) -2-oxo-2-thiazol-2-ylethylcarbamoyl]-4-oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1,2-a]pyrazin-3-y1) -acetic acid 0550 (1-carbamimidoyl-piperidin-4-ylmethyl) -2-oxo-2-thiazol-2yl-ethylcarbamoyl]-4-oxo-2- (3-phenyl-propionyl) -octahydropyrrolo[1,2-ajpyrazin-3-yl) -acetic acid 0555 [6-[l-(1-carbamimidoyl-piperidin-3-ylmethyl) -2-oxo-2-thiazol-2yl-ethylcarbamoyl]-4-oxo-2- (3 -phenyl -prop ionyl) -octahydropyrrolo[1,2-a]pyrazin-3-yl) -acetic acid 0560 (3-Guanidino-propylcarbamoyl) -4-oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[1, 2-alpyrazin-3-yl) -acetic acid 0565 (3-Guanidino-propylcarbamoyl) -4-oxo-2- (3-phenyl-propionyl) octahydro-pyrrolo[1, 2-a]pyrazin-3-yl) -propionic acid :0575 4-Oxo-2-(3-phenyl-propionyl)-octahydro-pyrroo[,2-a]pyrazile-6- 2-thiazol-2 -yl-ethyl]-methyl -amide 40 0580 ([1-carbamimidoyl--piperidin-4-ylmethyl) -2-oxo-2-thiazol-2-ylethyl]-methyl-carbamoyl) -4-oxo-2- (3-phenyl-propionyl) -octahydropyrroloti, 2-a]pyrazin-3-yl]-acetic-acid 0585 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-ajpyrazine-6carboxylic acid (1-carbamimidoyl-piperidin-3-ylmethyl) -2-oxo- 2-thiazol-2 -yl-ethyll-methyl -amide :0590 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid (3-guanidino-propyl) -methyl-amide 0595 2- (Naphthalene-2-carbonyl) -4-oxo-octahydro-pyrrolojl, 2a]pyrazine-6-carboxylic acid [4-guanidino-l- (thiazole-2carbonyl) -butyll-amide si 0625 (2-Methyl-benzylidene) -but-3-enoyl]-4-oxo-octahydropyrrolol, 2-a]pyrazine-6-carboxylic acid (1-carbamimidoylpiperidin-3-ylmethyl) -2-oxo-2-thiazol-2-yl-ethyl]-amide 0630 (2-Methyl-benzylidene) -but-3-enoyl]-4-oxo-octahydropyrroloi, 2-ajpyrazine-6-carboxylic acid (1-carbamimidoylpiperidin-4-ylmethyl) -2-oxo-2-thiazol-2-yl-ethyl]-anide 0635 2- (2-Benzylidene-pent-3-enoyl)-4-oxo-octahydro-pyrrolojl,2a]pyrazine-6-carboxylic acid (3-guanidino-propyl) -amide 0625 (2-Methyl-benzylidene) -but- 3-enoyl] -4 -oxo-octahydropyrrolo[1,2-alpyrazine-6-carboxylic acid (1-carbamimidoylpiperidin-3-ylmethyl) -2-oxo-2-thiazol-2-yl-ethyl]-amide 0602215Mty-enyiee bt3eoy]4ooothdo 06 rro2o[2- 2-a yrabzyine-boxyii acid [1-o(1-cab mdopiperidin-4-ylmethyl) -2-oxo-2-thiazol-2-yl-ethyl]-amide 0635 2- (2-Benzylidene-pent-3-enoyl)-4-oxo-octahydro-pyrrolo[1,2alpyrazine-6-carboxylic acid (3-guanidino-propyl) -amide 0645 4-Oxo-2- (3-phenyl--propionyl) -octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [4-imidazol-1-yl-l- (thiazole-2-carbonyl) -butyllamide 0670 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolojl, 2-alpyrazine-6carboxylic acid [3 (2 -amino- 6-chl oro-pyrimidin-4-yl) -1 (thiazole-2 -carbonyl) -propylli-amide 0675 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo1, 2-a]pyrazine-6carboxylic acid (6-amino-pyridin-2-yl) -1-(thiazole-2carbonyl) -propyl]-amide 0680 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid (2-amino-pyridin-4-yl) (thiazole-2carbonyl) -propyl]-amide .e 40 0685 4-Oxo-2- (3 -phenyl -prop ionyl) -octahydro-pyrrolo[1, 2-alpyrazine-6carboxylic acid [2-(2-amrfino-pyridin-4-yl)-l-(thiazole-2- 060carbonyl) -ethyl]-amide 069 4-Oxo-2- (3 -phenyl -prop ionyl) -octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid [2-(6-aiino-pyridin-2-yl)-1-(thiazole-2- 45 carbonyl) -ethyl]-amide 0695 2-[4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-a]pyrazine- 6-carbonyl]-3- (thiazole-2-carbonyl) 4-tetrahydroisoquinoline- 6-carboxamidine *0700 2-[4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrr10[l, 2-a]pyrazile .6-carbonyl]-3- (thiazole-2-carboflyl) 2,3,4-tetrahydroisoquinoline-7 -carboxamnidine 0705 N-[1-[4-Oxo-2- (3 -phenyl -propi ofl) -octahydro-pyrrolo[l, 2 (thiazole-2-carbonyl) -pyrrolidin- 3 -yl]guanidine 0710 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrralof1, 2-a~pyrazine-6carboxylic acid [1-(4-axino-cyclohexyl)-2-oxo-2-thiazol-2-ylethyl]-amide 0730 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[, 2-apyrazine-6carboxylic acid (3-aminomethyl-benzyl)-2-oxo-2-thiazol--2-ylethyl]-arnide 0745 4-Oxo-2- (3 -phenyl -prop ionyl) -octahydro-pyrrolo[1, 2-ajpyrazine-6carboxylic acid (3-guanidino-cyclohexylmethyl) -2-oxo-2thiazol 2-yl -ethyl]- amide 0755 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid (2-guanidino-cyclohexylmethyl) -2-oxo-2thiazol-2 -yl-ethyll-amide 0795 S-Oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a, 7-diazanaphthalene-4-carboxylic acid (1-carbamimidoyl-piperidin-4ylmethyl) -2-oxo-2-thiazol-2-yl-ethyl]-amide 0800 [4-[4-Guanidino-1- (thiazole-2-carbonyl) -butylcarbamoyl]--5-oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a, 7-diaza-naphthalen-6yl]-acetic acid 0810 3-[4-[4-Guanidino-1- (thiazole-2-carbonyl-butylcarbamoyl]-5-oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a, 7-diaza-naphthalen-6yl]-propionic acid 0815 5-Oxo-7- (3-phenyl-propionyl)-octahydro-2-thia-4a, 7-diazanaphthalene-4-carboxylic acid [3-guanidino-propyl]-amide 0820 5-Oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a, 7-diazanaphthalene- 4- carboxyl ic acid (1-carbamimidoyl-piperidin-3ylmethyl) -2 -oxo-2 -thiazol- 2-yl -ethyl] -amide 0830 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-a]pyrazine-6carboxylic acid (4-guanidino-1-thiazol-2-ylmethyl-butyl) -amide so 40 0835 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo1, 2-a]pyrazine-6carboxylic acid [4-guanidino-1-thiazol-2-yl-butyl) -amide More preferred compounds according to formula VIII

O..

o..O 45 include: 0335 2-Benzoyl-4-oxo-octahydro-pyrrolo1, 2-a]pyrazine-6-carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyll-amide 0. 50 06.50 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[1, 2-alpyrazifle-6carboxylic acid (2 -amino- imidazol- 1-yl) (thiazole-2carbonyl) -butyl]-amide 0655 4-Oxo-2 -phenyl -prop ionyl) -octahydro-pyrrolo[l, 2-alpyrazine- 6carboxylic acid (3 (2 -amino- methyl -pyrimidin-4-yl) 1- (thiazole-2-carbonyi) -propyl]-amide 0715 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid (4-amino-cyclohexylmethyl) -2-oxo-2-thiazol-2yl -ethyl]-amide 0720 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid (4-amino-benzyl) 2-oxo-2 -thiazol 2-yl -ethyl]amide 0725 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid [l-(4-aminomethyl-benzyl)-2-oxo-2-thiazol-2-ylethylj-amide 0735 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolofl, 2-ajpyrazine-6carboxylic acid (2-oxo-l-piperidin-4-ylmethyl-2-thiazol-2-ylethyl) -amide 0740 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo~l, 2-ajpyrazine-6carboxylic acid (2-oxo-l-piperidin-3-yl-2-thiazol-2-yl-ethyl)amide 0750 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid (4-guanidino-cyclohexylmethyl) -2-oxo-2thiazol-2 -yl-ethyl]-amide 0760 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-alpyrazine-6carboxylic acid (5-benzyl-thiazole-2-carbonyl) -4-guanidinobutylli-amide 0765 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[l, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (5-phenyl-thiazole-2-carbonyl) butyl]-amide 0770 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrido~l, 2-a]pyrazine-6carboxylic acid [4-guanidino-l- (thiazole-2-carbonyl) -butyll-amide 40 0775 5-Oxo-7-(3-phenyl-propionyl)-octahydro-2-thia-4a,7-diazanaphthalene-4-carboxylic acid [4-guanidino-l- (thia zole-2carbonyl) -butyl]-amide Goo* 0780 5-Oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a,7-diazanaphthalene- 4 -carboxyl ic acid [l-(4-carbamimidoyl-benzyl)-2-oxo- 2- thiazol-2 -yl -ethyl]-amide 0 *0000785 S-Oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a,7-diazao: naphthalene-4-carboxylic acid (3-carbamimidoyl-benzyl) -2-oxo- 79 2-thiazol-2-yl-ethylj-amide 5-Oxo-7-(3-phenyl-propionyl)-octahydro-2-thia-4a,7-diazanaphthalene- 4-carboxyl ic acid (l-carbamimidoyl-piperidin-3- 0 ylmethyl) -2-oxo-2-thiazol-2-yl-ethyl]-amide 0805 5-Oxo-7- (3-phenyl-propionyl) -octahydro-2-thia-4a,7-diazanaphthaiene-4-carboxyiic acid [4-guanidino-i- (thiazoie-2carbonyl) -butyi]-amide 0825 4-Oxo-2- (3-phenyi-propionyi) -octahydro-pyrrolo[1, 2-a~pyrazine-6carboxylic acid [4-guanidino-i- (hydroxy-thiazoi-2-yl-methyi) butyij-amide 0840 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrroio[1, 2-a]pyrazine-6carboxylic acid [4-methoxy-1- (thiazole-2-carbonyi) -butyi]-amide 0845 [6-[4-Methoxy-i- (thiazoie-2-carbonyi) -butyicarbamoyi]-4-oxo-2- (3phenyl -prop ionyl) -octahydro-pyrroio[1, 2-a]pyrazin-3-yl]-acetic acid 0850 (5-Methoxy-2- (14-oxo-2- (3-phenyi-propionyi) -octahydropyrrolo[i, 2 -a]pyraz ine 6-carbonyl] -amino) -pentanoyl) yl]-acetic acid 0855 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrroioli2-a]pyrazine-6carboxylic acid [4-amino-i- (thiazoie-2-carbonyi) -butyl-amide 0860 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrroio[i, 2-ajjpyrazine-6carboxylic acid [S-amino-i- (thiazoie-2-carbonyi) -pentyi]-amide 0865 4-Oxo-2- (3-phenyl-propionyl) -octahydro-pyrrolo[i, 2-alpyrazine-6carboxyiic acid [5-guanidino-i- (thiazoie-2-carbonyi) -pentyl]amide most preferred compounds according to formula VIII include: 0345 4-Oxo-2- (3-phenyl-propionyi) -octahydro-pyrroio[i,2-a]pyrazine-6carboxyiic acid [4-guanidino-i- (5-methyi-thiazole-2-carbonyi) 09 0 00 0 0 see Preferred compounds according to formula Ix include: 0890 3-Amino-4-oxo-2-phenyl-hexahydro-

I

pyrrolo[2, 3]thiazine-6- S carboxylic acid [1-(benzothiazole-2-

ST

carbonyl) -4-guanidino-butyl]-ainide %HN N 0 0 HN 0

SO

NM

J--NH

HN

0895 3-Amino-2-benzyl-4-oxo--hexahydropyrrolo[2, 3]thiazine-6carboxylic acid [1-(benzothiazole-2-

S

carbonyl) -4-guanidino-butyl)-amide

N

H2N j 0

S_

0 0 HN

_O

NH

,6NH

HN

0900 3-Amino-2-cyclohexyl-4-oxohexahydro-pyrrolo[2, 31thiazine-

S

6-carboxylic acid (benzothiazole- O 2-carbonyl) -4-guanidino-butyl]-amide

N

H2N 0 0 HN,,A 0 1<1N-0 e

S

NH

)=NH

H2N Preferred compounds according to formula X include: 0905 7-Benzyl-6-oxo-octahydro-pyrido[2,1- H

S

c][1,4]thiazine-4-carboxylic acid (1- NN,,,r, (benzothiazole-2-carbonyl)-4- 0 0 guanidino-butyl]-amide 0 NH J= NH

HN

0910 7-(4-tert-Buty1-benzy1)-6-oxo-

H

octahydro-pyrido[2, 41thiazine-

S

4-carboxylic acid (benzothiazole-

NN

2-carbonyl) -4-guanidino-butyl]-amide 0 0 0 N4-r'

(N

S

=NH

RP

0915 6-Oxo-octahydro-pyrido[2, 1- 4 ]thiazine-4-carboxylic acid [4guanidino-1- (thiazole-2-carbonyl) butyl]-amide 0925 7 -Benzyl-6-oxo-octahydro-pyrido[2, 1cJ[1, 4]thiazine-4-carboxylic acid [4guanidino-1- (thiazole-2carbonyl butyll-amide 0935 7 -Benzyl-6-oxo-octahydro-pyrido[2, 1- 4]thiazine-4-carboxylic acid [4guanidino-1- (thiazole-2carbonyl) butyl]-amide 0940 6 -Oxo-7-phenethyl-octahydropyrido[2, 4Jthiazine-4carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyl]-amide

C

S

S

S.

C

S.

S

S.

S

S..

S

0950 7-Benzyl-2,2, 6-trioxo-octahydro- 21>6_pyrido[2, 4]thiazine-4carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyl]-amide

H

0 NH S N 11L N H more preferred compounds according to formula X include: 925 7 -Benzyl-6-oxo-octahydro-pyrido[2, l-cI[1,4]thiazine-4-carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl)butyl]-amide; and94O 6- Oxo-7-phenethyl-octahydro-pyrido[2, 4]thiazine-4-carboxylic acid [4-guanidino-1- (thiazole-2-carbonyl) -butyl]-amide.

-7 Preferred compounds according to formula III include: 0960 4-oxo-1-(3-phenyl-propionyjjoctahydropyrrolo pyrimidine- 6- IN carboxylic acid(4-guanidino-1- r (thiazole-2-carbonyl) -butyl]-amide i H

NH

0965 4-oxo-1- (phenethylsulfony)- so octahydropyrrolo (1,2 ]pyrimidine-6carboxylic (thiazole-2-carbonyl) -butyl] -amide 0 N ,V 0S

NH

.6NH

H,N

For preparation of the compounds of formula (VII) various methods can be employed depending upon the particular starting materials and/or intermediates involved. The following scheme is one particular method of preparation.

0 0 t.o SCHEME I 0 0 R, STEP- I 0 0 R4 0 b STEP-2 STEP-3' g 4~ Y- 0 0 J STEP -4' STEP-3 B NH 2

D-

0 0 STEP-4 c 9* a a a a a a a a a a a a a..

.0

R

20 Step 1: The alkylation of a is done with appropriate bases according to the procedures described in Evans et al (J.

Am. Chem. Soc., 1981, 103, 2127; ibid, 1982, 104, 1737; Aldrichimica Acta, 1982, 15, 23) to give b.

Step 2: Compound h upon hydroboration and oxidation following conditions available in the literature (Synthesis, 1980, 151) results in the aldehyde c.

Step 3: The formation of adduct from aldehyde g with d is done by stirring the reactant in aromatic solvents e.g. benzene or toluene in presence of catalytic amount of suitable acid e.g, p-toluenesulfonic acid.

Step 3': The inter conversion of aldehyde c to aldehyde a is readily achieved by appropriate protection deprotection protocals found in T. Greene, Protective Groups In Organic Synthesis, (John Wiley Sons, 1981).

Step 4: The cylization of adduct to f may readily be achieved b:o: by appropriate Lewis acids e.g, trimethyl aluminum in suitable solvents e.g. dichloromethane, the methodology found in T. Greene, supra.

Step 4': Alternatively, the compound can be derived from the treatment of aldehyde a with d in presence of suitable aromatic solvents e.g, benzene.

35 Step The ester function of the bicyclic intermediate of formula f is then subjected to ,0 hydrolysation using an appropriate agent such as HC1 in an appropriate solvent such as ethyl ether to yield to the free carboxylic acid. The resulting compound is then coupled to R 1 H with a peptide coupling agent such as BOP in an appropriate solvent such as DMF to yield to a bicyclic coupled compound of formula (VIII). Suitable conditions for peptide bond formation are well known in th art of peptide chemistry. For example see Principles of Peptide Synthesis, Bodanszky Springer-Verlag, Berlin, Heidelberg, New York, Tokyo 1984; and The PeDtides, Analysis. Synthesis, Bioloy., Vol. l.edited by Gross E., and Meienhofer Academic Press New York, San Francisco, London, 1979.

For preparation of the compounds of formula (VIII) various methods can be employed depending upon the particular starting materials and/or intermediates involved. The following scheme is one particular method of preparation.

w 9 0 e e* 9* HN 1 Pg

OR

21 0 SCHEME 2 STEP I

OH

N

Pg O21 0 b STEP 2 j

N

Pg O21 0 -ozonolysis STEP 3 a.

a. a a a a a a 9 a a STEP 4

OR

2 1 a. 9 a U *99a 9*9* a a a a a.

a a 0* a a..

R

1

H

STEP 6 wherein; Pg is a nitrogen protecting group; each of R 20 and R 2 1 is independently a C,1 alkyl; and X, R, R. and R, are as previously defined.

The process in scheme 2 is briefly described as follows: STEP 1: The amino and carboxylic functions of the unsaturated compound of formula are protected with appropriate protecting groups. A variety of protecting groups known for reactive functional groups and suitable protection and deprotection protocols may be found in T. Greene, Protective Groups In Oraanic Synthesis, (John Wiley Sons, 1981). The appropriate protecting group to use in a particular synthetic scheme will depend on many factors, including the presence of other reactive functional groups and the reaction conditions desired for removal. The unsaturated compound of formula is easily obtained by methods and protocols known to chemist skill on the art.

The protected unsaturated compound of formula is subjected to appropriate conditions to allow cyclisation using an appropriate reagent such as mercuric acetate in an inert solvent such as tetrahydrofuran (THF) to yield to a protected amino alcohol of formula STEP 2 The protected amino alcohol of formula is oxidized using an appropriate oxidizing agent such as sulfur 30 trioxide pyridine complex in an appropriate solvent such S.: as diclhoromethane or dimethylformamide to yield to a protected amino aldehyde of formula Alternatively, intermediate can be made by the ozonolysis of a compound of formula prepared according to Collado et S. 35 al, J. Org. Chem.,1995, 60:5011.

STEP 3 The protected amino aldehyde of formula is coupled with an amino acid alkyl ester of formula by first forming the imine followed by contacting the obtained imine with an appropriate reagent such as sodium triacetoxy borohydride NaBH(OAc), to yield to a cyclic intermediate of formula STEP 4 The cyclic intermediate of formula is functionalized at the amino position to yield to the amino substituted cyclic intermediate of formula Conditions appropriate for such reactions are well known in the art and will depend on the nature of the Rssubstituent.

STEP The amino protecting group of the cyclic intermediate of formula is removed under appropriate conditions and the resulting compound is then subjected to appropriate condition for internal ring closure such as low heat in an inert solvent or as a raw compound to yield to a bicyclic intermediate of formula The bicyclic intermediate of formula can also be obtained by hydrolysing the ester function of the cyclic intermediate of formula 25 to the free carboxylic acid followed by standard peptide coupling using an appropriate coupling reagent such as benzotriazole-l-yloxy-tris- (dimethylamino)phosphonium hexafluorophosphate (BOP) in an Sinert solvent such as dimethyl formamide (DMF).

STEP 6 The ester function of the bicyclic intermediate of formula is then subjected to hydrolysation using an appropriate agent such as HC1 in an 35 appropriate solvent such as ethyl ether to yield to the free carboxylic acid. The resulting compound is then coupled to R,H with a peptide coupling agent such as BOP in an appropriate solvent such as DMF to yield to a bicyclic coupled compound of formula (VIII). Suitable conditions for peptide bond formation are well known in th art of peptide chemistry. For example see Principles of Peptide Synthesis, Bodanszky Springer-Verlag, Berlin, Heidelberg, New York, Tokyo 1984; and The Peptides, Analysis. Synthesis, Biology, Vol. l.edited by Gross E., and Meienhofer Academic Press New York, San Francisco, London, 1979.

For preparation of the compounds of formula (IX) various methods can be employed depending upon the particular starting materials and/or intermediates involved. The following scheme is one particular method of preparation.

SCHEME3 MeO R y MeO

R

5 Y Me 8

R

4 T MeO Re STEP1 R4 OH R R H 2

N,,(CH

2 )n 0 O R 20 0 a O c b Y= S-Pg NH-Pg STEP 2

R

R Y R STEP 3 R5 dR R4) N (CHn (CH2)n 0 0 OR 20 e 0 wherein: Pg is a sulfur or amino protecting group; L is a leaving group; each of R 2 and R 2 is independently a alkyl; and

R,,

R

4 and R, are as previously defined.

The process depicted in scheme 3 is briefly described as follows: STEP 1: The carboxylic acid compound is coupled to the cyclic amine compound with a peptide coupling agent such as benzotriazol-l-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) in the presence of a base such as n-methylmorpholine in an appropriate solvent such as dimethylformamide (DMF) or dichloromethane

(DCM)

to yield to an amido compound of formula Suitable conditions for peptide bond formation are well known in th art of peptide chemistry. For example see Principles of Peptide Synthesis, Bodanszky Springer-Verlag, Berlin, Heidelberg, New York, Tokyo 1984; and The Peptides, Analysis. Synthesis, Biology. Vol. l.edited by Gross E., and Meienhofer Academic Press New York, San Francisco, London, 1979.

STEP 2 The compound of formula is subjected to appropriate conditions to allow internal cyclisation to yield to a bicyclic intermediate of formula For example, acid mediated cyclisation using p-toluenesulfonic acid or TFA 30 in an appropriate solvent such as dichloroethane.

STEP 3 The ester function of the bicyclic intermediate of formula is subjected to hydrolysis 35 using an appropriate agent such as lithium hydroxide (LiOH) in an appropriate solvent such as tetrahydrofuran (THF) to yield to the free carboxylic acid. The resulting 46 compound is then coupled to RH with a peptide coupling agent such as BOP in an appropriate solvent such as DMF to give compound Suitable conditions for peptide bond formation are well known in the art of peptide chemistry.

For example see Principles of Peptide Synthesis, Bodanszky Springer-Verlag, Berlin, Heidelberg, New York, Tokyo 1984; and The Peptides, Analysis. Synthesis. Biology. Vol.

1_edited by Gross and Meienhofer Academic Press New York, San Francisco, London, 1979.

For preparation of the compounds of formula various methods can be employed depending upon the particular starting materials and/or intermediates involved. The following scheme 4 is one particular method of preparation.

S**

*e e** *9 ee *o o SCHEME 4 STEP 1 0 R a

R

4 0

OR

2 1 b 0 H, 8 OR, )STEP 2

NH

2 STEP 3

R

1

H

STEP4

S

S

*5

S

S S *5

S

S.

5

S

6**S

S

55.5 S S .555

S*

S S 55 S 5S .5

S

S

.55 wherein: 5each of and is independently a C 1 6 alkyl; and B, R 1 1

R

3 ,f R 4 and R, are as previously defined.

The process depicted in scheme 4 is briefly described as follows: STEP 1: The halogenated compound of formula is converted to a halomethyl ketone of formula using an appropriate reagent,such as diazomethane in an inert solvent such as diethyl ether at a temperature of about -25 0 C to about 0°C.

The resulting mixture is then treated under acidic conditions to yield to the halomethyl ketone of formula STEP 2 The halomethyl ketone of formula is coupled with an amino acid alkyl ester of formula with an appropriate base such as sodium cyanoborohydride in an organic solvent such as methanol (MeOH) to yield to a cyclic intermediate of formula STEP 3 The cyclic intermediate of formula is treated under acidic conditions using an appropriate acid such as camphorsulfonic acid in an appropriate solvent such as toluene to yield to a bicyclic intermediate of formula Goes 25 STEP 4 The ester function of the bicyclic intermediate of formula is subjected to hydrolysation S 30 using an appropriate reagent such as LiOH to yield to the free carboxylic acid. The resulting compound is then coupled to RH with a peptide coupling agent such as BOP o* in an appropriate solvent such as dimethylformamide to yield to a coupled bicyclic compound of formula 35 Suitable conditions for peptide bond formation are well known in th art of peptide chemistry. For example see Principles of Peptide Synthesis, Bodanszky Springer- Verlag, Berlin, Heidelberg, New York, Tokyo 1984; and The Peptides. Analysis. Synthesis. Biology. Vol. l.edited by Gross and Meienhofer Academic Press New York, San Francisco, London, 1979.

Compounds of the present invention are further characterized by their ability to inhibit the catalytic activity of thrombin, which is demonstrated in the assay as follows. Compounds of the present invention may be prepared for assay by dissolving-them in buffer to give solutions ranging in concentrations from 1 to 100p.M. In an assay to determine the inhibitory dissociation constant, for a given compound, a chromogenic or fluorogenic substrate of thrombin would be added to a solution containing a test compound and thrombin; the resulting catalytic activity of the enzyme would be spectrophotometrically determined. This type of assay is well known to those skilled in the art.

The compounds of the present invention may be used as anti-coagulants in vitro or ex vivo as in the case of contact activation with foreign thrombogenic surfaces such as is found in tubing used in extracorporeal shunts. The 25 compounds of the invention may also be used to coat the surface of such thrombogenic conduits. To this end, the compounds of the invention are obtained as lyophilized powders, redissolved in isotonic saline and added in an amount sufficient to maintain blood in an anticoagulated state.

C

The therapeutic agents of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers. The proportion of each carrier is 35 determined by the solubility and chemical nature of the compound, the route of administration, and standard pharmaceutical practice. For example, the compounds may be injected parenterally; this being intramuscularly, intravenously, or subcutaneously. For parenteral administration, the compound may be used in the form of sterile solutions containing other solutes, for example, sufficient saline or glucose to make the solution isotonic. The compounds may be administered orally in the form of tablets, capsules, or granules containing suitable excipients such as starch, lactose, white sugar and the like. The compounds may also be administered sublingually in the form of troches or lozenges in which each active ingredient is mixed with sugar or corn syrups, flavouring agents and dyes, and then dehydrated sufficiently to make the mixture suitable for pressing into solid form. The compounds may be administered orally in the form of solutions which may contain colouring and/or flavouring agents.

Physicians will determine the dosage of the present therapeutic agents which will be most suitable. Dosages may vary with the mode of administration and the particular compound chosen. In addition, the dosage may vary with the particular patient under treatment.

When the composition is administered orally, a larger 25 quantity of the active agent will typically be required to produce the same effect as caused with a smaller quantity given parenterally.

To further assist in understanding the present invention, the following non-limiting examples of such thrombin inhibitory compounds are provided. The following examples, of course, should not be construed as specifically limiting the present invention, variations presently known or later developed, which would be within S. 35 the purview of one skilled in the art and considered to fall within the scope of the present invention as described herein. The preferred compounds as of the -7/ present invention are synthesized using conventional preparative steps and recovery methods known to those skilled in the art of organic and bio-organic synthesis, while providing a new a unique combination for the overall synthesis of each compound. Preferred synthetic routes for intermediates involved in the synthesis as well as the resulting anti-thrombotic compounds of the present invention follow.

a a.

ao a EXAMPLE 1 H

O

Boc-1 N N-

O

CH3 1. Zn/Cu couple; ultrasound BocN N

C

I

A

CH

3 2. [o-CH 3

C

6

H

4 3 P]2PdCl 2 CH 4-iodobenzonitrile

C

NC

A solution of tert-butyloxycarbonyl-iodo-alanine-N,0dimethylamide (2.68 g, 7.5 mmol) Org. Chem. 1992, 57, 3397-3404) in dry benzene (30 mL), and dry N,Ndimethylacetamide (2.0 mL) was added to a dry nitrogen-purged round bottom flask charged with zinc-copper couple (0.90 g).

The resulting mixture was sonicated under nitrogen until no starting material remained (as judged by TLC). Bis(tri-otolylphosphine)palladium dichloride (0.35 g, 0.40 mmol) was added followed by 4 -iodobenzonitrile (1.72 g, 7.5 mmol). The resulting mixture was stirred under a nitrogen atmosphere with heating, allowed to cool, ethyl acetate (100 mL) was added, and the mixture filtered into a separatory funnel. Sequential washing with aqueous HC1 (50 mL; 0.1N), distilled H 2 0 (3 x mL), drying over Na,SO,, filtration, and concentration under reduced pressure yielded the crude product. Flash chromatography over silica gel (light petroleum-ethyl acetate gradient) afforded the purified compound.

0 0 N N OCH3 N Oc Boc. N 0ocH 1. Zn/Cu couple; ultrasound B c N l A I

CH

3 2. [o-CH3CH4)3P]2PdCl2 H C3 S .3-iodobenzonitrile

.*I

CN

A solution of tert-butyloxycarbonyl-iodo-alanine-N,Odimethylamide (2.68 g, 7.5 mmol) Org. Chem. 1992, 57, 3397-3404) in dry benzene (30 mL), and dry N,Ndimethylacetamide (2.0 mL) was added to a dry nitrogen-purged round bottom flask charged with zinc-copper couple (0.90 g).

The resulting mixture was sonicated under nitrogen until no starting material remained (as judged by TLC). Bis(tri-otolylphosphine)palladium dichloride (0.35 g, 0.40 mmol) was added followed by 3-iodobenzonitrile (1.72 g, 7.5 mmol). The resulting mixture was stirred under a nitrogen atmosphere with heating, allowed to cool, ethyl acetate (100 mL) was added, and the mixture filtered into a separatory funnel. Sequential washing with aqueous HC1 (50 mL; 0.1N), distilled H 2 0 (3 x mL), drying over Na,SO,, filtration, and concentration under reduced pressure yielded the crude product. Flash chromatography over silica gel (light petroleum-ethyl acetate gradient) afforded the purified compound.

0

H

Boc/00 "J

N

CH

3 1. Zn/Cu couple; ultrasound 2. [o-CH 3

C

6 H4) 3 P]2PdCl 2 2-iodobenzonitrile A solution of tert-butyloxycarbonyl-iodo-alanine-N,Odimethylamide (2.68 g, 7.5 mmol) Org. Chem. 1992, 57, 3397-3404) in dry benzene (30 mL), and dry N,Ndimethylacetamide (2.0 mL) was added to a dry nitrogen-purged round bottom flask charged with zinc-copper couple (0.90 g).

The resulting mixture was sonicated under nitrogen until no starting material remained (as judged by TLC). Bis(tri-otolylphosphine)palladium dichloride (0.35 g, 0.40 mmol) was added followed by 2-iodobenzonitrile (1.72 g, 7.5 mmol). The resulting mixture was stirred under a nitrogen atmosphere with heating, allowed to cool, ethyl acetate (100 mL) was added, and the mixture filtered into a separatory funnel. Sequential washing with aqueous HC1 (50 mL; 0.1N), distilled HO (3 x mL), drying over Na2SO,, filtration, and concentration under reduced pressure yielded the crude product. Flash chromatography over silica gel (light petroleum-ethyl acetate gradient) afforded the purified compound. 9.

9 *9 9 p

**WO

a 9 9..

9 0

CH

3

NC#:Q)

1. NH 2 OH, DIEA/EtOH 2. H 2 Pd/C/EtOH:HOAc To a solution of tert-butyloxycarbonyl-para-cyanophenylalanine-N,O-dimethylamide (1.33 g, 4.0 mmol) in dry ethanol (20 mL) was added hydroxlyamine hydrochloride (0.416 g, 6.0 mmol), and diisopropylethylamine (1.02 mL, 6.0 mmol).

The mixture was refluxed and then cooled. The precipitate was -7+ filtered, washed with cold ethanol, diisopropylether, dried with MgSO concentrated under reduced pressure, and used directly in the next step. The semi-solid was suspended in a mixture of acetic acid (20 mL), and dry ethanol (40 mL) with warming. Subsequently, Pd/C catalyst (0.30 g, 10% Pd) was added, and hydrogen was bubbled through the mixture with warming. The hydrogenation was continued until no starting material could be detected as judged by TLC. The catalyst was removed by filtration, the solution was concentrated under reduced pressure (50 mL), HC1 (50 mL, 1 N) was added, and the mixture was concentrated once again to 50 mL. The solution was chilled overnight yielding the title compound.

0 0 Boc- N NOCH3 1. NHOH, DIEA/EtOH Boc N N ,OCH3 CH 2. H2, Pd/C/EtOH:HOAc A

HCH,

NC

H2N NH To a solution of tert-butyloxycarbonyl-meta-cyanophenylalanine-N,O-dimethylamide (1.33 g, 4.0 mmol) in dry ethanol (20 mL) was added hydroxlyamine hydrochloride (0.416 g, 6.0 mmol), and diisopropylethylamine (1.02 mL, 6.0 mmol).

The mixture was refluxed and then cooled. The precipitate was filtered, washed with cold ethanol, diisopropylether, dried with MgSO, concentrated under reduced pressure, and used directly in the next step. The semi-solid was suspended in a mixture of acetic acid (20 mL), and dry ethanol (40 mL) with warming. Subsequently, Pd/C catalyst (0.30 g, 10% Pd) was added, and hydrogen was bubbled through the mixture with warming. The hydrogenation was continued until no starting material could be detected as judged by TLC. The catalyst was reduced pressure (50 mL), HC1 (50 mL, 1 N) was added, and the mixture was concentrated once again to 50 mL. The solution was chilled overnight yielding the title compound.

oI-

NH

o a s *hnllnn-~-iehlmd (13 r drcl ntenx tp h eisldwssseddi 7 0 0 H

H

Boc N OCH 3 1. NH0OH, DIEA/EtOH Bc- N OCH3 CH 2. H 2 Pd/C/EtOH:HOAc

I

CH if^Y"

CH

3 CN

NH

2

NH

To a solution of tert-butyloxycarbonyl-ortho-cyanophenylalanine-N,O-dimethylamide (1.33 g, 4.0 mmol) in dry ethanol (20 mL) was added hydroxlyamine hydrochloride (0.416 g, 6.0 mmol), and diisopropylethylamine (1.02 mL, 6.0 mmol).

The mixture was refluxed and then cooled. The precipitate was filtered, washed with cold ethanol, diisopropylether, dried with MgSO concentrated under reduced pressure, and used directly in the next step. The semi-solid was suspended in a mixture of acetic acid (20 mL), and dry ethanol (40 mL) with warming. Subsequently, Pd/C catalyst (0.30 g, 10% Pd) was added, and hydrogen was bubbled through the mixture with warming. The hydrogenation was continued until no starting material could be detected as judged by TLC. The catalyst was removed by filtration, the solution was concentrated under reduced pressure (50 mL), HC1 (50 mL, 1 N) was added, and the mixture was concentrated once again to 50 mL. The solution was chilled overnight yielding the title compound.

0 0 H H

H

Boc N, N Lithium thiazole/THF Boc

CH

3

H

2 N I

H

2

N

(30 mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) *00 dropwise at -780 C, and the solution stirred. tert- Butyloxycarbonyl-para-amidino-phenyladimethylamide (1.15 g, 3.3 mmol) in THF (15 mL) was then added dropwise, and the resulting mixture stirred. The reaction was quenched with 9 saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

O

H

BcN .OCH 3 H Bocl, N Lithium thiazole/THF Bock I A

CH

3

N

HN NH 2 HN NH 2 To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous THF mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -780 C, and the solution stirred. tert- Butyloxycarbonyl-meta-amidino-phenylalanine-N,O-dimethylamide (1.15 g, 3.3 mmol) in THF (15 mL) was then added dropwise and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

S

*r S

S

S

*SSS

S

S

S

S..

S

Boc' 0CH CH3 Lithium thiazole/THF To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous THF (30 mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -78° C, and the solution stirred. tert- Butyloxycarbonyl-ortho-amidino-phenylalanine-N,0-dimethylamide (1.15 g, 3.3 mmol) in THF (15 mL) was then added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted 77 with ethyl acetate (150 mL) and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

Boc' 1. H 2 RaNi/EtOH:NH 0

H

I3 Boc" N N C H2N

OCH

3

H

2

N=

a.

a a a

S.

a, tert-Butyloxycarbonyl-para-cyano-phenylalanine-N,Odimethylamide (1.33 g, 4.0 mmol) was dissolved in ethanol saturated with ammonia (30 mL), and sponge Raney Ni (100 mg) added. The solution was shaken under H 2 at room temperature psi). The solution was filtered through celite, and concentrated under reduced pressure to yield a clear residue.

The residue was dissolved in ethyl acetate (250 mL), and washed with 1 N NaOH (2 x 50 mL), and brine (2 x 50 mL). The solution was dried with MgSO filtered, and concentrated under reduced pressure.

0 0 Boc N OCH 3 1. Hz, RaNi/EtOH:NH 3 Boc N N

CH

3

OCH

3

NC

H

2

N

tert-Butyloxycarbonyl-meta-cyano-phenylalanine-N,0dimethylamide (1.33 g, 4.0 mmol) was dissolved in ethanol saturated with ammonia (30 mL), and sponge Raney Ni (100 mg) added. The solution was shaken under H 2 at room temperature (40 psi). The solution was filtered through celite, and concentrated under reduced pressure to yield a clear residue.

The residue was dissolved in ethyl acetate (250 mL), and washed with 1 N NaOH (2 x 50 mL), and brine (2 x 50 mL). The solution was dried with MgSO filtered, and concentrated under reduced pressure.

0

H

Boc N "OCH3 N CH 3

CN

1. H 2 RaNi/EtOH:NH 3

,NCH

OCH3 tert-Butyloxycarbonyl-ortho-cyano-phenylalanine-N,0dimethylamide (1.33 g, 4.0 mmol) was dissolved in ethanol saturated with ammonia (30 mL), and sponge Raney Ni (100 mg) added. The solution was shaken under H at room temperature psi). The solution was filtered through celite, and concentrated under reduced pressure to yield a clear residue.

The residue was dissolved in ethyl acetate (250 mL), and washed with 1 N NaOH (2 x 50 mL), and brine (2 x 50 mL). The solution was dried with MgSO filtered, and concentrated under reduced pressure.

0

H

Boc- I H2N OCH,

H

3 CS NHZ O HgCI 2

/THF

a

C

CCC.

C.a C C *a Ca

S..

C

tert-Butyloxycarbonyl-para-aminomethyl-phenylalanine-N,0dimethylamide (1.00 g, 3.1 mmol) was dissolved in dry THF mL) under nitrogen with stirring. The solution was cooled, N,N'-bis-(benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol), and HgCl 2 (0.95 g, 3.5 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (200 mL), and filtered through celite. The filtrate was concentrated under reduced pressure.

Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the purified compound.

0 NZ H j| 0 BocN N CH 3

H

3 CS NHZ Boc Boc N

OCH

3 HgC 2 TlHF

C

NH

2

HN

ZHN" NZ tert-Butyloxycarbonyl-meta-aminomethyl-phenylalanine-N,0dimethylamide (1.00 g, 3.1 mmol) was dissolved in dry THF mL) under nitrogen with stirring. The solution was cooled, N,N'-bis-(benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol), and HgC1 2 (0.95 g, 3.5 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (200 mL), and filtered through celite. The filtrate was concentrated under reduced pressure.

Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the purified compound.

H I Boc N *cH 3

H

3 CS NHZ Bo N N C

I

*a

OCH

3 HgCI/RIF

OCH

3

IH

NH N N

***ZN

tert-Butyloxycarbonyl-ortho-aminomethyl-phenylalanine-N,0dimethylamide (1.00 g, 3.1 mmol) was dissolved in dry THF mL) under nitrogen with stirring. The solution was cooled, N,N'-bis-(benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol), and HgCl, (0.95 g, 3.5 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (200 mL), and filtered through celite. The filtrate was concentrated under reduced pressure.

Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the purified compound.

$0 0 N CH3

H

Boc N' 1. Lithium thiazole/THF Boc A

I

OCH

3 ZHNyNN HN NHZ

NZ

NZ

To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous THF mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -78° C, and the solution stirred. The protected amino acid (1.36 g, 3.3 mmol) in THF (15 mL) was then added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

Boc o oo 1. Lithium thiazole/THF -NHZ

ZHN"NZ

To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous THF (30 mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -78° C, and the solution stirred. The protected amino acid (1.36 g, 3.3 mmol) in THF (15 mL).was then added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

o H c BoN 1. Lithium thiazole/THF Boc' OCH3 I N NH Z

H

ZN

N

ZN

ZN

To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous

THF

mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -780 C, and the solution stirred. The protected amino acid (1.36 g, 3.3 mmol) in THF (15 mL) was then added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with magnesium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

0 o .Boc- N /OCH 3 1. Zn/Cu couple; ultrasound Boc N N/OC

I

S: I CH 3 2. [o-CH 3

C

6

H

4 3

P

2 PdC 2 CH 3 ,I 2 aee A solution of tert-butyloxycarbonyl-iodo-alanine-N,0dimethylamide (2.68 g, 7.5 mmol) Org. Chem. 1992, 57, 3397-3404) in dry benzene (30 mL), and dry N,Ndimethylacetamide (2.0 mL) was added to a dry nitrogen-purged round bottom flask charged with zinc-copper couple (0.90 g).

The resulting mixture was sonicated under nitrogen until no starting material remained (as judged by TLC). Bis(tri-o- S. tolylphosphine)palladium dichloride (0.35 g, 0.40 mmol) was added followed by 2-iodobenzonitrile (1.72 g, 7.5 mmol). The resulting mixture was stirred under a nitrogen atmosphere with heating, allowed to cool, ethyl acetate (100 mL) was added, and the mixture filtered into a separatory funnel. Sequential washing with aqueous HC1 (50 mL; 0.1N), distilled

H

2 0 (3 x mL), drying over Na 2

SO

4 filtration, and concentration under reduced pressure yielded the crude product. Flash.

chromatography over silica gel (light petroleum/ethyl acetate gradient) afforded the purified compound.

0 0 H

H

Boc N/ N 1. Lithium thiazole/THF Boc N A I

A

CH

3

H

2 N N

H

2 N N To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous

THF

mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -780 C, and the solution stirred. The amino acid- N,O-dimethylamide (1.07 g, 3.3 mmol) in anhydrous THF (15 mL) was then added dropwise and the resulting mixture stirred.

The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO 4 filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

0 0 HO

H

Boc N 1 OC H3 1 Zn/Cu couple; ultrasound BocN N OCI I

I

CH

3 2. [o-CH 3

C

6

H

4 3

P]

2 PdCl 2 CH 3 2 0 NC N 0 A solution of tert-butyloxycarbonyl-iodo-aianine-N,0dimethylamide (2.68 g, 7.5 mmol) Org. Chem. 1992, 57, 3397-3404) in dry benzene (30 mL), and dry N,Ndimethylacetamide (2.0 mL) was added to a dry nitrogen-purged round bottom flask charged with zinc-copper couple (0.90 g).

The resulting mixture was sonicated under nitrogen until no starting material remained (as judged by TLC). Bis(tri-otolylphosphine)palladium dichloride (0.35 g, 0.40 mmol) was added followed by 2 -iodobenzonitrile (1.72 g, 7.5 mmol). The resulting mixture was stirred under a nitrogen atmosphere with heating, allowed to cool, ethyl acetate (100 mL) was added, and the mixture filtered into a separatory funnel. Sequential washing with aqueous HC1 (50 mL; 0.1N), distilled H0 (3 x mL), drying over Na 2

SO

4 filtration, and concentration under reduced pressure yielded the crude product. Flash chromatography over silica gel (light petroleum/ethyl acetate gradient) afforded the purified compound.

H

H

BocN

OCH

3 1. NH 2 OH, DIEA/EtOH BocN

N

CH

3 2. H 2 Pd/C/EtOH:HOAc

H

NC :H 2 N N NC NN

NH

To a solution of tert-butyloxycarbonyl-(4-cyano)3pyridylalanine-N,O-dimethylamide (1.34 g, 4.0 mmol) in dry ethanol (20 mL) was added N,O-hydroxlyamine hydrochloride (0.416 g, 6.0 mmol), and diisopropylethylamine (1.02 mL, mmol). The mixture was refluxed and then cooled. The precipitate was filtered, washed with cold ethanol, diisopropylether, dried with MgSO concentrated under reduced pressure, and used directly in the next step. The semi-solid was suspended in a mixture of acetic acid (20 mL), and dry ethanol (40 mL) with warming. Subsequently, Pd/C catalyst (0.30 g, 10% Pd) was added, and hydrogen was bubbled through the mixture with warming. The hydrogenation was continued until no starting material could be detected as judged by TLC.

The catalyst was removed by filtration, and the solution was concentrated under reduced pressure (50 mL), HC1 (50 mL, 1 N) was added, and the mixture was concentrated once again to mL. The solution was chilled overnight yielding the title compound.

*0

H

4 N N' 1. Lithium thiazolle/THF o N N

CH

3 N

J

H

2 N

H

2

N

N

NH

NH

*N NH To a solution of thiazole (1.28 g, 15.0 mmol) in anhydrous

THF

mL) was added n-BuLi (1.6 M/hexane, 8.9 mL, 13.9 mmol) dropwise at -78° C, and the solution stirred. The amino acid- N,O-dimethylamide (1.16 g, 3.3 mmol) in anhydrous THF (15 mL) was then added dropwise, and the resulting mixture stirred.

The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO 4 filtered, and concentrated under reduced pressure. The crude material was purified on silica gel ethyl acetate/hexane), and concentrated under reduced pressure.

Boc N NCH 1. H 2 PtOzAcOH Bo cN C NoQ

HN

tert-Butyloxycarbonyl-3-(4-pyridyl)alanine-N,0-dimethylamide (4.50 g, 14.4 mmol) was dissolved in acetic acid (100 mL), and PtO 2 (100 mg) added. The solution was shaken under H, until gas uptake ceased. The solution was filtered through celite, and concentrated under reduced pressure yielding tertbutyloxycarbonyl-3-(4-piperidyl)alanine-N,O-dimethylamide.

The residue was dissolved in ethyl acetate (250 mL), washed with 1 N NaOH (2 x 50 mL), brine (2 x 50 mL), dried with MgSO filtered, and concentrated under reduced pressure to yield the title compound.

0 o Boc: CH 1. H 2 PtO 2 /AcOH B N N CH 1 I o

OCH

3 OCH 3 CocH tert-Butyloxycarbonyl-3-(3-pyridyl)alanine-N,0-dimethylamide (4.50 g, 14.4 mmol) was dissolved in acetic acid (100 mL), and PtO 2 (100 mg) added. The solution was shaken under H, until S: gas uptake ceased. The solution was filtered through celite, and concentrated under reduced pressure yielding tertbutyloxycarbonyl-3-(3-piperidyl)alanine-N,0-dimethylamide.

The residue was dissolved in ethyl acetate (250 mL), washed with 1 N NaOH (2 x 50 mL), brine (2 x 50 mL), dried with MgSO filtered, and concentrated under reduced pressure to yield the title compound.

0 H 0 BocNH NCH 3 1. H 2 PtO 2 /AcOH Bo S OCH A I

OCH

tert-Butyloxycarbonyl-3-(2-pyridyl)alanine-N,O-dimethylamide (4.50 g, 14.4 mmol) was dissolved in acetic acid (100 mL), and PtO, (100 mg) added. The solution was shaken under H 2 until gas uptake ceased. The solution was filtered through celite, and concentrated under reduced pressure yielding tertbutyloxycarbonyl-3-(2-piperidyl)alanine-N,0-dimethylamide.

The residue was dissolved in ethyl acetate (250 mL), washed with 1 N NaOH (2 x 50 mL), brine (2 x 50 mL), dried with MgSO 4 filtered, and concentrated under reduced pressure to yield the title compound.

0 NZ o BocN CH3 HCS NHZ BOC N N IC HN OCH 3 HgCI 2 /THF OC 3

OCH

3 ZHN N

NZ

tert-Butyloxycarbonyl-3-(4-piperidyl)alanine-N,O-dimethylamide (1.00 g, 3.2 mmol) was dissolved in dry THF (10 mL) under nitrogen with stirring. The solution was cooled, N,N'-bis- (benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol), and HgC1, (0.95 g, 3.5 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (200 mL), and filtered through celite. The filtrate was concentrated under reduced pressure.

Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the title compound.

0 NZ BocNH CH H 3 CS NHZ Boc

OCH

3 HgCI 2 /THF

CH

OCH

N

K)

H N ZN NHZ tert-Butyloxycarbonyl-3-(3-piperidyl)alanine-N,0-dimethylamide (1.00 g, 3.2 mmol) was dissolved in dry THF (10 mL) under nitrogen with stirring. The solution was cooled, N,N'-bis- (benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol), and HgCl 2 (0.95 g, 3.5 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (200 mL), and filtered through celite. The filtrate was concentrated under reduced pressure.

Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the title compound.

0

NZN

H(benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol) BocN1, 5 3 H 3 CS NolZ d. T so on ws suspended in ethyl acetate (200 and filtered through

SOCH

3 HgCIj/HF

OCH

3 celite. The filtrate was concentrated under reduced pressure.

a

N

y N H Z a

ZN

tert-Butyloxycarbonyl-3r s(2-piperidyl alanine-N, -dimethylamide (1.00 g, 3.2 mmol) was dissolved in dry THF (10 mL) under nitrogen with stirring. The solution was cooled, N,N'-bis- (benzyloxycarbonyl)-S-methyl-isothiourea (1.14 g, 3.2 mmol), and HgCl 2 (0.95 g, 3.5 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (200 mL), and filtered through celite. The filtrate was concentrated under reduced pressure.

Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the title compound.

Boc/' 1. Lithium thlazole/THF To a solution of thiazole in anhydrous THF (1.23 g, 14.4 mmol) was added n-BuLi (1.6 M/hexane, 8.4 mL, 13.4 mmol) dropwise at -780 C and the solution stirred. The guanidylated 4piperidylalanine derivative (2.00 g, 3.2 mmol) in anhydrous THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO filtered, and concentrated under reduced pressure.

O0 H o Boc- N -1 H 1. Lithium thiazole/THF Bo N

OCH

3

N

ZN NHZ Z NHZ To a solution of thiazole in anhydrous THF (1.23 g, 14.4 mmol) was added n-BuLi (1.6 M/hexane, 8.4 mL, 13.4 mmol) dropwise at -78° C with stirring. The mixture was stirred at -78° C for 1 S h. The guanidylated 3-piperidylalanine derivative (2.00 g, 3.2 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO filtered, and concentrated under reduced pressure.

1. Lithium thiazole/THF 'I NHZ

ZN

To a solution of thiazole in anhydrous THF (1.23 g, 14.4 mmol) was added n-BuLi (1.6 M/hexane, 8.4 mL, 13.4 mmol) dropwise at -78° C with stirring. The mixture was stirred at -78° C for 1 h. The guanidylated 2-piperidylalanine derivative (2.00 g, 3.2 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO,, filtered, and concentrated under reduced pressure.

1. H 2 PtO 2 /AcOH 0

H

aJO NO 1-CH 3

H

2

N.,

a *ae.

a a a.

a a..

a tert-Butyloxycarbonyl-para-nitro-phenylalanine-N,Odimethylamide (13.88 g, 39.3 mmol) was dissolved in acetic acid (100 mL), and PtO, (100 mg) added. The solution was shaken under H 2 until gas uptake ceased. The solution was filtered through celite, concentrated under reduced pressure, taken up in H20 (150 mL), and lyophilized. The semi-solid was dissolved in ethyl acetate (350 mL), washed with 1 N NaOH (3 x mL), and brine (3 x 50 mL). The solution was dried with MgSO,, filtered, and concentrated under reduced pressure yielding the title compound.

0

H

Boc/ C 1. H2, PtO 2 /AcOH

NB

OCH

3 0 2 N

H

2 tert-Butyloxycarbonyl-meta-nitro-phenylalanine-N,0dimethylamide (13.88 g, 39.3 mmol) was dissolved in acetic acid (100 mL), and PtO, (100 mg) added. The solution was shaken under H 2 until gas uptake ceased. The solution was filtered through celite, concentrated under reduced pressure, taken up in H 2 0 (150 mL), and lyophilized. The semi-solid was dissolved in ethyl acetate (350 mL), washed with 1 N NaOH (3 x mL), and brine (3 x 50 mL). The solution was dried with MgSO 4 filtered, and concentrated under reduced pressure yielding the title compound.

0 o OCHOCH BocN N ,H3 0 2 H2H I OCH 3 tert-Butyloxycarbonyl-ortho-nitro-phenylalanine-N,0dimethylamide (13.88 g, 39.3 mmol) was dissolved in acetic acid (100 mL), and PtO 2 (100 mg) added. The solution was Sshaken under H2 until gas uptake ceased. The solution was filtered through celite, concentrated under reduced pressure, taken up in HI0 (150 mL), and lyophilized. The semi-solid was dissolved in ethyl acetate (350 mL), washed with 1 N NaOH (3 x 50 mL), and brine (3 x 50 mL). The solution was dried with MgSO 4 filtered, and concentrated under reduced pressure yielding the title compound.

0

O

HH H I BocN NCH3 1. Z-C, NaHCO3/THF:H 2 0 Boc

OCH

3 2. Lithium thiazole/THF

N..

H

2 N

NHZ

1. tert-Butyloxycarbonyl-3-(cis/trans-4aminocyclohexyl)alanine-N,O-dimethylamide (1.00 g, 3.0 mmol) was dissolved in saturated aqueous sodium bicarbonate, and THF mL, with stirring. The solution was cooled and a solution of benzyl chloroformate (0.43 mL, 3.0 mmol) in THF mL) was added dropwise. Excess solid sodium bicarbonate was added, the THF was removed under reduced pressure, and the remaining aqueous phase was poured into ethyl acetate (250 mL), and mixed thoroughly. The aqueous phase was discarded and the remaining solution was washed with saturated aqueous sodium bicarbonate (2 x 50 mL), 4 N aqueous sodium bisulfate (2 x 50 mL), and brine (2 x 50 mL). The solution was dried with MgSO 4 filtered, and concentrated under reduced pressure.

The semi-solid was chromatographed on silica gel (ethyl acetate/ hexane).

2. To a solution of thiazole (1.16 g, 13.7 mmol) in anhydrous THF was added n-BuLi (1.6 M/hexane, 8.0 mL, 12.8 mmol) dropwise at -78° C and the solution stirred. The above protected amino acid amide (1.41 g, 3.0 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO,, filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

Boc N1. Z-CI, NaHCOFHF:H 2 0 N Bocd-

OCH

3 2. Lithium thiazole/HF

*V

H2 ZHN 1. tert-Butyloxycarbonyl-3-(cis/trans-3- Cl1 aminocyclohexyl)alanine-N,0-dimethylamide (1.00 g, 3.0 mmol) was dissolved in saturated aqueous sodium bicarbonate, and THF mL, with stirring. The solution was cooled and a solution of benzyl chloroformate (0.43 mL, 3.0 mmol) in THF mL) was added dropwise. Excess solid sodium bicarbonate was added, the THF was removed under reduced pressure, and the remaining aqueous phase was poured into ethyl acetate (250 mL), and mixed thoroughly. The-aqueous phase was discarded and the remaining solution was washed with saturated aqueous sodium bicarbonate (2 x 50 mL), 4 N aqueous sodium bisulfate (2 x 50 mL), and brine (2 x 50 mL). The solution was dried with MgSO filtered, and concentrated under reduced pressure.

The semi-solid was chromatographed on silica gel (ethyl acetate/ hexane).

2. To a solution of thiazole (1.16 g, 13.7 mmol) in anhydrous THF was added n-BuLi (1.6 M/hexane, 8.0 mL, 12.8 mmol) dropwise at -78° C and the solution stirred. The above protected amino acid amide (1.41 g, 3.0 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO 4 filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

o H H BocN CH 1. Z-CI, NaHCO 3

/THF:H

2 0 o NBo OCH3 2. Lithium thiazole/THF

A

NH

2

ZNHZ

S 1. tert-Butyloxycarbonyl-3-(cis/trans-2aminocyclohexyl)alanine-N,0-dimethylamide (1.00 g, 3.0 mmol) was dissolved in saturated aqueous sodium bicarbonate, and THF mL, with stirring. The solution was cooled and a S solution of benzyl chloroformate (0.43 mL, 3.0 mmol) in THF (10 mL) was added dropwise. Excess solid sodium bicarbonate was added, the T

H

F was removed under reduced pressure, and the remaining aqueous phase was poured into ethyl acetate (250 mL), and mixed thoroughly. The aqueous phase was discarded and the remaining solution was washed with saturated aqueous S sodium bicarbonate (2 x 50 mL), 4 N aqueous sodium bisulfate (2 x 50 mL), and brine (2 x 50 mL). The solution was dried with MgSO 4 filtered, and concentrated under reduced pressure.

The semi-solid was chromatographed on silica gel (ethyl acetate/ hexane).

2. To a solution of thiazole (1.16 g, 13.7 mmol) in anhydrous THF was added n-BuLi (1.6 M/hexane, 8.0 mL, 12.8 mmol) dropwise at -780 C and the solution stirred. The above protected amino acid amide (1.41 g, 3.0 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO 4 filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

H H i° O NZ BocN NH 1. H 3 CS NHZ BoN oCH HgC/IHF

N

2. Lithium thiazole/THF ZN N1

H

2 N ZHN NWy

H

1. tert-Butyloxycarbonyl-3- (cis/trans-4aminocyclohexyl)alanine-N,0-dimethylamide (2.0 g, 6.1 mmol) was dissolved in dry THF (20 mL) under nitrogen with stirring.

The solution was cooled to 00 C, N,N'-bis-(benzyloxycarbonyl)- S-methyl-isothiourea (2.18 g, 6.1 mmol), and HgCl, (1.81 g, 6.7 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (300 mL), and filtered through celite. The filtrate was concentrated under reduced pressure. Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the purified product.

2. To a solution of thiazole (2.32 g, 27.3 mmol) in anhydrous THF was added n-BuLi (1.6 M/hexane, 15.9 mL, 25.4 mmol) dropwise at -78° C and the solution stirred. The above guanidylated amino acid (3.88 g, 6.1 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous S ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO,, filtered, and concentrated under reduced pressure. The crude S material was purified on silica gel (ethyl acetate/hexane), S and concentrated under reduced pressure.

e* 0 NZ 0 NBoc- JNH3 1. H 3 CS NHZ Boc I I N

OCH

3 HgC/THF YH2j 2. Lithium thiazole/THF

ZHN

1. tert-Butyloxycarbonyl-3- (cis/trans-3aminocyclohexyl)alanine-N,O-dimethylamide (2.0 g, 6.1 mmol) was dissolved in dry THF (20 mL) under nitrogen with stirring.

The solution was cooled to 0° C, N,N'-bis-(benzyloxycarbonyl)- S-methyl-isothiourea (2.18 g, 6.1 mmol), and HgC12 (1.81 g, 6.7 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (300 mL), and filtered through celite. The filtrate was concentrated under reduced pressure. Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the purified product.

2. To a solution of thiazole (2.32 g, 27.3 mmol) in anhydrous THF was added n-BuLi (1.6 M/hexane, 15.9 mL, 25.4 mmol) dropwise at -78° C and the solution stirred. The above guanidylated amino acid (3.88 g, 6.1 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

S

S**

S

SSS

0 NZ H

AI

Boc N 1. H 3 CS NHZ BocN HgCI/THF A 2. Lithium thiazole/THF

H

2 H.%.N ZN NHZ 1. tert-Butyloxycarbonyl-3-(cis/trans-2aminocyclohexyl)alanine-N,O-dimethylamide (2.0 g, 6.1 mmol) was dissolved in dry THF (20 mL) under nitrogen with stirring.

The solution was cooled to 0° C, N,N'-bis-(benzyloxycarbonyl)- S-methyl-isothiourea (2.18 g, 6.1 mmol), and HgC12 (1.81 g, 6.7 mmol) added. The solution was concentrated under reduced pressure, the remaining residue was suspended in ethyl acetate (300 mL), and filtered through celite. The filtrate was concentrated under reduced pressure. Flash chromatography over silica gel (hexane/ethyl acetate gradient) afforded the purified product.

2. To a solution of thiazole (2.32 g, 27.3 mmol) in anhydrous THF was added n-BuLi (1.6 M/hexane, 15.9 mL, 25.4 mmol) dropwise at -78° C and the solution stirred. The above guanidylated amino acid (3.88 g, 6.1 mmol) in THF (15 mL) was added dropwise, and the resulting mixture stirred. The reaction was quenched with saturated aqueous ammonium chloride. The mixture was diluted with ethyl acetate (150 mL), and the organic layer washed with saturated aqueous a. ammonium chloride (2 x 50 mL), brine (50 mL), dried with MgSO filtered, and concentrated under reduced pressure. The crude material was purified on silica gel (ethyl acetate/hexane), and concentrated under reduced pressure.

4 Example 2 Synthesis of Intermediates O 1. n-BuLi

AO

2.

SC1Ph 1 THF/-78C 2 (4S,5R)-3-(l-oxo-3-phenylpropyl)-4-(phenyl)-5-(methyl)-2oxazolidone A solution of 10.0 g (1.0 equiv., 56.4 mmol) of (4S, 5R)- 4 -phenyl-5-methyl-2-oxazolidone in 250 mL of dry THF, stirred at -78 0 C under argon, was treated dropwise with n-butyllithium (1.6 M in hexane, 1.1 eq., 38.8 mL). After stirring for 30 min., 8.4 mL equiv., 56.4 mmol) of hydrocinnamoyl chloride was introduced dropwise over a 10 minute period. The resulting mixture was warmed to 0°C, stirred for an additional hour, and quenched with saturated ammonium chloride. The solvent was removed in vacuo and the 15 resulting white solid dissolved in ethyl acetate and ddH 2 0. The aqueous phase was removed and extracted with two additional portions of ethyl acetate. The extracts were combined, washed with saturated sodium chloride, dried over sodium sulfate, and the solvent was removed in 20 vacuo to afford a white crystalline solid (2)(in 91% yield).

m.p.95-96.5; [a]D-35.8 CH 2 C12); [a]D-26.6 (c=1.018,

CH

2 C1 2 I1HNMR (CDC1 3 5 0.89(d, 3H, CH3I, J=6.6 Hz), 3.00-3.05 2H), 3.26-3.34 2H), 4.73-4.78 1H), 5.64, d, 1H, J=7.4 Hz), 7.22-7.46 0 0 1. LHMDS p Me Ph THF/-78 °C Me Ph 2 3 A solution of 5.0 g (1.00 equiv., 16.2 mmol) of in 100 mL of dry THF, was cooled to -78 0 C. Enolization was achieved with 17.8 mL (1.1 equiv., 17.8 mmol) of lithium bis-trimethylsilylamide, which was added dropwise via syringe. The solution was stirred for 30 min. before 4.45 mL (3.0 equiv., 48.5 mmol) of allyl iodide was introduced and the reaction warmed to -15 0 C. After 1 h the reaction was quenched with saturated ammonium chloride and extracted (3X) with ethyl acetate. The organic phase was washed with sodium metabisulfite, dried over sodium sulfate and the solvent removed in vacuo to afford an offcolour oil. Purification was achieved by flash chromatography on silica gel using a stepwise gradient (15:1, 12:1, 10:1) to yield a colourless oil Ao [a]D 47.5 (c=3.12, CH 2 Cl 2 9.

20 1HNMR (CDCl 3 8 0.82 3H, CH 3 2.31-2.40 (m, 1H, RCH=CHCH2), 2.49-2.57 1H, RCH=CHCH 2 2.84-3.00 2H, Ph-CH2), 4.32-4.37 1H, CH-(N)CO), 4.53-4.58 1H, CH 3 5.03-5.13 2H, ABX, CH=CH 2 5.21 (d, 1 H, Ph-CH, J=7.1 Hz), 5.81-5.89 1H, CH=CH 2 7.20-7.42 25 10H, ArH); 13C (CDCl 3 6 14.4, 36.2, 38.2, 43.9, 54.7, 78.4, 117.1, 125.4, 126.3, 128.2, 128.5, 129.0, 133.1, 134.8, 138.9, 152.4, 174.9.

o O 0 O I INI-'. 1. BH3/THF/cPC j 0 I 1 2. MCPBA/CHC3/RT

I

Me h Me Ph

OH

3 4 A sample of the allyl compound, (4.75 g, 13.6 mmol) in THF (100 mL) was treated with 13.6 mL (1.0 equiv., 13.6 mmol) of a 1.0 M solution of borane-tetrahydrofuran complex at 0 C and stirred for 2h. The solvent was evaporated and chloroform (100 mL) added via syringe.

Oxidation of the organoborane was achieved by the addition of 4.7 g (2.0 equiv., 27.2 mmol) of 3 -chloroperoxybenzoic acid at 0°C, with warming to ambient temperature and stirring for an additional hour. The organic phase was washed with 5% Na 2

CO

3 ddH20, and dried over sodium sulfate. Due to the instability of the alcohol, a quick column was performed to remove the extreme polar and nonpolar material which originated from the 3chloroperoxybenzoic acid. The alcohol was obtained in a yield of 0 e0 0 0 .S [aID 39.3 (c=1.038,

CH

2 C1 2 0 HNMR (CDC1 3 8 1.07 3H, CH 3 -CH, J=6.5 Hz), 1.81-1.93 3H, CH 2 2.10-2.19 1H, CH 2 3.10-3.17 2H, Ph-CH 2 3.873.90 2H, CH20H), 4.43-4.49 (m, 1H, CH-CO), 4.70-4.75 1H, CH 3 5.36 1H, Ph-CH, O" 25 J=7.1 Hz), 7.41-7.63 10H, Ar;H); o o *ooo 0 0 0 O AJL.O 1. PCC/mol. sieves N-O Me "Ph 2. CH 2 C1 2 /RT Me Ph OH 4 H O 4 l To a solution of the alcohol (1.0 g, 2.7 mmol), dissolved in dichloromethane (27 mL), 876 mg (1.5 equiv., 4.1 mmol) of pyridinium chlorochromate and 1.0 g of 4 angstrom molecular sieves were introduced and the mixture changed from bright orange to a black colour. The reaction was monitored by TLC and after 30 min., if starting material remained, additional molecular sieves were added. The solution was filtered through celite and the solvent was evaporated. The residue was dissolved in ethyl acetate and washed with saturated sodium chloride.

If the orange colour persisted in the organic phase, additional filterings through celite pads were performed.

The aldehyde was obtained in a quantitative yield as a transparent, colourless oil *f 1 HNMR (CDC1 3 8 0.84 3H, CH 3 -CH, J=6.6Hz), 1.87-1.94 1H, CH 2 -CH(H)-CHO), 2.04-2.13 1H, CH 2

-CH(H)-CHO),

20 2.45-2.50 2H, Ph-CH 2 2.79-2.85 (dd, 1H, CH 2

-CHO,

J=13.3 2.92-2.99 (dd, 1H, CH2-CHO, J=13.2 4.19-4.22 1H, CH-CO), 4.46-4.51 1H, CH 3 CH), 5.13-5.25 1H, Ph-CH), 7.20-7.39 10H, ArH), 9.69 1H, CHO); S• 25 1 13 C (CDC1 3 6 14.2, 23.8, 39.0, 41.2, 43.8, 54.9, 78.6, 125.3, 126.4, 128.2, 128.4, 128.5, 128.9, 132.8, 138.4, S152.4, 174.9, 201.1.

U 0 0 O U 1. PTSA/L-CYS Et-ester/ S0 mol. sieves N Me Ph PhH/RT Me Ph H O S NH

O

0 6 The aldehyde, (2.6 g, 7.10 mmol) was dissolved in benzene (70 mL) and a catalytic amount of ptoluenesulfonic acid was added, followed by 1.58 g (1.2 equiv., 8.52 mmol) of L-cysteine ethyl ester and 4 A molecular sieves. The reaction was allowed to stir overnight at ambient temperature followed be removal of solvent in vacuo. The residue was dissolved in chloroform, washed with saturated sodium chloride, ddH 2 0, and dried over sodium sulfate. The solvent was removed in vacuo to afford a gummy solid 0 CO 2 -Et N Me3AI DCM, N S" room temp. s Me Ph H

H

S NH

CO

2 Et 15 6 7 2.0M trimethylaluminum in hexane (2.4mL, 4.8mmol, 3 equivalents) was added slowly to starting material (6) (800mg, 1.61mmol) stirring in anhydrous dichloromethane under argon, using ovendried equipment. After stirring overnight, HPLC indicated that the reaction had gone to completion.

99 I O The mixture was quenched with excess methanol, then filtered on a short silica gel column (washing through with excess 10% methanol in ethylacetate. Evaporation gave 784 mg of crude material that was purified using 2:1 hexane:EtOAc on a silica gel column affording 258mg (0.81mmol being a 50% yield) of pure compound 6Sbenzylhexahydro-5-oxo-5H-thiazolo[3,2-a]pyridine-3R-ethyl ester as a white/yellow solid.

'H NMR (CDC13) d 1.28-1.31 3H), 1.72-1.81 3H), 2.10-2.13 1H), 2.66 (dd, 1H, J= 11.5 and 6.0 Hz), 3.29-3.34 2H), 4.19-4.29 2H), 4.88 (dd, 1H, J= and 5.0 Hz), 5.22 (dd, 1H, J= 8.0 and 6.0 Hz), 7.18-7.23 3H), 7.28-7.31 2H).

O

COEt GO\ H 2 0CO H LiOH/ THF/ H20

N

S

S

H H 7 8 S 20 LiOH.H20 (48mg, 1.12mmol) in 10mL of water was added to starting material (240mg, 0.76mmol) dissolved in 10mL of dioxane. After 1 hour, TLC in 1:1 Hexane:EtOAc showed no starting material. The reaction was quenched with 10% citric acid, then extracted twice with dichloromethane. Drying and 25 evaporating the combined organic layers gave 354mg of crude product. This was redissolved in dichloromethane, then precipitated by adding excess hexane. The product was filtered to give 200mg (0.68mmol being a 90% yield)) of an off-white solid, also known as 5H-thiazolo[3,2-a]pyridine-3R-carboxylic acid.

'H NMR (CD30D) d 1.71-1.82 3H), 2.12-2.17 1H), 2.67 (dd, 1H, J= 14 and 11 Hz), 2.77-2.81 1H), 3.30-3.40 0l0 3H), 4.81 (dd, 1H, J= 8.5 and 4.9 Hz), 5.16 1H, J= Hz), 7.18-7.31 O o

(COCI)

2 OH

C

9 Oxalyl chloride (25 g, 0.197 mol) was cooled to O°C and cyclohexane propionic acid (20 ml, 0.14 mol) was added. This was left to stir overnight. The resultant mixture was distilled to give an 84% yield of the colorless liquid cyclohexyl propionic acid chloride.

H O 1. n-BuLi/THF -78oC N HN O N 0 '0 Cr

CH

3 Ph 2. CH, Ph

CI

11 10 12 The chiral auxiliary (11) (13.6g, 76.7 mmol, 1 eq) was dissolved in dry THF and cooled to -78 0 C. Then n-BuLi (52.8 mL, 84.4 mmol, 1.2 equiv.) was added and left for mins (dark orange solution). The acid chloride (10)(13.4 20 g, 76.6 mmol, 1 eq) was then added and left to stir overnight. Work-up was done by quenching with saturated

NH

4 C1 extracting with ethyl acetate, washing the extracts

S

with water and brine, drying over sodium sulphate and concentration. A fast column, with dry loading, (6:1 S" 25 hexane ethyl acetate) was run to purify the product. This afforded in a white solid (12) which was recrystallized 101 i from ether and hexane to give the title compound in 78% yield.

[a]D=-20.1(c=l,EtOH); MP/BP mp=90.5-91.5oC 1H NMR (CDC13) d 0.86-1.10 5H), 1.18-1.30 4H), 1.54-1.75 7H), 2.86-2.97 2H0, 4.70-4.76 1H), 5.65 1H, J= 7.2 Hz), 7.28-7.42 O O O O 0[ 0 1. LIHMDS THF -78 0

C

N 0 N 0 C 2. N- r CH Ph

CH

3 Ph CH3 Ph 12 13 The starting material (12)(9.13g, 29 mmol, leq) was dissolved in dry THF and cooled to -78 0 C, after which LiHMDS (31.9 mL, 31.9 mmol, 1.1 eq) was added dropwise over 40 mins. Then, 30 minutes later, allyl bromide mL, 86.9 mmol, 3 eq) was added slowly over 10 mins. The mixture was left to warm overnight. Work-up included quenching with sat. ammonium chloride, extraction with ethyl acetate, washing with 10% sodium thiosulphate, decolourising with charcoal, drying over sodium sulphate and concentration in vacuo. The product was obtained as a yellow oil (13) in 96% yield.

25 [(]D-+9.5(c-1.0,EtOH) H NMR (CDC13) d 0.92-1.10 5H), 1.10-1.39 1.63-1.75 6H), 2.27-2.42 2H), 4.01-4.14 1H), 4.76-4.85 1H), 5.00-5.07 2HO, 5.65 1H, J= 7 Hz), 5.64-5.88 1HO, 7.27-7.46 102 (03 1. BH(CH(CH 3

)CH(CH

3 2 2 N 0 N 0 2. PCC/ DCM reflux(Ih) CH, Ph CH, Ph 0 13 14 2-methyl-2-butene was added dropwise to borane dimethylsulphide complex at -12°C. The reaction was maintained at this temperature for 15 minutes and then it was warmed to 0 C, after which it was stirred for 2 hours.

The disiamyl borane was then added to a mixture of the starting material 13 in THF using a double-ended needle at 0 C. The mixture was then stirred for 2 hours after which the solvents were removed and the residue dissolved in dichloromethane. It was carefully added to a suspension of pyridinium chlorochromate in dichloromethane contained in a flask equipped with a reflux condenser. After the initial exothermic reaction had subsided, the mixture was refluxed at 50°C for 1 hour. The dark brown liquid was dissolved in ethyl acetate and filtered through Florisil.

~The black residue of PCC was extracted with ethyl acetate and also filtered through the same Florisil pad.

20 Concentration of the filtrates resulted in a 78% yield of a yellow gummy product (14).

*e [a]D=-17.8(c=1.245,EtOH) 25 'H NMR (CDC13) d 0.89-1.18 5H), 1.20-1.47 8H), S 1.60-1.74 6H), 1.83-2.00 1H), 2.48-2.53 2H), 3.90-4.10 1HO, 4.12-4.16 1H), 4.76-4.80 1HO, 5.67 1H, J= 7Hz), 7.27-7.46 5H), 9.77 1H).

iL Toluiene p-TSAI mol. sieves HS NH 2

-COPE

N 0 CH 3

P

OH

3 .Ph The resulting aldehyde (14) (7.7g crude, 20.8 mnol 1 equiv.) was dissolved in 75 mL of toluene. To the solution was added a catalytic amount of ptoluenesulphonic acid (50 mg), 10 g of 4A molecular sieves, and L-cysteine ethyl ester (3.87 g, 20.8 mmol, 1 equiv.) The mixture was stirred overnight, filtered and concentrated. The residue was then purified by silica gel chromatography (6:1 hexane:ethyl acetate) afforded 6.36 g of the product (15) in 61% yield.

[(X1D=-48.3 (c=1.095,EtOH) 'H NMR (CDCl3) d 0.84-0.98 (in, 4H), 1.11-1.38 (mn, 7H), 1.50-1.90 (in, 10H), 2.80-2.99 (mn, 1H), 3.24-3.34 (in, 1H), 3.77-4.29 (mn, 4H1), 4.46-4.81 (mn, 2H), 5.66 lH, J= 7Hz), 7.27-7.46 (mn, 9 9 9. 9 9* *9 9* *9 9 9 9 9 9 9 9* .9 9* *9 a..

0

OH

3

P

Me 3

AII/DCM

000 RT -rS 0

COPE

C0 2 0t 104 l0 The starting material (15) (1.97 g, 3.9 mmol, 1 equiv.) was dissolved in 20 mL of dry dichloromethane and cooled to 0°C. Trimethylaluminum (5.9 mL, 11.8 mmol, 3 equiv.), was added dropwise and the mixture was left stirring overnight. After complete reaction as evidenced by HPLC, methanol was added until a yellow solid mass was formed.

Dichloromethane was added to dissolve the solid and the whole mixture was stirred for 15-30 minutes and then filtered. The residue after concentration in vacuo was run through a quick column (6:1 hexane:ethyl acetate), to remove auxilary and as many of the polar decomposition products as possible, affording in a 50% yield of a yellow oil (16).

'H NMR (CDC13) d 0.83-0.98 2H), 1.09-1.38 1.57-2.00 11H), 2.12-2.18 1H), 2.49-2.54 m, 1H), 3.10 (dd, 1H, J= 11 and 6 Hz), 3.27 (dd, 1H, J= 11.5 and Hz), 4.11-4.25 2H), 4.88 (dd, 1H, J= 11.0 and Hz), 5.14 (dd, 1H, J= 10 and 6 Hz).

S LIOH H 2 0-Diox S N 10C RT N 0 COEt 0 CO,H 2 2 S16 17 The starting material (16) (0.95 g, 2.9 mmol, 1 equiv.) was dissolved in 10 mL of dioxane. The solution was cooled to 10 C, and to it was added LiOHH20(0.123 g, 2.9 mmol, 1 eq.) dissolved in 10 mL of water. The bath was removed and the mixture was stirred at room temperature 30 for 1 hour. TLC showed complete reaction and the solvent was evaporated under vacuum. The remaining aqueous layer was washed with ether acidified with 10% citric acid, and extracted with dichloromethane The I 0 combined extracts were dried over sodium sulphate and concentrated to give a white solid which was recrystallized from ether. Concentration of the filtrate and purification by silica gel column chromatography (2:1 hexane: ethyl acetate) resulted in more product (17) with a m.p. of 198.2-199°C.

'H NMR (DMSO-d6) d 0.78-0,93 2H), 1.11-1.27 1.34-1.36 1H), 1.51-1.56 1H), 1.60-1.75 1H), 1.82-1.87 1H), 2.15-2.18 1H), 2.37-2.41 1H), 3.03 (dd, 1H, J= 11.5 and 5.5 Hz), 3.35-3.38 2H), 4.83 (dd, 1H, J= 9 and 4 Hz), 4.95 (dd, 1H, J= 8 and 5.5 Hz).

0o 0OH CHN, ra H 3

N

Cbz Cbz N N Cbz

N

HN NH HN '-NH N I I HIN NH Cbz Cbz Cbz 18 19

S

BOC-DiCbz Arg (18) (7.6 g, 14.0 mmol) was dissolved in anhydrous THF (40 mL) and cooled to 0 C. Triethylamine (2.2 mL) was added followed by 14.5 mmol of a 1M toluene solution of isopropyl chloroformate via a syringe. The reaction was allowed to stir at 0 C for 30 minutes then quickly filtered. The white solid was discarded. To the e* filtrate was bubbled freshly prepared diazomethane until S the color of the solution turned yellow. The reaction 25 mixture was allowed to stand overnight in a well ventilated fumehood which facilitated the discharge of excess diazomethane. Dry ether was added to precipitate the diazoketone. The product was filtered and dried under vacuum to give light yellow fluffy solid (4.6 g, 58%).

o7 Diazoketone (19) (ig, 1.77 mmol) was dissolved in THF mL) and to this solution was added 1M HC1 in ether (20 mL) at 0°C. The reaction was allowed to stir at ambient temperature overnight during which time a white precipitate was formed. Further precipitation was achieved by adding ether. Filtration and drying the solid afforded the product (20) (1.02 g 100%).

'H NMR (DMSO-d6) d 1.65-1.77 3H), 2.06-2.50 1H), 3.86-3.90 2H), 4.29 1H), 4.76 1H, J= 18Hz), 4.95 1H, J= 18 Hz), 7.35 2H), 7.36 2H), 7.35- 7.41 10H), 8.71 (br s, 3H), 10.1 (br s, 2H).

"C NMR (DMSO-d6) d 23.7, 26.4, 47.2, 47.9, 56.2, 68.0, 69.3, 128.6, 128.7, 128.8, 128.9, 135.2, 135.9, 153.4, 157.4, 198.9.

0 o N 0 N HO b O NHCbz NCbz HN ci 0 S17 20 21 To mimetic (17) (0.422 g, 1.42 mmol) in THF (50 mL) at 0 C and in presence of N-methyl morpholine (0.19 mL), was slowly added 1M toluene solution of isopropylchloroformate (1.71 mL) The reaction was allowed to stir at 0 C for 25 minutes then treated with aminochloromethylketone (20) in small portions. Once the addition was complete the reaction was further stirred for 15 minutes followed by addition of N-methyl morpholine (0.19 mL). The reaction was stirred at ambient temperature for 3 hours, then extracted with ethyl acetate followed by washing with brine and 10% aqueous citric acid. Removal of organic solvent gave a white foam (21) (1.03 g, 96%) which was used further without purification.

1 H NM~R (CDC13) d 0.07-0.97 (in, 1H), 1.15-1.41 (in, 7H), 1.62-1.91 (mn, 10H), 2.10-2.16 (in, 1H), 2.43-2.48 (in, 1H), 2.74-2.80 (in, 1H), 3.01-3.07 (mn, 1H), 3.87-3.94 (in, 1H), 4.11-4.19 (in, 2H), 4.60-4.66 (in, 1H), 4.74-4.86 (mn, 2H), 5.09-5.24 (mn, 4H1), 7.30-7.39 (mn, 10H), 7.95 1H, J= 8 Hz), 9.4 (br s, 1H), 9.56 (br s, 1H).

0: *108 EXAMPLE 3 0 8 NH T HN NHTos 1. TFA/DCM 2. DMF/ BOP O CO2H Ph 0

N

0

HN~

1

NH,

(N-t-BOC-N-tosyl)butyrylketoarginine (240mg, 0.515 mmol), was deprotected using 30% TFA in dichloromethane. The deprotected arginine derivative, was coupled with the mimetic (100 mg, 0.343 mmol) in DMF under basic conditions (Et 3 N, pH using BOP reagent (228 mg, 0.52 mmol) as the dehydrating agent. The reaction was typically complete within 2-4 hours. Extraction with ethyl acetate followed by successive washing with brine and aqueous citric acid yielded the crude product. The crude product was purified by column chromatography affording 180 mg of pure product. This product was then treated with HF to remove the tosyl group. Purification of the isolated deprotected product by HPLC afforded BCH-2737.

EXAMPLE 4 p p p p p p O* pp..

pfp p p p f p p ***ft f ft ft *ft ft *ft f tf HS-CH2-CO2H O NHCbz 0 ,NCb NMM/THF/RT 2. BBR3/DCM

CI

IO

Chloromethylketone (21) (0.188 g, 0.245 mmol) was dissolved in THF (10 mL) treated with NMM (0.036 mL) followed by mercapto acetic acid (0.02 mL, 0.299 mmol).

The reaction was stirred at ambient temperature overnight.

Extraction of the reaction mixture with ethyl acetate followed by successive washing with brine and 10 aqueous citric acid and evaporation of organic solvent gave the crude product which was purified by column chromatography to give foamy solid as the product (0.125 g, 62%).

This protected precursor (0.125 g, 0.154 mmol) was dissolved in DCM (5 mL) and cooled to -78 0 C. A 1M DCM solution of BBr, (1.54 mL, 1.54 mmol) was slowly added. The reaction was stirred at ambient temperature for 5 hours, then cooled to -78°C again and treated with anhydrous methanol (2mL). The reaction was brought to room temperature and stirred for 2 additional hours. The solvents were removed under reduced pressure and the residue was partitioned between ether and water. The water layer was collected, lyophilized and the final product (23) Sobtained as a powder after HPLC purification and lyophilization.

S 25 The products of the reactions described above can be isolated in the free form or in the form of salts. In addition, the products can.be obtained as pharmaceutically acceptable acid addition salts by reacting one of the free bases with an acid. In a similar manner, the product can 30 be obtained as pharmaceutically acceptable salts by reacting one of the free carboxylic acids with a base.

S* Likewise, treatment of the salts with a base or acid results in a regeneration of the free amide.

9 11 1 EXAMPLE A general method of synthesizing compound of formula II or Hs~~COd~e NH2 CH3CN No NaCNBH3 RT 16 hrs

KHMDS/THF

Tosyl N3 OH

I

NN

CONC"COA

NH (Mj.ce r r r e r r c 1. same procedure as for 2Q CO-NH CC2C O2M 2. deprotection as NHin eCxample 3 CBz NH Nl12CBZ )-NH COCH2CH2CO2Me

NH

I I 2 EXAMPLE 6 Synthesis of STEP 1 Synthesis of 2-Benzyloxycarbonylamino-4-hydroxybutyric acid tert-butyl ester

NHZ

(Cyclohexyl) 2 NH.HOOC XCOtB CO~t-Bu 0 1) oa NMM, THF 2) NaBH 4 MeOH

NHZ

HO

CO

2 t-Bu 0S 0

S

0

S

*0 To a solution of the protected aspartic acid (1)(Bachem, 2.50 g, 4.95 mmols) in 50 mL of dry tetrahydrofuran (THF), at -10 0 C, under N 2 was added N-methylmorpholine (109 pL, 0.2 eq) and isopropyl chloroformate M/toluene 384 pL, 1.1 eq). The soluion was stirred at 0 C for 60 min. In another flask, NaBH 4 (375 mg, 2 eq) was suspended in a dry 5:1 mixture of THF/MeOH (50 mL), 20 at -78 0 C, under N 2 This suspension was stirred at -78 0

C

for 30 min. The mixted anhydride solution was then added to the NaBH 4 suspension dropwise via canula, and the final solution was stirred at -78 0 C for 3 hr. Acetic acid (2.8 mL, 10 eq) was then added and the solution was S1: warmed to r.t. (30 min). The solvents were evaporated, the residue taken up in EtOAc and washed with sat.aq.

NaHCO 3 (2x) and brine. The organic layer was dried over MgSO 4 the solids were filtered and the solvent evaporated to give 1.53 g (4.95 mmols, 100%) of the alcohol as a clear oil.

1 H NMR (CDC1 3 400 MHz) 5 7.40-7.31 5H, ArH), 5.63 1H, J=7.3, NH), 5.13 (AB system, 2H, J=12.2, CH 2 Ph), 4.43 1H, 3.69 2H, 2.17 1H, H-3), 1.63 1H, 1.48 9H, t-Bu).

STEP 2 2-Benzyloxycarbonylamino-4-iodobutyric acid tert-butyl ester NHZ PhNP 2

NHZ

HO CO 2 t-Bu Imidazo1, -10 0 C I CO 2 t-Bu

CH

3 CN/EtO (3) 20 To a solution of the alcohol (1.53 g, 4.95 mmols) in a 1:1 mixture of CH 3 CN/Et 2 O (50 mL), at -10 0 C, under N 2 were added successively imidazole (607 mg, 1.8 eq) and Ph 3 P (2.21 g, 1.7 eq). Iodine (2.14 g, 1.7 eq) was then added in small portions over a period of 15 min. After the addition was completed, a white precipitate formed and the solution was brown. It was stirred at -10 0 C for min. It was then poured in Et20 and the organic phase was washed with sat.aq. Na 2 S0 3 sat.aq. CuSO 4

H

2 0 and dried over MgSO 4 The solids were filtered and the 30 solvent evaporated to give a yellow oil that was purified by flash chromatography (silica gel, 5% to 20% EtOAc/ Hex). The iodide was obtained in 83% yield (1.71 g) as a clear oil.

1 H NMR (CDC13, 400 MHz) 6 7.41-7.31 5H, ArH), 5.35 (bd, 1H, J=7.3, NH), 5.13 2H, CH 2 Ph), 4.30 1H, H- 3.22-3.12 2H, 2.42 1H, 2.20 (m, 1H, 1.48 9H, t-Bu).

STEP 3 Synthesis of 2-Benzyloxycarbonylamino-4-hexenoic acid tert-butyl ester NHZ -MgBr, Cul NHZ I CO 2 t-Bu THF, -78 0 C CO 2 t-But (4) To a suspension of Cul (2.27 g, 5 eq) in dry THF (20 mL), at -78 0 C, under N 2 was added slowly a 1.OM solution in THF of vinyl magnesium bromide (23.4 mL, 9.8 eq). The solution was then warmed up to -100C for 30 min (it turned then black) and cooled back to -78°C. A solution of the iodide (1.00g, 2.39 mmols) in dry THF (3.5 mL) was then added slowly to the cuprate solution. The reaction mixture was stirred at -78°C for 2.5 hr.

Sat.aq. NH 4 Cl (50 mL) was added and the mixture was brought back to room temp. with vigorous stirring. It was then poured in Et20 and stirred for 5 min. The dark suspension was filtered through a cintered funnel and the phases were separated. The aqueous phase was extracted with Et 2 O (2x) and the combined organic extracts were 30 dried over MgSO 4 The solids were filtered, the solvents evaporated and the crude oil purified by flash chromatography (silica gel, 5% AcOEt/Hex) to give 0.51 g of the pure alkene 1 H NMR (CDC1 3 400 MHz) 5 7.37-7.31 5H, ArH), 5.80 1H, 5.33 1H, J=7.8, NH), 5.12 2H,

CH

2 Ph), 5.05 1H, J=17.2, 5.01 1H, J=10.4, 4.30 1H, J=7.4, 2.16-2.08 2H, H-4), 1.92 1H, 1.74 1H, 1.48 9H, t-Bu).

STEP 4 Synthesis of l-Benzyloxycarbonyl-5-hydroxymethyl- 2 pyrrolidinecarboxylic acid tert-butyl ester

HO

S1) Hg(OAc) 2

THF

I 2) NaHCO 3 KBr 2COt-Bu 3) NaBH 4 0 2

N

Ot-Bu 0 To a solution of the alkene (4)(50 mg, 0.157 mmol) in dry THF (3.1 mL), at under N 2 was added mercuric acetate (75 mg, 1.5 eq). The solution was stirred at r.t. for 18 hr after which it was cooled down to 0 0

C.

Sat.aq. NaHCO 3 (2 mL) was then added and the mixture was 20 stirred at 0°C for 30 min. KBr (0.11g, 6 eq) was added S. and the mixture was stirred at r.t. for 2 hr. It was then poured in H 2 0/Et 2 O and the phases were separated.

The aqueous phase was extracted with Et20 (2x) and the combined organic extracts were dried over MgSO 4 The 25 solids were filtered and the solvents evaporated. Oxygen (02) was bubbled into a suspension of NaBH 4 (3.3 mg, 0.55 eq) in dry DMF (0.4 mL) for 1 hr, and to this was added dropwise (syringe pump, 3 mL/hr) a solution of the 'organomercurial bromide in DMF (3.1 mL) with continuous introduction of 02. The bubbling was continued for 1 hr and Et20 (5 mL) was added. The grey suspension was 115 filtered through Celite and the filtrate was evaporated.

The residue was chromatographed (silica gel, 6:4 Hex/EtOAc) to give the pyrrolidinol (30 mg, 57%) as a clear oil.

1 H NMR (CDC13, 400 MHz) 8 7.37-7.28 5H, ArH), 5.22- 5.09 2H, CH 2 Ph), 4.30 (dd, 1H, J=1.4, 8.3, 4.24 1H, 3.70-3.57 3H, CH 2 2.25 1H), 2.13 1H), 1.92 1H), 1.70 1H), 1.34 9H, t-Bu).

STEP Synthesis of l-Benzyloxycarbonyl-5-carboxy-2pyrrolidinecarboxylic acid tert-butyl ester OH 0 S0 3 -Pyr, EtN H ZN DMSO/CHC 0 0 C ZN Ot-Bu Ot-Bu .4 4 4 *r 4 *e4 4 .4 4* 4 4 4.4 To a solution of the alcohol (50 mg, 0.149 mmol) and Et 3 N (62pL, 3 eq) in dry CHCl 2 (0.8 mL) is added slowly, under N 2 at 0°C, a solution of S0 3 -Pyridine complex (71 20 mg, 3 eq) in dry DMSO. The solution was stirred at 0 C for 30 min and 10% citric acid (2 mL) is added. The pH is brought to 4 with 1M NaOH and the aqueous phase is extracted with Et2O The combined organic extracts were dried over MgSO 4 The solids were filtered and the 25 solvents evaporated to give a crude oil which was purified by flash chromatography (silica gel, 7:3 Hex/EtOAc). The pure aldehyde was obtained as a clear oil (45 mg, 116 11 7 1H NNR (CDC1 3 1 400 M4Hz) 5 9.68 9. 56 (ds, 1H, CHO), 7.36-7.29 5E, ArH), 5.23-5.11 2K, CH 2 Ph), 4.57- 4.39 (in, 2H, H-2, 2.30-1.97 Cm, 4H, H-3, 1.47 1.36 (2s, 9H, t-Bu).

STEP 6

NH

2

BOCHN

CO Me 2 MgSO 4

CH

2 C1 2 OBut 2) NaBH(OAC) 3 AcOH, THF

HN\

Z -N- CO Me 2

BOC

OBu t The pyrrolidine-aldehyde is coupled with the protected diamino-propionic acid by first forming the imine (8) (mgSO 4 1 CH 2 Cl 2 Isolation of the imine is done by filtration of the MgSO 4 and evaporation of the solvent.

15 The crude imine is then treated with NaBH (OAc), and actic acid (AcOH) in THF for 15 hours to obtain the amine (8) after extrative work-up.

STEP 7 HN Z -N-

CO

2

M

ZNH

BOC

1) H 2 ,Pd/C /MeOH OBut HN H -N- CO 2 Me

INH

BOC

117 11 The CB7 protecting group of the amine is removed by hydrogenation with palladium on charcoal 10% as a catalyst in methanol (MeOH). The catalyst is filtered and the MeOH evaporated to give the crude diamine that can be used without any purification.

STEP 8

H

HN Heat HN H-N> H N

N

CO Me O OBut

N

2 OBu ,NH 0 0 BOC N H

BOC

The cyclisation is done by heating the crude oil from step 7, neat slightly above the boiling point of methanol.

The bicyclic lactam (10) is purified by flash chromatography.

*1 11 9• e a 9 9* 9 .9 ao a.° 118 STEP 9

H

HN

N aBut 0 cod1 Pyridine -OBut IeNH

BOC

(11) The secondary amine of the bicyclic lactam (10) is protected as an amide using benzoyl chloride in pyridine.

Evaporation of the pyridine and extractive work-up give.

the bicyclic lactam-amide (11).

STEP HC1/Et 2

O

0 H N

OH

0 NH 0 NH3 OBut 0 a a.

a. a a .0* a a a a a a a a a a.

0* a a.

a.

a a..

.NH

BOC

(12) The BOC and t-butyl ester protecting groups of bicyclic lactam amide (11) are removed under acidic conditions (Ed1 in ethyl ether (Et 2 The amine salt (12) precipitates out of solution and is collected by filtration.

119 110~ STEP 11

C

6

H

5

CH

2 OCOC1

COOH

K

2 C0 31

CH

3

CN

0

H

N

N

OH

0 ZHN0

C)NH

3 (12) (13) The primary amine of compound (12) is protected with a CBZ group by reacting it with benzyl chioroformate in acetonitrile (CH 3 CN) with K 2 C0 3 as a base. Extrative workup gives fully protected carboxylic acid (13) which can be use for step 12 without further purification..

STEP 12 S. S S

S.

S 5555

S

0 H

H-

NN

HN N 4Z OH 0 ZHN 0BOP, EINiPr 2 DtF (13) Th a b xl c a i 1 )i.c u l d w t e z t i z l Eteacarboni cid (13)l ictt E)gvs cupledd as a solid which is purified by chromatography.

120 STEP 13

ZHN

S H 2 Pd/C 10% MeOH

HN

HN NH 2 (16) The two CBZ(Z) protecting groups of compound (15) are removed by catalytic hydrogenation with Pd/C 10% as a catalyst. The catalyst is filtered and the solvent is evaporated to give the amino-guanidine (16).

a p S a a a S a p* p a

S.

a. a p a a.

a a..

EXAMPLE 7 SYNTHESIS OF COMPOUND STEP 1

OH

S

N

HN

z 0 0 0 1) BGPA'F NMMv a a a a a a a a a a a a.

a..

H )OH (4) z 0 (2) 4-methylmorpholine (NI4M) was added to a solution of the carboxylic acid (1.7g, 4.9mmol, 1.Oeq), 4hydroxyproline (5.39mmol, 1.leq), and BOP reagent 15 (2.17g, 4.9mmol, 1.Oeq) in anhydrous DMF (l0mL) at room temperature. The reaction mixture was stirred at room tenperature over night, quenched with brine (5OmL) and ethyl acetate (lO0mL). The organic layer washed with 122 aqueous citric acid 2x50mL), sodium bicarbonate 2x50mL) and brine (50mL). The resulting organic layer was dried over anhydrous magnesium sulfate, filtered and the solvent evaporated. The crude residue was purified by flash chromatography ethyl acetate-hexanemethanol). l.lg of pure product was recovered 48% yield.

STEP 2

OH

S S S1) triethylarrine N N2a N HN HN o o To a solution of 4-hydroxyproline derivative (4 (115mg, 240umol, 1.Oeq) in dichloromethane (10 mL, anhydrous) at 0°C is added triethylamine (72mg, 720umol, 3.0eq) and methanesulfonyl chloride (28mg, 240umol, l.Oeq) and the reaction mixture is stirred at room temperature. The mixture is then quenched with an aqueous solution of 20 ammonium chloride and extracted with ethyl acetate. The organic layer is washed with 10% citric acid and brine, dried, filtered and the solvent is evaporated to dryness yielding compound 25 STEP 3 S

S

SN 1) H(QOAc) 2

N

NHN CF300CH/OoC HN HNz Z 0 C 0 2)Nc H 4 0 (6) The enamine (l.Oeq) is treated with mercuric acetate (1.1 eq) in THF. The solvent is evaporated to dryness and the residue dissolved in methanol. The resulting organomercurial is reductivly cleaved with sodium borohydride (1.3eq). The resulting crude lactam thioether is purified by flash chromatography on silica gel affording compound STEP 4

S

*C S

N

HN N (6)

Z

A (7) O To a solution of the lactam thioether (l.Oeq) in dry dichloromethane N-chlorosuccinimide (l.Oeq) is added at 0 0 C. The reaction mixture is warmed to room temperature.

When the reaction shows no more starting material the solid is filtered and the solvent evaporated to dryness.

The crude material is use without any further purification for step STEP C1 S S N P OJLi HN N Z 0 0 THF Z 0 0 0 (8) To a solution of the alpha-chlorothioether (l.Oeq) in THF (anhydrous) a solution of phenylcuprate (l.Oeq) (prepared according to litterature procedure) is added at low temperature. When the reaction mixture shows no starting chlorothioether, brine and ethyl acetate is added. The organic layer is dried, filtered and evaporated to dryness to afford the desired product STEP 6

S

SU LOH

S

N-I

HN

N

HN

O H 2 0/ HF Z OH

O

(8) (9) 15 The isolated bicyclic lactam is hydrolysed with one ;equivalent of lithium hydroxyde in a 1:1 mixture of THF and water. The mixture is stirred at room temperature for 1 hour. The crude mixture is extracted with ether and the resulting solution is poured into 10% citric acic aqueous solution and extracted with dichloromethane to yield the corresponding carboxylic acid 125 STEP 6 1

S

N

HN

Z 0 OH H 3 N S0

H

3 Ns

NH

J=NH

NHCBZ

H

2

I

2) BBr 3

CH

2 C1 2 The crude carboxylic acid is coupled with benzythiazole keto arginine in DMF using BOP as the coupling reagent in the presence of diisopropylethylamine.

Extraction with EtOAc gives a solid that is purified on silica gel to give the protected amide. The CBZ protecting group is removed with BBr, in dichloromethane at room temperature finally gives the bicyclic benzothiazole keto arginine inhibitors 15 The following compounds are produced accordingly with the execption that the appropriate substitution of products were made in order to obtain the final compounds.

COMPOUND #11 es

S

S

S.

5

S

S

0 0S55

S

S..

0O

S

S I z 7 (11)

NH

J=NH

H 2N COMPOUND #12

S

S

S S

S.

S

5** (12) EXAMPLE 8 STEP 1 1. a-12=N=N,THF/Et~er,-15 0

C

2. Hai~ber

CH

3 Commercially available glutaric acid monomethyl ester chloride (20 ml,0.144 mol) was disolved in 40 ml of dry tetrahydrofuran (THF) and cooled to -150C. Excess diazomethane freshly prepared in 300 ml of Ether was introduced via cannula at -150C to the solution. The mixture was left to warm up to room temperature overnight.

Excess diazomethane was evacuated from the flask with a current of argon. To bring the reaction to completion, ml of 1 N HC1 in Ether was added at 0°C and left to warm up to room temperature for 5 hours. The volume of the solvent was reduced and then washed with 2x 5% NaHCO 3 dried over Na 2

CO

3 and evaporated to give crude chloromethylketone (20.46 g 79%) wich was used in the next step without further purification.

1H NMR (CDCL 3 400 MHz) d 1.16-1.2 1.83-1.9 2.27-2.35 2.6-2.64(t,1H), 3.6 4.04 (s,2H).

STEP 2 OO CCHCH 3C HHa. H-COs-OET CH 3 COONN NaCNBH 3

HN

MeOH, R.T S

OCH

3 2 0 OCH 3 o 3 9.* 9 Crude chloromethylketone (2)(10.04 g, 56.15 mmol) was 25 disolved in 300 ml of dry MeOH. Sodium acetate (2 eq, 9.21 g, 112.3 mmol) was added followed by L-Cysteine ethyl ester hydrochloride salt (1.3 eq, 13.55g, 72.98 mmol) and sodium cyanoborohydride (1.4 eq, 4.9 g, 78.59 128 1 I mmol). The heterogeneous mixture was left to stir at room temperature for 2h30 min. 200 ml of methanol (MeOH) was then added to disolve all the solid and the pH was brought to 2 with 1N HC1 .The mixture was then basified with saturated NaHCO 3 until pH= 8. MeOH was evaporated and the remaining aqueous solvent was washed with ethyl acetate and dichloromethane. Solvents were combined, dried over Na 2

SO

4 and evaporated. The crude residue was purified by silica gel flash column chromatography using a gradient.of eluents ethyl acetate hexane in the following ratios: 5:5, 6:4, 7:3) giving cyclic compound 1 H NMR (CDCl 3 400MHz) of compound d 1.21-1.27 (t, 3H, J=7.06 Hz), 1.41-1.48 2H), 1.65-1.73 2H), 2.28-2.39 4H), 2.57-2.63 1H, J=10.9), 2.72- 2.76 (dd, 1H, J=10.7 Hz), 2.8-2.86 1H), 3.6-3.64 4H, .J=2.55 Hz), 3.63 3H), 4.13-4.2 2H) 13 C NMR (CDC13, 400 MHz) 13.078, 19.888, 28.326, 31.133, 32.741, 35.277, 50.462, 56.394, 59.149, 60.188, 69.713, 170.182, 172.52 9e O0 OCH 2

CH

3

H

HN Toluene/H

S

N

R eflux O OCH2CH, 3

A

129 i Cyclic compound (913mg, 3.32 mmol) was disolved in ml of dry Toluene. (lS)-(+)-10-Camphorsulfonic acid 92 mg, 0.39 mmol) was added and the mixture was left to reflux for 4 days. When all starting material was shown to be consumed (by TLC), the mixture was worked up by evaporation of solvent, dissolving residue in ethyl acetate and washing with 2 x 5 NaHC03. The Ethyl acetate layer was dried over Na 2 S0 4 and evaporated. The crude residue was purified by silica gel flash column chromatography using 60 ETOAC 40 %Hexane followed by ETOAC 30 Hexane giving 62.5% of Bicyclic compound 1H NMR (CDC1 3 400MHz) of compound d 1.27-1.31 (t, 3H, J=7 Hz), 1.5-1.6 IH), 1.72-1.87 2H), 2.02- 2.1 1H), 2.33-2.46 2H), 2.52-2.59 2H), 2.83-2.88 (dd, 1H,J=14 4 Hz), 3.14-3.18 1H), 3.78-3.85 1H), 4.2-4.27 2H, J= 3.9 Hz), 5.9- 5.92(t, 1H, J=3.4 Hz).

STEP H

H

N LiOH-H 2 0, T HF/H 2 0 O N OC -7R.T 0: OCH 2

CH

3 O OH Bicyclic (366mg,1.5 mmol) was dissolved in 25 ml of THF and 5 ml H 2 0 Lihium hydroxide. monohydrate (1.1 eq, 25 7.05 mg, 1.68 mmol) was added in 2.3 ml of H 2 0, at 0 C and the mixture was left to stirr at 0°C for 1 hr and at room temperature for 3 hrs. THF was then evaporated and the remaining aqueous mixture was acidified by addition of Citric acid until pH=2. Extraction of aqueous mixture 130

I

with 2 x CH 2

CL

2 and 2 x ETOAC, drying of combined organic layers with Na 2

SO

4 and evaporation gave a crude residue wich was purified by silica gel flash column chromatography using 70 ETOAC 30 %Hexane followed by 4.7 HOAC Ethyl acetate giving the pure acid in 54 yield 16 of starting material(4) was recovered.

1 H NMR (MeOD, 400 MHz) of compound d 1.57-1.69 (m, 1H), 1.70-1.80 1H), 1.81-1.89 1H), 2.05-2.12 1H), 2.35-2.5 2H), 2.51-2.66 2H), 2.86- 2.91 (dd, 1H, J=13.8, 4 Hz), 3.12-3.17 1H), 3.3- 3.32 1H), 3.78-3.84 1H), 5.76-5.78(t, 1H, J=3.53 Hz).

13C NMR (MeOD, 400 MHz) d 17.052, 27.07, 28.928, 31.382, 32.096, 51.016, 55.138, 170,088, 171.24 STEP 6

H

S

S 1)LiHMDS,-78 0

C

:N

the resulting solution 2 B is str,- 78 C irred RT 8C for 1 hour.

SBenzyl bromide (0.26 m, 2.22 mol) is then added and the mixture is allowed to reach room temperature and stirred To a solution of lithium bis (trimethylsilyl) amide (5ml of 1M THF solution, 5mmol) in THF (10 ml) is added at -78'C a solution of the carboxylic acid (500mg, 2.32 mmol).

\the resulting solution is stirred at -78°C for 1 hour.

Benzyl bromide (0.26 ml, 2.22 mmol) is then added and the mixture is allowed to reach room temperature and stirred for 15 hours. The mixture is then poured into 10% HCl ml) and extracted wiht diclhoromethane (4x 60ml). The combined organic phases are dried over MgSO, and the 131 solvent remove by evaporation to yield to the crude alkylated amide STEP 7 o 1) H N N H S S

H

H St

NH

N )NH S NHCBZ N

O

o OH O 0 S2) BBr 3

CH

2 C1 2

NH

=NH

7 H N The crude aklylated amide is coupled with benzythiazole keto arginine in DMF using BOP as the coupling reagent in the presence of diisopropylethylamine.

Extraction with EtOAc gives a solid that is purified on silica gel to give the protected amide. The CBZ protecting group is removed with BBr, in dichloromethane at room temperature finally gives the bicyclic benzothiazole keto arginine inhibitors The following compound is produced accordingly with the exeption that the appropriate substitution of products were made in order to obtain the final compounds.

COMPOUND #8 132 a a 132 0

~NN

NNH

)=NH

HN

2 EXAMPLE 9 Determination of K, Values for Heterocyclics The affinity of inhibitors for thrombin was measured according to the procedures described in (DiMaio et al, J.

Bio. Chem., 1990, 265:21698) Inhibition of amidolytic activity of human thrombin was measured fluorometrically using Tos-Gly-Pro-Arg-AMC as a fluorogenic substrate in mM Tris-HC1 buffer (pH 7.52 at 37 0 C) containing 0.1 M NaCI and 0.1% poly(ethylene glycol) 8000 at room temperature, and (Szewczuk et al., Biochemistry, 1992 31:9132).

The hydrolysis of the substrate by thrombin was monitored on a Varian-Cary 20001 m spectrophotometer in the fluorescence mode (XeX 383 nm, Xem 455 rim) or on a 20 Hitachi F2000 T fluorescence spectrophotometer 383 nm, 455 nm), and the fluorescent intensity was calibrated using AMC. The reaction reached a steady-state within 3 minutes after mixing thrombin with the substrate and an inhibitor. The steady-state velocity was then measured for a few minutes. The compounds of this invention were also pre-incubated with thrombin for 20 minutes at room temperature before adding the substrate. The steady-state was achieved within 3 min and measured for a few min. The kinetic data (the steady-state velocity at various concentrations of the substrate and the inhibitors) of the competitive inhibition was analyzed using the methods described by Segel (1975). A non-linear regression program, RNLIN in the IMSL library (IMSL, 1987), LMDER in MINPACK library (More et al., 1980) or MicrosoftTM Excell T M was used to estimate the kinetic parameters (Km V. and Ks).

dTT assay The fibrin clotting assay was performed in 50 mM Tris HCl buffer (pH 7.52 at 37 containing 0.1 M NaCI and 0.1% poly(ethylene glycol) 8000 with 9.0 x 10-10 M (0.1 NIH unit/mL) and 0.03 of the final concentrations of human thrombin and bovine fibrinogen, respectively, as reported elsewhere (Szewczuk et al.,supra). The clotting time was plotted against the inhibitor concentrations and the IC.

0 was estimated as the inhibitor concentration required to double the clotting time relative to the control. Results are summarized in Tables 1 and 2 below.

Fibrin Clot Assay 4 9. 9 The fibrin clot assay was performed essentially as 25 described by Krtenansky et al, FEBS, 1987, 211:10. A o: serial dilution of the inhibitor was prepared in 50 mM tris HCl buffer (pH7.8 at 23 containing 0.1M NaCI and 0.1% polyethylene glycol 8000. Human plasma collected in 3.8% sodium citrate, blood/anticoagulant 9:1) 30 was added to microtiter wells (microtiter plate, Falcon) containing l00pL of various inhibitor dilutions. The a.* S" solution was mixed after which 50pL of human thrombin (inM final conc.) was added and mixed for 15 seconds. The 4.

turbidity of the clot was immediately monitored by microplate autoreader (Dynateck MR 5000) at 405nm and recorded every 3 min. The maximal turbidity in the 134 13 absence of inhibitors was reached within a 60 min. IC.

0 values were calculated at 30 minutes as the inhibitor concentration that gave half the optical density of the control.

Platelet Acrreaation and Secretion Rat blood was collected into ACD (6/1 v/v) by cardiac puncture. Suspensions of washed platelets were prepared as described by Ardlie et al, (Br. J. Haematol. 1970, 19:7 and Proc. Soc. Exp. Biol. Med., 1971,.136:1021). The final suspending medium was a modified Tyrode solution (NaC1 138mM, KC1 2.9mM, HEPES 20mM, NaHPO 4 0.42mM, NaHCO, 12mM, CaCl 2 ImM, MgC1, 2mM, 0.1% glucose, 0.35% albumin, apyrase 1pL/mL pH Platelet counts were adjusted to 5000,000/pL.

To permit measurement of the extent of release of the contents of the dense granules, the platelets were labelled in the first washing solution with "C-serotonin (lpCi/lOmL of washing fluid) and release of 4Cserotonin was determined as described in Holmsen et al, (Enzymology, 1989, 169:206). Inipramine (5pM final cone.) was added to present the reuptake of released serotonin.

o* 2 Platelet aggregation was recorded at 37°C in an aggregometer (BioData PAP-4) at a stirring speed of 1,100 rpm by measuring the variations of light transmission.

l: Percentage of aggregation was determined 3 min. after the 30 addition of the stimulating agent (human thrombin 0.1IU/mL final cone.). Inhibitors were preincubated 1 minute at S37°C before addition of stimulating agent. IC, 0 values represent the concentration that was necessary to inhibit platelet aggregation or secretion to 50% of the control.

Arterial Thrombosis Model 135 FeCl 2 Induced Carotid Arterial Injury Model The FeCl 3 induced injury to the carotid artery in rats was induced according to the method described by Kurz, K.D., Main, Sandusky, Thrombosis Research 60; 269- 280, 1990 and Schumacher, W.A. et al. J. Pharmacology and Experimental Therapeutics 267; 1237-1242, 1993.

Male, Sprague-Dawley rats 375-410 g) were anesthetized with urethane 1500 mg\kg ip). Animals were laid on a 37 0 C heating pad. The carotid artery was exposed through a midline cervical incision. Careful blunt dissection was used to isolate the vessel from the carotid sheath. Using forceps, the artery was lifted to provide sufficient clearance to insert two small pieces of polyethylene tubing (PE-205 underneath it. A temperature probe (Physitemp' MT23/3) was placed between one of the pieces of tubing and the artery. Injury was induced by topical application on the carotid artery above the temperature probe of a small disc (3 mm dia.) of Whatman* No.1 filter paper previously dipped in a 35% solution of FeCl 3 The incision area was covered with aluminum foil in order to protect the FeC1, from degradation by light. The vessel temperature was monitored for 60 minutes after application 25 of FeCl 3 as an indication of blood flow. Vessel temperature changes were recorded on a thermister (Cole- Palmer Model 08533-41).

The time between the FeC1, application and the time at 30 which the vessel temperature decreased abruptly (>2.4 0

C)

was recorded as the time to occlusion of the vessel.

Inhibitor compounds were given as an iv bolus (mg/kg) followed immediately by an iv infusion (pg/kg/min. via femoral vein). The dose of inhibitor needed to double the time to occlusion in comparison to control animals in 136

I

which injury was induced in the absence of inhibitor was determined.

0 0 0 0 0 *000 0000 0* 00 0 00 0 000 137 Table 1 Antiplatelet activity jiM diT IC Plasma fibrin cmpd Ki p.M 0 Aggregation Secretion pM clot assay IC, jiM 0005 4 ND ND 47 >450 0010 4.6 21 19 89.5 >450 0015 16 >100 >100 162 >450 0020 2.2 18 14.2 22 >450 0025 53 >100 >100 >625 >450 0030 8.6 >100 >100 67 320 0035 34 >100 >100 319 >450 0040 19 >100 >100 207.5 >450 0045 74 ND ND 415 >450 0050 62 ND ND >450 0065 32.7 47.5 52 42 200 0070 4.4 22 2.1 25 78 :0080 0.048 0.4 0.38 0.375 ND :.0090 0.031 ND ND 0.33 ND :::0095 26 ND ND *0100 .19 ND ND 165 ND *supension of washed platelets from rats aInhibitory dissociation constant for human (x-thrombin 138 Table_2 Compound ki dTT Route M.O.T (nM) (nM) ivb+inf (min)+/-sem 0220 18 0225 550 0.75-50 23+/-7 235 0245 5 0.5-30 27+/-3 8 0.75-50 22.6+/-2.6 0250 40 350 0.25-20 23+/-8 0.75-50 22+/-3 0295a 1500 0.75-50 20+1-l 0295b 5000 520 0.75-50 19+/-2.7 0240 18 0.75-50 17+1-2.6 0210 8 0.75-50 20.13+/-3.4 0255 500 0260 16 0.75-50 14.83+/-0.2 0305a 220 0305b 12000 0265a 4 0.75-50 21.5+/-10 0265b 18 0.75-50 14.83+/-2.3 0285 10 0.75-50 11.33+/-1.34 150 031 5a+b 0315b 10 0.75-50 30.33+/-8.4 0335 25 138 0.75-50 45.8+/-14.2 0.5-30 41.5+/-7.27 0.25-20 27.5+/-11,3 0340 0.6 0.25-20 36+/-9,6 0.75-50 42.25+/-11 ,9 0345 2 0.75-50 50+/-5.86 0915 1600 075015+/-1,3 0935 120 9.

.9 9 9 99e

ILLO

Table 2 (continued) Compound Ki dTT Route M.O.T (nM) (nM) ivb+inf (min)+/-sem 0925a+b 10 0.75-50 19.6+/-0.2 0925b 0925a 7 0.75-50 20.3+/-3.5 0940a 16 0.75-50 15.2+/-0.82 0940b 160 0950a 150 1 0 a early eluting on RP HPLC single isomer b late eluting on RP HPLC single isomer a b mixture a a 9* a a a a a a a a *aaa a.

a.

a a a.

a a..

Claims (3)

1. A compound of formula R2 Y A R \E 1 R 12 (I) wherein R12 is attached to either D or E, in the S. 1 0 alternative, and is represented by the structure: S I 15 wherein: A is selected from (CH-R,) 1 S, SO, SO,, O and NR, wherein R 8 is hydrogen, C 6 alkyl optionally interupted with 1 or 2 .heteroatoms; C,66 aryl, C,3 cycloalkyl or heterocyclic ring or a hydrophobic group; B is selected from S, SO 2 O, NH, -CH= and CR 6 ,R wherein R, and R, are independently selected from hydrogen and C1 alkyl provided that when A is S, SO, SO,, O, or NR,, then B is CR 6 R 7 D is selected from (CH-R,) 0 2 wherein R, is hydrogen, C,_ alkyl or and CH with a double bond to B when B is or -CH=; E is selected from CH2 and CH substituted with the provided that only one of D and E is substituted with -C (0)R; X is selected from O, or CH-R,; S Y is selected from O, S, SO, SO,, N-R, and CH-R, provided \\melbOI\homeS\Pclarke\eep\specis\40628-95 biochen cm.doc 29/03/99 142 that when X is N-R, then Y is CH-R. or 0, and when X is 0 then Y is CH-R 8 Z is selected from 0, S and H 2 R, is one of formula VIa to VId: RIIIN p T I n R N Via K 07Vb G NIG I N P T Vic N P T ViMn wherein: RI, is hydrogen or C 6 alkyl; K is a bond or -NH-; G is C 1 4 alkoxy; cyano; -CH,-NH 2 -C (NH) -NH- C (NH) -NH 2 -CH 2 N 2 a C, cycloalkyl or aryl substituted with cyano, -NH, -CH 2 -NH 2 -CN)N 2 -NH- C(NH) -NH 2 or -CH 2 -NH-C (NH) -NH 2 or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted *..with cyano, -NH 2 -CH 2 -NH 2 C(H)-H2 -NH-C(NH)-NH 2 or 25 -CH NH-C (NH) -NH2 U is cyano, -NH 2 -C (NH) -NH 2 or -NH-C(NH)-NH 2 P is a bond, or a bivalent group: OH nH J is C 16 alkylene optionally substituted with OH, NH 2 and C 1 6 alkyl and optionally interrupted by a heteroatom. selected from 0, S and N; n is 0 or 1; and \\melb~l\home$\PCarke\Keep\specis\46 2

8-. 95 biochem cm.doc 29/03/99 143 T is a heterocycle selected from the group consisting of: xs R .Ir X, S7-R' xX, X, wherein X 10 X 11 and X 1 2 are each independently selected from the group consisting of N, or C-X 7 where X, is hydrogen, C_ 1 4 alkyl, or C, 5 8 aryl; X, and X 13 are each independently selected from the group consisting of 0, S, or CH-X; and R' is hydrogen, CL-, alkyl optionally carboxyl substituted, carboxyl, -C_ alkyl-CO,-C, 1 alkyl, C 6 0 aralkyl, C, 7 cycloalkyl, aryl or an aromatic heterocycle. R 2 is selected from H and CI_, alkyl optionally substituted 20 with C 6 aryl, a 6 member heterocycle or a C 37 cycloalkyl ring; R, is selected from H, NR,R 7 and alkyl; and R 4 and R 5 are independently selected from H; NR,R 7 C,_ 6 aryl or C 37 cycloalkyl optionally substituted with alkyl; 25 C.- 1 alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NR,R 7 or a aryl, heterocycle or C 37 cycloalkyl group optionally substituted with halogen, hydroxyl, Cl_, alkyl; an amino acid side chain; and a hydrophobic group, and wherein an alkyl is a straight or branched, saturated or unsaturated chain of carbon atoms. 2. A compound according to claim 1, wherein T is selected from the group consisting of: \\melbO1\home$\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 -144- N N R'I R' R IN/ N N NN N 6-.aakyC- Neece from or wherein R' is hydrogen, 0,16 alkyl optionally carboxyl substituted, carboxyl, -0O-1 alkyl-C0 2 -cl- 16 alkyl, 06-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle. 3. A compound according to ayoo claims 2, wheei T 3i wheei fR n R sahdopoi ru selectedfrm \\melb~l\home$\PClarke\Keep\specia\40628-95 biochem cv.doc 29/03/99 145 from C 1 2 0 alkyl, C 2 20 alkenyl or C 2 2 a ikynyl optionally interrupted by a carbonyl group, C -1 aryl, C 3 7 cycloalkyl, C 6 20 aralkyl, C 6 2 0 cycloalkyl substituted C 120 alkyl, wherein the aliphatic portion is optionally interrupted by a carbonyl group and the ring portion is optionally substituted with C 16 alkyl; and a hydrophobic amino acid side chain. compound according to claim 4, wherein R 3 is H. 6. A compound according to any one of claims 1 to wherein Z is 0. 7. A compound according to any one of claims 1 to 6, 15 wherein R 2 is H. 9 9 9 *9 9* 9 .9 9 99 9 9 9 *9*9

999. 9 9999 99*9 *99* 9**9 9~ 9 9999 9* 999w 9999 4 9 9 9 .9.9 99 9 99 99 9 A compound of the formula (VII): wherein 25 is one of formula VIa to VId: n G VIb RI >)O-8 Vic N P "I N (J~n U 1-3 N P J)n U wherein: R 11 is hydrogen or C 1 alkyl; \\nielbol\home$\PClarke\KeeP\specis\40628-95 biochen czn.doc 29/03/99 146 K is a bond or -NH-; G is C1-4 alkoxy; cyano; -C(NH)-NH; -NH- C(NH)-NH,; -CH 2 -NH-C(NH)-NH 2 a C, cycloalkyl or aryl substituted with cyano, -NH 2 -CH,-NH, -NH- C(NH)-NH 2 or -CH,-NH-C(NH)-NH 2 or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -CH,-NH 2 -NH-C(NH)-NH, or -CH 2 -NH-C (NH) -NH 2; U is cyano, -NH 2 -C(NH)-NH 2 or -NH-C(NH)-NH P is a bond, or a bivalent group: Oq H o I C:. J is C16 alkylene optionally substituted with OH, NH2 and C,6 alkyl and optionally interrupted by a heteroatom selected from O, S and N; n is 0 or 1; and 20 T is a heterocycle selected from the group consisting of: a. 25 x wherein XT II- X, X 5 X 0 ,F X 11 and X 12 are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, alkyl, or C 5 8 aryl; X, and X, 3 are each independently selected from the group consisting of O, S, or CH-X,; and R' is hydrogen, C 1 1 6 alkyl optionally carboxyl substituted, carboxyl, -CO-16 alkyl-CO 2 -C 16 alkyl, C6- 20 aralkyl, C 3 cycloalkyl, aryl or an aromatic heterocycle. R, is H or C1_, alkyl; R, is selected from H, NRR, and C_16 alkyl; and \\melb0l\homeS\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 147 R 4 and R 5 are independently selected from H; NRR,; C 16 aryl or C_7 cycloalkyl optionally substituted with C 1 6 alkyl; C 16 alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRR, or a C, 61 aryl, heterocycle or C 37 cycloalkyl group optionally substituted with halogen, hydroxyl, C, 6 alkyl; an amino acid side chain; and a hydrophobic group, and wherein an alkyl is a straight or branched, saturated or unsaturated chain of carbon atoms, and wherein an alkyl is a straight or branched, saturated or unsaturated chain of carbon atoms, and wherein an alkyl is a straight or branched saturated chain of carbon atoms. 9. A compound according to claim 8, wherein T is 15 selected from the group consisting of: a a. a a aoa a a *o a.* a a. a o ro oeo N R' NN a RT -7 N--N R. 1 7 S S N-N N N H:\PClarke\Keep\specis\40628-95 biochem cm.doc 23/04/99 148 and R' is hydrogen, C 16 alkyl optionally carboxyl substituted, carboxyl, -C0-6 alkyl-CO 2 -C 1 -H alkyl, C 6 2 0 aralkyl, C3 cycloalkyl, aryl or an aromatic heterocycle. 10. A compound according to claim 9, wherein T is selected from: N s R/ and R' is hydrogen, C11, alkyl optionally carboxyl substituted, carboxyl, -C alkyl-CO,-C 1 6 alkyl, C, 20 aralkyl, C, 3 cycloalkyl, aryl or an aromatic heterocycle. 15 11. A compound according to any one of claims 8 to 10, wherein R 2 and R, are H. 12. A compound according to any one of claims 8 to 11, wherein R 4 is C016 alkyl optionally interupted with a 20 heteroatom or a carbonyl, and optionally substituted with a C ,16 aromatic, C,7 cycloalkyl or heterocycle ring wherein the ring is optionally substituted with CF, or oxo. 13. A compound according to any one of claims 8 to 25 12, wherein R 5 is H. 14. A compound according to claim 8, wherein: R, is H; R 4 is C, 16 alkyl optionally interupted with a heteroatom or a carbonyl, and optionally substituted with a C,61 aromatic, C,3 cycloalkyl or heterocycle ring wherein the ring is optionally substituted with CF 3 or oxo; and RS is H. 15. A compound according to claim 8, selected from: 0085 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazolo[3,2- a]pyridine-3R-carboxamido (propylcarbo methoxy \\melbOl\home$\Pclarke\Keep\specis\40628. 9 5 biochem cm.doc 29/03/99 149 ketoarginine); and 0105 6S-cyclohexylmethylhexahydro-5-oxo-5H-thiazolo 3,2- a]pyridine-3R-carboxamido (a-benzothiozolo keto arginine). 16. A compound according to claim 1, of formula (VIII): R' c wherein RL is one of formula VIa to VId: Via .Q( S)0-7 G R,,Nb lb )0-8 K\ G *9 a9 a a a (J)n S N P Vc y (J)n U )1-3 R 2 is H or C alkyl; 30 R, is selected from H, NRR, and C, alkyl; and R 4 and Rs are independently selected from H; NR,R; aryl or cycloalkyl optionally substituted with alkyl; C alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRR, or a C aryl, heterocycle or C,1, cycloalkyl group optionally substituted with halogen, hydroxyl, alkyl; an amino acid side chain; and a hydrophobic group. \\mwlbOl\homS\Sieona\K..p\40628 95 a~m.nded.doc 22/07/99 150 wherein: R 11 is hydrogen or C1-6 alkyl; K is a bond or -NH-; G is C 1 -4 alkoxy; cyano; -NH 2 -C(NH)-NH; -NH- C(NH)-NH,; -CH 2 -NH-C(NH)-NH,; a C, cycloalkyl or aryl substituted with cyano, -CH 2 -C(NH)-NH -NH- C(NH)-NH, or -CH,-NH-C(NH)-NH,; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -CH,-NH -C(NH)-NH 2 -NH-C(NH)-NH 2 or -CH, 2 -NH-C (NH) -NH,; U is cyano, -C(NH)-NH, or -NH-C(NH)-NH; P is a bond, or a bivalent group: OH C H or e 3 J is C 16 alkylene optionally substituted with OH, NH 2 and C16 alkyl and optionally interrupted by a heteroatom selected from O, S and N; n is 0 or 1; and is a heterocycle selected from the group consisting of: I s x, R A X13 x x X '"2x X wherein X 5 X 1 0 X 11 and X, 2 are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, C 1 alkyl, or aryl; X, and X, 13 are each independently selected from the group consisting of S, or CH-X,; and R' is hydrogen, Cz- 1 alkyl optionally carboxyl substituted, carboxyl, -CO- 1 alkyl-CO, 2 -C, 16 alkyl, C6-20 \\melbOl\homeS\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 151 aralkyl, cycloalkyl, aryl or an aromatic heterocycle. 17. A compound according to claim 16, wherein T is selected from the group consisting of: S Sr7 N' S S e S. S So S a• *5 S* S S S S S S *5 aoo, oo a r a oe ft a a e eeel a r NN N N -N N--N N--N 0 C' N *6/N "<t 25 and R' is hydrogen, C, alkyl optionally carboxyl substituted, carboxyl, -CO. alkyl-CO,-Cl- alkyl, C6- 20 aralkyl, cycloalkyl, aryl or an aromatic heterocycle. 18. A compound according to claim 17, wherein T is selected from: \\melbOl\homeS\Simeona\Keep\40628 95 anmnded.doc 22/07/99 152 s ,/s SN R' is hydrogen, C -1 alkyl optionally carboxyl substicuted, carboxyl, -Co alkyl-CO 2 -C- 16 alkyl, C5,- aralkyl, C, 7 cycloalkyl, aryl or an aromatic heterocycle. 19. A compound according to any one of claims 16 to 18, wherein R, and R, are both H. A compound according to any one of claims 16 to 19, wherein R, is H or C 1 alkyl substituted with COOH. 21. A compound according to any one of claims 16 to wherein R 2 R, and R are H and R is C' 16 alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRP, or a CC, 1 aryl, heterocycle or cycloalkyl group optionally substituted with halogen, hydroxyl or C,,alkyl. 22. A compound according to claim 16, wherein: R 2 R, and R, are H 25. R is COg-alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NRR 7 or a aryl, heterocycle or cycloalkyl group optionally substituted with halogen, ~hydroxyl or alkyl. 23. A compound according to claim 16, selected from: 0345 4 -Oxo-2-(3-phenyl-propionyl)-octahydro-pyrrolo[1,2- a]pyrazine-6-carboxylic acid thiazole-2-carbonyl)-butyl]-amide; and 0340. 4-Oxo-2-(3-phenyl-propionyl) -octahydro-pyrrolo(1,2- a]pyrazine-6-carboxylic acid [4-guanidino-1-(thiazole-2- carbonyl)-butyl]-amide. \\melb011homS\Simeona\Keepl40625 95 -mnded.doc 22/07/9 153 24. A compound according to claim 1, of formula (IX) R, 0 wherein Y is selected from 0, S, SO, S02, N-R5 and CH-R8; R, one of formula VIa to VId: VIC )K GG G o /TV l d P (J)n T r N Vid Vi M I R, is H or alkyl; R, is selected from H, NR,R, and alkyl; and. R4 and R, are independently selected from H; NR, aryl or C,-7 cycloalkyl optionally substituted with Ci-, alkyl; C1-16 alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, NR,R, or a aryl, heterocycle or cycloalkyl group optionally substituted with halogen, hydroxyl, alkyl; an amino acid side chain; and a hydrophobic group; I. R, is hydrogen, alkyl optionally interrupted with 1 or 2 heteroatoms; aryl, C, cycloalkyl or heterocyclic ring or a hydrophobic group; and n is 1 or 2. \\melbOl\ho.mS\Simeona\Keep\ 4 0 62 8 95 am*nded.doc 22/07/99 154 is hydrogen or C alkyl; K is a bond or -NH-; G is C 4 alkoxy; cyano; -CH-NH,; -C(2NH)-NH,; -NH- C -CH,-NH-C(NH)-NH,; a C, cycloalkyl or aryl substituted with cyano, -NH2, -CH2-NH2, -C(NH)-NH 2 -NH- C (NH)-NH, or -CH 2 -NH-C(NH)-NH 2 or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -C (NH)-NH 2 -NH-C (NH)-NH 2 or -CH 2 -NH-O (NH) -NH 2 U is cyano, -NH 2 -C (NH)-NH, or -NH-C (NH) -NH 2 P is a bond, or a bivalent group: Off .selected from 0, S and N; XC X 30 X11 RI R 30 x, J is 016 a ikylene otionlly substituted with OH, NH 2 and 3 5 wherein alkyl and optionally interrupted by a heteroatomly selected selected from the group consisting of N, or C-X where X iN; n is 0 or 1; and S T is a heterocycle selected from the group consisting of: aa. N A 5 'heei X 5 XI 0 X 11 and X, are each independently selected from the group consisting of N, or C-X 7 where X 7 is \\melbOl\homeS\PClarke\Keep\secis\40628- 95 biochem cm.doc 29/03/99 155 X0, X, and are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, C 4 alkyl, or aryl; X and are each independently selected from the group consisting of O, S, or CH-X,; and R' is hydrogen, C alkyl optionally carboxyl substituted, carboxyl, -C 0 alkyl-CO,-CI- alkyl, C aralkyl, C,- 7 cycloalkyl, aryl or an aromatic heterocycle. 25. selected s- A compound according to claim 24, wherein T is from the group consisting of: a. a a a a. a a a a R" N NN N-N N NN N/ N S S I. N N and R' is hydrogen, C,1, alkyl optionally carboxyl substituted, carboxyl, -C 0 alkyl-CO,-C_, alkyl, C,,O aralkyl, C,. 7 cycloalkyl, aryl or an aromatic heterocycle. 26. A compound according to claim 25, wherein T is selected from: \\melb01\hom$S\Simeona\Keep\ 4 0628 95 amended.doc 22/07/99 156 N and R' is hydrogen, alkyl optionally carboxyl substituted, carboxyl, aikl-CO,-C alkyl, C 0 lky -CO -L6alk l' 6-20 aralkyl, cycloalkyl, aryl or an aromatic heterocycle. 27. A compound according to any one of claims 24 to 26, wherein R, and R, are both H. 28. A compound according to any one of--claims 24 to 27, wherein R, is H, NRR, or alkyl substituted COOH. 29. A compound according to any one of claims 24 to 28, wherein RS is aryl, C,- 2 aralkyl, or C1_1. alkyl sijbstituted with C, 7 cycloalkyl'. 30. A compound.according to claim 24, where-in n is 1; R2, R, and R, are H; and s0 R* is C6_ 1 6 aryl, C 2 aralkyl, or C 1 1 alkyl substituted with 7 cycloalkyl. 31. A compound according to claim 24, selected from: 0890 3-Amino-4oxo-2-phenyl-hexahydro-pyrrolo(2,l- bi (1,3]thiazine-6-carboxylic acid [1-(benzothiazole- 2 carbonyl)-4-guanidino-butyl]-amide; 0895 3-Ami no--ben zyl 4oxo-hexahydro -pyrro lo[ b](1,3]thiazine-6-carboxylic acid (l-(benzothiazole-2- carbonyl) -4-guanidino-butyll -amide; and 0900 3-Amino-2-cyclohexyl-4-oxo-hexahydro-pyr b] l,3]thiazine-6-carboxylic acid [l-Cbenzothiazole- 2 carbonyl) -4-guanidino-butyl -amide. 32. A compound according to claim 1, of formula \\melbOl\hom.S\SjU.ona\K..p\40623 95 aonded.doc 22/0799 157 0 O wherein 0 B is selected from S, SO,, O, NH, -CH= and CRP wherein R, and R, are independently selected from hydrogen and alkyl provided that when A is S, SO, SO,, O, or NR,, then B is CRR; R 1 is one of formula VIa to VId: 151 ,k via VI o-8 K 0-7 K G G N P Vic N P T Vid S00 UU *3 U 2 R, is H or C- alkyl; R, is selected from H, NRR, and C, alkyl; and R 4 and R. are independently selected from H; NR,R; aryl 00:. or 7 cycloalkyl optionally substituted with alkyl; C, j alkyl optionally interrupted by one or more heteroatom or carbonyl group and optionally substituted with OH, SH, N RR, or a arvl, heterocycle or cycloalkyl group optionally substituted with halogen,. hydroxyl, C alkyl; an amino acid side chain; and a hydrophobic group. wherein: S RU is hydrogen or C alkyl; \\melb~o\hogmS\SimonA\KeD\40528( 9S mended.doc 22/07/99 158 K is a bond or -NH-; G is C 1 4 alkoxy; cyano; -CH 2 -C(NH) -NH- C(NH)-NH,; -CH,-NH-C(NH)-NH,; a C, cycloalkyl or aryl substituted with cyano, -NH 2 -CH 2 -C(NH) -NH, -NH- C(NH)-NH 2 or -CH,-NH-C(NH)-NH,; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -CH, 2 -NH 2 -C (NH) -NH-C(NH) -NH, or -CH,-NH-C (NH) -NH 2 U is cyano, -C(NH)-NH, or -NH-C(NH)-NH 2 P is a bond, or a bivalent group: 0K OHH "c,oH a CH or J is C_ alkylene optionally substituted with OH, NH, and C 1 alkyl and optionally interrupted by a heteroatom selected from O, S and N; n is 0 or 1; and T is a heterocycle selected from the group consisting of: rxs xf x R A' SS I X x x wherein X 5 X 10 X, and X 1 are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, C1- alkyl, or aryl; X, and X, 3 are each independently selected from the group consisting of O, S, or CH-X,; and R' is hydrogen, C 11 alkyl optionally carboxyl substituted, carboxyl, alkyl-C0 2 -C 116 alkyl, C6-2 0 \\melb01\homeS\PClarke\Keep\specis\40628-95 biochem cm.doc 29/03/99 159 aralkyl, C 7 cycloalkyl, aryl or an aromatic heterocycle. 33. A compound according to claim 32, wherein T is selected from the group consistina of: NN~ N Nf ~0, N S FI I N N .N and R' is hydrogen, C,,,alky1 optionally carboxyl substituted, carboxyl, alkyl-CO 2 -C 1 -1 alkyl, C 6 20 aralkyl, C,- 7 cycloalkyl, aryl or an aromatic heterocycle. 34. A compound according to claim 33, wherein T is selected from: FV N R'_ and R' is hydrogen, C 1 16 alkyl optionally carboxyl substituted, carboxyl, alkyl-CO 2 -C 1 -1 alkyl, aralkyl, C 3- cycloalkyl, aryl or an aromatic heterocycle. \\melbl\hom*S\Siqona\K**p\40628 95 amtnded.doc 22/07/99 160 A compound according to any one of claims 32 to 34, wherein R 2 and R, are both H. 36. A compound according to any one of claims 32 to 35, wherein R 4 is Ci,1 alkyl substituted with C 6 aryl. 37. A compound according to any one of claims 32 to 36, wherein R, is H. 38. A compound according to claim 33, wherein B is S; R R, and R s are H; and R 4 is C_16 alkyl, alkenyl or alkynyl substituted with C, aryl optionally substituted with Cj_, alkyl, alkenyl or alkynyl. 39. A compound according to claim 33 wherein B i" S; R 2 R, and R 5 are H; and R 4 is Ci1 alkyl, alkenyl or alkynyl substituted with C6 16 aryl optionally substituted with alkyl, alkenyl or alkynyl. 40. A compound according to claim 32, selected from: I o 25 925 7-Benzyl-6-oxo-octahydro-pyrido[2,-c] [1,4]thiazine-4- carboxylic acid[4-guanidino-l-(thiazole-2-carbonyl)butyl]- amide; and 940 6-Oxo-7-phenethyl-octahydro-pyrido[2,l-c] 4]thiazine- 4-carboxylic acid[4-guanidino-l- (thiazole-2-carbonyl) 30 butyl]-amide. 41. A compound according to any one of claims 1, 8, 16, 24 or 32 substantially as hereinbefore described with reference to any one of the examples. \\melb01\homeS\Simeona\Keep\40628 95 asended.doc 22/07/99 161 Dated this 23rd day of April 1999 BIOCHEM PHARMA INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent Attorneys of Australia sees *0 0* H:\PClarke\Kep\sPecis\40628-95 biochem cmndoc 23/04/99