CA2216859A1 - Thrombin inhibitors - Google Patents

Abstract

A compound which inhibits human thrombin and has the structure (I) such as (II).

Description

W 096/31504 P~l~ l lCO

TITLE OF THE INVENTION
THROMBrN INHIBITORS
=
BACKGROUND OF THE I:NVENTION
Thrombin i~i a .serine protea,se pre.~ent in blood pla~ma in the form of a precur.~or, prothrombin. Thrombin play.s a central role in the mechani.sm of blood coa~ulation by converting the .~olution plasma protein, fibrinogen, into in~oluble fibrin.
Edwards et al., J. Ame) . Chem. Soc.. (1992) vol. 114, pp.
1~54-63. de.~cribe.s peptidyl a-ketobenzox~zole.~ which are rever~iible inhibitor.s of the .~ierine protea.se.s human leul;ocvte ela.~ta~e ~nd porcine p;mcre~tic ela.sta.se.
European Publication ~63 ~4 de.~cribe.~ allalog.s of peptida.~e .~ub.~trate.s in which the nitro~en atom of the .~ci.s.~ile amide roup of the _strate peptide ha.~ been replaced by hydrogen or a _stituted c~rbonyl moiety.
Au.stralian Publication ~S6245677 ~I~;o de~icribe~ peptida~e inhibitor~ having an activated electrophilic ketone moiety such a~
fluoromethylene ketone or o~-keto carboxyl derivative~.
Thrombin inhibitor~ de.~cribed in prior publication.~; contain .~idech~in~ of ar~inine and Iy~;ine. The~e .~tructure.~ how low ~ielectivity for thrombin over other tryp.~in-like enzyme.~i. Some of them .~how toxicity of hypoten~sion and liver toxicity.
European Publication 601 459 de.~cribe.~i .sulfonamido ~5 heterocyclic thrombin inhibitor~. such a.s N-[4-[(aminoimino-methyl)amino~butyl~- I -[N-(2-n~phthalenyl~ulfonyl)-L-phenylalanyl]-L-prolinamide.
WO 94/29336 de.scribe.~ compound.~ which are u~eful a~
thrombin inhibitor~s.
f W O 96/31504 PCTnUS~'014C0 SUMMARY OF THE INVENTION
Compound~s of the invention have the following structure:
O

Q xg~l~A

S wherein A i.~ C orN:
Xl. X~ and X~ e;lch independently attached to ~ ring c;3rbon atom. ~re indepen(iently ~elected from the group con~ ting of hydrogell. C l ~ all;yl. ~nd C l ~ alkoxy;
Y. attached to ;I ring cart)on atom. i.~ H. NH2 or OH:
Z i~ -(CH2) 1 ~-:
R I, R~, and R~ are independently hydrogen .
phenyl, 1:~ mono- or di-halogenated phenyl, naphthy I .
biphenyl.
a 5- to 10-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~;y.~tem any rin~ of which may be .~aturated or un.~aturated. and which cor~ t~ of carbon atom.~; and from one to three heteroatom.~ ~elected from the group con.~ ting of N, O and S, C 1-4 alkyl, branched C l 4 alkyl, C3 7 cycloalkyl, C5 12 bicyclic alkyl, C 1 1-16 tricyclic alkyl, R4(CH2)n, - (R4)2(CH).

PC rluss6/o q4 co (R4)(oR4)CH, R40(CH2)n, or R 1 may be joined with R2 to form a four- to .seven membered carbon ring in which zero to two carbon atom~s may be _stituted with heteroatoms independently .selected from the li~t N. O and S, where n i.~i 1, 2, 3 or 4;

R3 i.~i hydrogen.
( R2) ~N, wherein R'' i.~ the .~iame or different.
R~ OCONH. provided R'~ ix not hydrogen.
R~CONH, HO(CH2)p, where p i.s 0, 1, 2. 3 or 4, R2 SO2NH, provided R'~ not hydrogen, or (R'~)mNCONH. where m i.~i 1 or 2. wherein R'~ the .~me or different;

20 R4 i.~; independently phenyl.
mono- or di-halogenated phenyl.
n~phthyl, biphenyl, ~ 5- to 10-membered mono- or bicyclic heterocyclic ring bicyclic heterocyclic ring ~iy~item any ring of which may be .~;aturated or un.~aturated, and which con~i~;t~i of carbon atom~ and from one to three heteroatom~ selected from the group con.~isting of N, O and S, -COR5, _oR67 C 1-4 alkyl, branched C l 4 alkyl, C l 4 alkoxy, CA 022168~9 1997-09-29 W O96/31~04 PCTnUS9G/0~0 C3 7 cycloaLkyl, Cs 12 bicyclic alkyl, or Cl 1-16 tricyclic alkyl;

S R5 is -OH, _oR6~
-N(R7)2, where R7 is same or different~ and D~N--where D is -CH2CH~-, -CH ~-O-CH~-. or -CH ~-NH-CH~-.

R6 is C 1- 1 ~Ikyl;

R7 i~i hydrogen or Cl 4 alkyl;
1~
G is (CH2)~1 where ~1 i.s I or 2; or NRICH2; and Q i~ SCH2, or (CH2)r where r i,s I or 2, and pharmaceutically acceptable salt.s thereof~
These compounds ~how selectivity for thrombin inhibition over inhibition of trypsin and other tryp.sin-like enzyme.~. Tryp.~in-like 2~ enzyme.s (such as trypsin~ thrombin, factor xa, kallikrein, pl;~smin, urokinase~ and plasminogen activator) are serine dependent enzymes th~t catalyze hydroly.sis at arginyl and Iysyl peptide bonds~
The invention includes a compo,sition for inhibiting los~ of blood platelets, inhibiting formation of blood platelet aggregates, 30 inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolu~s formation in a m;~mm~l, comprising a compound of the invention in a pharmaceutically acceptable carrier~ These compositions may optionally include anticoagulant,s, antiplatelet agents, W O 96/31504 PCTnUS9''0~1C0 and thrombolytic agent.s. The composition,s can be added to blood, blood product.s. or m~mm~lian organs in order to effect the de~;ired inhibitions.
The invention also includes a composition for preventing or 5 treating un~table angina, refractory angina, myocardial infarction, transient i!;chemic attack,s, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombo.~ . di.~.~;eminated intr~ ascular coagulation.
ocular build up of fibrin, and reocclusion or re~iteno~is of recanalized ve~;~el.~. in a mammal. compri~ing a compound of the invention in a 10 pharmaceutic~lly acceptable c~rrier. The.~;e compo~;ition~i may option~
include antico~gul~nt.~i. antiplatelet agent~. ~nd thrombolytic agent.~;.
The invention ~ io includes ~ method for reducing the thrombogenicity of ~ urface in ;~ m;~mmal hy ~tt;3ching to the ~urface.
either cov;llently or noncovalently. ~ compound of the invention.
1~
DETAILED DESCRIPTION OF THE lNVENTlON
Compound.~i of ~he invention have the following !itructure:

Q X3~ A
x2 20 wherein A i.~i C or N;
Xl, X2 and X3, each independently attached to a ring carbon atom, are independently ~elected from the group con~i~ting of hydrogen, Cl 4 alkyl, and Cl 4 alkoxy;
25 Y, attached to a ring carbon atom, is H, NH2 or OH;
Z i~ -(CH2)1-3-;
R l, R2, and R2 are independently hydrogen, phenyl, CA 022l6859 l997-09-29 W O96/31504 PCTnUS96/0~1C0 mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 10-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system ~ny ring of which m~y be satur~ted or uns~turated. ~nd which con.~ t.~; of c~rbon ~tom.~ ~nd from one to three hetero~toms selected from the group consisting of N. O ~nd S~
C l 4 ;llkyl~
br~nched Cl ~ ~Ikyl.
C~ 7 cyc lo;lll;yl.
C~ I ~ bicvclic ;llI;yl.
C l 1-1~ tricyc lic ~I~;yl, R4(CH2)n.
1~ (R4)~(CH)-(R4)(oR4)CH, R40(CH2)n, or R I m~y be joined with R2 to forrn ~ four- to .~even membered c;~rbon ring in which zero to two c~rbon atom.~ m~y be ~ub.~tituted with hetero~tom~i independently .~elected from the li~it N. O ~nd S.
where 1~ is 1. 2. 3 or 4;

R3 i~i _ hydrogen, (R'~ N, wherein R2 i.s the s;~me or different.
R2 OCONH, provided R2 is not hydrogen.
R'7CoNH, HO(CH2)p, where p is 0, 1, 2, 3 or 4, R2 S02NH, provided R2 is not hydrogen, or (R2)1nNCONH, where m is 1 or 2, wherein R2 is the same or different;
R4 i.s independently PCTrUS9~'0~0 phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 10-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring sy.stem any ring of which may be ~ turated or un.~i~turated. ;~nd which con~si~it.~; of carbon atom~ and from one to three heteroatom.~ ~elected from the group con~ iting of N, O and S, -COR5, _oR6~
Cl 1 ~Ikyl, branched CI-1 alkyl, C1 1 ~Ikoxy~
C~ 7 cycloalkyl, C5 12 bicyclic alkyl, or Cl 1-16 tricyclic alkyl;

R5 i.~
-OH.
_oR6~
-N(R7)2, where R7 is ~ame or different, and D~N--where D is -CH2CH2-, -CH ~-O-CH2-, or -CH~-NH-CH2-;
R6 is C l ~ alkyl;

R7 i~ hydrogen or Cl 4 alkyl;

G i.~ (CH2)4 where 4 i.s 1 or 2; or NRlCH2; and Q is SCH2, or CA 022l68~9 l997-09-29 PCTlU~r"~1C0 (CH2)r where r i,s 1 or 2, and pharrnaceutically acceptable salt~s thereof.
In one clas.s, compound.~ of the invention have the following 5 ~tructure:
o R 2 ~3 ,N >~ N ~ , x 1 x2 wherein A i.~; C orN;
X I ~ X2 and X3, each independently attached to a ring carbon atom~ are independently .selected from the group con.~i~ting of hydrogen and Cl 4 alkyl;
Y~ attached to a ring carbon atom, i.~. hydrogen, NH2 or OH;
R 1 R2, and R2 are independently hydrogen, 1 5 phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~ystem any ring of which may be ~aturated or un~aturated. and which consi~t.~ of carbon atom.~i and from one to three heteroatoms .selected from-the group consi.~ting of N, O and S, C l 4 alkyl, branched C 1_4 alkyl, C3 7 cycloalkyl, Cs 12 bicyclic alkyl, Cl 1-16 tricyclic alkyl, R4(CH2)n, CA 022168~9 1997-09-29 W 096/31504 PCT/u~3~n~co (R4)2(cH)~
(R4)(oR4)CH, R40(CH2)n, or R 1 may be joined with R2 to form a four- to seven membered S carbon ring in which zero to two carbon atom,s may be ~substituted with heteroatom.s independently .selected from the li.st N~ O and S.
where n i.s 1, 2. ~ or 4;

10 R3 is hydro en.
(R2)2N. wherein R'' i.s the ~,ame or different.
R2 OCONH. provided R'' i.s not hydrogen.
R2CONH.
HO(CH2)p. where p i.s 0. 1, 2, 3 or 4, R2 SO2NH. provided R2 i.s not hydrogen. or (R2)mNCONH. where m i.s 1 or 2. wherein R'7 i.s the same or different;

20 R4 i.s independently phenyl .
mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to-7- mernbered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~iy.stem any ring of which may be .saturated or un.saturated. and which con.si.sts of carbon atom.s and from one to three heteroatom.s .selected from the group con.si.sting of N, O and S, COOH, C l 4 alkyl, branched Cl 4 alkyl, C3 7 cycloalkyl, C5 12 bicyclic alkyl, or PCr/US~C;~ ~ ~ CO

C l 1-16 tricyclic alkyl;

G is (CH2)~1 where ~1 is I or 2; or NRICH2; and s Q is SCH2, or (CH2)r where r is 1 or 2.

and pharm;lceutic~llly ~cceptable salts thereof.
In ~ ~,uhcl;lss of this cl;l.ss. the compound.~ h;~ve the following xtructure:
o R~3 G'NYl~N/~ l=' X

wherein 1;~ Ai.sCorN:
X I ~nd X''. e~ch independently attached to ~ ring carbon ~tom. ~re independently selected from the group con~isting of H and Cl 4 alkyl;
Y. attached to ~ ring carbon atom. is hydrogen. NH2 or OH;
R I . R2. and R2 are independently hydrogen .
phenyl.
mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be ' saturated or unsaturated, and which consist~s of carbon atoms and from one to three heteroatoms ~selected from the group consisting CA 022l6859 l997-09-29 W O96/31504 PCTnUS~G/'~5G0 of N, O and S, C 1-4 alkyl, branched Cl 4 alkyl, C3 7 cycloalkyl, S C5 12 bicyclic alkyl, Cl 1-16 tricyclic alkyl, R4(CH2)n, (R4)2(CH), (R4)(oR4)CH, R40(CH2)n, or R I may be joined with R'' to forrn ~ four- to .~evell membered carbon ring in which zero to two c~rbon atom~ m;ly be ~ub.stituted with hetero~tom.~ independentl~ ~elected from the li.~it N. O ,Ind S.

15 where n i~ 1, 2, 3 or 4;

R3 i~
hydrogen, (R2)2N~ wherein R2 i~ the ~i~me or different, R2 OCONH~ provided R2 j~ not hydrogem R~CONH, HO(CH2)p, where p is 0. 1, ''~ 3 or 4, R2 SO2NH, provided R2 i~ not hydrogen, or (R2)mNCONH, where m i~ 1 or''. wherein R2 i~ the ~ me or different;

R4 i~ independently phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or -bicyclic heterocyclic ring .sy.stem any ring of which may be ~saturated or un.saturated, and which consists of carbon atom~s and CA 022l6859 l997-09-29 PCTrUS9''0~4G0 W 096/31~04 from one to three heteroatoms selected from the group consisting of N, O and S, COOH, C 1-4 alkyl, branched Cl 4 alkyl, C3 7 cycloalkyl, Cs 12 bicyclic alkyl, or Cl 1-16 tricyclic alkyl;

G is (CH2),1 where ~I i.s 1 or 2; or NR I CH2; and Q i.~ SCH2, or (CH2)r where r i.s I or 2, and ph~rrnaceutic;~lly acceptable .s~lts thereof.

In a group of this ~ubclass. compound.s of the invention h~ve the following structure:
o ~ G~o>J~ x1 wherein X 1 and X2, each independently attached to a ring carbon atom, are independently ~selected from the group consisting of hydrogen and Cl 4 aLkyl;
Y~ attached to a ring carbon atom, is hydrogen or NH2, R 1, R2, and R2 are independently hydrogen, phenyl, . CA 02216859 1997-09-29 W O96/31504 PCTrUS9''0~1C0 mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring sy,stem any ring of which may be saturated or un~iaturated, ~nd which con.si~t.~i of carbon atom.~i and from one to three heteroatom.s .selected from the group con~ ting of N. Oand S.
C l 4 alkyl, br;mched C 1-4 alkyl.
C3 7 cycloalkyl, C~ l ~ bicylic alkyl, Cl 1-16 tricylic ~Ikyl, R4(CH2)n.
(R-1)2CH. wherein R4 i.~i the .~,~me or different.
(R4)(oR4)CH, R40(CH2)n, or R 1 m;~y be joined with R2 to form ~ four- to .~ieven membered carbon ring in which zero to two c~rbon ;ltom~, m~y be .~iub.~tituted with heteroatom~ independently ~elected from the li.~,t N. O ~nd S.
where n i.~i 1, 2, 3 or 4;

R3 i.~
hydrogen.
(R2)2N. wherein R2 i.~ the .~,~me or different.
R2 OCONH, provided R2 i.~, not hydrogen.
R2CONH, HO(CH2)p, where p is 0, 1, 2, 3 or 4, R2 SO2NH, provided R2 i~ not hydrogen, or (R2)mNCONH, where m i.s 1 or 2, wherein R2 is the .same or different;

R4 i.~; independently PCTnUS96/014C0 phenyl, mono- or di-halo~enated phenyl, naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~iy~tem any ring of ~~hich may be ~aturated or un~aturated. and which con~i~itx of carbon atom~ and from one to three heteroatom.~ ~elected from the group con.~ ting of N. O ~nd S.
I 0 COOH.
Cl ~ yl.
branched CI 1 ~ll;yl.
C~ 7 Cyclo;~
C5-12 bicyclic ~lkyl. or C1 1-16 tricycIic alkyl;

G i.~i (CH2),~ where ~ i,s I or 2. or NRICH2: and Q i~ SCH2, or (CH2)r where r i.~ I or 2.

and pharmaceutically acceptable ~alt.s thereof.

In another group of thi.~ .~ubcla~. compound~ of the invention have the following ~;tructure:

R ~ ~ N>,l~ H ,~

wherein 30 Y, attached to a ring carbon atom, i~ H or NH2;

W O96/31504 PCTnUS96/04460 R 1, R2, and R2 are independently hydrogen, phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~y.stem any ring of which may be ~aturated or unxaturated. and which con~ t~ of carbon atom.~; ~nd from one to three heteroatom.~ .~elected from the group con~ ting of N. O and S.
C 1 4 ~Ikyl, branched Cl ~ ~ll;yl.
C3 7 cycloalkyl.
Cs 1~ bicyclic alkyl, Cl 1-16 tricyclic alkyl~
K-1 ( CH2)n, (R4)7CH. wherein R4 i.~i the .~iame or different, (R4)(oR4)CH, R40(CH~ )n, or R 1 m;~y be joined with R2 to form ~ four- to .~even membered carbon ring in which zero to two carbon atom.~i may be .~;ub.~tituted with heteroatom~i independently ~elected from the li.~;t N. O and S.

where n i~i 1, '', 3 or 4;

R3 i.~
hydrogen, (R2)2N, wherein R2 i,~i the ,~iame or different, R'~ OCONH, provided R2 i~ not hydrogen, R2CONH, HO(CH2)p, where p i,~ O, 1, 2, 3 or 4, R2 S02NH, provided R2 i~ not hydrogen, or (R'~)mNCONH, where m i,~ 1 or 2. wherein R2 i~ the ~ame or W O96/31504 PCTrUSS~v~G0 different;
R4 is independently phenyl, mono- or di-halogenated phenyl, S naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~y~item any ring of which m~y be ~iaturated or un~iaturated. and which con.si.st~i of carbon ~to n.s and from one to three heteroatom.s ~;elected from the group cc n.~i~ting of N, O and S.
COOH.
C 1-~ alkyl, branched C l 4 alkyl, C3 7 cycloalkyl, Cs 12 bicyclic alkyl, or Cl 1-16 tricyclic alkyl;

G i~s (CH2)y where y i.~i I or 2, or NRICH2; and Q i.~ SCH2, or (CH2)r where r i.s 1 or 2, -25 ~nd pharmaceutically acceptable ~alt.s thereof.

In another group of thi~ subcla~i, compound~i of the invention have the following structure:

R ~GN~N ~3 30 wherein - CA 022168~9 1997-09-29 PCT/US9''0 ~Gn Y, attached to a ring carbon atom, i~, hydrogen or NH2;

R 1, R2, and R2 are independently hydrogen, phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring ~iy.~item anv rin_ of which may be ~iaturated or un.~aturated. ;md which conxi.~it~i of carbon atom~i and from one to three heteroatom~ ,elected from the group con.~ ting of N~ O and S.
C 1-4 ;llkyl, branched CI ~ alkyl, C3 7 cycloalkyl, Cs 1 2 bicyclic alkyl, C l 1-16 tricyclic alkyl, R4(CH2)n, (R4)2CH, wherein R4 j,L~ the ~ame or different.
(R4)(oR4)CH, R4O(CH2)n, or R I may be joined with R'~ to form a four- to .~even membered carbon ring in which zero to two carbon ~tom.~ may be ~iub.~;tituted with heteroatom~; independently ~,elected from the li~it N, O and S, where n i.~i 1, 2, 3 or 4;

R3 i,L, hydrogen, (R2)2N, wherein R2 i~ the ~ame or different.
R2 OCONH, provided R2 i,~ not hydrogen, R2CONH, HO(CH2)p, where p i~ 0, 1, 2, 3 or 4, W O96/31504 PCTrUS9~'0~0 - lg -R2 SO2NH~ provided R2 i~ not hydrogen, or (R2)mNCONH, where m i.s 1 or 2, wherein R2 i~s the ~ame or different;

;~ R4 i.~ independently phenyl, mono- or di-halo~enated phenyl, naphthy k biphenyl .
a 5- to-7- nlembel-ed mono- or bicyclic heterocyclic rin~ or bicyclic heterocvclic ring .sy.~tem ~nv rin~ of which may be ~iaturated or un.~tur~ted. and which con.~ t~i of carbon ;Itom~ nd from one to three heteroatom.s .selected from tlle ~Troup con~ tin~
of N. O ~nd S.
1 5 COOH.
Cl 4~Ikyl.
branched C l ~ ~Ikyl.
C3 7 cycloalkyl.
C5 1~ bicyclic ~Ikyl. or Cl 1-16 tricyclic ~Ikyl;

G i.s (CH2),1 where ~ 1 or 2, or NRICH2; and Q i~i SCH2, or (CH2)r where r i~ I or 2.

and pharrnaceutically acceptable ~;alt~ thereof.
Some abbreviation.s that may appear in thi~ application are 30 ~ follow.s.

PCTnUS~6/O~C0 _ 19 _ ABBREVIATIONS

De~i~nation BOC (Boc) t-butyloxycarbonyl :~ HBT(HOBT or HOBt) l-hydroxybenzotriazole hydrate BBC reagent benzotriazolyloxy-bis(pyrrolidino)-carbonium hexafluorophosphate PyCIU l ~ l ~3.3-bis(tetramethylene)-chlorouronium hexafluorophosphate EDC l -ethyl-3-(3-dimethyl;lminoprop~ l) c~rbodiimide hydrochloride (BOC)~O di-t-butyl dic.lrbonate DMF dimethylforn1~mide Et7 1~ or TEA triethvl~mine lS EtOAc ethyl acetate TFA trifluoroacetic acid DMAP - dimethylaminopyridine DME dimethoxyethane BH3-THF Bor;~ne-tetr~hydrofur~n complex D-Phe(3 4-Cl2) D-3.4-Dichlorophenyl~l~nine D-3.3-dicha D-3.3-Dicyclohexyl~lanine Pro Proline Arg Arginine Gly Glycine D-3.3.-diphe D-3.3-Diphenyl;llanine The compound.s of the present invention may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomer.s, or enantiomers with all isomeric forms being included in 30 the present invention. A racemate or racemic mixture does not imply a 50:50 mixture of stereoisomers.
When any variable occur.s more than one time in any .
con.stituent or in formula I, its definition on each occurrence is independent of it.s definition at every other occurrence. Also, CA 022l68~9 l997-09-29 W O96/31504 PCTnUS9G/0~1~0 combinations of ,substituents and/or variables are permi,s,sible only if ~uch combination,s result in stable compounds.
As used herein except where noted. "alkyl" i~s intended to include both branched- and straight-chain saturated aliphatic S hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl~ Bu is butyl); "~lkoxy" repre.sents a alkyl group of indicated number of carbon atom~; attached through an oxygen bridge; "Halo", a.s u.sed herein. mean.s fluoro, chloro. bromo and iodo; and "counterion" is u~sed to represent a small, ~iingle negat;vely-10 charged species. such a~ chloride. bromide. hydroxide. acetate.trifluroacetate, perchlorate~ nitrate. benzoate. m;lleate. tartrate, hemitartrate~ benzene sulfon~te. ~nd the like.
Cyclic alkyl, bicyclic alkyl, and tricyclic al~;yl refer to saturated ring systems, including spiro systems, fused systems. ;md 15 bridged systems, unsubstituted or substituted with oxygen to form c~rbonyl carbon systems, or C1 2 alkyl.
The term heterocycle or heterocyclic, as u~ied herein except where noted. represents a stable 5- to 7-membered mono- or bicyclic or .~table 7- to 10-membered bicyclic heterocyclic ring .system any ring of 20 which may be .~aturated or un.s~turated, and which consi~t.~ of c~rbon atoms ~nd from one to three heteroatom.s selected from the group consisting of N, O and S. and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be ~luaternized, and including any bicyclic group in 25 which any of the above-defined heterocyclic ring.s is fu~ed to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which result,~ in the creation of a stable .~itructure.
Example.~ of such heterocyclic elements include piperidinyl. piperazinyl~
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl.
30 azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, i.soxazolyl, isoxazolidinyl, morpholinyl. thiazolyl, thiazolidinyl, isothiazolyl, ~uinuclidinyl, i,sothiazolidinyl. indolyl, ~luinolinyl, iso~luinolinyl, CA 022168~9 1997-09-29 W O 96131504 PCTnUS9''0~C0 benzimidazolyl, thi~ 7.Oyl~ benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same a.s 5 morpholinyl.
The pharmaceutically-~cceptable .~alt~ of the compound.~ of Formul~ I (in the form of water- or oil-.soluble or di.sper.~ible product.~) include the conventional non-toxic xalt.s or the ~uatemary ammonium ~;alt.s which are formed. e.g.~ from inorganic or organic acid~ or ba~e.s.
10 Examples of such acid addition salts include acetate. adipdte~ alginate.
a~spdrt;lte. benzoate. benzene.sulfonate. bi~iul1'ate. ~utyrate. citrate, camphorate. camphor.sulfondte~ cyclopentanepropionate. digluconate.
dodecyl~ulfate. ethane.~iulfonate. fumarate. glucoheptanoate, glyceropho.~phate. hemisulfate~ heptanoate. hexanoate, hydrochloride, 15 hydrobromide. hydroiodide, 2-hydroxyethane.~ulfonate, Iactate, maleate, methane.sulfonate. ~-naphthalenesulfonate, nicotinate. oxalate, pamoate.
pectinate, per~ulfate, 3-phenylpropionate, picrate. pivalate, propionate.
,~uccinate. tartrate, thiocyanate, tosylate. and undec;3noate. Ba~e ~ialts include ammonium salt~, alkali metal .~alt~ ~iuch a~ .~odium and pota~.~ium 20 .~alt.s. alkaline earth metal .salt.s ~such a~ calcium and magne~;ium salt.s,.salt.s with organic ba.se.s .such a.s dicyclohexylamine .~alt.~. N-methyl-D-glucamine. and ~ialt.~i with amino acid~ .~uch a~i arginine. Iy~iine, and ~;o forth. Al~o, the ba.~ic nitrogen-containing group.~ may be ~uaternized with ~;uch agent.s a~ lower alkyl halide.s, ~uch a.~ methyl, ethyl, propyl, 25 and butyl chloride. bromide.~ and iodide.~; dialkyl ~iulfate.s Iike dimethyl.diethyl, dibutyl and diamyl sulfate.s. Iong chain halide~; ~uch a~ decyl, lauryl, myristyl and .stearyl chlorides, bromide.s and iodide.s, aralkyl halide.s Iike benzyl and phenethyl bromides and others.
Amide couplings used to form the compound,s of this 30 invention are typically performed by the carbodiimide method with reagent~ such a,s dicyclohexylcarbodiimide, or l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide. Other method.~ of forming the amide or peptide bond include, but are not limited to the synthetic route.s via an acid chloride, azide. mixed anhydride or activated e,ster. Typically, W O96/31504 PCTnUS9''011~0 ~solution phase amide coupling are performed, but ,solid-pha~se ~synthe,si~s by classical Merrifield techni~lues may be employed instead. The addition and removal of one or more protecting group~ is al~so typical practice.
S The compound.s shown in the tables below are exemplary compound~ of the pre~sent invention~ having Ki (uM) for human thrombin ~Ia in the range between 0.0000~ and 100:

PCTnUS9~/O~C0 O
~R
N\FO
¢~/ N--SO2~ _~
Cl R Scheme N

--N--~NH2 ~=N
H--~NH2 2 H--~NH2 H ~\~

--N--~\ N
H
N

.

CA 022l6859 l997-09-29 PCTnUS~''014C0 ~ ~ NH

R~ R2 Scheme N

BOC _N ~NH2 N

H--~NH2 \>= N
BOC _ N ~ N H2 2 ~=N
H ~~ N H2 2 Pcrlus~o~1cn wo 96/31504 ~> NH

R Scheme N

BOC --N ~ N H2 N

H ~ N H2 ~=N
BOC --N ~ NH2 2 ~=N
H \~ N H2 2 _ BOC --H--~N 2 PCT/US3.'~11G0 TABLE ~ (CONT'D) R1HN~
Oo~R

Rl R2 H --NH--CH2~

H --NH--CH2~N

H /=<
--NH--CH2cH2cH2~N

H --NH--CH2cH2~N

W O96/31504 . PCTnUS9''0~1C0 TABLE 3 (CONT'D) ~ ~ 2 CH2--S~2-- --NH--CH2{~ NH2 'l O~CH2--S~2-- --NH--CH2{, ~--NH2 CH3CH2--O--C--CH2-- --NH--CH2~NH2 W O96/31504 PCTrUS9f'0~4C0 - 2g -TABLE 3 (CONT'D) R1HN~
o CH2-S02- -NH-CH ~ NH2 CH2-S02- -NH-CH2 ~ NH2 CH2-S02- -NH-CH2 ~ NH2 ~ CH2- -NH-CH2 ~ NH2 N N

w o 96al504 . PCTrUS9"0~1'0 TABLE 3 (CONT'D) o Rl R2 BOC --NH--CH2~NH2 N

H --NH--CH2~NH2 PCTrUS9''~ C0 - ~
R1 HN--~N~ NR2 O O

o (CH3)3C--O--C----CH2~NH2 1~l /=\
(C H3)3C--O--C----C H2~N

H --CH2~NH2 H CH2~NH2 N

PCTrUS9"0~4C0 TABLE 4 (CONT'D) Cl ,~
- ~, NH R2 ~ O

Rl R2 H CH2~N

H--C H2C H2C H2~N

~=<
HCH2CH2~N

PCr/US~'0~1 ~CO
wo 96/31504 TABLE 4 (CONT'D) ,~
- ~ H
R1 HN ~N~ NR2 O O

Rl R2 C H2SO2 --C H2~ N H2 (CH3)3C--O--C--CH2-- --CH2~NH2 Il /=\
HO--C--C H2 --C H2~ /,~ N H2 CH3-O--CH2cH2-- --CH2~NH2 (CH3)3C-O--C-- --CH2~ NH2 CA 022l6859 l997-09-29 PCT/~ o41co TABLE 4 (CONT'D) ~1 R1 HN ~N~, NR~

(CH3)3C--O--C-- --CH,~NH2 o CH3 (CH3)3C--O--C-- --CH2~NH2 (CH3)3C--NH--C--CH2-- --CH2{~NH2 R ~
(CH3CH2)2N--C - CH2 --CH2~ NH2 PCT/U~i5~ CO

TABLE 4 (C:ONT'D) ~, R1 HN ~N~ NHR2 ~ C H2--S~2 --C H2~ N H2 CH3CH2--O--C--CH2 --CH2~=~NH2 CH3-O-C--CH2-SO-- --CH2--~ ~ NH2 0~
CH3CH2--O--C--CH-- --CH2~NH2 S N

PCTrUS9f'0~fO

- 3~ -TABLE 4 (CONT'D) - ~ H
R1 HN ~ ~ NR2 ~ O

CH3CH2--O--C--CH --CH2CH2CH2 ~N

~CH2--S~2-- --CHZcH2cH2 ~N

PCTnUS~6/O~C0 0~0 R 1 H N ~N~, N H R2 Rl R2 CH3)3C--O--C - CH2-- --CH2--~NH2 i~l --C H2~ N H2 (CH3)3C--N,--C - CH2-- N

(CH3--CH2~N--C--CH2-- --CH2~NH2 H --CH2~NH2 PCrlUS~/O~ ~CO
wo 96/31504 TABLE ~

0~0 "~
RlHN~N~T"NHR2 H--C H2~ ,~ N H2 (CH3)3C--O--C-CH2-- --CH2~NH2 (CH3--CH2~N--C--CH2-- --CH2~NH2 0/ \N--C - C H2-- --C H2~ N H2 PCTrUS9''011~0 TABLE S (CONT'D) 0~0 R 1 HN ~ N~, N H R2 ~N--C - C H2-- --C H2~ N H2 /--\ I I
HN N--C - C H2-- --CH2~ N H2 S . .

CA 022l6859 l997-09-29 W 096/31504 . PCT/U~/0~1C0 R~

O N ~

Cl CL H
F F H

Cl Cl CH3 CA 022l6859 l997-09-29 PCI/US9''0~ 1C0 Compound,~, of the invention can be prepared according to the general procedure.s outlined below:

SCHEME I
s HO ~\
~co CISO2~
~/ NH2 Cl~
Cl HO

,~/ NH--SO,~ ,~
Cl EDC+HOBT, ~,NH

W O 96/31~04 PCTrUS96/04460 o ~OC H3 ~/ N--SO
Cl 1 N LiOH/MeOH

~\~\OH

,[~1' H ~ J~3 Cl NH2 / \ BH3.THF NH2 ¢~N BH3.THF / \ )~

H N ~ / \ N
NH2 ~ NH2 ~J coupling agent \/~

W O96/31S04 PCTrUS9~ CO

N

~/ NH--S~2~ ~

Cl~ or H ~ N H2 ~/ N--SO2 ,~

Cl/~

WO96/31504 . PCTnUS9"0~0 . .

H2, BOC ~ 0,~

HN----~OH
BOC ~

EDC+HOBT H-Pro-OMe- HCI

HN'----~ Pro-ocH3 BOC ~

1 N LiOH/MeOH

- CA 022l6859 l997-09-29 PCTrUS3~'0~1C0 a~ ~

HN /--1~ Pro--OH
O
BOC

/coupling\
/ agent H Nr ~ BH3~TH ~\ BH3-TH~ CN

~iN / \~
N ~ - H N

W O96/31504 PCTnUS96/04460 O
~N"--~NH2 ~>""' NH ~~ ~NH2 H
~ BOC
TFA/C H2CI2 (1 :1 ) TFA/C H2CI2 ( 1 :1 ) H~NH2 =o ~ NH2 PCI~/IJS~"0~ 0 s c~nEr~nE 3 -HN----Tf OH
BOC ~

EDC+HOBT H-Pro-OMe- HCI
~, HN ~ Pro-OCH3 BOC ~

1 N LiOH/MeOH

W O 96/31504 PCT~US~6/O~C0 , - 47 -', ~

BocHN~~ Pro-OH

EDC HOBT I~N EtOH, HCI~N CucN ~N
Et3N ~CH3 Pd/C/H2 ~CH3 DMF ~CH3 NoH2 CN Br Boc ~ ~ N~''~

PCTrU~9. 0~4C0 - 4g -~NH ~NH2 or N

[~/ BOC ~ NH/~NH2 TFA/CH2CI2 (1 :1 ) BOC
TFA/CH2CI2 (1:1 ) ~NH~NH2 ~ ~-NH--~ Z

PCTnUS36/O~G0 S~HEME 4 ,~ EDC, HOBT. Et3N

OH H-Pro-OMe- HCI
BocHN'~f Cl Cl LiOH/H20/DME -/~

BocHN~Nf~,OCH3 BocHN~N~,OH

~ EDC. HOBT, Et3N

BocHN ~ N J~NH H N~
O O

EtOAc, HCI (g) W O96/31504 PCTnUS~'0'1~C0 Et3N, DMF
B ~~~
O \CI~N~/

Et3N, DMF \
Cl ~f NH~N~

~H ~o CA 022168',9 1997-09-29 W O96/31504 PCTrUS96/04460 <~~ THF, Et3N THF, n-BuLi O~ \~OH
Il ~ O

,L ~
Bn 0~ ~ KHM DS. THF

< ~ LiOOH. H20, TYF ~~

EDC, HOBT, H-Pro-OMe~ HCI
Et3N

PCTnUS~6N4~C0 SCHEME 5 (CONT'D) LiOH/H20/DME

N3~N~,OCH3 N3/~N~,OH
~ O O o H2N~ EDC, HOBT, Et3N

0~0 N~NH ~N
O o CH3 EtOH, HCI
PdtC~

CA 022l6859 l997-09-29 PCTnUS9G/011C0 SCHEME S (CONT'D) 0~0 H2N~H
O o CH3 -Et3N DMF/ fo \

/Br~

Et3N . DMF\ ~If 0~0 NH2 ' ~N~' N ~ N~, N ~ ~ \
- ~~

~ H~ ~ CH~

W O 96t31504 PCTrUS9~v~ GO

EXAMPLE I

Preparation of BOC-D-3,3-Dicyclohexylalanyl-6-(aminopyridin-3-yl)methyl-L-prolineamide Step A: Prepar;~tion of ~-Amino-~-aminomethvl,n~ridine A 300-ml fla.~k wa~ dried in an oven and cooled down in ~
dry nitrogen atmo.~phere. The fla.~k wa.~ e~uipped with a rubber.syringe cap and a magnetic .~tirring bar. The tla.sk Wcl.S irnrner~ied in a ice-water bath ~ and 21 ml (21 mmole) of 1.0M borane .solution in THF wa~
introduced into the reaction fla.~ik. followed by 0.3 ml of THF. Then 6-Aminonicotinamide (4~0 mg, 3.06 mmole) in l 0 ml of THF wa.
introduced. The re~ulting mixture wa~ tirred for 10 min. ;~nd 5 ml o~
6N HCI W;l~i added .~lowly~ and then 15.0 ml of H~O and 100 ml of 1:~ MeOH wa~ introduced~ and .~tirred continually over night. and filtered~
evaporated in ~ u(l to give product a~ a white .solid which wa.s further purified by chromatography u~;ing two column.s of 40 g ~ilica gel 60 (E.
Merck) each and eluting with n-Butanol-HOAC-H2O (4~ ); Fraction.s containing product were combined to give 2~5 mg (76~c yield) of product.
EI+: I 23 TLC: Rf=0.5 1. ~ilica gel. n-Butanol-HOAC-H ~O (4:1:2);

Step B: Boc-D-3.3-Dicha-OH ( 1 -2) A .~olution of BOC-D-3.3-Diphe-OH (2.0 g. 5.~s mmol) in 50 ml acetic acid/10 ml H2O wa.s hydrogenated at 62 p~;i OIl a Parr apparatu.s over 400 mg of Ir black cataly~t. After 24 h~ a .~econd portion of cataly.st wa~ added and the reaction continued for a ~econd 24 h interval. The reaction wa.s filtered through a Celite pad, and the filtrate wa~; added with 150 ml of H20 and filtrated again to give 2.0 g of BOC-D-3,3-Dicha a~s white ~olid (97% yield).
FABMS: 354 HPLC: retention time 24.3 min; Cl~, 95%A to 5%A over 30 min, A=0. 1 %TFA-H2O, B=0. 1 %TFA-CH3CN

W O96/31S04 PCTnUS9610~4C0 Step C: Boc-D-3.3-Dicha-Pro-OMe ( 1-3) To a solution of Boc-D-3,3-Dicha-OH (1.77 g, 5.0 mrnol) and H-Pro-OMe-HCI (0.91 g, 5.5 mmol) in 12 ml of DMF wa.~, added 4.6 ~ (6.0 mmol) of HOBt~H2O, the pH of the ~iolution wa~ adjusted to ~
S (moi~t narrow pH paper)~ and EDC (6.47 ~, 6.76 mmol) was added with ma~netic ~itirrin~;. After 3.5 hr~ the reaction wa~i yuenched by the addition of 10 ml of water. After l;eeping the mixture at room temperature for ~ hrs, the solvents were evaporated at reduced pre~ iure and the residue was dis~solved in EtOAc-H20. A~lueou~; KHSO.1 wa.
added to thi~i two-pha~ie mixture and the layer~ were ~eparated. The or(~anic layer was extracted with NaHCO~ aturated NaCI. and dried over MgSO.1. The ~iolvent wa.~i evaporated to ~ive product a.~ ~ white .~iolid which wa~i further purified ~ chromato~raphy u~iin~ two column~
ot 600 ~ ~iilica gel 60 (E. Merc~;) each and eluting with EtOAc-hexane (2:~). Fraction~ containing product were combined to ~ive 2.26 g (97%
yield) of product.
In ~ ~iimilar manner are prepared the followin~:
N-(benzyl~ulfonyl)-D-3.4-Dichloro-Phe-Pro-OMe (2-2). by couplin~ of N-(benzyl~;ulfonyl)-D-~.4-Dicllloro-Phe-OH (2-1 ) with H-Pro-OMe~HCI.
BOC-D-3. 3-Diphe-Pro-OMe (3-1), by couplill~ of Boc-D-3.3-Diphe-OH with H-Pro-OMe-HCI.

Step D: Boc-D-3.3-Dicha-Pro-OH
A sample of Boc-D-3,3-Dicha-Pro-OMe (1.76 g, 3.~s mmol) di~solved in 100 ml of 1:1(v/v) MeOH/H2O wa~i treated with 2.2 N
LiOH (2.2 ml) in portions over 1.5 hrs. keepin~ the pH at 12-13. After 3.5 hn~,. the reaction .~olution wa,~; adju~ted to pH 7 with dilute KHSO4 solution, 100 ml of EtOAc and 50 ml of ~I2O were added, and the ayueous layer was further adjusted to pH 2 with KHSO4 solution. The or~anic layer wa~ separated and wa~ihed twice with 50% ~;aturated NaCI
~,olution, dried over Na2SO4, and evaporated in v~c~u~ to give 1.64 ~, (96% yield).
. FABMS: 451 , CA 022l6859 l997-09-29 W O 96/31504 PCTrUS9"0~0 HPLC: retention time 26.4 min; Cl ~, 95%A to 5%A over 30 min.
A=O. I %TFA-H20, B=O. l %TFA-CH3CN

In a ~similar manner are prepared the following:
N-(benzyl~sulfonyl)-D-3,4-Dichloro-Phe-Pro-OH, by hydroly.~is of N-(benzyl.sulfonyl)-D-3~4-Dichloro-Phe-OMe with LiOH.
BOC-D-3,3-Diphe-Pro-OH, by hydroly~ of Boc-D-3.3-Diphe-OMe with LiOH

Step E: Preparation of BOC-D-3,3 -Dicyc lohex vlalanyl -6-(~minopvridin-3-vl')methvl-L-proline~lmide A .~iolution of 113 mg (0.75 mrnol) of Boc-D-3~3-Dich~l-Pro-OH. 6'' mg (0.50 rnrnol) of 6-Amino-'-~minomethylpyridine. 43 mg (0.7~ mmol) of HOBT~ 5-1 mg (0.7~ mmol) of EDC in 1.7 ml anh.
1 ~ NMP wa~ treated with DlEA to PH g.S. and the re.~iulting .~iolution .~itirred ~t room temp. in ~n N2 atmo.~iphere for ~S h. The re~ction w~.
diluted with 3X it.~ volume of water, ~nd the su.~ipen~iio~ itirred vigorou~;ly at room temp. for 15 min. The ~u~ipen~ion wa.~ filtered. the re.~;idue purified by preparative HPLC u~iing ~ trifluro~cetic ;lcid (0.1~)-CH3CNgradient. Lyophilizationofpurefr~ction.~gave 135m~
(97%) of product a.~ a trifluoroacetic ~cid hydr~te .~ialt.
Anal. CHN: C~lH49N504 ~1.75 CF3C02H-0~90 H20.
rAB MS: M+ 1 = 556.
HPLC: 99% pure @214. retention time.~=27.7 min, (Vydac Clg, gradient of 95%A/B to 5%A/B over 30 min, A=0.1%TFA-H20.
B=O. l %TFA-CH3CN

Preparation of D-3,3-Dicyclohexylalanyl-N-(6-aminopyridin-3-yl)methyl-L-prolineamide A solution of 122 mg (0.22 mmol) BOC-D-3,3-dicyclohexylalanyl-N-(6-aminopyridin-3-yl)methyl-L-prolineamide in 10 ml of 50% TFA/CH2C12 was stirred for 20 min, and the TFA was W O96/31~04 PCTIUWG/04~C0 removed under reduced pressure and the product purified by preparative HPLC using a TFA(0.1 %)-CH3CN gradient. Lyophilization of pure fraction~ gave 96 mg (96%) of the title compound as a trifluoroacetic acid hydrate salt.
S Anal. CHN: C26H41 N502 ~2.70 CF~C02H-0.55 H~O.
FAB MS: M+l = 456.
HPLC: 99% pure @214. retention time~=16.5 min, (Vydac Cl~, gradient of 95%A/B to 5%A/B over 30 min, A=0.1~TFA-H20, B=0.1 ~TFA-CH3CN

Prep~ration of N-(benzyl.~iulfonyl)-D-3,4-dichlorophenyl~l~nyl-N-(6-~minopyridin-3-yl)methvl-L-prolineamide 1~
Step A: Preparation of N-(henzyl.~ulfonvl)-D- ..~ -CI~Phe-()H('~- I ) (D)-3.4 -C12PheOH (1.4 ~ 6.0 mmol) ua~ di.~i~olved in 4~s mL diox~ne by addition of 6 ml 1 N NaOH. The re.~ulting ~olution wa.~i treated dropwi~e with phenylmeth~ne~ulfonyl chloride with r~pid 20 .~itirring at room temperature. After 2.5 hour. the ~lueou~i layer wa~
further adju.~ited to pH 2 with KHSOl ~;olution. 150 ml of EtOAc were ;~dded. The org~nic layer wa.~ .~ep~rated ~nd wa.~hed twice wi~h ~turated NaCl ,~olutioll, dried over Na2SO4, ~nd evaporated i/? l'~C'UO to give 2.31 g (~0% yield).
2~ FAB MS: M+l = 4g5 HPLC: 97% pure @214. retention time.~=20.1 min, (Vydac Cl~, gradient of 95 %A/B to 5 %A/B over 30 min, A=0.1 ~,TFA-H20, B=0.1 '7OTFA-CH3CN
~0 Step B: Preparation of N-(benzyl~ulfonyl)-D-3.4-dichlorophenyl-alanyl-N-(6-aminopyridin-3-yl)methyl -L-prolineamide A .~iolution of 121 mg (0.25 mmol) of N-(benzyl~ulfonyl)-D-3,4-C12Phe-Pro-OH, 62 mg (0.50 mmol) of 6-Amino-3-aminomethylpyridine, 43 mg (0.2~ mmol) of HOBT. 54 mg (0.2g W O96131504 PCTrUS~G/0~1G0 - 5g -mmol) of EDC in 1.7 ml anh. NMP was treated with DIEA to pH g.5, :md the resulting solution stirred at room temp. in an N2 atmosphere for ~ h. The reaction was diluted with 3X its volume of water~ and the .~iuspension stirred vigorously at room temp. for 15 min. The 5 .~uspension was filtered~ the residue purified by preparative HPLC using a trifluro~cetic acid (0.1 %)-CH3CN gradient. Lyophilization of pure fraction.~ g;lve 140 mg (95%) of product a.~i a trifluoroacetic acid hydrate salt.
Anal. CHN: C~7H ~9N~O~SICl~ ~'7.35 CF~CO~H-0.9~5 H~O.
10 FAB MS: M+l = ~S90.
HPLC: 99~ pure @21~. retention times=l~.~ min. (Vyd~c C
gradient of 9~,A/B tc- ~A/B over ~0 min A=O.I~TFA-H20, B=0. 1 '7fTFA-CH~CN

1 :S EXA MPLE 4 Prep;3ration of N-BOC-D-3.3-diphenylalanyl-N-(6-aminopyridin-3-yl)meth~ l-L-proline;3mide 110 mg. 0.25 mrnol of N-BOC-D-3,3-Diphe-Pro-OH ~nd 130 mg. 0 50 mmol of 6-Amino-3-~minomethylpyridine were coupled with hydroxybenztriazQle hydrate (43 mg, 0.2~ mmol) and EDC-HCI
(54 mg. 0.'2~ mmol) in 1.5 mL DMF at pH ~.~ with DIEA. The mixture wa.s ~stirred under N2 at room temperature overnight, then diluted with 10 mL of 10% a~lueous citric acid and extracted with CH2CI-~. The CH2C12 extracts were washed with a~ueou~i Na2co-~
dried (Na~SO4)~ filtered and concentrated i~? I'~IC'UO to give the crude Boc derivative of the title compound. and the product purified by prepar~tive HPLC u.~iing a TFA(0. 1 %)-CH3CN gradient. Lyophilization of pure fractions gave 122 mg (90%) of the white powder as a trifluoroacetic acid hydrate salt.
Anal.CHN: C31H37N5O4 ~1.75 CF3C02H-1.05 H2O.
FAB MS: M+ l = 544.

WO96/31504 PCTrUS~''01~60 _ 59 _ HPLC: 99% pure @214, retention time,s=lg.~ min, (Vydac C1~, gradient of 95%A/B to 5%A/B over 30 min, A=0.1%TFA-H20 B=O. 1 %TFA-CH3CN

EXAMPLE ~

Preparation of D-3.3-diphenylalanyl-N-(6-aminopyridin-3-vl)methyl-L-prolineamide A solution of (109 mg, 0.20 mmol) N-BOC-D-3.3-10 diphenylalanyl-N-(6-aminopyridin-3-yl)methyl-L-prolineamide in 10 ml of ~0~, TFA/CH2CI~ wa.s stirred for '70 min. the TFA wa.~ removed under reduced pre~i.sule. and the product purified by preparati~e HPLC
using a TFA(O. I '7~.)-CH~CN gradient. Lyophilizatioll of pure fractions ~ve ~7 mg (9~) of the title compound a.~ a trifluoroacetic ;3cid ydrate salt.
Anal.CHN: C26H29N~02-2.~0CFsC02H-1.35 H20.
FAB MS: M+ I = 444.
HPLC: 99% pure @214. retention time~i=13.9 min, (Vydac Cl~, gr~dient of 9~%A/B to ~%A/B over 30 min. A=O.I %TFA-H20.
20 B=O. I C~/oTFA-CH3CN

Prep~ration of BOC-D-3,3-Dicyclohexylalanyl-N-(6-amino-2.4-2~ dimethylpyridin-3-yl)methyl-L-prolineamide Step A: Preparation of 6-Amino-2,4-dimethyl -3-amino-methylpyridine A 300-ml flask was dried in an oven ~nd cooled down in ~
30 dry nitrogen atmosphere. The fla.sk was e~luipped with a rubbemsyringe cap and a magnetic stirring b~r. The fla.sk was immersed in a ice-water bath, and 21 ml (21 mmole) of l.OM borane ,solution in THF was introduced into the reaction fla.sk, followed by 0.3 ml of THF. Then 6-Amino-2,4-dimethyl-3-pyridinecarbonitrile (442 mg, 3.0 mmole) in 10 W O 96/31504 PCT/u~- ~n~co ml of THF wa~ introduced. The resulting mixture was ,stirred for 10 min, and 5 ml of 6N HCl wa~ added ,~lowly, and then 15.0 ml of H20 and 100 ml of MeOH was introduced, and stirred continually over night, and filtered, filtrate wa~ evaporated in vac~uo to give product a~ a white S solid which was further purified by chromatography u~ing a column,~ of 40 g ~iilica gel 60 (E. Merck) and eluting with n-Butanol-HOAC-H20 (4:1 :2). Fraction.~; containing product were combined to give 407 mg (90% yield).
EI+: 51 TLC: Rf=0.73, .~ilica gel~ n-Butanol-HOAC-H~O (4:1:2) Step B: Preparation of BOC-D-3.3-Dicyclohexylalanyl-N-(6-mino-'7. 1-dimethvlpyridin-3-vl)methvl-L-proline~lmide A .~iolution of 113 mg (0.'~5 mmol) of Boc-D-3.~s-Dicha-Pro-OH. 75.5 mg (O.SQ mmol) of 6-Amino-2.1-dimethyl-3-~minomethylpyridine (1-1), 43 mg (0.2~ mmol) of HOBT. 54 mg (0.2 mmol) of EDC in 1.7 ml ;~nh. NMP wa.~i treated with DIEA to PH ~.5~
and the re~iulting .~olution .stirred at room temp. in ~n N 7 atmo.~iphere for ~ h. The reaction wa.~ diluted with 3X it.~ volume of water. and the ~u.spen.sion .stirred vigorously at room temp. for 15 min. The u.~pen~ion wa~ filtered, the re.sidue purified by preparative HPLC u~ing ~l trifluroacetic acid (0.1%)-CH3CN gradient. Lyophilization of pure t'raction.~ gave 135 mg (93%) of product a.s a trifluoroacetic acid hydrate .~alt.
2:S An;~l. CHN: C33H53NsO4 ~1.55 CF3C02H~O.~sO H20.
FABMS: M+l =5~4.
HPLC: 99% pure @214, retention time.~=23.'2 min~ (Vydac Cl~, gradient of 95%A/B to 5%~A/B over 30 min, A=0.1%TFA-H20, B=O. l %TFA-CH3CN

W O 96/31504 . PCTru~ C0 Preparation of D-3,3-Dicyclohexylalanyl-N-(6-amino-~4-dimethyl-pyridin-3-yl )methvl-L-prolineamide A .solution of 17g mg (0.22 mmol) N-BOC-D-3,3-Dicyclohexylalanyl -N-(6-amino-2.4-dimethylpyridin-3-yl)methyl-L-prolineamide in 10 ml of 50% TFA/CH2C12 wa~; ~stirred for 20 min~ and the TFA wa~ removed under reduced pre.ssure and the product purified by preparative HPLC u~iing a TFA(0.1 ~)-CH~CN ~radient.
Lyophilization of pure fractions ave 102 mg (96~) of the title compound a.~i a trifluoroacetic acid hydrate .~alt.
Anal.CHN: C~ H-15N5O2 ~~.S0 CF~CO~H- I .35 H 7O.
FAB MS: M+ I = 4~4.
HPLC: 99~ pure @21~ retention time.s=16.7 min. (Vydac C
gradient of 95~A/B to S%A/B over 30 min. A=0.15~TFA-H20 B=0.1 %TFA-CH3CN

EXAMPLE ~

Preparation of N-(benzyl~;ulfonyl)-D-3,4-dichlorophellvlalanyl-N-(6-am ino- ~ .4-d imeth y lpvri din -3 -y l )meth v I -L-pro I i ne am i de A .~olution of 121 mg (0.25 mmol) of N-(benzyl~ulfonyl)-D-3,4-C12Phe-Pro-OH, 75.5 mg (0.50 mmol) of 6-Amino-2~4-dimethyl-3-aminomethylpyridine, 43 mg (0.2~ mmol) of HOBT, 54 mg (0.2 mmol) of EDC in 1.7 ml anh. NMP wa~ treated with DIEA to pH g.5~
and the re.sulting .~olution .~tirred at room temp. in an N2 atmo~iphere for ~S h. The reaction wa~i diluted with 3X it.s volume of water~ ~d the ~u.spen.sion ~tirred vigorou.sly at room temp. for 15 min. The .su.spen.sion wa.s filtered, the re.sidue purified by preparative HPLC u.~ing a trifluroacetic acid (0.1%)-CH3CN gradient. Lyophilization of pure fraction.s gave 140 mg (95%) of white ,solid as a trifluoroacetic acid hydrate salt.
Anal. CHN: C79H33NsO4S I C12 ~ 1.6~ CF3C07H-0.60 H2O.
FAB MS: M+l = 61~.

W O96/31~04 PCTrUS~C/O~CO

HPLC: 99% pure @214, retention time,~=19.5 min, (Vydac C
gradient of 95%A/B to 5%A/B over 30 min, A=0.1%TFA-H20, B=O. 1 %TFA-CH3CN

Preparation of N-BOC-D-3.3-diphenvlalanyl-N-(6-amino-~.4-dimethvl~yridin-3-vl)methyl-L-,oroline amide 10~ m~,, 0.''5 mmol of N-BOC-D-3~$-Diphe-Pro-OH and 75.~ m,~ 0.50 mmol of 6-Amino-2,4-dimethyl-i-aminomethylpyridine were coupled with hydroxybenztriazole hydrate (43 m~ 0.~ mmol) and EDC-HCI (~4 mg~ O.''~S mmol) in 1~5 mL DMF ~t PH ~. with DIEA. The mixture ua,~i .stirred under N~ ~t room temperature overni~ht~ then diluted with 10 mL of 10~, aL~ueou.~ citric acid and extracted with CH~CI~. The CH2CI7 extract.~ were wa,~hed with a~ueou~ Na2C03~ dried (Na ~S04), filtered and concentrated i~1 I'CI~'UO to give the crude Boc derivative of the title compound~ and the product purified by preparative HPLC u~ing a TFA(O.l'J/~)-CH~sCN ~radient.
Lyophilization of pure fraction,~ gave 12~ mg (90%) of the white powder a~; a trifluoroacetic acid hydrate ,~alt.
Anal. CHN: C33H41N504 ~1.35 CF3CO~H-1.55 H20.
FAB MS: M+l = 572.
HPLC: 99% pure @214~ retention timç,~=19.3min~ (Vyd~c CI~S, gr~adient of 95%A/B to 5%A/B over 30 mhl~ A=O. 1 %TFA-H20, B=O. 1 %TFA-CH3CN

EXAMPLE I O

Preparation of D-3,3-diphenylalanyl-N-(6-amino-2,4-dimethylpyridin-3-yl)methvl-L-proline amide A ,~olution of (114 mg, 0~0 mmol) N-BOC-D-3,3-diphenylalanyl-N-(6-amino-2,4-dimethylpyridin-3 -yl)methyl-L-proline amide in 10 ml of 50% TFA/cH2cl2 wa,s ,~itirred for 20 min, and the TFA wa~ removed under reduced pre~;sure and the product purified by W O96/31S04 . PCTnUS9"0~0 preparative HPLC u~ing a TFA(O. l %)-CH3CN gradient. Lyophilization of pure fraction~ gave 92 mg (9g%) of the title compound a~ a trifluoroacetic acid hydrate .salt.
Anal.CHN: C2~H33N502 ~2.75 CF3C02H~2.90 H20.
5 FAB MS: M+l = 472.
HPLC: 99% pure @214. retention time~=14.2 min, ~Vydac Cl~, gradient of 95%A/B to 5%A/B over 30 min. A=0.1%TFA-H20.
B=O. I %TFA-CH~CN

Prepariltion of N-(t-butyloxy-carboxymethyl)-D--l-chlorophenyl-al~nyl-N-t~-aminc)pyridin-~-yl )methyl-L-I-rolille amide ~Srep A: Prepalation of 6-amino-~-(aminomethvl)pyridine To 6-aminonicotinamide (11.0 g. ~0.21 mmol) .su.~pended in 500 mL of THF W;l~ added .solid lithium aluminum hydride (6.~5 g, 167.3 mmol) portionwi.~ie. Thi.s mixture wa~, refluxed for 72 h. cooled to RT and Lluenched by the addition of water (4.71 mL). I N NaOH
(4.71 mL) and then water (19 mL). After I h of vigorou~s ~tirring the mixture wa,s filtered through celite and wa~shed with ~00 mL of THF:MeOH (~s5:15). The volatile.~ were removed i~ .'U~ and the .solid was purified by flash column chromatography (60 x 150 mm column of SiO2, EtOH/NH40H gradient elution 99:1, 9~i:2 97:3) to afford 3.70 g (37% yield) of a white .solid: TLC (EtOH:NH40H~ 99:1) Rf = 0.16: lH
NMR (400 MHz, CDC13) ~ 1.54 (br ~;~ 2H), 3.73 (.s~ 2H)~ 4.50 (br .s.
2H), 6.4~ (d~ J = ~.4~z~ lH), 7.42 (dd~ J = ~.3 and 2.3 Hz~ lH). 7.99 (d.
J = 1.6 Hz, lH).

~tep B: Preparation of N-Boc-D-4-chlorophenylalanyl-L-proline methyl e.ster To a .~olution of N-Boc-D-4-chlorophenylalanine (2.72 g, 9.07 mmol), L-proline methyl e.ster hydrochloride (1.50 g, 9.07 mmol), EDC (2.0~5 g, lO.g~4 mmol), and HOBt (1.51 g, lO.g~S4 mmol) in 15 CA 022168~9 1997-09-29 PCTrU~9G~4C0 WO g6/31504 mL of DMF at 0~C was added triethylamine (3.0 mL, 21.76 mmol).
This ~tirred 1 h at 0~C then lS h at RT and wa~ diluted with 200 mL of ethyl acetate and washed with sat'd NaHCO3 (1 x 25 mL), water (S x 25 mL), and brine (1 x 20 mL). The .~olution wa~; dried over MgSO4, S filtered and concentrated to an oil which w~i purified by flash column chromatography (40 x 150 mm column of Sio2, EtOAc:Hex gradient elution 1 :2~ to 1: 1,) to afford 3.24 g (~7% yield) of a white foam: TLC
(EtOAc:Hex. 2: 1) Rf = 0.6~; 1 H NMR (400 MHz~ CDC13) ~ 1.42 (~, 9H), 1.61-1.66 (m, lH), l.~g-2.02 (m, 3H), 2.~4-3.02 (m. 3H), 3.5g-3.67 (m. lH). 3.72 and 3.72 rotomer~ , 3H). 4.31 (dd~ J = 7.7 ~nd 3.
Hz. IH).4.62(dd.J= 1~.6and~.3Hz~ lH).~ 1 (d.J=~.4Hz. IH).
7.0~S-7.15 (m, 2H), 7.'73-7.26 (m. 2H).

Step C: Preparation of N-Boc-D-4-chlorophenyl~lanyl-L-proline carboxylic acid To ~ ~;olution of N-Boc-D-4-chlorophenylalanyl-L-proline methyl e~ter (4.9~ g, 12.12 mmol) in 45 mL of DME wa~ added LiOH
(1.16 g) di~ olved in 15 mL of water. AfterO.Sh the ~olution wa~
concentrated to lS mL acidified to pH 3 with 10% HCI ;md extracted with ethyl ~cetate (3 x 100 mL). The combined organic layer wa~
wa~hed with brine (1 x lS mL), dried over MgSO4. filtered and c oncentrated in ~~ac~uo to provide 5.05 g of a white foam: TLC
l~EtOAc:Hex. 2:1) Rf = 0.05; lH NMR (400 MHz, CDC13) ~ 1.41 (~'i, 9H), 1.69~ 0 (m, 2H), 1.~-1.94 (m, lH), 2.09 (.~, lH), 2.20-2.25 (m, lH), 2.77-2.~3 (m, lH), 2.92-2.99 (m, 2H), 3.55-3.63 (m, lH), 4.37 (d, J = S.~ Hz. lH). 4.62-4.64 (m, 1 H), 5.3~ (d. J = 6.2 Hz~ lH), 7.09-7.1g (m, 2H), 7.20-7.27 (m, 2H).
.
Step D: Preparation of 6-amino-3-(aminomethyl)pyridine-N-Boc-D-4-chlorophenylalanyl-L-proline amide To a .~iolution of N-Boc-D4-chlorophenylalanine-L-proline carboxylic acid (1.22 g, 3.07 mmol) in 5 mL DMF wa,~ added 6-amino-3-(aminomethyl)pyridine (0.434 g, 3.53 mmol), HOBt (0.501 g, 3.6g4 mmol), and EDC (0.706 g, 3.6~4 mmol), cooled to OQC and added W O 96/31504 PcT/u5~ co triethylamine (0.51 mL, 3.6g mmol). After l~S h the reaction mixture was concentrated, diluted into ethyl acetate (150 mL) and washed with saturated NaHCO3 (lx20 mL), water (Sx20 mL) and brine (lxl~ mL), dried over MgSO4, filtered and concentrated to an oil. Flash column chromatography (30xl~0 mm column of SiO2~ CH~C12/CH2C12 saturated with NH3/MeOH gradient elution 60:39:1 tO 60:3~S:2 to 60:35:~) provided 1.30 g of a yellowi.~ih foam: TLC (CH~C12/CH2C12 ~;aturated NH~s/MeOH, 60:35:5) Rf = 0.3~; IH NMR (~00 MHz. CDCI~) dl.36 (.~. 9H). 1.64-1.74 (m. 2H). 1.~5 (br.~. IH). ~.~5-'7.'77 (m. IH).
2.74-2.~7 (m. lH). 2.~s9-2.99 (m. 2H), 3.64-3.66 (m. IH). ~.17-1.31 (m.
2H). 4.1~ 0 (m. 4H). 5,?5 (d. J = 3.g Hz. IH). 6.~ (dd. J = ~.3 and 1.9 Hz. IH). 7.12 = 7.14 (m. 2H). 7.2~ = 7.37 (m. ~H). 7.91 (.~. IH).
Anal.CHN: C~H32N50~CI ~ 0.2 H20.
Calc. C 59.3~; H 6.46: N 13.~s5.
1~ Found C ~9.02; H 6.'~7: N 13.5~.

Step E: Preparation of N-D- 1 -chlorophenvl-~lanyl-N-(6-aminopyridin-3-vl)methvl-L-proline ~mide To a .~olution of N-BOC-D-4-chlorophenyl-alanyl-N-(6-aminopyridin-3-yl)methyl-L-proline amide (I .30 g. ~.59 mmol) di.~ ;olved in ethyl acetate (10 mL)~ cooled to ()~C wa~i bubbled in HCI (g) for 10 min. Reaction .~tirred ~n additional 0.~S h and wa~ concentrated i~1 U~. The re~idue wa~ tritrated with ethyl ~cetate and filtered to provide 1.19 g of a white .~olid: mp 19~-205~C. LRMS (M+) 402.
Step F: Preparation of N-(t-butyloxy-carboxymethyl)-D-4-chlorophenyl-alanyl-N-(6-aminopyridin-3-yl)methyl-L-proline amide To N-D-4-chlorophenyl-alanyl-N-(6-aminopyridin-3-yl)methyl-L-proline amide (0.~0 g, 1.6~ mmol) di~;olved in 7 mL of DMF wa.~ added tert-butyl bromoacetate (0.30 mL, 1.~s4 mmol) and triethylamine (0.~s6 mL, 6.21 mmol). After 4~ h the .~;olution was diluted with 100 mL of ethyl acetate and wa~hed with ~aturated NaHCO3 (lx20 mL) water (4x20 mL), brine (lx20 mL), dried over MgSO4 W O96/31504 PCTnUS9''0~1C0 filtered and concentrated to a yellow oil. Thi~ wa.s purified by fla~h column chromatography (30x150 mm column of SiO2, CH2cl2lcH2cl2 ~saturated NH3/MeOH 60:39:1, 60:3~:2, 60:37:3, 500 mL each) to provide a white .~olid. Recry.~tallization from ethyl acetate/hexane.~
afforded 0.232 g of a cry~it;llline .~iolid. mp 176-17~~C. Anal. CHN:
C26H~sNso4cl - 0.1 H2O
calc C 60.30: H 6.66; N 13.53.
found C 59.96: N 6.65, N 13.25.

Prepar~tion of N-(N'.N'diethyl-c~rboxymethyl)-D-4-chlorophenyl-lanyl-N-(6-amino-1-yridin-~-vl)methvl-L-prc)line amide A~ above in Ex~mple 12. Step F. replacement of tert-butyl bromoacetate with N. N-diethyl-2-bromoacetamide afforded the de~ired product. After fl~ih column chrom~tography the product w~ olated a~ ~ foam: TLC (CH~CI~/CH2C12 .~;~turated NH3/MeOH, 60:35:5) Rf =
0.36: I H NMR (400 MHz. CDC13). o 1.01 (t, J = 1.0 Hz. 3H)~ I .09 (t, J
= 7.0 Hz. 3H); 1.60-1.65 (m. lH)~ 1.69-1.~S3 (m, 2H). 2.00 (br .~;~ IH), 2.14-2.1 g (m, 1 H), 2.64-2.71 (m, 1 H), 2.g2 (d, J=7.5Hz. 2H). 3.09-3. l ~S
(m. 3H). 3.25-3.33 (m. 4H)~ 3.34-3.49 (m. 3H), 4.27 (d. J=S.~Hz. 1 H).
4.37 (br .~. lH), 4.52 (d. J=5.5Hz, lH), 6.44 (d. J=~s.SHz. lH). 7.12 (d, J
- =g.2Hz. 2H), 7.22=7.26 (m, 2H), 7.43 (dd, J=~.3 ~nd 2.1Hz. lH), 7.90 (d. J=14Hz. lH), g.l4 (dd, J=10.~S, 5.4Hz, lH).
Anal. CHN: C26H35N603CI ~ 0.35 CH2C12:
Calc C 5~.37: H 6.6~; N 15.30 Found C 5~.73; H 6.~7; N 14.93 W O96/31S04 PCTnUS96/04460 Preparation of N-(2R)-azido-3-(3,4-methylenedioxyphenyl)alanvl-N-(6-amino-2-methylpvridin-3-vl)methyl- L-proline amide Step A: Preparation of 6-amino-~-cyano-2-methylpvridine To a .solution of 6-amino-3-bromo-2-methylpyridine (70.0g 0.1069 mol) in 40 mL of DMF was added copper( I ) cynanide ( I 1.~0 g, 0.12~ mol). The reaction mixture was refluxed under argon for 10 h.
In cooled to ~0~C and poured into a .~iolution of NaCN (21.:~0 g) dissolved in 70 mL water. Thi.s .stirred for I h and was extracted with ethyl acetate (4 x 100 mL). The combined organic layer was w~.~ihed with 10~ NaCN (I x 75 mL)~ water (1 x 7~ mL) ~nd brine (I x ~0 mL) and dried over MgSO4, The solution wa.s~lltered. concentrated and the 1~ re.sidue was tritrated with ethyl acetate and hexane.~i to provide the product a.~ a tan solid (g.90 g): I H NMR (300 MHz, CDCl~) ~ 2.5~ (.s.
3H). ~.39 (br s, 2H), 6.35 (d, J = ~.55 Hz, IH), 7.~4 (d, J = ~.~4 Hz.
IH).

20 Step B: Preparation of the dihydrochloride of 6-amino-3-(aminomethyl)-7-methylpyridine To a solution of 6-amino-3-cyano-2-methylpyridille ( 10.3g ~ 7~.04 mmol) in 300 mL of ethanol/ methanol (3:1 ) and 30 mL of 6N
HCI was added 10% Pd/C (5.00 g). The solution was flushed with 2~ hydrogen and vigorou.sly .stirred. After 14 h the mixture wa.~ filtered through celite and washed with 500 mL of methanol. The volatile.~i were removed in l'(l~'UO to provide a yellow .solid: 1 H NMR (300 MHz, CDC13) ~ 2.59 (.~i, 3H), 4.12 (.~;, 2H). 4.~9 (~, 6H), 6.94 (d. J = 9.1 Hz.
lH), 7.96 (d,J = 9.2 Hz, lH).
Step C: Preparation of (4R)-3-(3-(3,4-methylenedioxyphenyl)-I -oxopropyl)-4-(phenylmethyl)-2-oxazolidinone To a ,solution of 3-(3,4-methylenedioxyphenyl)propionic acid (10.93 g, 0.0~63 mol) dissolved in THF wa,s added triethylamine CA 022168~9 1997-09-29 W O 96/31S04 PCT/u~5G~o~Go - 6~ -(8.22 mL, 0.05g9 mol) and cooled to -7~~C. Trimethylacetylchloride (6.93 mL, 0.0563 mol) was added dropwi~se and the reaction was warmed to 0~C for 0.5 h and recooled to -7~~C. To thi~ W;l~S added a .solution of (4R)-(3-phenylmethyl)-2-oxazolidinone (9.49 g, 0.0536 mol) and n-BuLi (22.5 mL, 2.5 M in hexane.~. 0.563 mol) in 150 mL THF
after .~tirring for I h at -7~~C via cannula. After 1 h the reaction wa~
warmed to 0~C and .~tirred an additional 2 h then ~luenched with 100 mL
of 1/2 .~at'd NH4CI. The aLIueou~; layer wa.s extracted with ethyl acetate (3 x 100 mL) and the combined organic layer wa.s wa.~hed with ~at'd NaHC03 ( I x 100 mL), water (1 x 100 mL). and brine (1 x 100 mL )~ dried over MgS04. filtered and concentrated to an oil. Recry.xtallization from ethyl acetate and hexane!i afforded 15.17 g (76~ vield) of white cry~talline product: mp ~6-~7~C.

Step D: Preparation of (3(2R),4R)-3-(2-azido-3-(3.4-methylene-dioxyphenyl)- I -oxopropyl)-4-(phenylmethyl)-2-oxazol idinone To a ~;olution of N-(4R)-3-(3,4-methylenedioxyphenyl)-1-oxopropyl)-~-(phenylmethyl)-2-oxazolidinone (7.59 g, 21.5 mmol) di.~.solved in THF and cooled to -7~~C wa.~ added KHMDS (46.0 mL. 0.5 M in toluene. 23.0 mmol) dropwi.~e. After I h a precooled .solution -7~~C) of tri~yl azide (7.46 g, 24.15 mrnol) di~iolved in THF (50 mL) wa.~i added via cannula. The reaction w~ yuench with glacial acetic acid (7.0 mL) after 3 minute.s and .~tirred at RT for 6 h. Saturated NH4CI
(50 mL) wa.s added and the a4ueou.~ wa~ extracted with ethyl acetate (2 x 75 mL). The combined organic layer wa.~ wa.shed with .~at'd NaHC03 (2 x 50 mL), water (1 x 50 mL), brine (1 x 50 mL), dried over MgS04, filtered and concentrated in vacuo. The solid W;l~i recry.stallized from ethyl acetate and hexane.s to afford 5.74 g of a yellow white .solid. mp 125-132; lH NMR (400 MHz, CDC13) o 2.~3 (dd, J=13.4 and 9.5Hz, lH), 2.94 (dd, J=13.6 and 9.2Hz, lH), 3.13 (dd, J= 13.7 and 5.1 Hz, 1 H), 3.33 (dd, J= 13.4 and 3.OHz, 1 H), 4.15-4.23 (m, 2H), 4.62-4.66 (m, lH), 5.19 (dd, J=9.1 and 5.1Hz. lH), 5.94 (.s, 2H), 6.76-6.~s2 (m, 2H). 7.20-7.36 (m, 5H).

W O 96/31504 . PCTnUS9C/O~0 Step E: Preparation of N-(2R)-azido-3-(3?4-methylene-dioxvphenvl)- I -propanoic acid To a ~solution of (3(2R),4R)-3-(2-azido-3-(3,4-methylene-dioxyphenyl)-l-oxopropyl)-4-(phenylmethyl)-2-oxazolidinone (5.743 g, 14.57 mmol) di.s.solved in 75 mL of dioxane cooled to 0~C wa.i added .~
solution of LiOH (0.419 g. 17.49 mmol) di.~.solved in water (25 mL) ;3nd 30% H20~ (~.27 mL). A*er 0.5 h ethyl acetate wa~ added (15 mL) .lnd the .solution wa.~ concentrated to 25 mL in l~a( uo. The a~lueou.s l.~yer wa.s acidified to pH 2 with 10% HCI and extr~cted w ith ethyl acetate (3x75 mL). the combined org.1nic layer wa.s wa.shed with water ( lx20 mL). brine ( I x'~0 mL). dried over M, SO4. filtered and concentrated to an oil. Thi~i materi~l wa.~ u..ed directly in the next .step.

Step F: Preparation of N-(2R)-azido-3-(3.4-methylene-l~S dioxyphenvl)alanvl-L-~roline methvl e.ster To ;~ .~olution of (2R)-~zido-3-(3.4-methylenedioxyphenyl)-I-propanoic ~cid (from above) di.~.solved in 30 mL of DMF was added L-proline methyl e.ster hydrochloride (''.53 g. 15.30 mmol), HOBt (2.06 g, 15.30 mmol). EDC (2.93 g, 15.30 mmol). cooled to 0~C and then ;idded triethyl~mine (4.46 mL. 32.06 mmol). After I h the reaction W;i.'.
w.~rmed to RT and .stirred 1~ h. then diluted with ethyl ~cetate (300 mL) and wa..hed with .sat'd NaHCO3 (lx50 mL), water (3x50 mL). brine (lx50 mL). dried over MgSO4. filtered and concentrated to an oil. The re.sidue W.l.S purified by fla.sh column chromatography (40 x 150 mm columnofSiO2.EtOAc:Hexgradientelution 1_'7 1000mL,to 1:1 500 mL) to ;~fford 3.26 g of waxy solid. TLC (EtOAc:Hex. 1:2) Rf = 0.17:
IH NMR (400 MHz~ CDC13) ~i 1.7~ 2 (m. lH), 1.97-2.40 (m, 2H).
3.02-3.15 (m. 3H), 3.64-3.74 (m. 4H), 3.~s4-3.X9 (m, lH), 4.40-4.52 (m.
lH), 5.92 and 5.93 rotamer~. (.s, 2H), 6.6~-6.75 (m, 3H).

W O96131504 PCTnUS~6/0~4C0 Step G: Preparation of N-(2R)-azido-3-(3,4-methylene-dioxvphenvl)alanvl-L-proline carboxvlic acid To N-(2R)-azido-3-(3,4-methylenedioxyphenyl)alanyl-L-proline methyl e.ster (0.~s4 g, 2.43 mmol) di.~olved in 6 mL of THF
cooled to 0~C wa.~ ~dded ~ .~olution of LiOH (0.62 g, '.5~ mmol) in 2 mL of water. After 0.5 h reaction w~ ~uenched by the addition of ethyl acetate (10 mL) and concentrated to a 3 mL volume. The .~olution wa.s acidified to pH 2 with 10% HCI and extracted with ethyl acetate (3x 25mL). the org~nic l~yer was wa~hed with water (Ix10 mL). and brine (I x 5 mL). dried over M~SO~. filtered and concentr;~ted to a white ~;olid (0.74~s g. 93~k ~ield). IH NMR (400 MHz. CDCI~) ~ I.XI-1.9~
(m. 3H). 2.25-2.2X (m. IH). 2.96-3.19 (nl. 3H), 3. 5-'.62 (m. 2H).3.X5 (dd, J = g.l and 6.~) Hz. IH). 1.49 (dd. J = 7.9 and ''.9 Hz. IH). 5.94 (.~.
2H). 6.6~-6.76 (m. 3H).
Step H: Preparation of N-(2R)-azido-3-(3,4-methylene-dioxyphenyl )~lanyl -N-(6-amino-2-methylpyridin-3-vl )methvl- L-proline amide To ~ ~olution of (2R)-azido-3-(3.4-methylenedioxy-phenyl)alanyl-L-proline carboxylic ~cid (0.319 g, 0.961 mrnol) di~olved in 4 mL of DMF wa.~ added 6-;~mino-3-(aminomethyl)-2-methylpvridine dihydrochloride (0.222 g. 1.05 mrnol). EDC (0.202 g~
1.05 mmol), and HOBt (0.142 g, 1.05 mmol), cooled to 0~C and added the triethylamine (0.42~ mL, 3.074 mrnol). The re~ction w~i warrned 2~ to RT after Ih and ~luenched after 16 h b~ diluting into 100 mL of ethyl acetate and wa.~hed with .~at'd NaHCO~ ( lx25 mL), water (4x25 mL), brine ( Ix20 mL). dr~d over MgSO4. filtered and concentrated to ;~
foam. Fla~h column chromatography (25x150 mrn column of SiO2, CH2C12/CH2C12 .saturated with NH3/MeOH gradient elution 60:3~:2 to . ~
60:37:3) provided 0.324 g of an oil: TLC (cH2cl2/cH2cl2 .~aturated NH3/MeOH, 60:30:10) Rf = 0.34; lH NMR (400 MHz, CDC13) ~ 1.61-1.79 (m. 2H), 2.01-2.07 (m, 2H), 2.35 (~, 3H), 2.g5-2.90 (m, lH), 3.01 (dd, J=13.4 and 6.6Hz, lH), 3.14 (dd, J=13.4 and g.4Hz, lH), 3.50 (t, J=2.7Hz. lH), 3.~3 (dd, J=~s.4 and 6.6Hz~ lH), 4.20-4.31 (m, 2H), 4.42 W O96/31504 PCTnUS~Gi~ 0 (~, 2H), 4.53 (dd, J=7.g and l.~Hz, lH), 5.93 (dd, J=2.0 and 1.3 Hz, lH), 6.29 (d, J=~.2Hz, lH), 6.65-6.74 (m, 4H), 7.27 (d, J=3.7Hz, lH).

Step I: N-(2R)-amino-3-(3,4-methylenedioxyphenyl)alanyl-N-(6-amino-2-methvlpyridin-3-yl)methyl- L-proline amide To N-(2R)-azido-3-(3.4-methylenedioxyphenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl- L-proline amide di.L,.L,olved in 10 mL of ethanol and 2.99 mL of 10% HCI wa.~; added 10~ Pd/C (0.30 g) ;~nd charged with hydrogen. After 2 h the re;3ction wa.'i flu.~ihed with ;3rgon and filtered throu~h celite wa.~ihing with ab.~olute ethanol. The ~iolution W;l.~; concentrated i~7 l'~l('U(~ and the oil w~, tritrated with ethvlLlcetate to afford 0.21 g white .L,olid. mp 172-l~g~C:
Anal~ i for- C22H27N~O4- 2HC1 0.95 ~ H20 calc C~ 51.76: H. 6.01: N, 13.59 t~und C. ~1.'>2: H. 6.14; N. 13.2~

Prep~r~tion of (2R)-N-(N-morphilino-2-~cet~mide)-3-(~s~4-methylene dioxyphenyl)alanyl-N-(6-;1mino-2-methylpyridin-3-yl)methyl-L-F~rolineLImide To a ~,olution of N-(2R)-amino-3-(3.4-methylelledioxy-phenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L-proline amide dihydrochloride (0.95 g, 0.1~4 mrnol) in DMF (2 mL) cooled to 0~C w~ dded N-morphilino-2-bromo;~cetamide (0.03~ g, 0.1~s4 mmol) and triethylamine (0.02~ mL. 0.202 mmol). The re~ction w~, w~rmed to RT over 3 h ;md after 19 h the volatiles were removed in ~~acuo. The re~idue wa~ purified by fla~;h column chromatography (15 x 150 mm column of SiO2, CH2C12/CH2C12 ~aturated with NH3/MeOH gradient elution 60:39:1; 60:3~s:2; 60:37:3; 60:36:4) to provide 0.009 g of a white ~olid: TLC (CH2C12/CH2C12 L,aturated NH3/MeOH,60:30:10) Rf=
0.15; lH NMR (400 MHz, CDC13) ~ 1.7~ 3 (m, 2H), 2.20-2.25 (m, lH), 2.35 (.'i, 3H), 2.71 (dd, J=16.1 and 7.3Hz, lH), 2.77 (d, J=7.3 Hz, 2H), 3.15 (d, J=14.5 Hz, lH), 3.29 (d, J=14.5 Hz, lH), 3.30-3.36 (m, W O 96/31504 PCTrUS96/04460 2H), 3.41-3.55 (m, 6H), 3.62-3.65 (m, 2H), 4.21-4.3~ (m, 4H), 4.57 (dd, J=7.~S and 2.6Hz, lH), 5.92 (dd, J=3.3 and 1.3Hz, 2H), 6.2~ (d, J=~.2Hz, lH), 6.62-6.73 (m, 4H), 7.26-7.2~ (m, 2H).

Preparation of (2R)-N-(N',N'-diethyl-2-acet~mide)-3-(3.4-methylene-dioxyphenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L-proline~mide The titled compound W;lS prepared Tin ~ manner .~,inlilar to that de~scribed in Example 16 u.sing N. N-diethyl-~-bromoacet;lmide in place of N-morphilino-2-bromoacetamide to ;3fford an amphorou.~ ,olid;
mp g5-100~C; TLC (CH2C12/CH2CI~ saturated NH~s/MeOH. 60:30:10) Rf = 0.39; -1 ~ Analy~,i.s for C2gH3 ~N6O5 ~ 0.45 H2O
calc C, 61.62; H. 7.00; N~ 15.40 found C, 61.65; H, 7.20; N, 15.12 (2R)-N-(N'-pyrrolidine-2-acetamide)-3-(3,4-methylenedioxy-phenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L-prolineamide Thi.s wa.s prepared in a manor .similar to that de.scribed in Example 16 u.sing N-pyrrolidine-2-bromoacetamide in place of N-morphilino-2-bromoacetamide to afford an amphorous ~solid; mp 7~-~2~C; TLC (CH2C12/CH2C12 .saturated NH3/MeOH. 60:30: 10) Rf =
0.32;
Analysi.s for C2~sH36N605 ~ 0.50 H20 calc. C, 61.64; H, 6.~4; N, 15.40 found C, 61.62; H, 6.77; N, 15.01 W O 96/31504 PCTnU~9-Oq1C0 -Preparation of N-BOC-D-3~3-diphenylalanyl-N-(6-amino-2-methyl-pyridin-3-yl)methvl-L-proline amide To a solution of N-BOC-D-3.3-diphenylal;lnyl-L-Pro-OH
(0.4g g, 1.096 mmol) in DMF (5 mL) cooled to 0~C was added EDC
(0.23 g, 1.205 mmol), HOBt (0.162 g, 1.205 mmol). the dihydro-chloride of 6-amino-3-(aminomethyl)-2-methylpyridine (0.253 g, 1.. 05 mmol) and then triethylamine (0.611 mL. 4.3~ mmol). The reaction was warrned to RT after lh ;md ~uenched after 16 h by dilutin~ into 100 mL of ethyl acetate and wa.shed with sat'd NaHCO ~ ( I x25 mL). water (4x25 mL). brine (Ix20 mL). dried over M~SO~. filtered and concentrated to a foam. Flash column chromatot~r~phy (25x150 rnrn column of SiO~, CH2C12/CH2C12 .saturated with NH~/MeOH gradient elution 60:3g:2 to 60:37:3) provided 0.525 ~ of a foam. The foam was dissolved in EtOAc (5 mL) ;md prepcipitated with hexanes (20 mL) to afford ~ white solid: TLC (CH2C12/CH2C12 .~ turated NH3/MeOH, 65:30:5) Rf = 0.47; 1 H NMR (400 MHz~ CDC13) ~ 1.24 (s. 9H), 1.42-1.4g (m~ IH). 1.57 (m~ 2H). 2.10-2.14 (m. IH). 2.3'7- (s. 3H), 2.50-2.57 (m, 1 H). 3.67 (dd. J=g.9 and ~s.6Hz. 1 H), 4.19-4.~0 (m~ 3H), 4.36 (dd.
J=11.3 ;~nd g.6Hz. 2H). 4.~4-4.90 (m. 2H). 6.32 (d. J=g.24Hz. lH), 7.16-7.41 (m. 13H).
CHN anal. for C32H39N504 ~ 0.45 H2O.
Calc C. 67.93; H, 7.11; N, 12.3g.
Found C. 67.92; H, 7.02; N, 12.42.

EXAMPLE l~S

Preparation of N-D-3,3-diphenylalanyl-N-(6-amino-2-methylpyridin-30 3-yl)methyl-L-proline amide To a ~solution of (0.452 g, 0.g 1 mrnol) dis,solved in ethyl acetate (10 mL), cooled to -7g~C wa.s bubbled in HCI (g) for 10 min.
Reaction .~tirred an additional 4 h at 0~C and was concentrated in V~lCUo.

W O96/31S04 PCTrUSS''~ CO

The residue was tritrated with ethyl acetate and filtered to provide 0.407 g of a white solid: mp 211-216~C, CHN anal. for C27H3 1N5O2- 0.70H20 and 1 .~S5 HCI
Calc. C, 60.32; H, 6.42; N, 13.03.
Found C, 60.32; H, 6.41; N, 12.7~S.

Kj for thrombin i,~ the inhibition con.~tant for the te~ited compound with human thrombin. Ki for tryp~in is the inhibition con~tant for the te.~ted compound with bovine tryp.~in. Rate con.stant.
I() were determined u.~ing the following in l'it~ procedure.<;.

IM l~jtr(~ ~t.~;~;LIV for deterrninin~ proteilla~ie inhibition As~iay.~ of hum~n a-thrombin and bovine tryp.sin were perforrned at '~5~C in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1~, PEG. Trvpsin a.~ay.~i al.so contained I mM CaCl2.
In a.s.~ay.~ wherein rate.s of hydroly~i.s of a p-nitroanilide (pna) ~trate were determined, a Thermomax 96-well plate reader wa.~ u~ied wa,~ u.~ed to mea.~ure (at 405 nm) the time dependent appearance of p-nitroani line . ,s~r-PR-pna (.~iarco.sine-Pro-Arg-p-nitroanilide) wa.s u~;ed to as~iay human a-thrombin (Km=125 ,uM) and bovine trypsin (Km=l~S ,uM). p-Nitroanilide .~ub.strate concentration wa~i determined from mea,~urement.~ of ab.sorbance at 342 nm u~iing an extinction coefficient of ~270 cm~l M- 1.
In certain ~tudie.s with potent inhibitor.~ (Ki < 10 nM) where the degree of inhibition of thrombin wa.s high, a more .~en.~itive activity a~i.say wa~i employed. In thi.s a.~.say the rate of thrombin catalyzed hydroly.~i.s of the fluorogenic .sub,~trate Z-GPR-afc (Cbz-Gly-Pro-Arg-7-amino-4-trifluoromethyl coumarin) (Km=27 IlM) wa.s determined from the increa.se in fluore.scence at 500 nm (excitation at 400 nm) a~ssociated with production of 7-amino-4-trifluoromethyl coumarin. Concentration,s of stock ~solution,s of Z-GPR-afc were determined from mea~surements of ab.sorbance at 3~0 nm of the 7-CA 022168~9 1997-09-29 W O96/31504 PCTnUS9''0~1C0 - 7~ -amino-4-trifluoromethyl coumarin produced upon complete hydrolysi.s of an ali~uot of the ,stock solution by thrombin.
Activity assay.s were performed by diluting a stock solution of substrate at least tenfold to a final concentration < 0.1 Km into a S .solution containing enzyme or enzyme e~luilibrated with inhibitor.
Times re~luired to achieve e~uilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (VO) or pre.~ence of inhibitor (Vi) v~ere measured. Assuming competitive inhibition. and that unity is negligible 10 compared Km/~S~ /e. and ~Il/e (where IS~ . and e respectivel~
represent the total concentrations. of substrate. inhibitor and enzvme).
the eLIuilibrium constant (Kj) t'or dissociation of the inhibitor ~rom the enzyme can be obtained from the dependence of VO/Vi on [Il ~ihown in eL~uation 1.
1~
Vo/Vi = I + [ Il/Ki ( I ) The activitie~; shown by this as.~ y indicate that the compound~i of the invention ale therapeutically u.seful for treating 20 variou.~ conditions in patients ~uffering from un.stable angina. refractory angina myocardial infarction. transient ischemic attackx atrial fibrillation, thrombotic stroke. embolic stroke~ deep vein thrombo~i.~i.
di.sseminated intravascular coagulation, and reocclusion or re.~teno.~is of recanalized ves.~iel.s.
'~S
Thrombin Inhibitors - Therapeutic U.se~
Anticoagulant therapy is indicated for the treatment and prevention of a variety of thrombotic conditions, particularly coronary artery and cerebrovascular disease. -Tho.se experienced in thi.~ field are 30 readily aware of the circumstances re~luiring anticoagulant therapy.
The term "patient" u.sed herein is taken to mean m~mm~l~; such as primates, including humans, sheep, hor.ses, cattle, pigs, dog~. cats, rats, and mice.

CA 022168~9 1997-09-29 W O96/31504 PCT/U~r''~1G0 Thrombin inhibition is u.seful not only in the anticoagulant therapy of individual.s having thrombotic condition~;, but is u.~eful whenever inhibition of blood coagulation i.s re~uired such as to prevent coagulation of .stored whole blood and to prevent coagulation in other S biological samples for testing or .storage. Thu~s, thrombin inhibitors canbe added to or contacted with any medium containin~ or su.spected of containing thrombin and in which it is desired that blood coagulation be inhibited, e.g. when contacting the mammal's blood with material selected from the group con.si.sting of vascular graft~s~ stents. orthopedic prothe.~ . cardiac pro.sthe.sis. and extracorporeal circulation .~y.~t~,m.
The thrombin inhibitor.s of the invention can be admini.stered in .such oral form.s as tablets. cap.sule~ (each of which include.s su.stained relea.se or timed relea.se formulation.s), pills, powder.
granule.~i. elixer~i, tincture.s, suspen.sions, syrups. and emulsion.s.
1~ Likewi.se. they may be ~mini.stered in intravenou.s (bolu~i or infu.sion), intraperitoneal, .subcutaneous. or intramuscular form. all u.sing forms well known to tho.se of ordinary .skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed a.~; an anti-aggregation agent. For treating ocular build up of '~0 fibrin, the compound~i may be administered intraocularly or topically a.s well a.~ orally or parenterally.
The thrombin inhibitor.s can be admini.stered in the form of ~ a depot injection or implant preparation which may be formulated in such a manner a.s to perrnit a sustained relea.se of the active ingredient.
2~ The active ingredient can be compre.ssed into pellets or small cylinders and implanted .subcutaneou.sly or intramuscularly as depot injection.s or implants. Implant.s may employ inert material.~ such a.s biodegradable polymer.s or .synthetic .silicone.s, for example. Sila.~tic. .silicone rubber orother polymers manufactured by the Dow-Corning Corporation.
The thrombin inhibitor.s can also be admini,stered in the form of lipo~;ome delivery .system,s, such as small llnil~mellar ve.sicles, large unilamellar vesicle.s and multilamellar vesicles. Liposome.~ can be formed from~ a variety of phospholipid.s, such a~ chole.sterol, .stearylamine or pho.sphatidylcholine~.

CA 022168~9 1997-09-29 W O 96/31504 PCTnUS9''0~0 The thrombin inhibitors may also be delivered by the use of monoclonal antibodies a,s individual carriers to which the compound molecule.s are coupled. The thrombin inhibitors may also be coupled with xoluble polymers as targetable drug carrier~s. Such polymers can 5 include polyvinlypyrrolidone. pyran copolymer. polyhydroxy-propyl-methacrylamide-phenol. polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl res,idues.
Furthermore. the thrombin inhibitor.s may be coupled to a class of biodegradable polymen~ useful in achieving controlled release of a drug.
10 for example. polylactic acid. poly~lycolic acid. copolymens of polylactic and polyglycolic acid. polyepsilon caprolactolle. polyhvdroxy butyric acid. polyorthoe.~iters. polyacetal.s. polydihvdropyr~n.~.
polycyanoacryl~te~i and cros.~, linked or amphipathic blocl; copolymers of hydrogel.s .
The do.sage regimen utilizing the thrombin inhibitors i.s selected in accordance with a variety of factor.s including type. species.
age. weight. sex and medical condition of the patient: the .~,everity of the condition to be treated; the route of admini.stration; the renal and hepatic function of the patient; and the particular compound or .salt thereof '~0 employed. An ordinarily ~,killed physician or veterinarian can re~dily determine alld pre.scribe the effective amount of the drug re~uired to prevent. counter, or arrest the progress of the condition.
- Oral do~sages of the thrombin inhibitors" when used for the indicated effects" will range between about 0.1 mg per kg of body '~5 weight per day (mg/kg/day) to about 100 mg/kg/day and preferably 1.0-100 mg/kg/day and mo.st preferably 1-~0 mg/kg/day. Intravenously, the mo.~t preferred do~e.~ will r~nge from about 0.01 to about 10 mg/kg/minute during a constant rate infu.sion. Advantageou.~,ly, the thrombin inhibitor.s may be admini.stered in divided doses of two, three, 30 or four time.s daily. Furthermore, they can be ~clministered in intrana.sal form via topical use of ,suitable intranasal vehicles, or via transdermal route.s, u.sing those forms of tran,sdermal skin patches well known to those of ordinary .skill in that art. To be ~clmini~tered in the forrn of a tran~sdermal delivery system, the dosage ~lministration will, CA 022l68~9 l997-09-29 W O 96/31504 PCTrUS9C/0~1C0 - 7~ -or course, be continuous rather than intermittent throughout the dosage regime.
For example, oral tablet.s can be prepared which contain an amount of active compound of between 100 and 500 mg, typically between 200 and 250 mg. Typically. ~ patient in need of thrombin inhibitor compound. depending on weight and metaboli.sm of the patient.
would be admini.stered between about 100 and 1000 mg active compound per day. For L~ patient re~luiring 1000 mg per day. two tablets containing 250 mg of active compound can be administered in the morning and two tLIblet~i containing 250 mg of active compound C~lll ~gain he administered in the evening. For ;~ p~tient re~uiring 500 mg per d;iy. one tablet cont;lining 250 mg of active compound c~n be admini.stered in the morning and one t~blet cont~ining 2~50 mg of active compound can ag~in he administered in the evening.
The thrombin inhibitors are typically ~dmini.stered ~.~ active ingredient.s in admixture with suitable pharmaceutic~l diluent.s.
excipient.s or carrier.~ (collectively referred to herein ~.~ "c~rrier"
m;~terials) .suitably ~elected with respect to the intended form of ~dministration. that i.s. oral tablet~. c~p~iule~i. elixer~s. ~yrup~ ~nd the like~
and consi~tent with convention pharmaceutical practice.s.
For in~tance. for oral admini~stration in the forrn of ~ t~blet or cap.sule. the active drug component can be combined with ~n oral.
non-toxic, pharm lceutically acceptable. inert carrier such as l~cto.se, .~,tarch, sucro~e, gluco~ie, methyl cellulo~e. m~gne.~ium stearate, dicalcium pho~;phate~ calcium .sulfate. m~nnitol, .sorbitol and the like; for oral administration in li~luid form. the or~l drug components can be combined with any oral, non-toxic, pharm;lceutically acceptable inert carrier such a.~ ethanol, glycerol, water and the like. Moreover, when de.sired or nece.s~sary, .suitable binder.s, lubricant.s, disintegrating agentx and coloring agent.s can al.so be incorporated into the mixture. Suitable binders include starch, gelatin, natural .sugLIr.s such a.s glucose or beta-lacto.se, corn-sweetener.s, natural and synthetic gum.s .such as acacia, tragacanth or sodium alginate, carboxymethylcellulo.~ie, polyethylene glycol, waxe~ and the like. Lubricants u.sed in these dosage forms W O96131504 PCTnU~9C~O~C0 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.
S The thrombin inhibitors can also be co-administered with .suitable anti-coagulation agents or thrombolytic agent~s such as plasminogen activators omstreptokinase to achieve synergi.stic effects in the treatment of various ascular pathologie~. For example. thrombin inhibitor.s enhance the efficiency of ti.~sue pl;3sminogen activator-medi~ted thrombolytic reperfu.sion. Thrombin inhibitors may be administered finst following thrombus formation~ and ti.ssue plasmino~en ~ctiv~tor or other plasminogen ~ctivator i.~ ~dmini.~itered thereafter. They m;~y also be combined w ith hepari~ pirin. or ~ ~rfarin.

Claims (20)

WHAT IS CLAIMED IS:

1. A compound having the following structure:

wherein A is C or N;
X1, X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of hydrogen, C1-4 alkyl. and C1-4 alkoxy;
Y, attached to a ring carbon atom, is H, NH2 or OH;
Z is; -(CH2)1-3-;
R1, R2, and R2' are independently hydrogen, phenyl, mono- or di-halogenated phenyl.
naphthyl, biphenyl, a 5- to 10-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S.
C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, C11-16 tricyclic alkyl, R4(CH2)n, (R4)2(CH), (R4)(OR4)CH, R4O(CH2)n, or R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and s.
where n is 1, 2, 3 or 4;
R

hydrogen, (R2)2N, wherein R2 is the same or different.
R2' OCONH, provided R2' not hydrogen.
R2CONH, HO(CH2)p, where p is 0, 1, 2, 3 or 4.
R2'SO2NH, provided R2 is not hydrogen, or (R2)m NCONH, where m is 1 or 2, wherein R2 is the same or different;

R4 is independently phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 10-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, -COR5, -OR6, C1-4 alkyl, branched C1-4 alkyl, C1-4 alkoxy, C3-7 cycloalkyl, C5-12 bicyclic alkyl, or C11-16 tricyclic alkyl;

R5 is -OH, -OR6, -N(R7)2, where R7 is same or different, and , where D is -CH2CH2-, -CH2-O-CH2, or -CH2-NH-CH2-:

R6 is C1-4 alkyl;

R7 is hydrogen or C1-4 alkyl;

G is (CH2)q where q is 1 or 2; or NR1CH2; and Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.

2. A compound of claim 1 having the following structure:

wherein A is C or N;

X1, X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of H and C1-4 alkyl;
Y, attached to a ring carbon atom, is H, NH2 or OH;
R1, R2, and R2' are independently hydrogen, phenyl, mono- or di-halogenated phenyl.
naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl.
C11-16 tricyclic alkyl, R4(CH2)n, (R4)2(CH), (R4)(OR4)CH, R4O(CH2)n, or R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1, 2, 3 or 4, R3 is H, (R2)2N, wherein R2 is the same or different, R2'OCONH, provided R2 is not hydrogen, R2CONH, HO(CH2)p, where p is 0, 1, 2, 3 or 4, R2'SO2NH, provided R2 is not hydrogen, or (R2)m NCONH, where m is 1 or 2, wherein R2 is the same or different;

R4 is independently phenyl, mono- or di-halogenated phenyl.
naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S.
COOH, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, or C11-16 tricyclic alkyl;

G is (CH2)q where q is 1 or 2; or NR1CH2; and Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.

3. A compound of Claim 2 which has the structure:

wherein A is C or N;
X1 and X2, each independently attached to a ring carbon atom, are independently selected from the group consisting of H and C1-4 alkyl;
Y, attached to a ring carbon atom, is H or NH2;
R1, R2, and R2' are independently hydrogen, phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S.
C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, C11-16 tricyclic alkyl, R4(CH2)n, (R4)2(CH), (R4)(OR4)CH, R4O(CH2)n, or R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1, 2, 3 or 4;
R3 is H, (R2)2N, wherein R2 is the same or different, R2 OCONH, provided R2 is not hydrogen.
R2CONH, HO(CH2)p, where p is 0, 1, 2, 3 or 4, R2'SO2NH, provided R2 is not hydrogen, or (R2)m NCONH, where m is 1 or 2 wherein R2 is the same or different;

R4 is independently phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, COOH, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, or C11-16 tricyclic alkyl;

G is (CH2)q where q is 1 or 2; or NR1CH2; and Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.

4. A compound of Claim 3 having the structure:

wherein X1 and X2, each independently attached to a ring carbon atom, are independently selected from the group consisting of H and C1-4 alkyl;
Y, attached to a ring carbon atom, is H or NH2;
R1, R2, and R2' are independently hydrogen, phenyl.
mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, C11-16 tricyclic alkyl, R4(CH2)n, (R4)2CH, wherein R4 is the same or different, (R4)(OR4)CH, R4O(CH2)n, or R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S.
where n 1, 2, 3 or 4;
R3 is H, (R2)2N, wherein R2 is the same or different, R2'OCONH, provided R2' is not hydrogen, R2CONH, HO(CH2)p, where p is 0, 1, 2. 3 or 4.
R2'SO2NH, provided R2 is not hydrogen. or (R2)m NCONH, where m is 1 or 2. wherein R2 is the same or different;

R4 is independently phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated. and which consisting of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, COOH, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, or C11-16 tricyclic alkyl;

G is (CH2)q where q is 1 or 2, or NR1CH2; and Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.

5. A compound of Claim 3 having the structure:

wherein Y, attached to a ring carbon atom, is H or NH2;
R1, R2, and R2' are independently hydrogen, phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, C11-16 tricyclic alkyl, R4(CH2)n, (R4)2CH, wherein R4 is the same or different, (R4)(OR4)CH, R40(CH2)n, or R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N.O and S.

where n is 1, 2, 3 or 4;

R3 is H.
(R2)2N, wherein R2 is the same or different.
R2' OCONH, provided R2' is not hydrogen, R2CONH, HO(CH2)p, where p is 0, 1, 2, 3 or 4, R2' SO2NH, provided R2' is not hydrogen, or (R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;

R4 is independently phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, COOH, C 1-4 alkyl, branched C 1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, or C11-16 tricyclic alkyl;

G is (CH2)q where q is 1 or 2, or NR1CH2; and Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.

6. A compound of Claim 3 having the structure:

wherein Y. attached to a ring carbon atom, is H or NH2;

R1 . R2. and R2' are independently hydrogen, phenyl.
mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to 7-membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, C1-4 alkyl, branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, C11-16 tricyclic alkyl, R4(CH2)n, (R4)2CH, wherein R4 is the same or different.
(R4)(OR4)CH, R4O(CH2)n, or R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N. O and S.
where n is 1, 2, 3 or 4;
R3 is H.
(R2)2N, wherein R2 is the same or different.
R2'OCONH, provided R2' is not hydrogen.
R2CONH, HO(CH2)p, where p is 0, 1, 2. 3 or 4, R2'SO2NH. provided R2' is not hydrogen' or (R2)mNCONH, where m is 1 or 2, wherein R2 the same or different;

R4 is independently phenyl, mono- or di-halogenated phenyl, naphthyl, biphenyl, a 5- to-7- membered mono- or bicyclic heterocyclic ring or bicyclic heterocyclic ring xystem any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, COOH, C 1-4 alkyl.
branched C1-4 alkyl, C3-7 cycloalkyl, C5-12 bicyclic alkyl, or C11-16 tricyclic alkyl;

G is (CH2)q where q is 1 or 2, or NR1CH2; and Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salt thereof.

7. A compound of Claim 3 selected from the group consisting of:

, , , and and pharmaceutically acceptable salts thereof.

8. A composition for inhibiting thrombin in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

9. A composition for inhibiting formation of blood platelet aggregates in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

10. A composition for inhibiting formation of fibrin in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

11. A composition for inhibiting thrombus formation in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

12. A method for inhibiting thrombin in blood in a mammal comprising administering to the mammal a composition of Claim 8.

13. A method for inhibiting formation of blood platelet aggregates in blood in a mammal comprising administering to the mammal a composition of Claim 9.

14. A method for inhibiting formation of fibrin in blood in a mammal comprising administering to the mammal a composition of Claim 10.

15. A method for inhibiting thrombus formation in blood in a mammal comprising administering to the mammal a composition of Claim 11.

16. A method for inhibiting thrombin in stored blood comprising administering to the mammal a composition of Claim 8.

17. A method for inhibiting formation of blood platelet aggregates in stored blood comprising administering to the mammal a composition of Claim 9.

18. A method for inhibiting formation of fibrin in stored blood comprising administering to the mammal a composition of Claim 10.

19. A method for inhibiting thrombus formation in stored blood comprising administering to the mammal a composition of Claim 11.

20. The use of a compound of Claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting thrombus formation, preventing thrombus formation, inhibiting thrombin, inhibiting formation of fibrin, and inhibiting formation of blood platelet aggregates, in a mammal.