AU680240B2 - Fibrinogen receptor antagonists - Google Patents

Description

WO 94/18981 PCT/US94/01881 -1- TITLE OF THE INVENTION FIBRINOGEN RECEPTOR ANTAGONISTS BACKGROUND-OF THE INVENTION The invention relates generally to modulating cell adhesion and to inhibiting the binding of fibrinogen and other proteins to blood platelets, and inhibiting the aggregation of blood platelets specifically to the IIb/IIa fibrinogen receptor site. Fibrinogen is a glycoprotein present in blood plasma that participates in platelet aggregation and in fibrin formation. Platelets are cell-like anucleated fragments, found in the blood of all mammals, that also participate in blood coagulation.

Interaction of fibrinogen with the IIb/IIIa receptor site is known to be essential for normal platelet function.

When a blood vessel is damaged by an injury or other causative factor, platelets adhere to the disrupted subendothethial surface. The adherent platelets subsequently release biologically active constituents and aggregate. Aggregation is initiated by the binding of agonists, such as thrombin, epinephrine, or ADP to specific platelet membrane receptors. Stimulation by agonists results in exposure of latent fibrinogen receptors on the platelet surface, and binding of fibrinogen to the glycoprotein IIb/IIla receptor complex.

Attempts have been made to use natural products and synthetic peptides to determine the mechanism of adhesion and platelet aggregation. For example, Rouslahti and Pierschbacher in Science, 238, 491-497 (1987), describe adhesive proteins such as fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor that are present in extracellular matrices and in blood. The proteins contain the tripeptide arginine-glycine-aspartic acid (RGD) as their glycoprotein IIb/lIa recognition site. These arginineglycine-aspartic acid containing tripeptides are recognized by at least one member of a family of structurally related receptors, integrins, which are heterodimeric proteins with two membrane-spanning subunits. The authors state that the conformation of the tripeptide s re I ~P~t PL ~i~s~RI1 Is WO 94/18981 PCT/US94/01881 -2sequence in the individual proteins may be critical to recognition specificity.

Cheresh in Proc. Nat'l Acad. Sci. 84, 6471-6475, (1987), describes an Arg-Gly-Asp directed adhesion receptor expressed by human endothelial cells that is structurally similar to the IIb/IIIa complex on platelets but is antigenically and functionally distinct. This receptor is directly involved in endothelial cell attachment to fibrinogen, von Willebrand factor, and vitronectin.

Pierschbacher and Rouslahti, in J. of Biol. Chem., 262, 17294-17298 (1987) hypothesized that the Arg-Gly-Asp sequence alone would be a sufficient signal for receptor recognition and binding and that, therefore, the conformation of the tri-peptide sequence would be determinative. Various synthetic peptides were produced and the authors concluded that the stereochemical conformation of Arg-Gly-Asp as influenced by enantiomeric substitutions or additions to this sequence significantly influenced receptor-ligand interaction. The authors further showed that cyclization of a decapeptide by forming a disulfide bridge between non-terminal residues Pen and Cys, rendered the peptide much less effective at inhibiting attachment to fibronectin.

In Proc. Nat'l Acad. Sci. 81, 5985-5988 (1984), the same authors describe tetrapeptide variants of the cell recognition site of fibronectin that retain attachment-promoting activity. Peptides having a tetrapeptide recognition site are described in U.S. Pat. Nos. 4,589,881 and 4,614,517. A number of large polypeptide fragments in the cellbinding domain of fibronectin have cell-attachment activity. For example, see U.S. Pat. Nos. 4,517,686, 4,661,111 and U.S. Pat. No.

4,578,079.

Ruggeri et al., Proc. Nat'l Acad. Sci 83, 5708- 5712 (1986) explore a series of synthetic peptides designed in lengths to 0 16 residues, that contain RGD and a valine attached to the aspartic, acid.

residue of RGD that inhibit fibrinogen binding to platelets. See also Koczewiak et al., Biochem. 23, 1767-1774 (1984); Ginsberg et al., J Biol. Chem. 260(7), 3931-3936 (1985); and Haverstick et al., Blood I I WO 94/18981 PCT/US94/01881 -3- 66(4), 946-952 (1985). Other inhibitors are disclosed in Eur. Pat.

Nos. 275,748 and 298,820.

A number of low molecular weight polypeptide factors have been isolated from snake venom.- These factors apparently have high affinity for the gplb/Illa complex. For example, Huang et J.

Biol Chem., 262, 16157-16163 (1987); Huang et al., Biochemistry 28, 661-666 (1989) describe the primary structure of the venom trigramin which is a 72 amino acid polypeptide that contains the RGD subunit.

Echistatin is another venom which has high affinity for the gpIb/IIIa complex. This polypeptide contains 49 amino acids and has the RGD subunit and various disulfide bridges. Gan et al., J. Biol. Chem., 263, 19827-19832 (1988). See also, Dennis et al., Proc. Nat'l Acad. Sci.

USA 87, 2471-2475 (1989). However, these snake venom factors also have high affinity for other members of the adhesive protein receptor family including the vitronectin and fibronectin receptors so are not selective for the gpllb/IIla complex.

While it is known that the tripeptide sequence Arg-Gly-Asp is present in certain polypeptides that can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin, the tripeptide Arg-Gly-Asp has low activity. At present, there is little understanding of how other amino acids coupled to this sequence influence binding specificity. U.S. Pat. No 5,023,233, assigned to Merck Co., Inc., discloses small cyclic hexapeptides which contain the sequence Arg-Gly-Asp and are useful platelet aggregation inhibitors.

U.S. Pat. No. 5,037,808 discloses the use of indolyl platelet-aggregation inhibitors which are believed to act by antagonizing interactions between fibrinogen and/or extracellular matrix proteins and the platelet gpllb/fIIa receptor. U.S. Pat. No. 5,037,808 discloses guanidino 3 peptide mimetic compounds that retain an Asp residue which inhibit platelet aggregation. The^ describes the.

use of antibody-poly-peptide conjugates wherein said polypeptides contain the Arg-Gly-Asp (RGD) sequence.

The a 1' iscloses the use of large cyclic peptides containing RGD flanked by proline residues which are I ~as L~LL QL~ '9k ~g 4 platelet aggregation inhibitors. The WO 9101331 discloses small cyclic platelet aggregation inhibitors which are synthetic cyclic pentapeptides containing the tripeptide sequence Arg-Gly-Asp and a thioether linkage in the cycle. The United States Patent 5,051,405 published May 2, 1991, also discloses the use of peptide and pseudopeptides such as N-amidino-piperidine-3-carboxylglycyl-L-aspartyl-L-valine that inhibit platelet aggregation and thrombus formation in mammalian blood. The application Eur. Pat. 445 796 discloses linear compounds which can include internal piperazinyl or piperidinyl derivatives. Eur. Pat. App. No. 91300179.8, assigned to Merck Co., Inc., and published on July 17, 1991, discloses linear polypeptide fibrinogen receptor antagonists.

United States Patent 5,256,812 discloses compounds of the R1-A-(W)a-X-(CH 2 )b-(Y)c-B-Z-COOR wherein R 1 is a guanidino or amidino moiety and A and B are chosen from specific monosubstituted aryl or heterocyclic moieties.

While a multitude of compounds or peptide analogs believed to inhibit platelet aggregation by inhibiting binding to a blood platelet by fibrinogen are known, the present invention provides novel fibrinogen receptor antagonists that have significant binding activity and are, therefore, useful for the reasons stated herein. A number of very serious diseases and disorders involve hyperthrombotic complications which lead to intravascular thrombi and emboli. Myocardial infarction, stroke, phlebitis and a number of other serious conuitions create the need for novel and effective fibrinogen receptor antagonists.

0* oe ,9 o a* ,a I) [N:\LIBAA]00368:VMJ ap Ir WO 94/18981 WO 9418981PCTTJS94/01881 SUMMARY OF THE [NVENTION Compounds of the invention have the formula o R 2 R 6 11 1 Q- (C H 2 -NR 5-C-R 8 I or o R 2 R 6 11 1 1

Q)-(CH

2 a-AB -uC- R'for example HN 0

NNH

N-N

HNSO

2 CHI: actvity. The compounds have fibrinogen receptor antagonist WO 94/18981 PCT/US94/01881 -6- DETAILED DESCRIPTION OF THE INVENTION 0

R

2

R

6

Q-(CH

2 C-N- R5-C-R 8 or

R

4

R

1 0 R 2 R6 11 I I

Q-(CH

2 )n-a-AB- C- R5-C R 8

R

1 wherein Q is NH NH II II

H

2 N-C- HN-C-NH- R 7 HN- or Q is a 4-9 membered mono- or bi-cyclic ring system containing 1, 2 or 3 heteroatoms chosen from N, O or S and either unsubstituted or substituted with R 8 AB is a fused ring stem sharing adjacent carbon and nitrogen atoms, wherein A is a 5, 6 or 7 membered saturated or unsaturated rirg containing 1, 2 or 3 heteroatoms selected from O, S or N; B is a 5, 6 or 7 membered saturated or unsaturated ring containing 1, 2 or 3 heteroatoms selected from O, S or N; ~llaa~sse g II WO 94/18981 WO 948981PT/US94/01881 -7 R I is H, C 1-4 alkyl, N(Rg)2, -N(R 8 )S02R 7

NR

8 CO2R 7

NR

8

C(O)R

7

N

8

C(O)N(R

7

)R

8

N(R

8 )SO2N(R 7 )Rg, N(R 8 )SO2N(R 8 )C(O)0R7,

C(O)N(R

7 or a cyclic group with R 6 as defined below;

R

2 is H, C 1-4 alkyl, C 1 4 branched alkyl, C 1 4 alkyl aryl, or aryl;

R

4 is H, C1-4 alkyl, Cl..4 branched alkyl, cyclic Cl-4 alkyl or CI-4 aikenyl;

R

5 is CH, -CH(CH2)n, a bond, or when R 5 is adjacent N(R 4 -C(CH2)n; 11 0 6 is COGH, CH2OH, C(0)NO1R1 C02R 9 tetrazole, acylsulfonamide, or

P(OH)

2 11 0 ,or a cyclic group with R I as defined below; wherein the cyclic group of R I with R 6 is 00

R

7 wherein y 0 or S;

R

7 is H, branched or Str it chain C1..4 substituted or unsubstituted alkyl, branched or straight chain lower alkenyl, Ci1 -4 aikylaryl, substituted aryl, or 5 or 6 membered heteroaryl containing 1, 2, or 3 N, S, or 0 heteroatoms Ry WO 94/18981 PCT/US94/01881 8wherein substituted alkyl is hydroxy substituted or C1-4 alkoxy substituted alkyl, and wherein substituted aryl is substituted by one, two or three of the following groups: halogen, C1-4 alkoxy, hydroxy, or C1-4 alkyl;

R

8 is H, branched or straight chain C1-4 alkyl;

R

9 is H, C1-4 alkyl or aryl; n is 0-7; n' is 0-3; and a is R7

-NC-

I or a bond, 0 and pharmaceutically acceptable salts.

In one embodiment, the compounds have the formula 0 R2 R 6

Q-(CH

2 )n-a-AB- C-N- R5-C-R 8 or

R

4 R1 O R 2

R

6 I I I Q- (CH 2 a-AB- C- R 5 -C R

R

1 ~-7 WO 94/18981 WO 9418981PCTIUS94/01881 9wherein Qis

NH

H

2 N AjsF;

(CH

2

NNH-

N

H

2 f\ NH RN~ J- I' i- (CH 2 )n R 8 N )-L

'-(CH

2 )n' R'N N 0 0 WO 94/18981 WO 9418981PCT11US94/01881 10

H

2 N- HN-C-

<N

HN

(CH

2 )n

H

2 N

(CH

2 )n.

NH

(CH

2 )n 8 2 N A- C

HNH

N H

H

2 N 1110/ or

N-

N

n =0-7; n' 0-3; R4= H, C 1-4 alkyl, C 1-4 branchied alkyl, cyclic C 1-4 alkyl or C 1-4 alkenyl; CH, -CH(CH2)n, or a bond; WO 94/18981 WO 9418981PCT/US94/01881

R

2 is H, C 1-4 alkyl, C 1-4 branched alkyl, Cl1 4 alkyl aryl, or aryl; Rl H, Cl1-4 alkyl, N(R8)2, -N(RS)SO2R7, NR 8 CO2R7, NR8C(O)R7, NR8C(O)N(R7)R8, N(RS)SO2N(R7)Rg, N(R8)SO2N(R8)C(O)0R7, C(O)N(R7)2, or a cyclic group with R 6 R6= COOHK CH2OH, C(O)N(R' C02R 9 tetrazole, acylsulfonamide, or

P(OH)

2 0 ,or a cyclic group with R 1 the cyclic group of R I with R 6 is N, 'N-R 7

R

Y" wherein y=0or S; R7= H, branched or straight chain Ci .4 substituted or unsubstituted alkyl, branched or straight chain lower alkenyl, Cl1-4 alkylaryl, substituted aryl, or 5 or 6 membered heteroaryl containing 1, 2, or 3 N, S, or 0 heteroatoms wherein substituted alkyl is hydroxy substituted or Ci -4 alkoxy substituted alkyl, and wherein substituted aryl is substituted by one two or three of the following groups: halogen, Ci1 4 alkoxy, hydroxy, or Ci1 -4 alkyl; RS H, branched or straight chain Cl1-4 alkyl; R9= H, Ci1-4 alkyl or aryl; I WO 94/18981 PCT/US94/01881 12a= 0

-N

I

R or a bond; A a 5, 6 or 7 membered saturated, partially saturated, or unsaturated ring containing 1, 2 or 3 heteroatoms selected from O, S or N; B a 5, 6 or 7 membered saturated, partially saturated, or unsaturated ring containing 1, 2 or 3 heteroatoms selected from 0, S or N; wherein A and B form a fused ring system sharing adjacent carbon and nitrogen atoms.

In the compounds of the present invention, the components having asymmetric centers occur as racemates, racemic mixtures, and as individual enantiomers and/or diastereomers. All isomeric forms are included in the present invention.

In one class of this embodiment, the compounds have the formula, 0 R2 R 6 I I I Q-(CH2)n- a-AB- C-N-R5-C-R8

R

4

R

1 I LI L~l ~Llb~ I~b WO 94/18981 WO 9418981PCTIUS94/01881 13 AB is selected from the group of 0 V wherein Vis Nor CR, and D is CH 2

CH

2 -0H 2

CH

2 C(R 7 2

CH

2 or

H

2 C OH 2

(OH

2

(OH

2 )n 0 N. wherein X =Nor OR x wherein R 3 ON, C(0)N(R 7 250 r(H2)n' N, or WO 94/18981 Wo 9418981PCTIUS94/01881 14 0 IN

N

0 N N (y N an

(H

2 C)n In a subclass of this class of this embodiment compounds are those having the formula 0O

R

2

R

6

Q-(CH

2 -N-C ~R' where AB is 0 wherein V is N or CH, N and D is 0H 2

-CH

2 or 2 C(R 4 2

CH

2 WO 94/18981 WO 9418981PCTIUS94/01881 15 In another class of this embodiment the compo unds have the formula 0

R

2

R'

II I R 1OR AB is selected from the group of wherein Vis Nor CR 7 and D is OH 2 0H 2 -0H 2 0H 2 C(R 7 2 0H 2 or

H

2 G OH 2 250 X'wherein X =Nor CR 3 wherein R 3 O N, O(O)N(R )R 8 300 -N O 0,or WO 94/18981 Wo 9418981PCT[US94/01881 -16 0 N N ,rN 0 100 N N and

(CH

2 )n- 0 NJ 'NkN wherein y 3 is 0 or H 2 y 3 In a subclass of this class of this embodiment, the compounds have the formula 0 R 2 R 6

Q-(CH

2 CR C R 8 0 a= R and AB is selected from

I

WO 94/18981 WO 9418981PCTIUS94/01881 17 v 0 r 4 wherein V is N or OR 7 and 0 isO-H 2 i CH 2 -C H 2

CH

2 O(R 7 2 0H 2 or

H

2 0 OH 2

(OH

2 )n'-(OH 2 )n and 0 wherein X =N or OR, 3 78 wherein R N, CO)N(R )R, 0 0,or WO 94/18981 WO 9418981PCTIUS94/01881 18 In another subclass of this class of this embodiment, the compounds have the formula 0 oR 2 R 6 If and AB is selected from 0 N N

,N

0 N

N

and (CH 0 N Nwherein y 3 is 0 or H 2 y 3

-M

WO 94/18981 WO 9418981PCTIUJS94/01881 -19- Specific examples of compounds of the invention are those selected from the following group of compounds and their pharmaceutically acceptable salts: NHS0 2 NHC0 2

CH

2 Ph,

<OH

NHSO

2

NH

2 H N N 0 0 N

COOH

HN

H HNS0 2

C

4

H

9 WO 94/18981 WO 9418981PCTIUS94/01881 20 HN00 C N -Nl 'NC0 2 0H 3 H HNSO 2

C

4

H

9

,COOH

COOH

H HNSO2 -NHC 4

H

9

,COOH

N o"I C0 2

C

2

H

OH:

3 0- 0 2

H

H HNSO

H

3 WO 94/18981 WO 9418981PCT[US94/01881 21

HN

HN 00 N

I

-N

H

WO 94/18981 WO 9418981PCT/1JS94/01881 22 HN 0P N- C0 2

H

K-,N'N

I

H HNS0 2 C H 3 HN 0 N -y00 2

H

N-N HINH HN 0 N-N C2

H

0 H0 2 C t 0 NH

H

HN 0 0 N

H

HN 0 0 CO H N\

H

0 HN 0 C02H~

H

0 N Et 2 PCTIJS94IOI881 WO 94/18981 23 H N" O 0

HNN

0 KIC N-TC0 2

H

H HNSO 2

C

4

H

9

H

HN

NNH

0 N C H No"

I

PU :O /0 Z os 'HO OSNH

H

WOO'~

H H 6HO OS NH

H

HZOO

N

'3H3-0

OSN

HZOO

,HI'OOS NH'

NH

N3H N H tlz 18681/t,6 OMA T88TO/t6Sfil~ld

I

WO 94/18981 WO 9418981PCTIUS94/01881 25 HN 0 0 N- C 0 2

H

0 Nd 2 H HNS0 2

H

3 Additional examples of compounds of the invention are 0 0 N e- 000H H HNS0 2

OH

HN 0 0 N -N I 00H N.~N H HNS0 2

~/C

HN 0 N 0 NN C0 2

H

H HNS0 2

H

HN 0 N 0

H

H HN S0 2 andH CI__ I WO 94/18981 PCT/US94/01881 -26- HN o N J 0 ,N 1CO02H H HNSO 2

H

3 The term "pharmaceutically acceptable salts" shall mean 1o non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts include the following salts: Acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, valerate.

The term "pharmaceutically effective amount" shall mean that amount of a drug or pharmaceutical agent that wili elicit the biological or medical response of a tissue, system or animal that is being sought by a researcher or clinician. The term "anti-coagulant" shall include heparin, and warfarin. The term "thrombolytic agent" shall include streptokinase and tissue plasminogen activator. The term "platelet anti-aggregation agent" includes, for example, aspirin, ticlopidine, and dipyridamole.

The term "alkyl" means straight or branched alkane, alkene or alkyne.

The term "aryl" means a 5-10 membered unsaturated mono- or bicyclic ring group.

~I blL~ sil I 1_ I~p~d la~ WIO 94/18981 PCT/US94/01881 -27- The term "heteroaryl" means aryl containing 1, 2, 3 or 4 heteroatoms.

The term "heteroatom" means N, O, or S.

The term "cyclic;,' unless otherwise more-specificaly defined, means mono- or bicyclic saturated ring groups having 5-10 members.

The term "heterocyclic" means cyclic containing 1, 2, 3 or 4 heteroatoms.

In the compounds of the invention, heteroaryl groups and heterocyclic groups contain no more than 2 0 atoms or 2 S atoms.

The term "alkoxy" includes an alkyl portion where alkyl is as defined above.

The terms "arylalkyl" and "alkylaryl" include an alkyl portion where alkyl is as defined above and to include an aryl portion where aryl is as defined above. The CO-n or Ci-n designation where n may be an integer from 1-10 or 2-10 respectively refers to the alkyl component of the arylalkyl or alkylaryl unit.

The term "halogen" includes fluorine, chlorine, iodine and bromine.

The term "oxy" means an oxygen atom. The term "oxo" means The term "thio" means a sulfur atom. Under standard nomenclature used throughout this disclosure, the terminal portion of the designated side chain is described first followed by the adjacent functionality toward the point of attachment. For example, a C1-5 alkyl substituted with C1-6 alkylcarbonylamino is equivalent to H O I II

C

1 -salkyl Cl_ 6 alkyl In the schemes and examples below, various reagent symbols have the following meanings: dL-~ya 88_ ~dl WOv 94/18981 PCT/US94/01881 28 BOC(Boc): Pd-C:

DMF:

DMSO:

CBZ:

CH2C12: CHC1 3 EtOH: MeOH: EtOAc: HOAc:

BOP:

t-butyloxycarbonyl.

palladium on activated carbon catalyst.

dimethylformamide.

dimethylsulfcxide.

carbobenzyloxy.

methylene chloride.

chloroform.

ethanol.

methanol.

ethyl acetate.

acetic acid.

benzotriazol-1 -yloxytris(dimethylamino)phosphonium, hexafluorophosphate.

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

EDC:

Oxone: potassium peroxymonosulfate LDA: lithium diisopropylamide DMA: N,N-Dimethylaniline HOBT: Hydroxybenzotriazole Therapeutic Treatment Compounds of the invention may be used for inhibiting integrin protein-complex function relating to cell attachment activity.

They may be administered to patients where inhibition of human or mammalian platelet aggregation or adhesion is desired.

Certain compounds of the invention are eliminated from circulation rapidly and are particularly useful in inhibiting platelet aggregation. Thus, these compounds may find utility in surgery on peripheral arteries (arterial grafts, carotid endaterectomy) and in cardiovascular surgery where manipulation of arteries and organs, 3 and/or the interaction of platelets with artificial surfaces, leads to platelet aggregation and consumption. The aggregated platelets may form thrombi and thromboemboli. They may be administered to these surgical patients to prevent the formation of thrombi and thromboemboli.

dL~Ld~ ~L WO 94/18981 PCT/US94/01881 -29 The compounds of the present invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures; suspensions, syrups, and emulsions; Likewise, they may be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, sublingual, intranasal or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an anti-aggregation agent.

Compounds of the invention may be administered to patients where prevention of thrombosis by inhibiting binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor is desired. They are useful in surgery on peripheral arteries (arterial grafts, carotid endarterectomy) and in cardiovascular surgery where manipulation of arteries and organs, and/or the interaction of platelets with artificial surfaces, leads to platelet aggregation and consumption. The aggregated platelets may form thrombi and thromboemboli. They may be administered to these surgical patients to prevent the formation of thrombi and t:';omboemboli.

Extracorporeal circulation is routinely used for cardiovascular surgery in order to oxygenate blood. Platelets adhere to surfaces of the extracorporeai circuit. Adhesion is dependent on the interaction between gplb/IIa on the platelet membranes and fibrinogen adsorbed to the surface of the circuit. (Gluszko et al., Amer. J.

Physiol., 252(H), 615-621 (1987)). Platelets released from artificial surfaces show impaired hemostatic function. Compounds of the invention may be administered to prevent adhesion.

Other applications of these compounds include prevention of platelet thrombosis, thromboembolism and reocclusion during and after thrombolytic therapy and prevention of platelet thrombosis, thromboembolism and reocclusion after angioplasty or coronary and other arteries and after coronary artery bypass procedures. They may also be used to prevent myocardial infarction.

~-~llsera~sllsss~b~ Lk eQ~ Il~p~ I I N Ov 9/18981 PCT/US94/01881 The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to-be' treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day and preferably 0.05-100 mg/kg/day and most preferably 0.1-20 mg/kg/day.

Intravencu. the most preferred doses will range from about 1 to about 10 Lg/kg/minute during a constant rate infusion. Advantageously, compounds of the present invention may be administered in divided doses of two, three, or four times daily. Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal y routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather that intermittent throughout the dosage regime.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are i; typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixers, syrups and the like, and consistent with convention pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, b ~la~--rr ~PPp~P~, WvO 94/18981 PCT/US94/01881 31 dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier-such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or betalactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene 1o glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate,.sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch methyl cellulose, agar, bentonite, xanthan gum and the like.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.

I

C -U s WOo 94/18981 PCTIUS94/01881 32- The compounds of the present invention can also be coadministered with suitable anticoz ulants, including antiplatelet agents such as heparin, aspirin, warfarin, dipyridamole and other compounds and agents'known to'inhibit blood clot formation, and thrombolytic agents such -as plasminogen activators or streptokinase, to achieve beneficial effects in the treatment of various vascular pathologies.

The novel compounds of the present invention were prepared according to the procedure of the following examples. The most preferred compounds of the invention are any or all of those specifically set forth in these examples. These compounds are not, however, to be construed as forming the only genus that is considered as the invention, and any combination of tle compounds or their moieties may itself form a genus. The following examples further illustrate details for the .preparation of the compounds of the present invention.

Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless otherwise noted.

In addition to the following preparative procedures, several examples of in vitro bioactivity of compounds within the scope of the present invention are indicated. To illustrate, one test which is used to evaluate fibrinogen receptor antagonist activity is based on evaluation of inhibition of ADP-stimulated platelets. Aggregation requires that fibrinogen bind to and occupy the platelet fibrinogen receptor site.

Inhibitors of fibrinogen binding inhibit aggregation. In the ADPstimulated platelet aggregation assay used to determine inhibition associated with the compounds claimed in the instant invention, human platelets are isolated from fresh blood, collected into acid citrate/dextrose by differential centrifugation followed by gel filtration on Sepharose 2B in divalent ion-free Tyrode's buffer (pH 7.4) containing 2% bovine serum albumin.

Platelet aggregation is measured at 37 0 C in a Chronolog aggregometer. The reaction mixture contains gel-filtered human platelets (2 x 108 per ml), fibrinogen (100 micrograms per ml (ug/ml)), I ~e~L BB c s WOo 94/18981 PCT/US94/01881 -33- Ca 2 (1 mM), and the compound to be tested. The aggregation is initiated by adding 10 mM ADP 1 minute after the other components are added. The reaction is then allowed to proceed for at least 2 minutes. The extent of inhibition of aggregation'is expressed as thepercentage of the rate of aggregation observed in the absence of inhibitor. The IC50 is the dose of a particular compound inhibiting aggregation by 50% relative to a control lacking the compound.

In the following examples, all temperatures are in degrees Celsius, unless otherwise indicated.

SCHEME A Freparation of sulfonamide intermediate compounds

CONH

2 NaOH

H

2 0/dioxane H2N- CO 2

H

SH n-C 4 HgSO 2

CI

CONH

2

(C

4

H

9

)SO

2 NH

CO

2

H

H

A2 1. Br, NaOH 2. Boc20, NaOH NH Boc

THF/H

2 0

(C

4

H

9 )S02NH CO 2

H

H

A-3 L-Asparagine-o(-butanesulfonamide (also N-(n-Butyl-sulfonyl)-Lasparagine) A-2 I' I g ~b ~118~ I WO 94/18981 PCT/US94/01881 -34- A solution containing L-asparagine (6.45 g, 48.9 mmol) and NaOH (2.0 g, 50.0 mmol) in 100 ml of 50% aqueous dioxane was cooled to 0° in an ice bath. To this rapidly stirred mixture, a solution of NaOH (2:2 g, 55.0 mmol) in 50 ml of water and neat butane sulfonyl chloride (7.0 ml, 53.9 mmol) were added alternately over a period of min. The reaction solution was concentrated to a volume of 50 ml at reduced pressure and aqueous residue was cooled, acidified with concentrated HC1, and extracted into ethyl acetate (3 x 100 ml). The organic extracts were dried over Na2SO4 and concentrated to a volume of approximately 50 ml, anhydrous ether (50 ml) was added and the resulting white precipitate was isolated by vacuum filtration yielding A-2, mp. 154-155°.

Boc-a-butane sulfonamido-P amino alanine (also 2(S)-(n-Butyl- 5sulfonylamino)-3-(N-Boc-aminopropionic acid) A-3 A solution containing NaOH (6.04 g, 151 mmol) in 50 ml was cooled to 0° and bromine (1.40 ml, 26.9 mmnol) was added.

The resulting solution was stirred at 00 for 5 min. Next, a cooled solution of A-2 (5.23 g, 20.7 mmol) and NaOH (1.66 g, 41.4 mmol) in 15 ml of H20 was added at once and mixture stirred at 00 for 5 min then heated to 800 for 15 min. The solution was then cooled tc 250 and acidified with 12N HCI (11 ml) and stirred until gas evolution ceased.

The solution was then made basic by the addition of 2N NaOH and ml of THF was added along with di-t-butyldicarbonate (9.0 g, 41.4 mmol). After stirring overnight at 250 the THF was removed at reduced pressure and the basic aqueous phase extracted with ethyl acetate (2 x 50 ml). The aqueous phase was then made acidic with KHSO4 and extracted with ethyl acetate (3 x 100 ml). The pooled acidic extracts were dried over Na2SO4 filtered and evaporated giving A-3 as a white solid, mp 111-112°.

-pap~p bla IIEP(IIII WO 94/18981 PCT/US94/01881

HCI

NH Boc NH 2

(C

4 H)SONH CO 2

(C

4 Hg)SO 2 NH C0M H H 3. A-4 2(S)-(n-Butylsulfonylamino)-3-aminopropionic acid (A-4) A solution of A-3 (3.83 g, 11.8 mmol) in 200 ml of ethyl acetate was cooled to HCI gas was bubbled through the solution for min. The solution was then warmed to 250 and stirred for 30 min then concentrated at reduced pressure to 50% of its volume and diluted with 100 ml of ether. The resulting white solid was collected by vacuum filtration giving A-4 as a solid.

Ethyl 2(S)-(n-Butylsulfonylamino)-3-amino propionate A solution of A-4 (1.0 g, 3.8 mmol) in 50 ml of anhydrous ethanol was saturated with HCI gas then heated at reflux for 3.0 h. The solvent was evaporated to afford pure A-5 as a white solid.

1 H NMR (300 MHz, CD30D) 8 4.38 2H); 4,32 1H); 4.23 (m, 2H); 2.85 2H); 1.65-1.45 4H); 1.3 2H); 0.96 3H).

Ib,~ L~ ~f ~L ~a Is~ L- I I L- WO, 94/18981 PCT/US94/01881 36-

H

2 N CO 2

H

NH

2 0 H NaOH

H

2 0/dioxane

CH

3

SO

2

C

H

2 N C02H H NHS 2

CH

3 0CH

H

2 N

NHSO

2 H 3

A-H

A-7 anH2SO4 Dioxane "CO 2 tBu

H

2 N

NH-SO

2 CH3 N-Tosyl-L-Asparainte L-Asparagine (10.0 g, 75.7 mmol) was placed in a 500 ml round bottom flask equipped with a magnetic stir bar and an addition funnel. IN Sodium hydroxide (85 ml, 1.1 eq.) was added. p-Toluenesulfonyl chloride (15.88g, 83.27 mmol) was dissolved in ethyl acetate (100 mi). This solution was added to the reaction flask with vigorous stirring. IN Sodium hydroxide (85 ml, 1. leq.) was placed in the addition funnel, then added dropwise with vigorous stirring over a 2 h period. The reaction mixture was stirred ~L II LLL~ L~b~ ~3~C~PI ~gC I L_ WOr 94/18981 PCT/US94/01881 37 an additional 2 h, at room temperature. The organic and aqueous layers were separated and the aqueous layer was washed with ethyl acetate (2x50 ml). The aqueous liquid was cooled to 0° then acidified with hydrochloric acid (cone:). A white crystalline solid was obtained.

Recrystallization from hot water yielded A-6.

1 H NMR (300 MHz DMSO-d6) 6 7.91 J=8.79 Hz, 1H); 7.64 (d, J=8.06 Hz, 2H); 7.32 d (overlapping), J=8.06 Hz, 3H); 6.87 br, 1H); 4.03 1H); 3.32 H20); 2.49 IH); 2.43 d, J=7.08, 15.38 Hz, 1H); 2.35 3H); 2.21 d, J=6.11, 15.38 Hz, 1H).

2(S)-Tosylamino-3-aminopropionic acid (A-7) A solution containing NaOH (22.0 g, 550 mmol) in 100 ml was cooled to 0° and bromine (5.03 ml, 97.5 mmol) was added dropwise. The resulting solution was stirred at 0° for 10 min., then a cooled solution of A-6 (21.5 g, 75.0 mmol) and NaOH (6.68 g, 161 mmol) in H20 (50 ml) was added in a single portion. After stirring at 0° for 20 min, the reaction was heated to 800 for 30 min, then cooled.

The cooled solution was adjusted to pH=7 with concentrated HC1 and the resulting white solid filtered to give A-7.

1 H NMR (300 MHz, DMSO-d6) 8 8.2-7.2 (br, 2H, (NH,COOH)); 7.70 J=8.18 Hz, 2H); 7.38 (J=8.18 Hz, 2H); 3.7-3.0 (br, 2H, (NH2, 3.12 J=4.76 Hz, IH); 2.99 d, J=4.64, 11.96 Hz, IH); 2.79 2 d, J=9.52, 11.96 Hz, 1H); 2.36 3H).

tert-Butyl-2(S)-(Toluenesulfonvlamino)-3-amino propionic acid (A-8) A -7 (5.0 g, 19.4 mmol) was suspended in Dioxane (100 ml) in a 1 liter pressure bottle. The bottle was cooled to -15 0 C and 3 isobutylene (100 ml) was condensed into the dioxane. Concentrated H2S04 (5 ml) was added and the bottle sealed and stirred at room temperature for 36 h. The bottle was opened, and the excess isobutylene carefully vented. The solution was diluted with ethyl acetate (200 ml) and washed with IN NaOH, (200 ml). The organic layer was Y~srar~ apl~-- ll~ prrr I 1- WOr 94/18981 PCT/US94/01881 38 dried (Na2SO4), filtered and evaporated to give A-8 as a white crystaline solid.

IH NMR (3CMH.z, CDCl3) 6 7.68 J 8.18 Hz, 2H); 735'-(d, J=- 8.18 Hz, 2H); 3.85 2.93-2.79 2H); 2.32 3H); 1.38 (s, 9H).

SCHEME 1 tBuO 2 C

H

2 NNH2 I EtOH N CI 0

II

C13COCOCC13 toluene

NHNH,

H

0 tert-Butyl 2-Hydrazinopyridine-3-carboxvlate (1-2 A solution of tert-butyl 6-chloro-nicotinate (735 mg, 3.44 mmol) in ethanol (5 mL) was cooled to 0° and treated with anhydrous hydrazine (2.75 g, 86 mmol) dissolved in ethanol (5 mL). This mixture was allowed to reach 250 and stirred 20 h and then warmed to 600 for 2 h. The mixture was dissolved in water and extracted with ethyl acetate. The organic portion was washed with water and brine, dried (Na2SO4), and concentrated to give 1-2 as an oil that was used directly in the next step.

r 4 ~ap

I

WO 94/18981 WO 9418981PCTJUS94/01881 -39 tert-Butyl [2,3-Dihydro-3-oxo- 1,4-triazolo-[4,3-a]pyridin-6-yl]carboxylate (1-3) 1-2 was dissolved in 35 mL of toluene and slowly added to a refluxing, solution of bis(trichloromethyl)carbonate (I1. g, 1.9 in toluene (35 rnL). This was further refluxeci for 1.2 h and cooled, added to water and extracted with ethyl acetate. The organic portion was dried (Na2SO4), concentrated, and flash chromatographed on silica gel to yield 1-..L IH NMR (DMSO-d6, 300 MHz): 5 1.58 7.25 I1H); 7.45 8.25 1H); 12.40 1H).

ICH

2

CH

2 1N tBuO 2

C

tBuO 2 C 14 CBZ N- N 2.TFA

(OH

2 2 1-3

CBZ

WO 94/18981 WO 948981PCIS94/01881 40 0 11 HN -CH 2

CH

2 OH CICOCH 2 Ph CBZ-ND CH 2

CH

2 0H NaHCO 3

CH

2 01 2

H

2 0 12, Ph 3

P

imidazole benzene

CH

2

CH

2

I

1-4 2(N-CBZ-Pip~eridin-4-vl)ethvl iodide (1-4) A mixture of (2-b ydroxyethyl)piperi dine] (Aldrich) g, 38.7 mmol), sat. NaHCO3 (50 ml) and C112C02 (150 ml) was treated with benzyl chioroformate (6.05 ml, 42.5 mmol). After stirring at room temperature for 2.5 h, the organic layer was removed and with H20 and brine, dried (Na2SO4), then filtered and concentrated to give the protected alcohol as a colorless oil.

IH NMR (300 MHz, CDCl3) 8 7.26 (in, 5H); 5.13 2H); 4.23 2H); 3.63 2H); 2.85 2H); 1.83 2H); 1.68 (in, 2H); 1.43 (in, 1H); 1.03 (mn, 2H); This protected alcohol (6.3 g, 23.9 inmol) was combined with Ph3P (7.0 g, 23.9 inmol), iodine (6.06 g, 23.9 inrol), and imidazole (1.95 g, 28.7 minol) in benzene (100 ml) and refluxed for L L _I~ WO 94/18981 PCT/US94/01881 -41 h. The solution was cooled, filtered and then concentrated. The residue was chromatographed on silica gel (1:1 ethyl acetate/Hexane) to give 1-4 as a white solid.

1H NMR (300 MHz, CDC13) 8 7.26 5H); 5.14 2H); 4.23 2H); 3.86 4H); 1.85 2H); 1.68 2H); 1.45 1H); 1.03 2H).

3-[[(2,3-dihydro-3-oxo-[2-(N-CBZPiperidin-4-yl)ethyl]-1,2,4triazolor4,3-al pyridin-6-yllcarboxylic acid 1-3 (350 mg, 1.6 mmol) dissolved in 20 ml of acetonitrile was treated with powdered potassium carbonate (630 mg) and heated to 600 for 20 h. The mixture was cooled to 250 added to water and extracted with ethyl acetate. The organic portion was dried (Na2SO4), concentrated, and flash chromatographed on silica gel (35% ethyl acetate in hexane) to give the desired ester as a yellow oil.

The crude tert-butyl ester was converted to the acid by treating with 15 mL of methylene chloride and 15 mL of trifluoroacetic acid at 0° and then warming to 250 for 1.2 h. The mixture was concentrated to dryness under vacuum, added to water and extracted with ethyl acetate. The organic portion was dried (Na2SO4), concentrated, and crystallized from ethylacetate/ether (25/1) to give as a pale yellow powder.

1 H NMR (DMSO-d6, 300 MHz): 5 1.05 2H); 1.45 1H); 1.75 4H); 2.79 3.95 4H); 5.05 2H); 7.30 6H); 7.50 (d, 1H); 8.25 1H).

~a I~ I1ICT-- vO 94/18981 PCT/US94/01881 -42-

HOOC-\

HO

2 C 1. HOBT, DIEA, ED C N H HCI.H2 N/v

C

O2Et N N 2. LOH N THF/HO20 O (CH 2 2

-N

CBZ

-N

0 (CH,)2

CBZ

3-[[[(2,3-Dihydro-3-oxo-2[2-(N-CBZ-Piperidin-4-yl)ethyl]- 1,2,4triazolo[4.3-alPyridin-6-vlcarbonylIaminolpropionic acid (1-6) A solution of 1-5 (75 mgs, 0.18 mmol) in dimethylformamide (1 mL) was treated sequentially with hydroxybenztriazole mgs, 0.26 mmol), diisopropyl ethyl amine (87 gl, 0.5 mmol), ethyl 2-aminopropionate hydrochloride (40 mgs, 0.26 mmol), and EDC mgs, 0.26 mmol). This mixture was stirred at 250 for 15 h. The mixture was dissolved in water and extracted with ethyl acetate. The organic portion was washed with water and brine, dried (Na2SO4), and concentrated to provide the desired ester.

This crude ethyl ester wa: dissolved in 5 mL of THF. 5 mL of water and treated with 0.37 mL of IN aqueous LiOH solution. This was stirred 3 h at 25°. The mixture was dissolved in water and extracted with ethyl acetate. The organic portion was washed with water and brine, dried (Na2SO4), and concentrated to a provide 1-6, mp 201-203° (dec).

WO 94/18981 WO 4/1981PCTI US94/01881 43 H0 2

C~-

Me 3 SH, CH 3

CN

NHN<I-)NC\

NN

1-7 3-14 [(2,3-Dihydro-3-oxo-2-(2-(piperidin-4-yl)ethyl] -1 ,2,4-triazolo- [4.3 -a-1pvridin-6-yllcarbonyll aminoipropionic acid (1 -7) A solution of 1-6 (64 mg, 0. 129 mmole) in acetonitrile ml) at 0' was treated with iodotrimethylsilane (107.0 mg, 0.533 mmole) and the reaction stirred 0.5 h. The reaction was quenc 'hed into water, extracted with diethyl ether and chromatographed on silica using EtOH/NH4OHIH2O (10111) to give upon concentration a white foam.

Crystallization from ethanol gave 1-7 as a white solid, mp 267-269'.

WO 94/18981 WO 9418981PCTIUS94/01881 44

C'CO

2 i-BU, Et 3

N

CH

2

C'

2 ,C0 2

H

H

2

NK>(

NHSO

2 C4Hq A-4 0 Me 3 SiI 1-1,,,C02H

CH

3

CN

NH

N H NHS 2

C

4 Hq

H

1-9 2(S)-[6-Butylsulfonyl)amino]-3[[[2,3-dihydro-3-oxo-2-[2-(N-CBZ- Piperidin-4-y1!A)e,,hyl]-1I,2,4-triazolo [4,3-aljPyridin-6-yl] carbonyl[amnino Propionic acid (1-9) was coupled to A-4 as described for 1-6 to provide 1-8.

lH NMR (300 M14z, DMSO d6) 8 8.26 I 8.50 IRH); 7.63 (d, 1H); 7.54 11H); 7.4-7.3 1 (in, 6H); 5.01 211); 4.10 (mn, 1H); 3.96 (in, 4H); 3.60 (in, 1H); 3.46 (mn, 111); 2.95 2H); 2.73 (brm, 2H); 1.71 (mn, 1.53 (mn, 2H); 1.46 (mn, 1H); 1.01 (in, 211); 0.83 3H).

WO 94/18981 WO 9418981PCTIUS94/01881 45 2(S)-[(n-Butylsulfonyl)amino] -3 [[[2,3-dihydro-3-oxo-2-[2-(piperidin-4yI)ethyl] -1 ,2,4-triazolo [4,3-a]Pyridin-6-yl] carbonyl]atmino propionic acid (1 -9) was, treated, with trimethylsilyl iodide in CH3CN as described for 1-7 to afford 1-9.

1 H NMR (300 Mliz, D20) 6 8. 10 I1H); 7.43 ILH); 7.15 ILH); 4.00 (in, 4H); 3.60 2H); 3.31 (in, 2H); 3.23 3.93 (in, 2H); 103.8 1 2H); 1.93 2H); 1.80 (in, 2H); 1.6-1.23 (mn, 7H); 0.85 3H).

SCHEME 2

HO

2 C

CH

3 0H CH 3 0 2

C

N OH 3 N OH 3 2-1 C32 NBS, BPO

"H

3 0 2

C

CC1 4 NI Br 2-3 Methvy1 6-ne thylpvyri dine 3-carboxv late (2-2) A solution of 6-methyl nicotinic acid, 2-1 (5 g, 36.5 inmol) in 100 ml of anhydrous methanol was placed in a 250 ml three neck flask equipped with a dropping funnel vertical condenser and CaCI2 drying tube. The reaction solution was cooled to -l15' in an ice acetone bath and SOC12 (5 ml, 69.1 innol) was acided dropwise. The solution was then heated at reflux for 3 h then cooled and the solvent removed at reduced pressure. The resulting white solid was treated with 60 ml of saturated NaHCO3 and extracted into CH2CI2 (3x50 ml). The pooled -bl ~I WO 94/18981 PCT/US94/01881 -46extracts were dryed (Na2SO4 filtered and evaporated at reduced pressure. The resulting oil crystallized on standing giving 2-2 as a white solid.

1 H NMR (300 MHz, CDC13) 8 9.05 J 1.4 Hz, 1H); 8.08 (dd, J=1.4 and 6.8 Hz); 7.19 J=6.8 Hz, 1H); 3.98 3H); 2.63 3H).

Methyl 6-Bromoethylpyridine-3-carboxylate (2-3) 2-2 (10.6 g, 71.5 mmol) was combined with NBS (12.73 g, 071.5 mmol), 100 mg benzoyl peroxide and 200 ml CC14 and refluxed under an inert atmosphere for 18 h. The reaction solution was cooled, filtered, and concentrated to a viscous orange oil which was flash chromatographed on silica gel using 20% ethyl acetate in hexane giving 1 the desired pyridyl bromide 2-3.

1H NMR (300 MHz, CDC13) 5 9.05 J=1.4 Hz,, 1H); 8.08 (dd, J=1.4 and 6.8 Hz); 7.19(d, J=6.8 Hz, 1H); 5.38 2H); 3.98 3H).

s~ a IruP----rsss-~qs WO 94/18981 WO 9418981PCTIUJS94/01881 47 BOON/

CH

2

CH-

2

NH-

2

CH

3 0 2

C

2-4,

CH

3

CN

CO

2

CH

3

BOCNQ-(CH

2 2

-NH

triphosgene DMA, toluene BoN 0 C I 0 Methyl 6-[2-(N-Boc-Piperidin-4-yl)ethylamino]-methylpyridine-3carboxvlate A mixture of 2-3 (1.0 g, 4.34 mmol), 2-4 (2.16 g, 10.0 mmol) and K2C03 (0.66 g, 4.4 mmol) in 100 ml of anhydrous CH3CN was placed in a 250 ml flask and refluxed for 3 h then cooled and filtered. The filtrate was concentrated at reduced pressure and chromatographed on silica gel using 10% CH3OI-/EtOAc as eluent to afford 2-5 as a yellow residue.

WO 94/18981 WO 9418981PCTIUS94/01881 48 1 H NMR (CDCI3) 859. 18 J= 1.4Hz, I 8. 15 (dd, J= 1.4 and 6.8 Hz, 1H); 7.39 J=6.8 Hz, 1H); 4.08 (br d, J=12 Hz, 2H); 3.98 2 H); 3.95 3H); 2.75 (overlapping m, 6H); 1.78 J=12.Hz, 2H); m, 1.4 9H); 0.98 (in, 21H).

Methyl 1 -(Chlorocarbonyl)-2,3-dihydro-3 -oxo-2-[[2-(N-Bocp2iperidin-4-vl)ethyllimidazo[ 1 5-alpyridin-6-vllcarboxylate (2-6) (480 mg, 1.27 inmol) was dissolved in 50 ml of toluene, N,N- dimethyl aniline (645 ml, 4.08 mm-ol) was added and the solution cooled to 00. To this, a solution of triphosgene 13 g, 3. 18 rnmol) in 15 ml toluene was added dropwise over 30 min. The solution was then heated to 100' for 1.5 1h then cooled, washed twice with IN RdI, water and brine (50 ml of each), dried over Na2SO4 and evaporated giving 515 mg of a yellow crystalline, solid 2-6.

I H NMR (CDCl3) 8 8.82 J=1.4Hz, I S. 25(d, J=6.89 Hz, I 7.91 J=6.89 Hz, 1IH) (dd, J= 1.4 and 6.8 H-z,1I H; 4.08 (br d, J= 12 Hz, 2H); 3.98 2 3.95 3H); 2.75 (overlapping mn, 6H); 1.78 J=12.Hz, 2H); 1.5 (over lapping mn, 4H); 1.4(s, 9H); 0.98 (mn, 2H).

0 BocNO ?7 Na CO 2

CH

3 Et 2

NH*HCI

(CH

2 N -~CH 2

CI

2 NaHCO 3 Ck 0 0 BOCNO (0H 2 2 -N -N C2H (Et)NP 0 2-7 I L. L-Csl WO 94/18981 PCT/US94/01881 -49 Methyl 1-[(N,N-Diethylamino)carbonyl]-2,3-dihydro-3-oxo-2-[[2-(N- Boc-piperidin-4-yl)ethyl]-imidazo[ ,5-a]pyridin-6-yl]carboxylate (2-7) g,,0.64 mmol) was dissolved in 100 ml of CH2C12 and diethylamine hydrochloride (105 mg, 0.97 mmol) was added along with 50 ml of saturated NaHCO3 solution. This biphasic mixture was stirred for 1 h, then the organic layer was separated and washed with citric acid then brine (50 ml), dried over Na2SO4 and evaporated giving 2-7.

1 H NMR (CDC13) 5 8.45 J=1.4Hz, 1H); 7.15 (dd, J=1.4 and 6.8 Hz, 1H); 6.78 J=6.8 Hz, 1H); 4.08 4H); 3.95 3H); 3.51 4H); 2.75 3H); 1.78 J=12.Hz, 2H); 1.5 (over lapping m, 4H); 1.4 9H); 1.1 6H); 0.98 2H).

tert-Butyl-l [[(N,N-diethylamino)carbonyl]-2,3-dihydro-3-oxo-2[2-(N- Boc-piperidin-4-yl)ethyl]imidazo[ 1,5-a]Pyridin-6-yl]carbonyl]amino propionate (2-8) 2-7 (315 mg, 0.64 mmol) was dissolved in 10 ml of CH30H, 15 ml H20 and 0.725 ml IN NaOH added and mixture stirred at room temperature for 3.5 h. The organic solvent was removed at reduced pressure and the aqueous residue acidified with citric acid and extracted with CH2C12. The organic extracts were washed with and brine then dried over Na2SO4 filtered and evaporated to give-the 2 desired acid.

1 H NMR (CDC13) 8 8.32 J=1.4Hz, 1H); 7.10 (dd, =1.4 and 6.8 Hz, 1H); 6.78 J=6.8 Hz, 1H); 4.08 4H); 3.51 4H); 2.75 3H); 1.78 J=12.Hz, 2H); 1.5 (overlapping m, 4H); 1.4 9H); 1.1 0.98 (2H).

6H); 0.98 2H).

%e~8slB~P I ~a~ WO 94/18981 WO 9418981PCTIUS94/01881 50 BocN C22N N C0 2 0H 3 (Et) 2 N P1. NaOH, CH 3 OH, H 2 0 0 2. CICO 2 -i -Bu, Et 3

N

HCI.H

2 N 2 tBU Z-1 0H 2 C1 0 0N

CA

BocN (CH 2 2

-N

0 2-8 N Et 2

HCI

EtOAc

(CH

2 2 -N ,.N (Et) 2

N

0 2-9 This acid (105 mg, 0.215 mmol) was dissolved in 10 ml of CH2CI2, Et3N (48m1, 0.47 mmol) was added and solution cooled to 30-10'. Next, isobutyl chioroformate (30 ml, 2.36 mmol) was added and mixture stirred at -10' for 30 min. To this a solution of (3-alanine tert-butyl ester hydrochloride (58.7 mg, 323 mmol) and Et3N (32 ml, 0.323 mrnol) in 10 ml of CH2CI2 was added and solution wafmed to room temperature. Reaction solution washed with 10% citric acid, WO 94/18981 Wo 9418981PCTIUS94/01881 51 and brine (10 ml each) and dried over Na2SO4, concentrated and chromatographed giving 2-8.

IH NM-R (CDCI3) 568.16 11-i); 7.02 Hz, 6..78 J=6.8 1H); 6.63 5.6Hz, 111); 4.08 (in, 4H); 3.51 (in, 6H); 2.75 (in, 2H); 2.45 (mn, 2H); 1.78 J=12.Hiz, 2H); 1.5 (overlapping m, 6H); 1.4 (s, 9H); 1.37 9H1); 1. 1 6H); 0.98 (in, 2H).

-[(N,N-Diethylamino)carbonyl] -2,3-dihydro-3-oxo-2[2-(piperidin-4vethvllimidazof 1 5-alpvridin-6-vllcarbonvllpropionic acid (2-9) 2-8 (100 mg, 0. 16 minol) was dissolved in 20 m1 of ethyl acetate anhydrous HCI was passed through the solution. at 0 0 C for 5 min then the mixture was stirred at room temperature for 1 h. The solvent evaporated at reduced pressure and the residue triturated with ethyl acetate and filtered to give 2-9.

III NMR (DMSO-d6) 6 8.90 (br s, 1H); 8.60 (br s, LH); 8.3 IH); 7.6 3H); 7.1 111); 6.89 IH); 4.08 (in, 4H); 3.51 (in, 6H); 2.75 (in, 2H); 2.45 (mn, 2H); 1.78 J=12.Hz, 2H); 1.5 (overlapping mn, 6H); 1. 1 6H); 0.98 (in, 2H).

HNa

CH

2

CH

2 OH B0C 2 0. BOO

CH

2

CH

2 OH 1,P3 imidazolea benzene 2-11 BOCNa

OH

2

CH

2 I 1. NaN 3 DMSO ON -HC22 2. Ph 3 P, THF 2-4 WvO 94/118981 PCT/US94101881 -52- 2-(N-Boc-Piperidin-4-vl)ethanol (2-11) 4-Piperidine-2-ethanol (2-10) (AlcLdch) (130 g, 1.0 mole) was dissolved in 700 mL dioxane, cooled to 0° and treated with 3 N NaOH (336 mL, LO mole), and di-t-butyldicarbonate (221.8 g, mole). The ice bath was removed and the reaction stirred overnight.

The reaction was concentrated, diluted with water and extracted with ether. The ether layers were combined, washed with brine, dried over MgS04, filtered and evaporated to give 2-11.

Rf 0.37 in 1:1 EtOAc/Hexances, ninhydrin stain.

1 H NMR (300 MHz, CDC13) 8 4.07 (bs, 2H); 3.7 (bs, 2H); 2.7 J 12.5 Hz, 2H); 1.8-1.6 6H); 1.51 9H); 1.1 (ddd, J 4.3, 12.5, 12 Hz, 2H).

2-(N-Boc-Piperidin-4-yl)ethyl iodide (2-12) 2-11 (10.42 g, 0.048 mol) was dc~solved in 400 ml benzene, imidazole (4.66 g, 0.068 mol), triphenylphosphine (15.24 g, 0.05 mol) and iodine (0.048 mol) were added at room temperature. After 6 hours the reaction mixture was filtered and the filtrate was evaporated to give a dark residue. This was purified by flash chromatography on silica gel eluting with 10% EtOAc-hexanes to give 2-12 as a yellow oil.

2-(N-Boc-Piperidin-4-yl)ethyl amine (2-4) To 2-12 (27.9 g, 0.082 moles) dissolved in DMSO (400 ml) was added sodium azide (5.01 g, 0.086 moles) as room temperature and the resulting solution was heated at 650 for 2 h. The cooled reaction mixture was diluted with 250 ml EtOAc, extracted with 2 x 100 ml portions of water 2 x 50 ml portions of brine and then dried (MgSO4).

Solvent removal provided the desired azide as a pale yellow oil, Rf (silica gel, 70% acetone/hexane).

This azide (19.3 g, 0.076 moles) in THF (400 (195 ml) was added triphenylphosphine (80.0 g, 0.305 moles) in one portion at room temperature. This was stirred at room temperature 3 'r-lc~J~l ~L41 I I WO 94/18981 PCT/US94/01881 -53 hours and the organic solvents were then removed in vacuo. The residue was acidified to pH 2 with 10% KHSO4 solution and this was extracted 4 x 100 ml portions of EtOAc. The organic extract was extracted withl2x. 100 mol portions of 10% KHSO4 and the aqueous phases were combined and the pH was adjusted to 10 with 2N NaOH.

This solution was extracted with 4 x 200 ml portions of CH2C12. These were combine dried (MgSO4) and the solvent was removed to give 2-4 as an oil. Kf 0.3 (silica gel, eluting with 10% CH30H in CHC13/NH3).

1 H NMR (300 MHz, CDC13) 8 4.05 (broad, 2H); 2.72 J 7.2 Hz, 2H); 2.62 2H); 1.64 J 12.2 Hz, 2H); 1.43 9H); 1.42-1.32 5H); 1.09 2H).

SCHEME 3

CO

2

H

SN 1. HCI, CHO3H COC H H0 2 C N 3 2. K 2

CO

3

,CH

3 CN H Br 2

CH

2

CH

2 Br H3C0 2 C N Br 3-1 3-2 Dimethyl I-(2-Bromoethyl)pyrazole-3,5-dicarboxylate (3-2) A solution of pyrazole-3,5-dicarboxylic acid (75 g, 431 mmol) in 11 of anhydrous methanol was treated with anhydrous HCI gas. The HCI addition was continued for 30 min after which, the solution was allowed to cool to room temperature and allowed to stand for 16 h. The solution was then heated at reflux for 3 h then cooled and the solvent removed at reduced pressure. The resulting white solid was ~-~PI~Pslesa~ s -C-~4Pe-~_ds-a c~s~b I~ WO 94/18981 PCT/US94/01881 54 treated with 600 ml of saturated NaHCO3 and extracted into CH2C12 (3x500 ml). The pooled extracts were dried (Na2SO4), filtered and evaporated at reduced pressure. The resulting white solid was recrystalized from methanol with the addition of anhydrous ether to give dimethyl pyrazole-3,5-dicarboxylate (3-la).

IH NMR (CDC13) 5 7.38 1H); 3.98 3H); 3.93 3H).

A solution of this ester (5.0 g, 27.2 mmol) in 150 ml of anhydrous acetonitrile was treated with K2C03 (5.2 g, 40.0 mmol) and 1,2-dibromoethane (25.0 ml, 291 mmol). The resulting mixture was heated to reflux under argon. After 25 min the reaction suspension was cooled, filtered and the filtrate evaporated to dryness at reduced pressure and placed on a high vacuum line for 12 h. The resulting white solid was recrystalized from hexane to give 3-2 as a white solid.

1 H NMR (CDC13) 8 7.38 1H); 5.03 J=8.2 Hz, 2H); 3.98 3H); 3.93 3H); 3.75 J=8.5 Hz, 2H).

C0 2

CH

3

H

3 CO2C N C BccN CCH 2

CHNH

2 2-4 DIEA, KI Br CH 3

CN

3-2 BocN (CH2)2-N CO 2

H

BooN N -L Y- WkO 94/18981 PCT/US94/01881 Methyl-[4,5,6,7-Tetrahydro-4-oxo-5-[2(N-Boc-Piperidin-4-yl)ethyl]pyrazolo[ 1.5-alpyrazin-2-yl1carboxvlate (3-3) A solution of 3-2 (14.0 g, 48.0 mmol), diisopropylethyl amine (25 ml, 144 mmol), Boc-4-aminoethylpiperidine (12.0 g,,52.6 mmol), and potassium iodide (2.39 g, 0.3 mmol) in 250 ml CH3CN was refluxed under N2 for 4.5 h then cooled, filtered and evaporated at reduced pressure. The resulting yellow residue was chromatographed on silica gel using EtOAc as eluent to give 3-3 as an off-white crystalline solid.

IH NMR (300 MHz, CDC13)5 7.15 1H); 4.29 J=7.0 Hz, 2H); 3.93 (br d, J=12 Hz, 2H); 3.76 3H); 3.61 J=5.3 Hz, 2H); 3.42 (t, J=7.3 Hz, 2H); 2.65 J=7.6 Hz, 2H); 1.55 J=12.5 Hz, 2H); 1.38 (m, 2H); 1.33-1.25 1H); 1.27 9H); 1.01 2H).

A solution containing LiOH (14.05 mg, 0.335 mmol) in ml H20 was added to a solution of 3-3 (90.81 mg, 0.223 mmol) in ml CH30H and the mixture was heated to 600 for 2.5 h then cooled, and the CH30H removed at reduced pressure. The remaining aqueous phase 2 was acidified with 10% aqueous citric acid and extracted with CH2C12 (2 x 50 ml). The pooled organic extracts were dried over Na2SO4 then evaporated to give the desired acid as a white solid.

1H NMR (300 MHz, CDC13) 8 7.43 1H); 4.48 J=7.0 Hz, 2H); 4.01 (br d, J=12 Hz, 2H); 3.77 J=5.3 Hz, 2H); 3.51 J=7.3 Hz, 2H); 2.71 J=8.3Hz, 2H); 1.72 J=12.5Hz, 2H); 1.53 2H); 1.42-1.37 (m, 1H); 1.35 9H); 1.10 2H).

~ll" ~sssssa~ P sr ~a~~r~sd~Y

I

WO 94/18981 WO 9418981PCT/U594/01881 56 BocN

(OH

2 2 1. LiOH, CH 3 OH, H 2 0 2. C1C02-I -Bu, NMM, THF

H

2

N

H NHS0 2

C

4

H

9

BOCN

(q"12)2

NH

HIV C2

NHSO

2

C

4

H

9 2(S)-[(n-Butylsulfonyl)amino-3[[ [4,5,6,7 -tetrahydro-4-oxo-5 42-(N-Bocpiperidin-4-yl)ethyl]pyrazolo-[ 1 ,5-a]pyrazin-2-yljcarbonyljjamino propionic -acid (3-4) .Isobutyl chioroforinate (1.75 ml, 13.35 mmol) was added to a cooled solution containing this acid (4.98 g, 12.72 mmol) and N-methyl morpholine (1.53 ml, 14.00 mmol) in 100 ml TI-IF This mixture was stirred under an atmosphere of dry nitrogen. After reacting for 1 h, HPLC analysis of an aliquot indicated that the reaction -C WO 94/18981 PCT/US94/01881 -57 was >90% complete. The N-methyl morpholine-HCI was removed by filtration and the filtrate poured into a solution containing A-4 (4.30 g, 16.54 mmol), diisopropylethylamine (4.27 ml, 33.10 rmmol) THF mly and H120 (20, ml). The THF was then removed from the reactionsolution at reduced pressure and the remaining aqueous portion acidified with sat. KHSO4 and extracted vith ethyl acetate (3 x 200 ml).

Pooled extracts were dried over Na2SO4, filtered, and concentrated giving a red colored oil from which 3-4 formed as a white solid.

1 H NMR (DMSO-d6) 5 8.31 J=6Hz, 1H); 7.62 J=8.5 Hz, 1H); 7.01 1H); 4.43 J=6.6 Hz, 2H); 4.11 1H); 3.92 J=12 Hz, 2H); 3.80 J=6.6 Hz, 2H); 3.51 J=7.3 Hz, 2H); 3.65 2H); 3.51 J 6.8 Hz, 2H); 2.96 J=7.2 Hz, 2H); 1.70 J=l 1 Hz, 2H); 1.53 (m, 2H); 1.60-1.49 (overlapping m, 5H); 1.40 9H); 1.28 J=7.1Hz, 2H); 1.05 2H); 0.79 J=7.1Hz, 3H).

2(S)-[(n-Butylsulfonyl)amino]-3-[[[4,5,6,7-tetrahydro-4-oxo,-5-[2- (piperidin-4-yl)ethyl]pyrazolo[ 1,5-a]pyrazin-2-yl]carbonyl]amino propionic acid monohydrochloride o 0 HN (CH2)2-N N H CO N-N H HNSO 2

C

4

H

9 A solution of 3-4 (278 mg, 0.437 mmol) in 30 ml ethyl acetate was cooled to 0° and HCI gas bubbled through for 3 min. The reaction mixture was warmed to room temperature, stirred for 30 min then taken to dryness on a rotary evaporator. The remaining white solid was recrystalized from ethanol/water (90:10) filtered and vacuum dried over P205 giving 3-5 as a white solid.

I" ~s~BI IBd~ WO 94/18981 WO 9418981PCT/TUS94101881 -58 1 H NMR (DMSO-d6) 8 8.95 (br s, 1H); 8.33 J=5.7 Hz, 1H); 7.64 (d, J=9 Hz, IH); 7.02 1H); 4.35 J=5.1 Hz, 2H); 4.10 (in, 1H); 3.81 (t, 1=5.2 Hz, 2H); 3.6-3.4 (in, 4H); 3.21 J=10.5 Hz, 2H); 2.95 J=7.8 H z, 2.891' (br 1.96 1=11IHz, 2H); l'.62LT2(over'appihg multiplets, 9H); 0.80 J=7.3H1z, 2H).

SCHEME 4 HCI H 2 N~>C2H H NCB C 3

NHCBZ

H HCI H 4-1 4-1la Methyl 2(S)-N-Benzyloxycarbonylamino- 3-aminopropi onate hydrochloride (4-l a) Commercially available 2(S)-N-benzy loxycarbonylamino- 3-aminopropionic acid (Fluka) was refluxed in methanolic HCl for 2.5 h then evaporated and the residue crystalized from methanol/ether to give 4-I1a as a white solid.

I H NMR (3 00 MHz, DMSO-d6) 8 7..63 (in, 5H); 5.93 IH); 5.15 (s, 2H); 4.56 (in, 1H); 3.95-3.83 (mn, 2H); 3.73 2H).

WO 94/18981 WO 9418981PCT[US94/01881 59 0 BooND (CH2)-N CC 2

H

H

2 N2<C2CH3 H

NHCBZ

a 00 B N H S.CO2CH3 BoCN (0H 2 2 -N H" HB

H

2 Pd/C

CH

3 0H BocNO NH,--7,C2CH3 H'

NH

2 Methyl-2(S)-[(CBZ)amino]-3[ ,6,7-tetrahydro-4-oxo-5-[2(N-Bocpiperidin-4-yl)ethyljpyrazolo[ 1 ,5-alpyrazin-2-yl]carbonyl] amino] propionate (4-2) A solution of 3-3~ (5.6 g, 14.0 mmol), NCX-Cbz-L-2,3diaminopropionic acid methyl ester hydrochloride (4-la) (4.5 g, 15.5 mmol), HOBT (2.37 g, 15.5 mmol), and Et3N (4.1 ml, 29.5 mmol) in ml anhydrous DMF was stirred under N2 for 48 h at room temperature. The DMF removed at reduced pressure and the residue dissolved in 700 ml ethyl acetate and washed successively with saturated NaHCO3 solution, H20, 10% citric acid, H20 and brine (1 x 100 ml WO 94/18981 Wo 9418981PCTIUS94/01881 60 each), dried over Na2SO4, filtered and evaporated. The resulting clear glass was chromatographed on silica gel using 3% CH3OH/C:42Cl2 as eluent to yield pure 4- as a white solid.

1 H NMR (.CDCl3) 857.43 (in, 5H1); 7.3 5 I 7. 18 1=6.5 Hz, 1lH); 5.98 1=6.8 Hz, 1H); 5.09 2H); 4.59 (in, LHI); 4.38 (mn, 2H); 4.10 (br d, J=12 Hz, 2H); 3.8 3H); 3.73 1=5.3 Hz, 2H); 2.71 J=8.3 Hz, 2H); 1.72 J=12.5 Hz, 2H); 1.53 (in, 2H); 1.42-1.37 (in, 1H); 1.35 9H); 1. 10 (mn, 2H).

Methyl -2 amino- 3- -tetrah ydro-4- oxo- 5- 2(N- Boc-piperidin- 4-vI~ethvllpvyrazolo[ 1 .5-alpvyrazin-2-vll carbonvl-iaininollpropionate (4-3) To 4-2 (6.3 g, 10.26 mniol) in 700 ml CH30H was added 15650 mg 10% Pd on C and the resulting mixture stirred under I atm of 15H2 for 48 h. The catalyst was removed by filtration through celite and the filtrate concentrated to give a colorless glass which was triturated with Et2O and filtered to affo~rd 4-3 as a white solid.

1 H NMR (300 MHz, CDCl3) 8 7.43 (mn, 5H); 7.35 I1H); 7. 18 IH); 5.98 1=6.8 Hz, 1H); 5.09 2H); 4.59 (in, 1H); 4.38 (in, 2H); 4. 10 (br d, J= 12 Hz, 2H); 3.81, 3H); 3.73 J=5.3 Hz, 2H); 2.71 1=8.3 Hz, 2H); 1.72 1=12.5 Hz, 2H); 1.53 (mn, 2H); 1.42-1.37 (in, 1H); 1.35 9H); 1.10 (mn, 2H).

I

WO 94/18981 WO 91891PTIUS94/01881 61 BocQ (0-H 2 2 ICj

AC

2 0

THF

BocN

(CH

2 2 0-

N

N, 0 C0C3 N N H ~>CO2CH3 00 2

CH

3 H NHCCH 3

NH

2 01 N

H

44-4 Methyl-2(S)-(Acetylamino)-3-[[[4,5,6,7-tetrahydro-4-oxo-5-[2-(N-Bocpiperi din -4-ylI)ethyllpyrazolo 1 ,5-ajjpyrazin-2-yljcarbonyl] amino]p2rop~ionate (4-4) Acetic anhydride (70 ml, 0.76 mmol), was added to a cooled (00) solution of 4-3 (350 mg, 0.69 mmol) in 10 ml THF. The resulting solution was allowed to warm to room temperature and stirred for 18 h, then concentrated, and the residue was dissolved in 50 ml ethyl acetate and washed successively with NaHCO3, H20, 10% KHSO4, and brine (25 ml each). The organic layer was dried over Na2SO4 and evaporated giving a colorless residue which was chromatographed on silica gel with 3% CH3OH/CH2Cl2 to yield 4-4 as a white solid.

IH NMR (CDCl3) 6 7.29 1H) 7.24 J=6.4 Hz, 1H); 6.81 J=7.6 Hz, I1-H); 4.79 (in, I 4.3 8 (in, 2H); 4. 10 (br d, J= 12 Hz, 2H); 3.81 3.80 (in, 2H); 3.73 J=5.3 Hz, 2H); 2.71 1=8.3Hz, 2H); 2.01 3H); 1.72 J=12.5 Hz, 2H); 1.57 (mn, 1.42-1.37 (in, 1H); 1.37 9H); 1.09 (mn, 2H).

WO 94/18981 WO 9418981PCTIUS94/01881 62 2-(S)-(Acety lamino)-3-[[[4,5,6,7 -tetrahydro-4-oxo-5 (piperi din -4yl)ethyl]pyrazolo[ 1,5-a]pyrazin-2-yl] carbonyl] amino propionic acid A solution of 4-4 (203 mg, 0.38 mmol),. 1 N LiON (0-76ml, 0.76 mmol), H20, CH3OH, and THF (5 ml each) was stirred overnight at room temperature. The organic solvents were removed at reduced pressure and the remaining solution was diluted with 25 ml made acidic with 10% KHS04, and extracted into ethyl acetate.

The ethyl acetate was washed with H20 and brine, dried over Na2SO4, filtered and evaporated to provide the desired acid, 1 H NMR (CDCl3) 5 7.93 (br, 1 7.81 (br, I 7.29 IR); 4.79 (in, I1H); 4.348 (in, 2H); 4. 10 (br d, J= 12 Hz, 2H); 3.80 (br in, 2H); 3.73 (br, t, 2H); 2.71 1=8.3Hz, 2H); 2.11 3H); 1.72 J=12.5 Hz, 2H); 1.57 2H); 1.42-1.37 (mn, 1H); 1.37 9H); 1.12 (mn, 2H).

HN

BocN 1. LiOH,CH 3

OH/H

2 0 0 2. HOI, EtOAc

N

0 0

NO

N- C2H -H NHCCH 3 N 0

I

N 0 NH,,7 >(C2CH3 H NHCCH 3 4-4 0 This acid (169 mg, 32.8 inmol) was dissolved in 50 ml ethyl acetate was cooled to 0' and treated with dry HCI for 30 min.

U

WO 94/18981 WO 9418981PCTIUS94/01881 63 The solvent was removed in vacuo and the residue triturated with anhydrous ether, filtered and dried over P205, to give 4-5 as a white solid, mp 150-156'.

4-3 H0CI 02 H NHCBZ 4-1 00 BocND (CH 2 2 -N CO 2

H

U NH. H NHCBZ 1. H 2 Pd/C0 2. HOI/EtOAc H(H)-N /N N N.=N H NH 2 204- 2(S)-[(Cbz-Amino)] [4,5,6,7-tetrahydro-4-oxo-5-12(N-Cbzpiperidin-4-yl)ethyllpyrazolo[ 1,5 -alpyrazin -2-yl] carbonyl] amino p2rop~ionic acid (4-6) 4-3 was coupled to Ncx-CBZ-L-2,3-diamino-propionic acid (Fluka) using the procedure described for 3-4 to provide 4-6, the doubly protected adduct.

2(S)-Amino-3-[[[4,5,6,7-tetrahydro-4-oxo-5-[2-(piperidin-4-y)ethvllpyrazolo[ 1 ,5-allpvrazin-2-vVlcarbonvll amino propionic acid, Treatment of 4-6 with H2 in the presence of Pd/C gave the desired acid, mp. 157'. The Boc group was then removed with HCl/EtOAc in standard fashion to give pure 4-7, mp. 195-198'.

I

WO 94/18981 WO 9418981PCTIUS94/01881 64 00 BocN (0H 2 2 -N CO 2

NCH

3

C'SO

2

C

4 Hq NHN- H~ NH 2 NMM, THF BocND

(CH

2 2 -N 00CO

CH

3 ,NH H~ NHS0 2

CH,

4-8 Methyl 2(S)-[(n-Butylsulfonylamino)] -3 ,6,7-tetrahydro-4-oxo-5- [2-(N-CBZ-piperidin-4-yl)ethyllpyrazolo[ 1,5 -a]pyrazin-2-yl] carbonylilaminopropionate (4-8) A solution of 4-3j (0.30 g, 0.61 nimol), n-butyl sulfonyl chloride 16 g, 0.91 mmol), and N-methyl morpholine in 50 ml of THF was stirred at room temperature for 12 h. The solvent was evaporated at reduced pressure and the resulting oil was dissolved in CH2Cl2 (50 ml) washed with 10% KHS03 (50 ml) then dried over Na2SO4, filtered and evaporated. The resulting residue was chromatographed on silica gel giving 4-8 as a colorless glass.

I H NMR (CDCl3) 8 8.31 J 6Hz, 1H); 7.62 J 8.5 Hz, LH); 7.01 1H); 4.43 J 6.6 Hz, 2H); 4.11 (in, 1H); 3.92 J 12 Hz, 2H); 3.83 3H); 3.80 J 6.6 Hz, 2H); 3.51 J 7.3 Hz, 2H); 3.65 (in, 2H); 3.51 J 6.8 Hz, 2H); 2.96 J 7.2 Hz, 2H); 1.70 J 11I Hz, 2H); 1.53 (in, 2H); 1.60-1.49 (overlapping m, 5H); 1.40 9H); 1.28 J 7.1 Hz, 2H); 1.05 (in, 2H); 0.79 J 7.1 Hz, 3H).

WO 94/18981 WO 9418981PCT[US94/01881 65 SCHEME CH302

CO

2

CH

3 HN-lq BrCH 2

CH

2

CH

2 Br

CH

3 0 2 C C0CH

N~

Br 1. H 2 Pd/C, EtOH 2. C 6

H

6 ref lux NaN 3

DMSO

250, 5 hr

CH

3 0 2 C C0CH N0

CH

0.

HN s CO 2

CH

3 N- N BocND -CH 2

CH

2

I

5-3 NaH, DMF

CO

2

CH

3 NaOH, CH 3 OH, H 2 0 WO 94/18981 WO 9418981PCTIUS94/01881 66 SCHEME 5 (CONTD) 0 HOBT, Et 3 N, CH 2 Ci 2 11 BocND (C

H

2 N 0 2

C

2

H"

H NHSO 2

C

4 Hq 0 0 BOCN (CH 2 2

-N'N

N-N

H NHSO 2

C

4 Hq 1. NaOH, CH 3 0H, H 2 0 2. HOI, EtOAc HND (CI NH C2 H' NHSO 2

C

4

H

9 I~ WO 94/18981 PCT/US94/01881 -67 Dimethyl 1-(3-Bromopropyl)pyrazole-3,5-dicarboxylate (5-1) Compound 5-1 was obtained as a white crystalline solid using 1,3-dibromopropane in the procedure described for 3-2.

1H NMR (CDCl3) 8 7.38 1H); 4.95 J=8.2Hz, 2H); 3.95 3H); 3.92 3H); 3.75 J=8.5 Hz, 2H) 2.51 2H).

Dimethyl 1-(3-Azidopropyl)pyrazole-3,5-dicarboxylate (5-2) A solution of 5-1 (1.0 g, 3.45 mmol) in 10 ml DMSO was treated with NaN3 (0.883 g, 13.8 mmol) and mixture stirred at 25 0

C

for 5 h. Next, the reaction mixture was diluted with 100 ml of H20 and then extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed with water (2x100 ml) and brine (1 xl00 ml), dried over Na2SO4 and evaporated to give 5-2 as a colorless oil.

Methyl-5,6,7,8-tetrahydro-4-oxo-4H-pyrazolo[ 1,5-a] [1,4]diazepin-2-yl]carboxylate (5-3) A solution of 5-2 1(851 mg, 3.25 mmol) in 100 ml absolute EtOH was treated with 100 mg 10% Pd on C and the mixture was shaken on a Parr hydrogenator at 45 Psi for 5 h. The catalyst was removed by filtration through celite and the filtrate was evaporated to give 800 mg of a colorless oil. NMR analysis showed this material to a mixture of 1-(3-aminopropyl) Dimethylpyrazole-3,5-dicarboxylate and the cyclic diazapineone. This mixture was dissolved in 50 ml of 2 benzene and refluxed for 15 h then evaporated. The resulting tan solid was recrystalized from CH2Cl2/hexane to afford 5-3 as a white solid.

m.p. 220-221°C. iH NMR (CDC13) 6 7.36 6.42 (br t, 1H); 4.58 J=8.0 Hz, 2H); 3.95 3H); 3.39 (dt, J=7.2 Hz, 2H); 2.31 (m, 2H).

2H).

L~ _PPP P'~ WO 94/18981 PCT/US94/01881 68 Methyl-5,6,7,8-tetrahydro-4-oxo-5-[2-(N-Boc-piperidin-4-yl)ethyl]-4Hpyrazolo[ 1.5-al] 1,41 diazepin-2-yl carboxvlate To a solution of 5-3 (175 mg, 0.83 mmol), in 50 ml DMF wasv added 60% NaH (36 mg, 0.91 mmol), the mixture was, stirred, under N2 at -15° for 30 min. To this mixture a solution of 2-(N-Boc- Piperidin-4-yl)ethyl iodide (283 mg, 083 mmol) in 25 ml DMF was added dropwise over 20 min. The resulting solution was stirred for min at -150 then warmed to room temperature and allowed to stir overnight. The DMF was evaporated at reduced pressure and the residue redissolved in ethyl acetate, filtered and chromatographed on silica gel using ethyl acetate as eluent to afford pure 5-5 as a glass.

1 H NMR (CDC13) 5 7.24 1H); 4.50 J=7.0 Hz, 2H); 3.93 (br d, J=12 Hz, 2H); 3.94 3H); 3.61 J=5.3 Hz, 2H); 3.42 J=7.3 Hz, 1 2H); 2.7 (br t, J=6.3 Hz, 2H); 2.3 2H); 1.55 J=12.5 Hz, 2H); 1.38 2H); 1.33-1.25 IH); 1.27 9H); 1.01 2H).

5,7,8-Tetrahydro-4-oxo-5-[2-(N-Boc-piperidin-4-yl)ethyl-4Hpyrazolo[1,5-a[1 ,4]diazepin-2-yl-carboxylic acid (5-6) A solution of 5-5 (166 mg, 0.395 mmol) in 10 ml was treated with IN NaOH (0.435 ml, 0.43 mmol). The resulting solution was stirred at room temperature for 18 h and then removed at reduced pressure. The remaining aqueous phase was acidified with 10% aqueous citric acid and extracted with CH2C12 (2 x 50 ml). The pooled organic extracts were dried over Na2SO4 then concentrated to give 5-6 as a white solid.

IH NMR (CDC13) 5 7.29 1H); 4.52 J=7.0 Hz, 2H); 4.12 (br d, J=12 Hz, 2H); 3.94 3H); 3.61 J=5.3 Hz, 2H); 3.42 J=7.3 Hz, 2H); 2.7 (br t, J=6.3 Hz, 2H); 2.3 2H); 1.55 J=12.5 Hz, 2H); 1.38 2H); 1.33-1.25 1H); 1.27 9H); 1.01 2H).

-r ~plr V O 94/18981 WO 9418981PCTIUS94/01881 69 Ethyl- (n-Butylsulfonyl)amino] 3- [5 ,6,7,89-tetrahydro-4-oxo-5- [2- (N-Boc-piperidin-4-yl)ethyl]-4H-pyrazolo[ 1 1 ,4]-diazepin-2vll carbonyll amino p2ropionoate (5-7) 5-6 (1,47 mg, 0.36 minol) in CH2Cl2/5..ml was treated with.

ethyl 2(S)-n-butanesulfonamido-3-amino-propionate (AL-5) (115 mg, 0.40 mmol), HOBT (49 mg, 0.36 mmol), and Et3N (0.10 ml, 0.724 mmol) in 50 ml CH2Cl2 and this solution was stirred under N2 for 18 h at room temperature. The reaction solution was washed successively with sat. NaHCO3, H20, 10% citric acid, H20 and brine (1 x 20 ml each), dried over Na2SO4, filtered and evaporated. The resulting clear glass was chromatographed on silica gel using 5% CH3OH/EtOAc as eluent giving pure 5-7.

1 H NMR (CDCl3) 5 7.32 I1H); 7.24 1=6.89 Hz, I1H); 5.54 J=7.2 Hz, 1H; 4.43(t J=7.8 Hz, 2H); 4.32 (in, 1H); 4.28 J=7.1 Hz, 2H); 4. 10 br d, J= 12 Hz, 2H); 3.85 (in, 2H); 3.61 J=5.3 Hz, 2H); 3.42 (t, 1=7.3 Hz, 2H); 3.03 J=7.1 Hz, 2H); 2.7 (br t, J=6.3 Hz, 2H); 2.3 (mn, 2H); 1.65 -1.45 (overlapping mn, 7H); 1.38 (in, 2H); 1.33-1.25 (in, lH); 1.37 9H); 1.30 1=7.4 Hz, 3H); 1.01 (in, 2H); 0.96 J=7.3 Hz, 3H).

2(S)-[(n-Butylsulfonyl)amino]-3- [[[5,6,7,8-tetrahydro-4-oxo-5-[2- (piperidin-4-yl)ethyl] -4H-pyrazolo[ 1,5-a] [1 diazepin-2-yl] carbonyl] propionic acid (5-8) To a solution of 5-7 (100 ing, 0.156 inmol) in 10 ml CH3OH, was added 1IN NaOH (160 ml, 0. 16 inmol) and H20, 10 Ml.

The resulting solution was stirred at room temperature for 3.5 h then removed at reduced pressure. The remaining aqueous phase was acidified with 10% aqueous citric acid and extracted with CH2Cl2 (2 x 50 ml). The pooled organic extracts were dried over Na2SO4 then evaporated to give the desired acid.

'H NMR (CDCl3) 8 17.24 1=6.8 Hz,1IH); 7.28 1 6.0 J=7.2 Hz, I1H); 4.43 1=7.8 Hz, 2H); 4.32 (in, ILH); 4. 10 (br d, J= 12 Hz, 2H); I _II WOr 94/18981 PCT/US94/01881 3.85 2H); 3.61 J=5.3 Hz, 2H); 3.42 J=7.3 Hz, 2H); 3.03 (t, J=7.1 Hz, 2H); 2.7 (br t, J=6.3 Hz, 2H); 2.3 2H); 1.65-1.45 (overlapping m, 7H); 1.38 2H); 1.33-1.25 IH); 1.37 9H); 1.01 n(m, 2H); 096 J=7.3 Hz, 3H).

This acid (89 mg) in 15 ml ethyl acetate was cooled to 0° and HC1; gas bubbled through for 3 min. The reaction mixture was warmed to room temperature, stirred for 30 min then taken to dryness on a rotary evaporator. The remaining white solid was triturated with ether, filtered and vacuum dried over P205 to give 5-8 as a white solid.

1H NMR (DMSO-d6) 8 8.95 (br s, 1H); 8.33 J=5.7 Hz, 1H); 7.64 (d, J=9 Hz, 1H); 7.02 1H); 4.35 J=5.1 Hz, 2H); 4.10 m, 1H); 3.81 (t, J=5.2 Hz, 2H); 3.6-3.4 4H); 3.21 J=10.5 Hz, 2H); 2.95 J=7.8 Hz, 2H); 2.81 (br m, 2H); 1.96 J=l Hz, 2H); 1.62-1.2 (overlapping multiplets, 9H); 0.80 J=7.3Hz, 2H).

~I r~aa rsaarP -ss~ *0 94118981 Wo941s~s1PCTIUS94/01881 '71 SCHEME 6 1. acetone, H 2 0 2. H 2 Pd/C EtOH 2-6 6- ,cis E-1 B, trans BooND (C H 2 2 1. NaOH, CH 3 OH, H 2 0

HCI

2. HN11,CA 6-1lA

TFA

0H 2 C1 2 BocND N H -,-CO 2 tBU NH C2H 6-3 WOr 94/18981 PCT/US94/01881 -72- Ethyl-3-oxo-2[2-(N-Boc-piperidin-4-yl)ethyl]octahydroimidazo- [1.5-alpyridin-6-yllcarboxylate A 6-1B) 2-6, (514 mg, 1.1 mmol) was dissolved in 25 ml of acetone ml.of H.20 was addedand the mixture heated to 60 0 C for 3.5 The..

acetone was removed at reduced pressure and the resulting yellow precipitate filtered. This crude material was dissolved in 100 ml of toluene and refluxed for 3 h. The toluene was evaporated giving a yellow solid.

This solid (500 mg, 1.11 mmol) was dissolved m 100 ml of ethanol 75 mg of 10% Pd on C was added and mixture shaken on Parr hydrogenator at 55 psi for 13 h. The catalyst was removed by filtration through celite and the solvent evaporated. The resulting colorless oil was chromatographed on silica gel using 70% ethyl acetate/30% hexane to give 268 mg of the cis reduction product 6-1 A along with 132 mg of the trans product 6-1B.

Isomer 6-1 A 1 H NMR (CDC13) 8 4.15 1H); 4.06 2H); 3.68 3H); 3.41 (m, 20 2H); 3.22 1H); 2.90 1H); 2.80 2H); 2.68 2H); 2.43 (m, 1H); 2.16 1H); 1.81 1H); 1.69 2H); 1.60 1H); 1.45 (s, 9H); 1.43 3H); 1.39 1H); 1.11 2H).

Isomer 6-lB 1 H NMR (CDC13) 8 4.34 IH); 4.06 2H); 3.69 3H); 3.40 (m, 3.36 1H); 3.30 1H); 3.11 1H); 2.89 1H); 2.84 (m, 1H); 2.67 2H); 2.63 1H); 2.30 1H); 1.69 2H); 1.67 (m, 2H); 1.60 1H); 1.45 9H); 1.41 1H); 1.09 2H).

WO 94/18981 WO 9418981PCTIUS94101881 73 tert-Butyl (±)-cis-[[3-oxo-2[2(N-Boc-piperidin-4-yI)ethyl] octahydroimidazof 1 5-a]pvridin-6-vllcarbonyllaminolpropionate (6-2) 6-l1A was hydrolyzed with IN NaOH in CH3OH/H20 as described for. 1-6 .to give, the desired. acid. This acid was coupled.with., -alanine t-butyl ester as described for 2-8, to provide 6-2.

IH NMR (CDC13) 8 6.32 1H); 4.06 (in, 2H); 3.98 (in, IR); 3.56 (in, 3.21 (in, 2H); 2.91 (in, 1H); 2.92 (in, 1H); 2.80 (mn, 2H); 2.40 (t, 2H); 2.23 (in, 1H); 2.09 (mn, 1H); 1.81 (in, 3H); 1.69 (in, 2H); 1.60 (in, I 1.45 18H); 1.43 (in, 3H); 1. 11 (in, 2H).

(±)cis-3-Oxo-2[2-(piperidin-4-yl)ethyl] octahydroimidazo[ I 6-vVI carbonvillaminop~ropionic acid (6-3) 6-2 (65.3 ing, 0.13 mimol) was dissolved in 10 ml of anhydrous CH2Cl2 and cooled to 0 0 C. Trifluoroacetic acid (0.200 ml) was added and solution stirred for 1 h then evaporated at reduced pressure to give pure 6-.

IH NMR (CD3OD) 8 4.06 (in, 2H); 3.91 (in, 1H); 3.56 (in, 5H); 3.21 (in, 2H); 2.91 (in, lH); 2.92 (in, 1H); 2.80 (mn, 2H); 2.40 2H); 2.23 (in, 1H); 2.09 (in, 1H); 1.81 (in, 3H); 1.68 (in, 2H); 1.60 (in, 1H); 1.28 (in, 3H); 0.91 (mn, 2H).

I

I

WO 94118981 WO 9418981PCTIUS94/01881 74 SCHEME 7

CISO

2 N HCO 2

CH

2 Ph

CH

2 01 2 Et 3

N

1. LIOH, THF 2. HCI, EtflAc HI',>-CO2H H' NHSO 2 NHC0 2

CH

2 Ph 7-2

H

2 Pd/C 2. HOI, EtOAc 0 0 HN (CH 2 2 -N NH CO 2

H

N-~N H NHS 2

NH

2 WO 94/18981 PCT/US94/01881 Methyl-2(S)-[(N-CBZ-Aminosulfonyl)amino]-3-[[[4,5,6,7-tetrahydro- 4-oxo-5-[2(N-BOC-piperidin-4-yl)ethyl]pyrazolo[ 1,5-a]pyrizine-2yllcarbonyllaminolpropionate (7-1) To- a' 0 solution of chlorosulfamylisooyanate (45.1 Il1, 0.508 mmol) in methylene chloride was added benzyl alcohol (53 ml, 0.508 mmol). The reaction was aged 90 min at 0° and a solution of 4-3 (250 mg., 0.508 mmol) in methylene chloride containing triethylamine (142 ml, 1.02 mmol) added. The reaction was allowed to warm to room temperature and stirred overnight (18 hr). The reaction was adjusted to a pH=3.0 with aqueous sodium bisulfate and the product was extracted with methylene chloride(3xl0 ml). The organic extracts were combined, concentrated and chromatographed on silica (eluent methylene chloride, 5% methanol) to give 7-1.

1H NMR (300 MHz, CDC13) 5 1.40 9H), 2.65 2H), 3:65 3H), 5.10 2H), 6.65 1H), 7.2-7.5 6H), 8.90 1H) 2(S)-[(N-CBZ-Aminosulfonyl)amino]-3-[[[4,5,6,7-tetrahydro-4-oxo-5- [2-(N-Boc-piperidin-4-yl)ethyl)pyrazolo[1,5-a]pyrazine-2-yl]carboxyl]amino propionic acid (7-2) To a solution of 7-1 (100 mg) in THF (5 ml) was added IN LiOH (0.6 ml) and the mixture stirred at room temperature for 18 h.

The reaction was quenched by addition of aq. sodium bisulfate and product extracted into ethyl acetate (2 x 15 ml). Concentration of the extracts gave the desired acid.

1 H NMR 1.4 9H), 2.6 (br, t, 2H), 7.1-7.2 (br.m, 5H), 7.3 1H).

This acid was dissolved in EtOAc, cooled to and treated with HCI (gas). The reaction mixture was concentrated and flushed with ethyl acetate to give 7-2, mp >2000 (dec.) CHN analysis Calc. C, 44.86; H, 5.92; N, 14.20 Found: C, 44.50, H, 6.09; N, 13.80 -PC ~e~ WO 94/18981 WO 9418981PCT/US94/01881 76 2(S)-(Aminosulfonyl)amino-3-[[[4,5,6,7-tetrahvdro-4-oxo-5-[2- (piperidin-4-yl)ethyljjpyrazolo[ 1 ,5 -a]Pyrazin-2-yl] carbonyl] aminopropionic acid (7-3) Asolution of 7- (70 mng), in methanol (20 ml) was treated with 10%Pd/C (35 mg) and the mixture hydrogenated (I atm.) overnight (18 hrs). The mixture was filtered and concentrated to give 46 mg of an oil. The oil was dissolved in ethyl acetate, cooled to 0' and HCl gas bubbled in over 30 min. Concentration of the reaction gave 7-3 a white solid, mp >2000, FAB MS, M+1=458.

WO 94/18981 WO 9418981PCTIUS94/01881 77-

SCHEMESS

C

2

H

5 0 2

C

HN C0 2 0 2

H

5

C

2

H

5 0 2

C

N/C0 2 0 2

H

Brj 8-2 NaH,.THF BrCH 2

CH

2 Br BocND -CH 2

CH

2

NH

2 KI, DIEA 1. LiOH, CH- 3 0H, H 2 0

CO

2

CH

3 2. EDO, HOBT, TEA HCI*H 2 N

,~.CO

2 tBu U HCI, EtOAc BocN, (C H 2 2 NH'--'-C2tBU

N/

0 0 HND -(CH 2 2 N NHl 'C2

I

WOQ 94/18981 PCT/US94/01881 -78- Diethyl 1-(2-Bromoethyl)pyrrole-2.4-dicarboxylate (8-2) A solution of diethyl pyrrole-2,4-dicarboxylate (5.50 g, 29.4 mmol) in tetrahydrofuran (200 ml) was cooled in an ice bath and a suspension of NaH (6.5 g, 68.6 mmol) in ml) was added in a stream. The reaction flask was warmed to room temperature. After stirring 1 h at room temperature 1,2dibromoethane (25.2 ml, 294 mmol) was added and the mixture was refluxed for 24 h. Water (50 ml) was added to the reaction flask. The mixture was rotary evaporated to remove tetrahydrofuran. Saturated 0sodium bicarbonate solution (100 ml) was added to the residue and the resulting solution was extracted with methylene chloride (4 x 50 ml).

The combined organic extracts were dried with anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was rotary evaporated to give a yellow solid. This material was recrystalized from hexane ethyl acetate 80:20 to give 8-2 as a yellow solid.

1 H NMR (DMSO-d6) 8 7.83 1H); 7.15 1H); 4.69 2H); 4.25 4H); 3.78 2H); 3.31 (H20); 1.29-1.22 6H).

Ethyl [4,5,6,7]-Tetrahydro-4-oxo-5-[2-(N-Boc-Piperidin-4-yl)ethyl]pyrrolol 1.5-apyrazin-2-ylJcarboxvlate (8-3) The alkyl bromide 8-2 (3.30 g, 10.4 mmol., 1.0 2-4 (3.52 g, 15.5 mmol., 1.5 potassium iodide (5.18 g, 31.2 mmol), diisopropylethylamine (5.42 ml, 31.2 mmol., 3.0 and acetonitrile ml) were combined. The suspension was heated to reflux for 24 h, and then rotary evaporated to remove acetonitrile. Saturated sodium bicarbonate solution (100 ml) was added, and the solution was extracted with ethyl acetate (5 x 50 ml). The combined organic extracts were dried with anhydrous sodium sulfate and concentrated to a brown oil.

The crude product was subjected to column chromatography using silica. The column was eluted with methylene chloride then'methylene chloride containing 1% methanol to give pure 8-3 as a white solid.

~es~J~ a(lSI I WO 94/18981 PCT/US94/01881 -79 IH NMR (CDC13) 6 7.32 1H); 7.29 1H); 4.28 2H); 4.19-4.00 4H); 3.7-3.6 2H); 3.6-3.5 2H); 2.68 2H); 1.8-1.7 br, 2H, H20); 1.6-1.4 m, 1 1H); 1.33 3H); 1.2-1.1 2H).

tert-Butyl [4,5,6,7]-tetrahydro-4-oxo-5[2-(N-Boc-piperidin-4y)ethyllpyrrolo[ 1.5-alpyrazin-2-vllamino propionate (8-4) 8-3 (620 mg, 1.54 mmol) lithium hydroxide monohydrate (160 mg, 4.00 mmol., 2.6 water (15 ml), and methanol (10 ml) were combined in a 50 ml round bottom flask equipped with a magnetic stir bar. The solution was stirred for 4 h at room temperature then heated to 900 for 1 h. Any remaining methanol was removed by rotary evaporation and the aqueous residue was acidified with 10% K2SO4 then extracted with ethyl acetate (4x50 ml). The combined organic extracts were dried with anhydrous sodium sulfate, filtered, and evaporated giving the desired acid as a white solid.

IH NMR (DMSO-d6) 5 7.51 2H); 6.84 1H); 4.19 2H); 3.90 br, 2H): .63 2H); 3.43 2H); 3.4-3.2 (H20); 2.6-2.8 (br, 2H); 1.66 2H); 1.43 2H); 1.36 9H); 1.1-0.9 2H).

This acid (150 mg, 554 mmol), EDC (117 mg, 0.609 mmol), 1-hydroxybenzotriazole (82.2 mg, 0.609 mmol), triethylamine (0.300 ml, 1.11 mmol., 4.0 p-alhnine-t-butyl ester (111 mg, 0.609 mmol), and methylene chloride (10 ml) were combined in a 100 ml round bottom flask equipped with a magnetic stir bar. The resulting solution was stirred at room temperature overnight. Solvent was rotary evaporated from the reaction flask and the resulting residue was subjected to column chromatography using silica. The column was eluted with methylene chloride, methylene chloride with 2% methanol then 4% methanoL Fractions containing.product were pooled-to.

provide 8-4 as a white solid.

IH NMR (CDC13) 8 7.32 1H); 7.04 1H); 6.58 1H); 4.2-4.0 4H); 3.7-3.5 6H); 2.68 2H); 2.51 2H); 1.70 3H, 1.53 2H); 1.45 9H); 1.21-1.0 2H). 1 L i 3- -e L ~I -yl c -IC -L ll II j 1 11 WO 94/18981 WO 9418981PCTIUS94/01881 4,5,6,7-Tetrahydro-4-oxo-5- [2-(piperidin-4-yl)ethylI 1,5-a]pyrazine-2vilcarbonvil1amino- propionic acid, T-he ester,8-4 (180 mg, 7 0.34-7 mmnol),,and~ethyl..acetate- S ml) were combined in a 50 ml round bottom flask. The suspension was cooled in an ice bath. Hydrogen chloride was bubbled through the suspension for 1.5 min. The reaction flask was warmed to room temperature, then solvent was removed by vacuum filtration giving as a white solid, mnp 248-249'.

IH NMR (DMSO-d6) 5 9.0-8.5 (br, IH, .05 (in, 1H); 7.42 1H); 7.05 1H); 4.15 (in, 2H); 3.62 (mn, 3.5-3.3 (in, 4H, H20); IL'~ 3.15 br, 2H); 2.85-2.7 (br, 2H); 2.5-2.4 (in, 4H); 1.81 2H); 1.6- 1.4 (in, 3H); 1.4-1.2 (in, 2H).

WO 94/18981 WO 9418981PCTrtJS94/01881 81 -SCHEME 9 BQCNH X. O2C2H5 H NH 2

CISO

2

NHC

4

H

9 9 -1 py, CH 2

CI

2 HOI

H

2 N C2C2H5 EtOAc H~ NHS0 2

NHC

4 Hq

-~C

2

C

2

H

BOCNH H x' N HS 0 2 -N H 4 H '9 9-2 EDO, HOBT DMF, a3-3

NHN

2 H~ NHS0 2

NHC

4

H

9 1 1. NaOMe, MeOH 2. HCI, EtOAc N HC 4

H

9 Ethyl 2S)-Amino-3-(N-Boc-amino)propionate (9-1) Commerically available 2(S)-3-diaminopropanoic acid (Fluka) (10 g, 96.2 mmol) was dissolved in absolute ethanol (200 ml) and the solution was saturated with anhydrous HCl gas then heated at reflux for 2.5 h. The solvent was removed and the residue WO 94/18981 PCT/US94/01881 82recrystallized from EtOH/Et20 to afford the ethyl/ester dihydrochloride as a hygroscopic white solid.

This material (5 g, 24.3 mmol) was suspended in CH2C12 (200'ml)' cooled to -50P. Next, triethylamine (7.0 mly 51 .mmol,) was,.

added and the mixture stirred for 5 min. A solution of di-tert-butyl dicarbonate (5.30 g, 24.3 mmol) in 100 ml CH2C12 was added dropwise over a 30 min period and the mixture stirred at -500 for 1.5 h, then warmed to room temperature. The solution was washed with water (2 x 100 ml), then dried (Na2SO4) and evaporated. The resulting residue was chromatographed silica gel (80:20 CH2CI2/CH30H) to afford pure 9-1.

1 H NMR (300 MHz, CDC13) 6.03 (brt, 1H); 4.35 1H); 3.85 (m, 2H); 3.23 2H); 1.21 3H).

Ethyl 2(S)-n-Butylaminosulfonylamino-3-(N-BOC-amino)propionate (9-2) A solution of 9-1 (500 mg, 2.15 mmol) in CH2C12 (10 ml) was treated with pyridine (261 ml, 3.23 mmol) and n-butylsulfoamoyl chloride (406 mg, 2.37 mmol). The solution was stirred at room temperature for 3 h, then poured onto silica gel and eluted with acetone/hexane to give pure 9-2 as a white solid.

IH NMR (300 MHz, CDC13) 5 5.43 1H); 4.9o, 1H); 4.40 (brs, 1H); 4.10 2H); 4.05 1H); 3.56 2H); 3.08 2H); 1.8-1.2 (overlaping multipets, 16H); 0.90 3H).

Ethyl 2(S)-(n-Butylaminosulfonylamino)-3-aminopropionate (9-3) A solution of 9-2 (612 mg, 1.65 mmol) in ethyl acetate ml) was cooled to -50 and anhydrous HCI was bubbled in for min. The reaction was concentrated and the product isolated by filtration to give 9-3 as a white solid.

I II II I -r L WO 94/18981 WO 9418981PCT[US94/01881 83 IH NMR (300 MHz, DMSO-d6) 8 5.82 lH); 4.56 (brs, IH); 4.20 2H); 4.02 (in, 1H); 3.45 (in, 2H); 3.01 (in, 2H); 1.9-1.36 (in, 7H); 0.93 3H).

[(n-Butylaininosulfonyl)amino] [[4,5,6,7-tetrahydro-4-oxo- [2-(piperidin-4-yl)ethyl]pyrazolo[ 1 ,5-a]pyrazin-2-yl] carbonyllamine propionate (9-4) Coupling or 9-3 with 3-.3 with EDC and HOBT in DMF as for 1-6 provided 9-4.

1 H NMR (300 MHz, CDC13) 8 7.29 1H); 7.19 lH); 5.43 1H); 4.41 2H); 4.26 2H); 4.20 (mn, 1H); 4.08 2H); 3.86 (in, 2H); 3.76 (mn, 2H); 3.60 2H); 3.08 2H); 2.68 2H); 1.78 2H); 1.6- 151.08 (mn, 8H); 1.43 9H); 0.92 3H).

2(S)-[(n-Butylaminosulfonylamino] -3 ,6,7-tetrahydro-4-oxo-5- (piperi din-4-yl) ethyl] pyrazol o[ 1, 5 pyrazin- 2-yl] c arbonyl1] aminop~ropionic acid Hydrolysis of 9-4 with NaOMe, isolation of the crude acid, and subsequent treatment with HCL in EtOAc as described for 5-7 provided 9-5 as a white solid, mp. 155-160'.

WO 94/18981 WO 9418981PCTIUS94/01881 84 SCHEME

HOI

CH

3 0 2 0. 0 N H 2

N

CH

2 Br K 2 00 3

CH

3

CH

3 0 2

CON

1CO N H, CO 2 tBu 10-1 0

CH

3 0 2 C N N\~NC02tBu N~ 1- 10-2 0 0

HO

2 C A 'N~C 2 C N 10-3

;O

2 tBU triphosgene toluene, DMA 1. NH40H,

CH

2

CI

2 00 G 2. MeO 2 C-S0 2 NEt 3

CH

2

CI

2 3. 1 N Na0H 1. EDC, HOBT, Et 3

N

BOCNO CH 2 CHpNH 2 2. TFA 0 HNa (CH 2 2 NH N A N C02H

NN

CON

-U

WO 94/18981 WO 9418981PCT[US94/01881 85 yridvyhrethvI1-5-alanine tert-butyl ester (10-0) A mixture of 2-3 (871 mg, 3.79 mmol), !3-alanine tertbutyl ester*HCl (2.7 g, 15 nunol), and K2/C.'03 (4.5 g, 30 mmol) in 100 ml of anhydrous CH3CN was .placed~.in~a, 250 mi flask and, refiuxed. for 3 h, then cooled and filtered. The filtrate was con'-entrated at reduced pressure and chromatographed on silica gel using EtOAc as eluent to afford 10-1 as a colorless glass.

I

1 H NMR (CDCl3) 8 9.18 J=1.4Hz, 1 8. 1(dd, J= 1.4 and 6.8 Hz, 1H); 7.39 J=6.8 Hz, 1H); 4.08 2 3.95 3H); 3.04 2H); 2.60 2H); 1.4 9H).

tert-Butyl-2-(2-carboxyethyl)-lI -chlorocarbonyl-3-oxo-2,3-dihydro- 1 5 -alpyri dine-6-carboxv late (1 0-2) 10-1 (800 mg, 2.71 mmol) was dissolved in 50 ml of toluene. N,N-dimethyl aniline (2.0 ml, 16.7 mmol) was added an solution cooled to 00. To this, a solution of triphosgene (1.7 g, 5.7 mmol) in 15 ml toluene was added dropwise over 30 min. The solution was then warmed to 25' and stirred for 3.0 h then washed twice with IN HCI, water and brine (50 ml of each), dried over Na2SO4 and evaporated giving 10-2 as a yellow crystalline solid.

1 H NMR (CDCl3) 8 8.83 1=1.4Hz, 1H); 8.25 J=6.8 Hz,- 1H); 7.82 (dd, J=1.4 and 6.8 Hz,lIH); 4.43 J=7.2 Hz, 2H); 3.98 3 2.75- J=7.2 Hz, 2H); 1.4 9H).

tert-Butyl-2-(2-carboxyethyl)- 1 -cyano-3-oxo-2,3 -dihydroimidazo- .5-allpyri dine- 6-c arboxy late (10-3) 10-2 (250 mg, 0.65 rnmol) was dissolved in 100 ml of 30CH2Cl2 10 ml of ammonium hydroxide was added and this biphasic mixture was stirred for I h then the organic layer separated and washed with 10% citric acid then brine (50 ml), dried over Na2SO4 and evaporated.

1311 a 41 VO 94/18981 PCT/US94/01881 -86- This residue (150 mg, 0.42 mmol) was dissolved in 100 ml of CH2C12, 355.7 mg of Methoxycarbonylsulfamoyl-triethylammonium hydroxide, inner salt (Burgess reagent, 1.48 mmol) was added in three portions- over a 2 h period. The.resultingsolution, was stirred at room temperature for an additional hour and then concentrated and chromatographed on silica gel using 1:1 hexane/ethyl acetate as eluent giving the nitrile in quantitative yield. This material was subjected to saponification using 1N LiOH to give the desired carboxylic acid 10-3.

1H NMR (CDC13) 6 8.75 1H); 7.43 1H); 7.18 1H); 4.23 2H); 2.85 3H); 1.4 9H).

3-[1 -cyano-3-oxo-6-2-(piperidin-4-yl)ethylcarbamoyl)-2,3-dihydro- Simidazorl.5-alpyridin-2-yl]propionic acid (10-4) 10-3 (170 mg, 0.51 mmol) was dissolved in 10 ml of CH2C12, Et3N (71 ml, 0.51 mmol) was added along with HOBT (69.3 mg, 0.51 mmol), EDC (98.6 mg, 0.52 mmol) and 2-4 (117.2 mg, 0.51 mmol). The mixture was stirred under N2 for 18 h then washed with 10% citric acid, H20 and brine (10 ml each) and dried over Na2SO4, concentrated and chromatographed giving a yellow solid (215 mg, 0.43 mmol). This material was deprotected using trifluoroacetic acid in CH2C12 to give the 10-4 TFA salt as a yellow solid, 173°.

1 H NMR (300 MHz, DMSO d6) 8 1.75 1H); 7.43 1H); 7.31 1H); 7.15 1H); 4.23 2H); 3.41 2H); 3.23 2H); 2.83 (m, 2H); 1.85 2H); 1.53 2H); 1.41 1H); 1.32 2H).

d ~Q t'l 7 WO 94/18981 WO 9418981PCT/1JS94/01881 87 SCHEM 11I C0 2 0H 3

CH

3 0 2 C0 N F-i B r HCI.H 2 N

CA

K

2 00 3

CH

3

CN

0

CH

3 0 2 C N 2 tBu N-

NJ

1. LIOH, H 2 0, CH 3 0H 2.BOcN

-CH

2

OH

2

NH

2 2-4 EDC, HOBT 11-1 3. HCI/EtOAc HND

CH

2

CH

2 -NHI\ N CO2H 11-2 Methyl [4,5,6,7 -tetrahydro-4-oxo-5 (tertbutyl propionyl)]pyrazolo rl.5-alpvyrazin-2-yllcarboxvlate (11-1) (1.4 g, 4.8 n-uol), P-alanine tert-butyl ester-HC1 (0.90 g, 5 mmol), and potassium carbonate (0.78 g, 5.28 mmol) in 150 ml CH3CN was refluxed under N2 for 4.5 h then cooled, filtered and evaporated at reduced pressure. The rmsulting yellow residue was

-M

VVOg 94/18981 PCT/US94/01881 88 chromatographed on silica gel using 2% CH30H/CH2C12 giving the diester 11-1 as a colorless glass.

tH NMR (CDC13) 8,7.31 2H); 3.93 3.61 2H); 2.71 2H); 2.35 2H); 1.23 9H).

-4,5,6,7-Tetrahydro-4-oxo-.2(2-(piperidin-4.yl)ethylcarbamoyl)acid (11-2) A solution containing LiOH (145 mg, 3.41 mmol) in 10 ml was added to a solution of the ester 11-1 (1.0g, 3.1 mmol) in ml CH30H and the mixture was heated to 60 0 C for 2.5 h then cooled and the solvent removed at reduced pressure. The remaining residue was acidified with 10% citric acid and extracted CH2C12 (2 x 100 ml).

The pooled organic extracts were washed with H20, dried and evaporated to afford the desired acid as a colorless glass.

1 H NMR (CDC13) 5 7.21 1H); 4.48 2H); 3.63 2H); 2.71 2H); 2.32 2H); 1.23 9H).

20 This acid (500 mg, 1.62 mmol) was dissolved in 10 ml of CH2Cl2, HOBt (220 mg, 1.62 mmol) was added along with EDC (309 mg, 1.62 mmol), and 2-4 (356 mg, 1.63 mmol). The mixture was stirred under N2 for 16 h then washed with 10% citric acid, H20 and brine (10 ml each) and dried over Na2SO4, concentrated and' 2 chromatographed giving a colorless foam. This material was deprotected using HCI in ethyl acetate, to give the HCl salt of 11-2 as a white solid. MP= 192-194°C.

1 H NMR (300 MHz, DMSO d6) 8 9.0 (br, s, 1H); 8.35 1H); 6.99 (s, 1H); 4.38 J 6.0 Hz, 2H); 3.83 J 5.5 Ht, 2H); 3.64 J 7.1 Hz, 2H); 3.30-3.1 4H); 2.85-2.65 2H); 2.54 J 7.1 Hz, 2H); 1.85-1.75 br, 2H); 1.60-1.20 (overlapping m, Is r L I PCTIUS94/01881 0 WO 94/1898 1 89 SCHEME 12 C0 2 0H 3 Br~j-

NDO-CH

2

CH

2 i" 4

,H

2

K

2 C0 3

CH

3

CN

CO

2

CH

3 12 -2 1. LiOH, H 2 0, CH 3 0H 2.

H

2 N l.C2u H NHSO 2 O O H 3 8 A-8 N

Q-

OH

3 I TFA, CH 2

CI

2 NH_1<C2 H NHS0 2 K) OH 3 N QO 12-4

-M

'WO 94/18981 PCT/US94/01881 2-(4-Pvridvl)ethylamine (12-1) A solution of NH4C1 in 200 ml of H20 was placed in a 1L Flask. 4-Vinyl pyridine (56.4 ml, 0.52 mol) was added along with 150 ml CH30H and on the mixture heated at 600 for 18 h. The reaction solution was cooled to 0° in an ice bath and made basic by the addition of 30% NaOH. The basic solution was extracted with CH2C12 (5 x 100 ml) and the pooled extracts dried, then evaporated. Vacuum distillation of the residue afforded 12-1 as a colorless liquid.

1H NMR (300 MHz, CDC13) 5 8.53 3 6.1 Hz, 2H); 7.25 J 6.1 H2, 2H); 3.02 2H); 2.77 2H); 1.4 (brs, 2H).

Methyl [4,5,6,7-tetrahydro-4-oxo-5-[2-(pyridin-4-yl)ethyl]pyrazolo- S1.5-alpyrazin-2-yl]carboxylate (i 2-2) A solution of 3-2 (1.4 g, 4.8 mmol), 4-(2-aminoethylpyridine) (0.645 g, 5.28 mmol), and potassium carbonate (0.78 g, 5.28 mmol) in 150 ml CH3CN was refluxed under N2 for 4.5 h then cooled, filtered and evaporated at reduced pressure. The resulting yellow residue was redissolved in 50 ml of DMF and treated with NaH (200 mg of a 60% oil dispersion) and heated at 900 for 3 h then concentrated at reduced pressure and chromatographed on silica gel using CH30H/CH2Cl2 giving the ester a 12-2 as a pale yellow glass (0.91g, mmol, 68%).

1H NMR (CDC13) 8 8.32 2H); 7.52 2H); 7.34 IH); 4.48 2H); 3.91 3H); 3.61 2H); 2.71 2H) 2.35 2H).

tert-Butyl 2(S)-[(p-toluenesulfonyl)amino]-3-[[[4,5,6,7-tetrahydro-4oxo-5-[2-(4-pyridyl)ethyl]py itzolo[ ,5-a]pyrazin-2-yl]carboxylate (12-3) A solution coiaining LiOH (130 mg, 3.05 mmol) in 10 ml was added to a solution of 12-2 (910 mg, 3.0 mmol) in 10 ml and the mixture was heated to 600 for 2.5 h then cooled and the solvent removed at reduced pressure. The remaining residue purified I II lr Is ''WO 94/18981 PCT/US94/01881 -91 by ion exchange chromatography on Dowex-50W resin to affording the desired acid as an off-white solid, mp i87°.

This acid (300 mg, 0.78 mmol) was suspended in 50 ml of anhydrous DMF, A-8 (293 mg, 81 mmol), EDC (150 mg, 0.78 mmol), HOBt (105 mg, 0.78 mmol) and N-methyl morpholine (87 ml, 0.78 mmol) were added and the resulting clear solution was stirred at 25 0

C

for 19 h. The solution was diluted with 100 ml of EtOAc, washed successively with sat. NaHCO3, H20, and brine (25 ml), dried over Na2SO4 and evaporated to provide 12-3.

1H NMR (300 MHz, DMSO-d6) 5 8.63 2H); 7.80 2H); 7.58 (d, 2H); 7.29 1H); 6.93 1H); 5.95 2H); 4.40 2H); 4.08 1H); 3.86-3.74 4H); 3.35-3.20 2H); 3.10 2H); 1.30 9H); 2(S)-[(p-Toluenesulfonyl)amino]-3-[[[4,5,6,7-tetrahydro-4-oxo-5-[2-(4pyridyl)ethyl]pyrazolo[1,5-a]pyrazin-2-yl]carboxylic acid*TFA salt (12-4) 12-3 was deprotected using TFA in CH2C12 and purified by reverse phase chromatography to give 12-4 as its TFA salt, mp 182- 185°.

1 H NMR (300 MHz, DMSO-d6) 8 8.72 (br d, 2H); 8.19 IH); 8.00 1H); 7.81 2H); 7.58 2H); 7.19 2H); 6.85 1H); 4.40 2H); 4.01 1H); 3.83-3.76 4H); 3.48 1H); 3.26 1H); 53.10 2H).

~1 I I I=I L 1~6~C PCTfUS94O1881 WO 94/18981 92 SCHEME 13 NaH, DMF ,O0H 3 N ot,B r O N 0 Q N I 00H 3 13-1 1. Li0H, H 2 0ITHF 2.

CA

H NHS0 2

K>--H

A-8 HOBt, EDO, 0H 2 C1 2 N C2t-OH H NHS0 2 Q H 0 13-2

TFA

0H 2 01 2 'T WO 94/18981 WO 9418981PCTFIUS94/01981 93 SCHEME 13 (CONT'D.)

CQ

2 H1

IHSO

2 O0--H 3 13-3 1. SH 2 Pyridine/Et 3

N

2. 0H 3 1, acetone 3. NH 4 CI, CH 3 0H

H

2 N 6 N CO H K>NN H 6 0 ;.HS0O 2 y OH 13-4 Methyl-S ,6,7 ,8-tetrahydro-4-oxo-5-[3 (cyanophenyl)methyl-4-Hpyrazolor 1 .5-a [14 A.4lzpin-9-vll -ab lt (1-1 A solution of (3.02 g, 14.5 mmol) in 60 ml anhydrous DMF was cooled to 0 0 C and treated with NaH (60% in oil) (636 mg, 15.98 mmol). The resulting mixture was stirred at 00 for 1.5 h, then a solution of 3-cyariobenzyl bromide (3.11 g, 15.89 mmol). in 50 ml, of., DMF was added dropwise. The resulting mixture was stiffed at 25' for 1 8h then diluted with 200 ml EtOAc and washed with H20 (3 x 100 ml) and brine (100 ml). The organic layer was dried (NaSO4), filtered and 'i WO 94/18981 PCT/US94/01881 -94evaporated. The resulting solid was recrystallized from CH2C12/CH30H to give 13-1 as a white solid.

IH,NMR (CDCI3) 7.85.(s, H);.7,78 J 8 Hz, 1H);.7.59 J 8.

Hz, 1H); 7.46 1H); 7.30 1H); 4.80 1H); 4.43 J 8 Hz, 2H); 3.89 3H); 3.38 J 8 Hz, 2H); 2.09 2H).

tert-Butyl 2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tet i:ydro-4oxo-5-[(3-cyanophenyl)methyl]-4H-pyrazolo[1,5-a][1,4]diazapin-2-yl]carbonvllamino]propanoate (13-2) A solution of ester 13-1 (1.5 g, 4.04 mmol) in 100 ml THF was treated with IN LiOH (5.1 ml, 5.1 mmol) and 100 ml H20 and stirred at 250 for 1.5 h. The THF was removed at reduced pressure and the aqueous residue acidified with 1N HC1. The resulting precipitate was filtered and dried in vacuo to give the desired product as a white solid.

1 H NMR (CDC13) 8 7.95 1H); 7.73 1H); 7.53 1H); 7.43 1H); 7.30 1H); 4.85 2H); 4.43 2H); 3.31 2H); 2.08 (m, 2H).

The above acid (1.0 g, 3.23 mmol) was combined with A-9 (1.24 g, 3.54 mmol), HOBt (480 mg, 3.54 mmol); EDC (641 mg, 3.54 .mmol) in 100 ml CH2C12. N-methyl morpholine (403 p1l, 3.83 9mmol) 2 was added and the resulting solution stirred at room temperature for 16 h, then was washed successively with sat. NaHCO3, 10% KHSO3 and brine (100 ml each), then dried over Na2SO4, filtered and evaporated.

The residue was chromatographed on silica gel (EtOAc) to give 13-2 as a white solid.

1 H NMR (300 MHz, CDC13) 8 7.73 J 6.8 Hz, 2H); 7.68 1H); 7.65 1H); 7.51 1H); 7.37 J 6.8 Hz, 2H); 7.20 1H); 7.18 1H); 5.63 J 6.5 Hz, 1H); 4.80 2H); 4.78 1H); 4.45 (t, I LIL kl 0 WO 94/18981 PCT/US94/01881 2H); 3.85 1H); 3.08 1H); 3.40 2H); 2.43 3H); 2.19 (m, 2H); 1.65 9H).

2(S)-,[(p-toluensulfonyl)amino],3-[[[5,6,7,8rtetrahydro-4-oxo-5-[(3cyanophenyl)methyl]-4H-pyrazolo[ 1,4]diazapin-2-yl]carbonyl]aminolpropanoate (13-3) A solution of 13-2 in CH2C12 (15 ml) was treated with ml of TFA. The solution was stirred at 00 for 2.5 h then evaporated Sgiving 13-3 as a colorless solid.

1 H NMR (300 MHz, CDC13) 8 7.73 J 6.8 Hz, 12H); 7.65 IH); 7.65 1H); 7.50 1H); 7.31 J 6.8 Hz, 2H); 7.3 1H); 7.28 1H); 6.15 J 6.5 Hz, 1H); 4.80 2H); 4.63 1H); 4.43 (t, 2H); 3.82 1H); 3.65 1H); 3.48 2H); 2.43 3H); 2.19 (m, 1 2H).

2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[(3amidinophenyl)methyl]-4H-pyrazolo[ 1,5-a] 1,4]diazapin-2-yl]carbonyllaminolpropanoic acid (13-4)_ 13-3 (400 mg, 0.73 mmol) was dissolved in 10 ml of a 4:1 mixture of pyridine and Et3N. The solution was saturated with SH2 and stirred until the nitrile could no longer be detected by HPLC The excess SH2 was removed by passing a stream of nitrogen through the solution. The remaining solution was then evaporated -and the residue triturated with IN HCI and filtered giving a yellow solid. This material was dissoved in 15 ml of acetone and treated with CH3I (250 gl) and then heated to 500 until the thioamide could no longer be detected by HPLC (2 The solvent and excess CH3I were evaporated and the residue redissolved in CH30H containing (NH4)2CO3 (144 mg, 1.14 mmol). The solution was heated at 500 for 12.5 h then evaporated, and 13-4 was isolated by preparative reverse phase chromatography.

11 I 1 0 WO 94/18981 PCTIUS94/01881 96 IH NMR (300 MHz, DMSO-d6) 859.38 2H1); 9.17 2H); 8.19 1H); 8.16 2H); 7.78 1H); 7.75 (in, 2H); 7.63 (in, 111); 7.60 2H); 7.21 2H); 6.93 111); 4.81 211); 4.40 2H); 3.93 (mn, PCTIrUS94IO1881 0 WO 94/18981 97 SCHEME 14 0

N-N

0 NaH, DMF' Br(C H 2 3

-CI

14-1 1. NaN 3

DMSO

2. LiOH, H 2 0/THF 14-2 1. EDC, HOBt, DMF NMM

H

2 N CO0 2 t Bu H NHSO 2 0 H 3 2. 10% Pd onC, H 2 H ,"NCO~tu H"NHSO 2 Q H 3 J-4- PCTIUS94/01981 14WO 94/18981 98 SCHEME 14 (CONT'D.)

OH

3

HN

INOH

3

H

2

N

DMF, H 2 0, DIPEA 2. TFA, 0H 2 C1 2

H--N(CH

2 3 -1

H

2 N

H

NHSO

2 Q O-H 3 14-4

H

2 N- (C H 2 3 H COAtU H"l

NHSO

2 K>

OH

3 14-3 CN1)?-S-CH,-HI

N

DMF, Et 3

N

2. TFA, 0H 2 C1 2 0 H C0 2

H

H NHSO 2 O

OH

3 14-5 I I WO 94/18981 WO 9418981PCTIVS94/01881 99 Methyl-S ,6,7,8-tetrahydro-4-oxo-5-(3 -chloropropyl)-4H-pyrazolo- [1 .5-ai[ 1 4idiazep~in-2-vllcarboxylate (14-1) 5-3 (2.0 g, 9.5 mmol) was alkylated with l-chloro-3-bromo m,, 1 0.5. minol). was described for 13- 1 Ao. give jjj as, a.

white solid.

I H NMR (300 MHz, CDCl3) 5 7.28 IlH); 4.58 2H); 3.93 3H); 2.78 2H); 2.68 2H); 2.46 2H); 3.27 (in, 2H); 2.18 (in, 2H).

5,6,7 ,8-Tetrahydro-4-oxo-5-(3 -azidopropyl)-4H-pyrazolo diazep~in-2-vl] carboxylic acid (14-2) A solution of this chloride (909 mg, 3.2 inmol) and NaN3 (620 mg, 9.5 mmol) in DMF (15 ml) was stirred at room temperature 36 h. The solution was diluted with ethyl acetate (50 ml) the washed H20 (3 x 50 ml), then dried (Na2SO4), filtered and evaporated to give the azide as a white solid. This material was hydrolyzed in the usual manner to afford 14-2 as a white solid.

I H NMR (300 MHz, DMSO-d6) 8 7.00 IH); 4.41 2H); 3.52 2H); 3.41 2H); 3.26 2H); 2.20 (in, 2H); 1.80 (in, 2H).

tert-Butyl 2(S)-(p-Toluenesulfonylamino)-3-[5 ,6.7 ,8-tetrahydro-4-oxo- 5-(3-aminopropyl)-4H-pyrazolo diazepin-2-yllcarboxyl)ainino)propionate (14-3) The acid 14-2 was coupled with A-9 as described for 13-2 to give the desired product as a white solid. This material was dissolved in ethanol and residual over 10% Pd on C under a H2 atmosphere to give 1.4-3 as a white solid.

1 H NMR (300 MHz, DM80 -d6) 8.18 I1H); 7.68 2H); 7.23 (d, 2H); 6.98 1H); 4.4 (in, 3H); 3.93 2H); 3.48-3.2 (mn, 6H); 2.78 (t, 2H); 2.43 3H); 2.69 (mn, 2H); 1.86 (in, 2H); 1.08 9H).' I WO 94/18981 WO 9418981PCTIUS94/01881 -100 2(S)-(p-Toluensulfonylamino)-3-[5,6,7 ,8-tetrahydro-4-oxo-5-(3-guanidinopropyl)-4H-pyrazolo[ 1,5-a] 1,4] diazepin-2-yl] carbonyl)amino)prop~ionic acid (14-4) A solution of mg, Q.1I mmol.) inDMF (5 ml} Was..

with DJPEA (90 glt, 0.5 ml) and 3 ,5-dimethylpyrazole- 1carboxamidine (30 mg, 0.5 mmol) and heated at 80'C for 12 h. The solution was evaporated and the residue purified by chromatography on neutral al'aininia (CH2Cl2ICH3OH/NH4OH, 80/2011) to give the desired product as a white solid. This material was deprotected with TFA in the usual manner and purified by preperative reverse-phase chromatography to give (14-4) as a white solid.

IH NMR (300 MHz, D20) 8 8.2 1H); 7.58 2H); 7.18 2H); 4.38 2H); 3.51 (in, 5H); 3.45 2H); 3.2 (in, 1H); 3.18 (in, 2H); 2.10 3H); 2.08 (in, 2H); 1.8 (mn, 2H).

2(S)-(p-Toluenesulfonylainino)-3-[5 8-tetrahydro-4-oxo-5-[3 [N- (imidazolin-2-yl)aminolpropyll-4H-pyrazolo[ 1,5-a] diazepin-2vi] carboxyl Iamino]ipropioni c acid (14-5) A solution of 14-3 was reacted with 2-methylthio-2imidazoline hydroiodide. using the procedure described in 14-4. The crude material was deprotected with TFA and 14-5 isolated by preperative reverse-phase chromatography.

1 H NMR (300 MHz, DMSO-d6) 8.20 I1H); 8. 10 21H); 7.60 2H); 7.21 (d 3H); 6.86 1H); 4.46 2H); 4.01 (in, 2H); 3.8-3.5 (overlapping in, 8H); 2.23 3H); 2.10 2H); 1.80 2H).

PCTIUS941S1 I. WO 94/18981 101 SCHEM 1. Isobutylchioroformate NMM, THE~

H

2 N C0 2 H 2. Et 3 N, THF, DIEA HN NNHSO 2 Q0

CH

3 NH C2 w NHSO 2

OH

3 15-1

HOI

CH

2 C1 2 0 0 HN

(CH

2 2 -N'L NH C2

H

N-N HNHSO 2 K0 OH WIIO 94/18981 PCT/US94/01881 102- 2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(N- B OC-piperidin-4-yl)ethy -4H-pyrazolo 1,4] diazepin-2-yl]carbonyllaminolpropionic acid A,solution of.56 (5.0 g, 12.3 mmol) in THF (15,0 ml) was cooled to 0-10 0 and N-methylmorpholine (2.11 ml, 19.2 mmol) was added via syringe. After mixing 20 min., isobutyl chloroformate (2.38 ml, 18.2 mmol) was added dropwise via syringe, and the resulting solution was stirred for 0.5 h to afford the desired mixed anhydride.

A-7 (7.00 g, 27.1 mmol), THF (125 ml), and Sdiisopropylethylamine (4.71 ml, 27.1 mmol) were combined in a 500 ml round bottom flask with a magnetic stir bar. Water was added in small portions until a clear solution resulted. The resulting solution was cooled in an ice bath. The mixed anhydride suspension was added in a single portion to the solution of 9 with vigorous mixing. After 20 min.

stirring the reaction solution was concentrated to remove THF. The remaining aqueous material was acidified with 10% potassium bisulfate and the resulting precipitate was filtered to give white solid.

This material was subjected to flash column chromatography using silica (EM Science, 230-400 mesh, 10 x 20 cm).

The. column was eluted with methylene chloride:methanol:ammonium hydroxide 98:2:0.2, 95:5:0.5, 90:10:1, then 85:15:1.5 to give the pure 15-1 as a white solid.

1-H NMR (DMSO-d6) 8 8.23 J 3.40 Hz, 1H); 7.64.(d, J 8.20 Hz, 2H); 7.32 J 8.20 Hz); 7.2-7.0 (br, 1H); 6.86 1H); 4.36 (t,J 6.70 Hz, 2H); 3.89 br, J 12.21 Hz, 2H); 3.59 1H); 3.47 J 7.08 Hz, 2H); 3.5-3.1 br, 5H, H20); 2.8-2.6 (br, 2H); 2.33 3H); 2.17 J 6.47 Hz, 2H); 1.66 br, J 11.97 Hz, 2H); 1.55-1.45 (m, br, 3H); 1.37 9H); 1.1-0.9 br, 2H).

-I I' LL ~s 0, WO 94/18981 PCT/US94/01881 103- 2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2- (piperidin-4-yl)ethyl]-4H-pyrazolo-[ 1,4]diazepin-2-yl]carbonyl]aminolpropionic acid (15-2) 15-1 (7.42, g,.,1J.48 mmol), was. placed,in. a. IL round bottom flask equipped with a magnetic stir bar. Methylene chloride was added and the reaction mixture was cooled to 0-50. Hydrogen chloride was bubbled through the suspension with stirring. After about 2 min.

the solid went into solution, and soon afterward a second precipitate formed. After bubbling gas through the suspension for an additional 1 min. the reaction flask was warmed to room temperature. After min. the contents of the reaction flask were concentrated. The resulting white solid was the hydrochloride salt of 15-2 and by HPLC analysis was of >99% purity.

This hydrochloride salt of 15-2 was subjected to ion exchange chromatography using Dowex 50X8-200 ion exchange resin (110 g, 4.11 meq/g). The resin was prepared by washing with water, methanol, water, 6N hydrochloric acid, and water (500 ml each). At this time the eluent was pH 7. The hydrochloride was dissolved in water (30 ml) and then applied to the top of the column. The column was eluted with water. The pH of the eluant became strongly acidic.

When the pH of eluant returned to 7, the column was eluted with ammoniun hydroxide:acetonitrile:water 50:25:25 Portions containing U.V. active material were combined then concentrated at high vacuum. The resulting white foam was dried for 8 h on the-high vacuum to provide 15-2.

1 H NMR (DMSO-d6) 5 9.0-8.5 (br, 1H); 8.19-8.16 1H); 7.67 J 8.18 Hz, 2H); 7.32 J 8.18 Hz, 2H); 6.89 1H); 4.38 J 6.84 Hz, 2H); 3.75-3.65 br, 1H); 3.46 br, 2H); 3.5-3.1 br, 8H, H20); 2.77 br, J 11.36, 2H); 2.35 3H); 2.17 J 6.47 Hz, 2H); 1.80 br, J 12.7 Hz, 2H); 1.53-1.42 br, 3H); 1.33-1.24 (m, br, 2H).

L L 111~ 1 I I WO 94/18981 WO 9418981PCT/US940188,t 104- Using the methods set forth previously, particularly in Schemes 3 and 4, the following compounds of Table 1 were prepared.

H NHR

-R

MP-M salt form S0 2 /a OCH 3 rK 0 S0 2

H

3 115-1 20 TFA 110-120 TFA 160-165 zwiterion 195-198 zwiterion So 2 180-188 HOI 0 1k C1 150-156 HOI

<NH

0 0 0 195-200 zwiterion 121 -1 24

TFA

WO 94/18981 W094/8981PCTIUJS94/01881 105 TABLE 1 (CONTID) -R mP( 0 C) salt form 210-212 TFA

SO

2

OH

3 85-95 TFA S02

C

S02

N

175-180 TFA 115-120 HOI 78-80 TFA s -,NH T 0---P 200 (dec) 210 (dec)

HCI

S0-"NH 2 Additional compounds, prepared according to procedures analogous to those of the exemplary procedures described above, are shown in the following tables: WO 94/18981 WO 9418981PCTIUS94/01881 106 TABLE 2 HN 0a

N

R

H00 2

H

N H H0''

B

R

0

N

0 2

H

5 0 N

H

H

O H 3 B m9(LI- H 145-152 H H 168-170 H UM 3 H 164-167 0H

,H

H H 110-135 -ON H H 180-188 0, WO 94/18981 PTU9/18 PCTIVS94101881 107 TABLE 2 (CONTOD' A B R 0 N

ICH

CAH

H NHS0 2

C

4 Hq 156-1 0 LKN C3

H

NHS0 2

C

4 Hq 134-140 0L<H

CH

3 0,' 0'

LN

2

D

NHSO

2

C

4 Hq 110-1 130-1 N HS0 2 0 4 Hq

NHSO

2

C

4

H

9 128-1 0 L NH H SO 2 \/&CHS 8-19 185-190 A~ I WO 94/189~81 CJS9018 PCTfUS94/01881 -108 TABLE 3 n 2 2 1 0 0 m 0 1 1 NH-(CY1 2 )mCH;:'9 2

HJ

-mp (OC) 110-115 115-1 121-123 135-1 41 140-1 WO 94/18981 WO 9418981PCT(US94/01881 109 TABLE 4 HN H0H~C2

R

H

6

R

H

H

NHSO

2

C

4

H

9 N HSO 2

C

4 Hq amide stereochemistry relative to H6 cis trans

NH*

chemical shift (PPM) 6.35 7.38 cis trans 6.31 7.29 "H6" refers the hydrogen group at position 6 of the bicyclic structure I I WO 94/18981 WO 9418981PCTIUS94/01981 110- TABLE M P (00)

OH

3 S0 2 0 4

H

9

SO

2

H

3 S0 H 3 S02\a

CI

0 1 OH 110-116 186 164-1 71 164-1 135- 140 not determined H S0 2 C H 3 H I x 173-174 185-1 89 178-179 H H WO 94/18981 WO 9418981PCTIUJS94101881 TABLE 6 HN 0 N- yC2 1H H 248-249 151H

NHSO

2 -a

H

3 175-18r) 1

H

3 H 118-122 1O 3 NHS0 2 C H 3 155-160 2 Ph H 137-139 2 Ph NHS0 2 H 3 185-188 2 H NH0 a C3192-194 HNHC0j- Ph 176-178 302 H

NHSO

2

O

3

H

7 168-170 2 H NHS0 2

C

2

H

5 17-4

Claims (3)

112- WHAT IS CLAIMED IS: 1. A compound having the formula 0 R2' R 6 Q-(CH 2 C-N- Rs-C- R 8 R 4 R 1 O R 2 R 6 II I I Q- (CH 2 a-AB- C R-C R R 1 wherein Q is NH HN-C H 2 N-C- NH II H 2 N-C-NH- SRHN- or Q is a 4-9 membered mono- or bi-cyclic ring system containing 1, 2 or 3 heteroatoms chosen from N, O or S and either unsubstituted or substituted with R S AB is a fused ring system sharing adjacent carbon and nitrogen atoms, wherein A is a 5, 6 or 7 membered saturated or unsaturated ring containing 1, 2 or 3 heteroatoms selected from O, S or N; B is a 5, 6 or 7 membered saturated or unsaturated ring containing 1, 2 or 3 heteroatoms selected from O, S or N; ~IIII~I~ a ~qlpl~ll. I--1 I I I R I is H, Cl1-4 alkyl, -N(R 8 )S02R 7 NRSCO2R 7 NR 8 (O)R7, NRSC(O)N(R 7 )RS, N(Rg)SO2N(R 7 )R 8 N(Rg)SO'2N(RS)C(O)0R 7 C(O)N(R 7 or a cyclic group with R 6 as defined below; R 2 is H, C1-4 alkyl, CI-4 branched alkyl, CI-4 alkyl aryl, or aryl;. R 4 is H, C 1-4 alkyl, CI1-4 branched alkyl, cyclic C 1 -4 alkyl or C 1-4 alkenyl; R 5 is CH, -CH(CH2)n, a bond, or when R 5 is adjacent N(R 4 -C(CH2)n; 0 R 6 is COOH, CH2OH, C(0)N(R 7 C02R 9 tetrazole, acylsulfonamide, or IIH) .0 ,or a cyclic group with R 1 as defined below; wherein the cyclic group of R I with R 6 is R82 0 08 0 7 4 R 7 is H, branched or straight chain Ci 1 4 substituted or unsubstituted alkyl, branched or straight chain lower aikenyl, C 1 4 alkylaryl, substituted aryl, or 5 or 6 membered heteroaryl containing 1, 2, or 3 N, S, or 0 heteroatoms wherein substituted alkyl is hydroxy substituted or C1-4 alkoxy substituted alkyl, and wherein substituted aryl is substituted by one, two or three of the following groups: halogen, Cl.4 alkoxy, 114 hydroxy, or C 1 4 alkyl; R 8 is H, branched or straight chain Ci1- alkyl; R 9 is H, C 1 4 alkyl or aryl; n is 0-7; and a is -NC 'I or a bond, and pharmaceutically acceptable salts; wherein the terms alkyl and aryl are each as hereinbefore defined. 2. A compound of claim 1 having the formula 0 R 2 R 6 11 1 1 Q-(CH 2 n-a-Ab-C-N-R 5 -CR 8 I I R4 R, 0* C S C. C 0 6000 0 S.C S. C C C CC S. S S .6CC o R 2 R 6 11 1 1 Q--(CH 2 )--a-AB-C-R 5 '-R 8 wherein S. C *CO S S C *6 S *0 S CS. S CC C S0 CC r'.\Iib,,a]00804:JVR PCTIUS94/01881 ,I WO 94/18981 115 Qis NH H 2 N I (CH 2 )<INyNH- N R 8 1 2 N NH -(CH 2 )n (CH 2 R 8 N N ;o0 R 8 N 0 N WO 94/18981 PTU9/18 PCTIUS94/01881

116- N ,N :CH 2 N N HN (CH 2 )n H 2 (CH 2 )n' NH (CH )n H 2 NN H H 2 N N N== NH H 2 N ll DZ I or N n 0-7; n' 0-3; R= H, C1-4 alkyl, C1-4 branched alkyl, cyclic C1-4 alkyl or CI-4 alkenyl; CH, -CH(CH2)n, or a bond; R 2 is H, C1-4 alkyl, CI-4 branched alkyl, C1-4 alkyl aryl, or ary,'; WO 94/18981 WO 9418981PCTIUS94/01881 -117- R H, C-4 alyl, N(Rg)2, -N(Rg)S02R 7 NR 8 CO2R 7 NR 8 C(O)R 7 NRgC(O)N(R 7 )Rg, N(RS)SO2N(R 7 )Rg, N(R 8 )SO2N(RS)C(O)0R 7 C(O)N(R 7 or a cyclic group withR6 R 6 =COGH, CH2OH, C02R 9 tetrazole, acylsulfonamide, or P(OH) 2 1 0 or a cyclic group with RI; wherein the cyclic group of R I with R 6 is R7~~ N- Rr Y wherein y =0 or S; R7= H, branched or straight chain Ci -4 substituted or unsubstituted alkyl, branched or straight chain lower alkenyl, Cl1-4 alkylaryl, substituted aryl, or 5 or 6 membered heteroaryl containing i, 2, or 3 N, S, or 0 heteroatorns wherein substituted alkyl is hydroxy substituted or Cl1 -4 alkoxy substituted alkyl, and wherein substituted aryl is substituted by one two or three of the following groups: halogen, C1-4 aikoxy, hydroxy, or Ci 1 4 alkyl; R 8 H, branched or straight chain Cl1.-4 alkyl; R= H, Cj-4alkyl or aryl; I_ WO 94/18981 PCT/US94/01881 -118- a= 0 K. N R 7 R or a bond; A a 5, 6 or 7 membered saturated, partially saturated, or unsaturated ring containing 1, 2 or 3 heteroatoms selected from O, S or N; and B a 5, 6 or 7 membered saturated, partially saturated, or unsaturated ring containing 1, 2 or 3 heteroatoms selected from 0, S or N; wherein A and B form a fused ring system sharing adjacent carbon and nitrogen atoms; and racemates, racemic mixtures, enantiomers and diastereomers thereof. 3. A compound of Claim 2 having the formula 0 R2 R 6 j I I Q-(CH2)n-a-AB-C-N- Rs-C- R 8 R 4 R 1 wherein AB is II I s 119 0 DN~ wherein V is N or OR, and D is OH 2 0H 2 -OH 2 0H 2 0(R 7 2 0H 2 or H 2 C OH 2 (CH 2 (OH 2 )n wherein X N or OR* 3 wherein R 3 ON O(O)N(R 7 )R 8 0 *6* S S .555 55S555 Cl 2 Xv' herein n' 0-3, or N R 8 WO 94/18981 WO 9418981PCT[US94/01881 120 0 and 0 K N (H 2 C)n 4. A compound of Claim 3 wherein AB is N 0 N\ wherein V is N or OH, and D is CH 2 -CH 2 or CH 2 C(R 4 2 CH 2 121 A compound of Claim 2 having the formula o RI R' Q-(CH 2 C-R5-C-R 8 wherein AB is 1 0 V D 0* 0 9 S *900 0 0 0 0 *90e **00 09 00000 0000 00 00 wherein V is N or CR 7 and D is CH 2 CH 2 -C H 2 CH 2 C(R 7 2 0H 2 or H 2 C OH 2 0 N 'k N- x wherein X N or CR, wherein R 3 ON, rI(0)N(R 7 L 2N 0 ,or 0 -(CH2)n' ().wherein n' is 0-3; WO 94/18981 WO 9418981PCTITJS94/01881

122- N" (C H 2 )n' ;and 0 AN N wherein y 3 is 0 or H 2 y 3 6. A compound of Claim 5 wherein 0 and 1 4 WO 94/18981 WO 9418981PCT/LUS94/01881 123 AB is selected from Z/O wherein V is N or OR 7 and D is OH 2 CH 2 -CH 2 0H 2 0(R 7 2 0H 2 or H 2 0 OH 2 and wherein X N or OR 3 wherein R 3 CN, O(O)N(R 7 N 0 or 0 (CH 2 )n. *0 94/18981 Wo 9418981PCT/US94/01881 124 7. A compound of Claim 5 wherein a a bond; and AB is selected from 0 0 and (CH 2 )n' 0 AN y 3 wherein y 3 is 0 or H 2 8. A compound of Claim 2 which is selected from 0 0 e OH NI N HSO 2 NHCO 2 CH 2 Ph, NVO 94/18981 VVO 9418981PCTIUS94/01881 125 N N-N HND NHSO 2 NH 2 HN0 N N~COOH N-~N H HN 0 O IN N yCOCH N-N HI H HNSO 2 C 4 H 9 HN 0 0 N 2 0H 3 Nn-/ N -N IH' H HNSO 2 C 4 Hq .COOH I WO 94/18981 WO 9418981PCTIUJS94/01881 126 N 1 COOH No ,CO 2 H N-N HN S0 2 C 4 Hq 9 PCTIUS94/01881 W~O 94/18981 127 HNC HN 0 0 N 'y u N OH 3 HN 0 ~N /OO C02H PCr[US94/01081 WO 94/18981 128 C0 2 H INH 2 HN 0 002 N N H 0' HN 0 C0 2 H NN-e- N~ ~H H-NSO 2 C 4 H 9 PCT/US94/01881 WO 94/18981 129 C02H H N 0C0 2 H N ~HNS0 2 C 4 Hq 0 N Et 2 HNC 0 NI C02H NH H0 2 C N NCG HNa H02 WO 94/18981 PCTtUS94/01881 130 CO 2 H H~ INSO 2 O 4 ,C0 2 H CH 3 A I HHNSO 2 a C H 3 N HN H ,and PCTIUS94/01881 WO 94/18981 131 HN 0 N 0 N C0 2 H OH 3 9. A compound of Claim 8 selected from the group consisting of ,COOH _,CO0H HNSO 2 /Cl H N3 N >12 C02H H HNSO 2 H 3 C0 2 H HN SO 2 OH 3 d A fibrogen receptor antagonist having a fused ring system sharing adjacent carbon and nitrogen atoms, substantially as hereinbefore described with reference to any one of the Examples. 11. A pharmaceutical composition, comprising a compound of any one of Claims 1 to 10 and a pharmaceutically acceptable carrier. *12. A method for inhibiting the binding of fibrinogen to blood platelets in a mammal, comprising administering to the mammal a pharmacologically effective amount S0. of a compound of any one of Claims 1 to 10 or of a composition of Claim 11. 13. A method for inhibiting the aggregation of blood platelets in a mammal, comprising administering to the mammal a pharmacologically effective amount of a compound of any one of Claims 1 to 10 or of a composition of Claim 11. 14. A method for preventing thrombus or embolus formation in a mammal, comprising administering to the mammal a pharmacologically effective amount of a 15 compound of any one of Claims 1 to 10 or of a composition of Claim 11. o 15. A method for treating thrombus or embolus formation in a mammal, comprising administering to the mammal a pharmacologically effective amount of a compound of any one of Claims i to 10 or of a composition of Claim 11. 16. A method for inhibiting the aggregation of blood platelets in a mammal, comprising administering to the mammal pharmacologically effective amounts of a thrombolytic agent, a compound of any one of Claims 1 to 10 or of a composition of Claim 11, and an anticoagulant. 17. A method of Claim 16 wherein the thrombolytic agent is tissue plasminogen activator or streptokinase and the anticoagulant is heparin. 18. A method for preventing or treating thrombus or embolus formation in a mammal, comprising administering to the mammal pharmacologically effective amounts of a thrombolytic agent, a compound of any one of Claim:, 1 to 10 or of a composition of Claim 11, and an anticoagulant. RA8 0- tNi\liba]00804:JVR "s ~IP -~LI 133 19. A method of Claim 18 wherein the thrombolytic agent is tissue plasminogen activator or streptokinase and the anticoagulant is heparin. Dated 1 May, 1997 Merck Co. Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 9g [N:\Iibaa]00804:JVR INTERNATIONAL SEARCH REPORT fIntenational application No, PCTIUS94/O1 881 A. CLASSIFICATION OF SU13JECT MATITER :A61K 31/55, 31/415, 31/495; C07D 471/04, 487/04 US CL :514/221,249,300,303; 540/502; 544/350; 546/121 According to International Patent Classification or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) U.S. :514/221,249,300,303; 540/502; 544/350; 546/121 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Please See Extra Sheet. Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A USDA 5,095,018 (Kelley) 10 March 1992. 1-22 A USIA 5,166,154 (Skiles et al) 24 November 1992. 1-22 A,P USA 5,278,161 (Branca et 11 January 1994. 1-22 Further documents are listed in the continuation of Box C. See patent family annex. Special categories of cited docu~ments: -r later document palisad atr the insereatioosl filial date or priority dute and not in conflictwith the apsiLicatioa but cited to understavid the documtendeflning the gene?,! state of the ait which is not considered pricxpie or thaMr unierlying the invention to be pan of particular relevance E ealie docmen pulishd o or lte th intmatocalrun.X. document of particuiar relva=ce the cihmed inveoim canot be adlr dcumea ublihedon r atertheintenatona fiingdueconsidered novel or canno be considered to involve an inventive step U documnent which may throw doubts on priority claim(s) or which i when the document is taken slowe cited to establish the publication dute of another citation or ote dacuanet of particular relevance; the chimed invention cannot be special reason (am specified) considered to involve ra inventive step whita *ah document is document referring to an omal disclosure, se, exhibition or other combined with one or mome other such docnnarm, iusch comobination melkalbe@n3 obvious to a person skilled an the an documen publihed prior to the internationad filing dule bt iter than documnent member of the same petat family the priority date claimed Date of the actual completion of the international search Date of mailing of the international qearch r 09 MAY 1994 JUN 017 1994 Name and mailing address of the ISA/US Authorized officer Commissioner of Patents and Trademrarks Box PCTROBERT T. BOND Washington. D.C. 20231 Facsimile No. (703) 305-3230 Telephone No. (0)308-1235 Form PCT/15A/210 (second sheet)(July 1992)* INTERNATIONAL SEARCH REPORT [trainlplcto~, International application No, PCTIUS94/01881 B. FIELDS SEARCHED Documentation other than minimum documentation that are included in the fields searched: RING INDEX PLUS 1+11 +111 SUPPS. CHEMICAL ABSTRACTS VOL. 56 -VOL. 117 DIAZEPINODIAZOLE 1962 -DECEMBER 1992 Form PCTIISAI21O (extra shect)(July 1992)*