CA2246811A1

CA2246811A1 - Macrocyclic peptides useful in the treatment of thrombin related disorders

Info

Publication number: CA2246811A1
Application number: CA002246811A
Authority: CA
Inventors: Michael N. Greco; Bruce E. Maryanoff
Original assignee: Individual
Current assignee: Janssen Pharmaceuticals Inc
Priority date: 1996-02-20
Filing date: 1997-02-19
Publication date: 1997-08-21
Also published as: AU717024B2; US5888971A; IL120239A0; NO983800L; JP2000504728A; NZ331447A; AU2052697A; TW517062B; ZA971419B; EP0932619A1; NZ501877A; WO1997030080A1; NO983800D0

Abstract

Compounds of Formula (I) and Formula (III) which are useful in the treatment of thrombin and trypsin related disorders.

Description

WO 97/30080 PCT/US97tO2575 MACROCYCLIC PEPTIDES USEFUL IN THE TREATMENT OF
THROMBIN RELATED DISORDERS

This invention relates a series of macrocyclic peptides, interme~i~tes 5 used in their manufacture and pharmaceutical compositions containing them. The compounds are inhibitors of serine proteases, particularly ~-thrombin and may be used in a variety of thrombin related disorders such as venous thrombosis and arterial thrombosis.

BACKGROUND OF THE INVENTION

With a rapidly aging population, dise~ses of the v~cular system are of great concem to our society. Arterial thrombosis is the major cause of death in the form of heart 2ltlac~s and strokes, while venous thrombosis is 15 ~ssoci~ted with pulmonary embolism which occurs after surgery or extended periods of inactivity.
Thrombin is a multifunctional serine protease whose role in thrombosis and hemostasis has been documented by a number of sources (See Qener~lly. Tapparelli, ~L TjPS 1993, 14, 366-76). Thrombin acts as 20 a procoagulant through proteolytic cleavage of fibrinogen to form fibrin and as an anticoagulant through activation of the protein C pathway (followed by inactivation of coagulation factors V and Vlll. ) The concentration of active thrombin is limited by a number of feedback mechanisms involving endogenous factors and proteins. In addition to protein C, antithrombin lll is 25 another regulating protein which forms a complex with endogenous heparin.
This complex binds to active thrombin, thus inactivating it.
Current anticoagulant therapy consists of three classes of compounds: heparins, coumarins and low molecular weight heparins.
These drugs act indirectly to limit the concentration of active thrombin.
30 Heparins and low molecular weight heparins interact with antithrombin lll and the coumarins inhibit a number of vitamin K dependent coagulation factors. Although these drugs are prescribed for diseases associated with venous thrombosis and arterial thrombosis, their use is limited. They have a number of side effects, a slow onset of action and only the coumarins are 3~ orally active (warfarin and dicumarol).
Indirect thrombin inhibitors have been shown to be less effective at controlling associated diseases than direct thrombin inhibitors. Thus the search for orally active direct thrombin inhibitors is underway in a number of W 097~W PCTnUSg7~2S75 laboratories. These efforts have produced a nurnber of acyclic peptidyl compounds which directly inhibit thrombin. PPACK, argatroban, (O-NAPAP, hirulog-1 and DUP 714 are examples of these inhibitors . Many of these compounds lack useful oral activity, and many have a poor selectivity 5 for thrombin versus other serine proteases. Therefore, a need remains for new direct thrombin inhibitors.
When compared to acyclic peptides, cyclic peptides have a number of structural features that havs been link~d to changss in the biological activity of simple peptides. Due to the absence of polar end groups and a relatively 10 rigid structure, cyclic peptides are hypothesized to be more membrane permeable and less susceptible to peptid~ses. Potentially one could inco,,uorale the structure of simple pepticles, within rigid, non-polar macrocyclic framework, to produce active bioavailable compounds.
Cyclotheonamide A (CtA) is a cyclic peptide which was isolated from 15 Theonell~ sp, a marine sponge. It inhibits a variety of serine proteases particularly thrombin and trypsin.

~OH

O~, Nl~ o H2N~N ' ~ > ~0 cyclotheonamide A (CtA) Although this molecule inhibits thrombin (Ki# ca.1-2 nM), it is a scarce natural product which is difficult to extract from its natural source. In addition, CtA is not an optimal candidate for treating thrombin-related disorders as its selectivity for thrombin over trypsin does not favor thrombin. The invention 25 described below claims a novel macrocyclic peptides that inhibit thrombin at nanomolar levels and exhibit reasonable selectivity for thrombin over trypsin.

The invention relates to new compounds of the Formula I
~~ NH~ ~ (C~H2)m O :~\,, NH~,A

HN
H2N~NH
I

wherein:

mis2to 12;
A is / ~ \

(CH2)n~~
N~C

\ / a where the amido carbonyl is bound to B and the a aminomethine is bound to the depicted ring carbonyl, R3 is hydrogen, hydroxy or Ct salkoxy, n is 1 or2 and a is 0 or 1 ;
B is / R6~, R4~
~ ~ b WO 97~N~80 rCTnUS97/02S7S

where the amido carbonyl of B is bound to the depicted ring methylenes and the methine is bound to A, R4 is selected from the group consisting of any of hydrogen, C1 salkyl, carboxyC1 salkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine, bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phenyl substituents are C~.salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 salkyl, diphenylC1 2alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), Rs and R6 are each hydrogen or taken together with the carbon to which each is attached to form a carbonyl, and b is 0 or 1 ;
G is / R8 E~R9\
~N,%

where the amine of G is bound to the ring methylenes and the methine is bound to the depicted amide, R7 is independently selected from the group consisting of hydrogen, C~ salkyl, carboxyC1 salkyl, phenyl, substituted phenyl (where the WO 97~0080 PCT~US97102575 phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 s alkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phenyl substituents are C1 salkyl, carboxy C1 ~alkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 salkyl, diphenylC1 2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), E is carbon or C(CH2)q-, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25, Rg and Rg are hydrogen or taken together with the carbon of E to form a carbonyl, and g is 0 or 1 ;
and pharmaceutically acceptable salts thereof.

An additional aspect of the invention relates to novel compounds of the Formula II which are intermediates in the synthesis of compounds for the Formula I, ~~ NH.G-(C~H2)m ~
HN
HzN~ NH
II
wherein:
R~ is hydroxy;

R2 is hydrogen;

W O97~HO80 rCTnUS97/02S7S

m is 2 to 12;

A is (CH 2 ) n~N_~

/ a where the amido carbonyl is bound to B and the a aminomethine is bound to the depicted ring carbonyl, R3 is hydrogen or C1 5alkoxy, n is 1 or2, and 15 aisOor1;
B is 6~, R4~
~ ~ b where the amido carbonyl of B is bound to the depicted ring methylenes and the methine is bound to A, R4 is selected from the group consisting of hydrogen, C1 salkyl, carboxyC1 salkyl, phenyl, substituted phenyl (where the phenyi substituents ars C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phenyl W O 97/30~ PCTrUS97~2~75 substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkYlcarbonylamino~ C1 salkoxy~ fluorine bromine or chlorine), 3-pyridylC1 5alkyl, 4-pyridylC1 salkyl, diphenylC1 2alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkYlcarbonylamino~ C1 salkoxy~ fluorine, bromine or chlorine), Rs and R6 are each hydrogen or taken together with the carbon to which each is attached to form a carbonyl, bisOor1;
G is / R8 E~Rg\

where the amine of G is bound to the ring methyl~nes and the methine is bound to the depicted amide, R7 is selected from the group consisting of hydrogen, C1 5alkyl, carboxyC1 salkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phsnyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 salkyl, diphenylC1 2alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, W O97~0080 PCT~US97~2575 carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcsrbonylamino, C1 salkoxy, fluorine, bromine or chlorine), -5 E is carbon or C(CH2)q-, where q is O to 12, with the proviso that the sum of q and m cannot exceed 25, R8 and Rg are each hydrogen or taken together with the carbon of E to form a carbonyl, 10 gisOor1;
or pharmaceutically acceptable salts thereof.

.

W 097/30080 PCT~US97~2575 Yet another aspect of the invention relates to novel thrombin inhibitors of the Formula III.

Wf=N~LH (CH2)m ~~HN~~

HNJ
H2NbNH
III
5 wherein:

m is2to 12;

W is nitrogen, sulfur or oxygen;
A is ~_ \
(CH2)n~~ _a ~_~N

/ a where the amido carbonyl is bound to B and the a aminomethine is bound to the depicted ring carbonyl, R3 is hydrogen, hydroxy or C1 salkoxy, 20 nis1 or2;

a is 0 or 1 ;

WO97~K~80 PCTA~S97~2S75 Bis / R6~, R4~
J~ / b where the amido carbonyl of B is bound to the depicted ring methylenes and the methine is bound to A, R4 is selected from the group consisting of hydrogen, C1 salkyl, carboxyC1 salkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1.salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy. fluorine bromine or chlorine), 3-pyridylC1,salkyl, 4-pyridylC1 salkyl, diphenylC1 2alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), Rs and R6 are each hydrogen or taken together with the carbon to which they are attached to form a carbonyl, bisOor1;

G is R8 Rg \
/N,%

/ g where the amine of G is bound to the ring methylenes and the methine is bound to the depicted amide, R7 is independently selected from the group consisting of hydrogen, C1 salkyl, carboxyC1 salkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 5alkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 5alkylamino, hydroxy, C1 5alkylcarbonylamino, C1 5alkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 5alkyl (where the phenyl substituents are C1.5alkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C~ salkylamino, hydroxy, C1 5alkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 5alkyl, diphenylC1 2alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 5alkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), E is carbon or C(CH2)q-, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25, R8 and Rg are each hydrogen or taken together with the carbon of E to form a carbonyl, g is 0 or 1 ;
or pharmaceutically acceptable salts thereof.

WO 97/30080 PCT~US97102S75 The terms used in describing the invention are commoniy used and known to those skilled in the art. However, the terms that could have other meanings are defined. ~Independently" means that when there are more than one substituent, the substitutents may be the same or different. The term "alkyl"
5 refers to straight, cyclic and branched-chain alkyl groups and "alkoxy" refers to O-alkyl where alkyl is as defined supra. "CBZ" refers to benzyloxycarbonyl.
"BOC" refers to ~-butoxycarbonyl and "Ts" refers to toluenesulfonyl. "DCC"
refers to 1 ,3-dicyclohexylcarbodiimide, "DMAP" refers to 4-N'N-dimethylaminopyridine and "HOBr refers to 1-hydroxybenzotriazole hydrate.
10 "FMoc" refers to N-(9-fluorenylmethoxycarbonyl). Amino acid refers to compounds where the amino group and the carboxy group are on different carbon atoms. The terrn a-amino acid, refers to compounds where both the carboxy and the amino group are attached to the same carbon atom. The stereochemistry of this carbon is indicated by the terms "D and L" where D
15 indicates right-handed chirality.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention may be prepared by a number of 20 synthetic schemes, where the macrocyclic ring members A, B, and G dictate the appropriate synthesis. The starting protected mono and di-peptides are either known or readily synthesized by standard techniques known in the art.
See Bodansky, M. Pract~ce of Pepbde Synthes~s; Springer Verlag, 1984. All syntheses include a series of peptide coupling reactions, where the 25 macrocycle is built, oxidized and deprotected.
As illustrated, Scheme I may be used to prepare a compound of Formula I where m is 7;
A is (CH2)n~ ~
~ ~N~

where R3 is hydrogen and n is 1;

..

B is R6~

R4~f 5 where R4 is 2-methyl-1-propyt and Rs is taken together with R6 to form a carbonyl. A t~nown ~-protected a-amino acid la, is coupled at room temperature to a known C-protscted amino acid Ib, using HOBT/DCC in an inert solvent, such as DMF, CH3CN or THF, over 5-24 h. Although HOBT/DCC is the preferred coupling agent other agents can be used and 10 include: BOP, BOP-CI and PyBrOP. The protecting groups are chosen in order to permit selective removal, where the favored protecting groups are CBZ for nitrogen and I-butoxycarbonyl for carboxy. However, other well known protecting groups may be substituted and are described in Green, Theodora r~t~ling Groups in Org~nic Synthesis; John Wiley & Sons, New 15 York, 1981. As illustrated the CBZ group is removed by hydrogenation at approximately 20 psig using Pd(OH)2/C as a catalyst. However, other conditions may be used such as catalytic transfer hydrogenation using Pd/C
and formic acid. The resulting C-protected di-peptide Ic, is coupled to an N-protected aliphatic amino acid, Id, followed by removal of the N-protecting 20 group to give amine le. As illuslld~ , the CBZ serves as the N-protecting group and Pd/(OH)2 is the hydrogenation catalyst. However either the protecting group or the reaction conditions may be modified as previously described. Interrnediate le is coupled to 6-[[imino~4-methylbenzenesulfonyl)-amino]methyllamino]-2-(R,S)-[[2-(trimethylsilyl)ethoxy]methoxy~-3(S)-25 [9-phenylmethoxycarbonyl)-amino~hexanoic acid, ( Maryanoff ~!. Journa/
of the American Chemical Society 1995, 1 f 7, 1225-39) using HOBT/DCC
at room temperature for 4-24 h in an inert solvent and deprotected with Pd(OH)2 to give the arginine derivative If. The I-butoxycarbonyl and SEM
protecting groups are removed with TFA and the resulting intermediate is 30 coupled at room temperature with BOP-CI and DMAP in an inert solvent such as Cl 12CI2 to give the hydroxy macrocyclic derivative ~. Compound is oxidized using the Dess-Martin periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger such as anisole, thioanisole, pentamethylbenzene, 35 dimethytsulfide or cresol to give a compound of Formula I.

W097/3~ PCTnUS97/0257S

This scheme may be used to form the compounds of the invention where m is 2-12, A is (CH2)n-~ ~
~_~N~

where n is 1 or 2 and R3 is hydrogen or C1 5 alkoxy, and B is O~
N-H
R4~

10 where R4 is hydrogen, C1 salkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 5alkyl, carboxy C1.5alkoxycarbonyl, carboxamido, amino, C~ salkylamino, hydroxy, C~ salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 5alkyl (where the phenyl substituents are C1 5alkyl, carboxy 15 C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C
salkylcarbonylamino, C1 5alkoxy, fluorine bromine or chlorine), 3-pyridylC1 5alkyl, 4-pyridylC1 5alkyl, naphthyl, substituted naphthyl or diphenylC1 2alkyl. For example to prepare compounds where m is 2-12, the illustrated reactant ld, 8-~N-benzyloxycarbonyl)aminooctanoic acid, is 20 replaced with an analog of "m" methylenes such as 6-(N-benzyloxycarbonyl)aminohexanoic acid. To prepare a compound where A
is (CHz)n~ ~
J_~N~

wo 971~0080 rCTlUS97/0257S

R3 is hydrogen and n is 2, simply replace Ib, D-Pro-O-I-Bu, with D-pipecolinic acid -I-butyl ester To prepare a compound where B is 0~
N-H
R4~

and R4 is 3-pyridylmethyl, replace I~ N-BOC-D-leucine with N-BOC-D-3-pyridyl-alanine. When active aromatic substituents are desired such as hydroxy, amino or carboxy, those compounds may be prepared as protected derivatives where the protecting groups well known in the art are described 10 in Green, Theodora Protecting Groups in Organic Synthesis; John Wiley &
Sons, New York, 1981. For example to prepare a compound where R4 is 4-hydroxybenzyl, a I-butyldimethylsilyl group is used as protecting group and removed with HF in the last step of the scheme.

W 097~0080 PCT~US97/02575 SCHEME I

CBZN~OH
H 0 ~ N~
~ O C02-tBu ~ / Ic ,N_~ ~
H C02-tBu / (Id) Ib ~

~ C02H
O N~ SEM0~ NHCBZ
(CHz)7 ~CO2~-Bu(CH2)3NHCN(NH)Ts Ie (CH2)~N' )--NH \-~' (CHZ~)~LN ,~ C02~-Bu ~ ~ HNS~NH

HN~NH 1E \ (CH2~)~N\~)--~N~

HN
H2N~NH

" .

W O 97/30080 PCTfUS97102575 Another method of synthesis, illustrated by Scheme II, may be used to prepare a compound of Formula I where m is 4;
Ais o J

where R3 is hydrogen and n is 1;
10 Bis R6~

~J

where R4 is benzyl and Rs is taken together with R6 to form a carbonyl; and G is R8 ~Rs ~E~ .NCx~

20 where E, Rg and Rg are taken together to form a carbonyl and R7 is 4-chlorobenzyl.

A known N-protected -a-amino acid IIa, is coupled at room temperature to a known C-protected amino acid nb~ using HOBT/DCC in an 25 inert solvent, such as DMF, CH3CN or THF, over ~-24 h. Although HOBT/DCC is the preferred coupling agent other agents may be used and include: BOP, BOP-CI and PyBrOP. The protecting groups are chosen in order to permit selective removal, where the favored protecting groups are CBZ for nitrogen and ~-butoxycarbonyl for carboxy. However, other 30 protecting groups well known in the art may be substituted and are W 097/30080 PCTnUS97/02S75 described in Green, Theodora, rl.)t~ting Groups in Organic Synthesfs;
John Wiley & Sons, New York, 1981. As illustrated the ~-butoxycarbonyl group is removed with TFA to give the N-protected di-peptide ~. This - intermediate is coupled to an ~-protected di-peptide ~, followed by removal 5 of the N-protecting group to give amine ~. As illustrated the CBZ serves as the ~I-protecting group and Pd/(OH)2 is the hydrogenation catalyst.
However either the protecting group or the reaction conditions may be modified as previously described. Intermediate Ile is coupled to 6-[[imino[4-methylbenzenesulfonyl)-amino]methyl]amino]-2-(R,S)-[[2-1 0 (trimethylsilyl)ethoxy]methoxy]-3(S)-~9-phenylmethoxycarbonyl)-amino]hexanoic acid, ( Maryanoff ~1- Joumal of the American Chemical Society 1995, 117, 1225-39) using HOBTtDCC at room temperature for 4-24 h in an inert solvent and deprotected with Pd(OH)2 to give the arginine derivative IIf. The ~-butoxycarbonyl and SEM protecting groups are 15 removed with TFA and the resulting intermediate is coupled at room temperature with BOP-CI and DMAP in an inert solvent such as CH2CI2 to give the hydroxy macrocyclic derivative ~. Compound 11~ is oxidized using the Dess-Martin periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger to give a 20 compound of Formula I.
This scheme may be used to form the compounds of the invention where m is 2-12, A is (CH2)n-~ ~
~_~N~

2~
where R3 is hydrogen or C1 salkoxy, B is 0~
N-H
R4~

30 where R4 is hydrogen, C1 ~alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 5alkyl, carboxy C1 sallcoxycarbonyl, carboxamido, amino, C~ salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where th~ phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C
salkylcar~onylamino, C1 salkoxy, fluorine bromine or chlorine), 3-5 pyridylC1 salkyl, 4-pyridylC1 salkyl or diphenylC1 2alkyl, and G is o ,NSS

where R7 is hydrogen, C1 salkyl, phenyl, substituted phenyl (where the 10 phenyl slJ~stituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phenyl substituents are C1 5alkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C
15 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 5alkyl, naphthyl or diphenylC1 2alkyl. For example to prepare compounds where m is 2-12, the illustrated reactant IIb, ~aminopentanoic acid -~-butyl ester is replaced with an analog of "m"
methylenes such as 7-aminoheptanoic acid -~-butyl ester To prepare a 20 compound where A is ~h o CN~
and B is N~H

replace the illustrated reactant IId with 3-pyridylalanine-pipecolinic acid (0-~Bu) A compound where G is W 097/~ PCTAJS97~257S
-,NSS

and R7 is butyl can be prepared by replacing Ila with N-BOC-D-norleucine.
When the aromatic substituents hydroxy, amino or carboxy are desired, those compounds may be prepared as protected derivatives where the 5 protecting groups well known in the art are described in Green, Theodora Protecting Groups in Organic Synthesis; John Wiley & Sons, New York, 1981. For example to prepare compound where R4 or R7 is 4-aminobenzyl, an allyloxycarbonyl group is used as protecting group and removed with tetrakis(triphenylphosphine)palladium(0) at the end of the scheme.

SCHEME II

Cl~ ,,.N~CcOB2zH Cl~ H, IIa CBZHN~N~ ~COzH
H2N~ C02-~,-Bu / nc (CH2 IIb ~/ ~ N~

~L H ~3 0 CO2-tBu CI~NH2 ~ C02-t-Bu _~ ~jr~-(C-2~o ~ CO2-t-Bu Ts IIf Cl~ o~_H_(CHz~
0~ j H ~~ '13 H,N~NH

HN
H2N~NH

WO 97/30080 PCTIUSg7/02575 Yet another method, illustrated by Scheme III, iS used to prepare a compound of Formula I where m is 3;
A is where R3 is hydrogen and n is 2;

B is R6~

R4~
~J

where R4 is benzyl and both Rs as well as R6 are hydrogen. A known N-prot~cted aldehyde ~, is reductively aminated at room temperature to a 15 known Ç-protected amino acid ~, using NaB(OAc)3H in an inert solvent, such as CH2CI2 or (CH2)2CI2, over 2-16 h. The protecting groups are chosen in order to permit selective removal, where the favored protecting groups are Fmoc for nitrogen and ~-butoxycarbonyl for carboxy. However, other protecting groups may be substituted as previously discussed. The 20 free amine of the resulting product IIIÇ is protected as the CBZ and the Fmoc group of the other amine is cleaved with an anhydrous base such as piperidine to give IIId. Intermediate IIId is coupled to 6-[[imino[4-methylbenzenesulfonyl)-amino~methyllamino]-2-(R,S)-~2-(trimethylsilyl)ethoxylmethoxy]-3(S)-[9-fluorenylmethoxycarbonyl)-25 amino]hexanoic acid, ( Maryanoff et al. Joumal of ~he Amencan Chemical Society 1995, 117, 1225-39) using HOBT/DCC at room temperature for 4-24 h in an inert solvent and deprotected with Pd(OH)2 to give the arginine deriYative IIIe. The Fmoc group is removed with an organic base and the l-butoxYcarbonyl and SEM protecting groups are removed with TFA. The 30 resulting intermediate is coupled at room temperature with BOP-CI and DMAP in an inert solvent such as CH2CI2 to give the hydroxy macrocyclic derivative ~f. Compound Illf iS oxidized using the Dess-Martin periodinane W 097~X0 PCTnUSg7~2S7S

in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger to give a compound of Formula I.

- - This Scheme lll may be used to form the compounds of the invention 5 wheremis2-12,Ais (CH2)n~ ~
~_~N--SC

where R3 hydrogen or C1 5alkoxy and n is 1 or 2, and B is H~
N-H
R4~

where R4 is hydrogen, C1 5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1 5alkyl, carboxy C1 5alkoxycarbonyl, 15 carboxamido, amino, Cl salkylamino, hydroxy, C1 ~alkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 5alkyl or diphenylC1 2alkyl. For example to prepare compounds where m is 2-12, the illustrated reactant ~" is replaced with an analog of "m" methylenes such as 5-(N-9-20 fluorenylmethoxycarbonyl)aminopentaldehyde To prepare a compoundwhere A is ~h o CN~

25 and B is ~H

W O97~n~ PCT~US97~2575 replace the illuslraled reactant IIIb with norleucine-proline-(O-~-Bu). When active aromatic substituents are desired such as hydroxy, amino or carboxy, those compounds may be prepared as protected derivatives where the 5 protecting groups well known in the art are described in Green, Theodora Protecting Groups in Organic Synthesis; John Wiley & Sons, New York, 1981. For example to prepare compound where R4 is 4-carboxybenzyl, an methylester is used as protecting group and removed with aqueous LiOH
prior to oxidation with Dess Martin periodiane.

~o 97J30080 2246811 1998 08 SC~EIUIE III PCr/lJS97/0257~;
- - tmOC~ ~C~2~)3 H C~lo ~ ~noc-N ~c~2)4 Z-IBu ~ ,~

H2~(c~l2)4 CBZ
0~"~

2-tBu (Cl~ ,CB2 o~H N)~' r' C~--C02t-BU

TS N~
(C~2)4N BZ~ ~,,/
~0 IIN
H~ lLf Ts ~ \~ (C~2)4 ~'0 ~2 N~

W O 97~0080 rCT~US97~02575 Another method of synthesis is illustrated by Scheme IV may be used to prepare a compound of Formula I where m is 5;

A is (CH2)n~~
~_~N~C

where R3 is hydrogen and n is 1; and Rg E~Rg ~ ~N~S
Gis R7 H
where E, R8 and Rg are taken together to ~orm a carbonyl and R7 is phenyl.

A known N-protected amino acid ~a. is coupled at room temperature to a known C-protected amino acid ~, using HOBT/DCC in an inert solvent, such as DMF, CH3CN or THF, over 5-24 h. Although HOBT/DCC is the preferred coupling agent other agents be used and include: BOP, BOP-CI
and PyBrOP. The preferred protecting groups are CBZ ~or nitrogen and I-butoxycarbonyl for carboxy; however, other protecting groups may be substituted as discussed previously. As illustrated the ~-butoxycarbonyl group is removed with TFA to give the ~I-protected di-peptide IVc. This intermediate is coupled to an C-protected di-peptide IVd, followed by removal of the Dl-protecting group to give amine IVe. As illustrated the CBZ
seNes as the ~-protecting group and Pd/(OH)2 is the hydrogenation catalyst. However either the protecting group or the reaction conditions may be modified as previously described. Intermediate IVe is coupled to 6-[[imino[4-methylbenzenesulfonyl)-aminolmethyl~amino~-2-(R,S)-[[2-(trimethylsilyl)ethoxy]methoxy]-3(S)-[9-phenylmethoxycarbonyl)-amino]hexanoic acid, ( Maryanoff ~1 Joumal of ~he American Chemical Society 1995, 117, t225-39) using HOBT/DCC at room temperature for 4-24 h in an inert solvent and deprotected with Pd(OH)2 to give the arginine derivative ~. The ~-butoxycarbonyl and SEM protecting groups are W 097~ 0 rCTAUS97~257S

removed with TFA and the resulting intermediate is coupled at room temperature with BOP-CI and DMAP in an inert solvent such as CH2Cl2 to give the hydroxy macrocyclic derivative ~. Compound ~ is oxidized using periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger to give a compound of Formula I.

This Scheme IV may be used to torm the compounds of the invention where m is 2-12, A is (cH2)n~

and G is -~ ,N~x where R7 is hydrogen, C1.5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1.salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, 20 C1 ~alkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 salkyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C
salkylcarbonylamino, C~ salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 salkyl naphthyl or diphenylC1 2alkyl. For 25 example to prepare compounds where m is 2-12, the illustrated reactant ~, 6-(N-CBZ)aminohexanoic acid is replaced with an analog of "m" methylenes such as 7-(N-CBZ)aminoheptanoic acid. To prepare a compound where A
is ~ O
CN~

WO 97~0080 PCT~US97/02S75 replace the illustrated reactant ~ with D-pipecolinic acid t-butyl ester. A
compound where G is O
,N%

and R7 is butyl can be prepared by replacing I~ with D-norleucine. When active aromatic substituents are desired such as hydroxy, amino or carboxy, thoss compounds may be prepared as protected derivatives where the p~uteoting groups well known in the art are described in Green, Theodora 10 Protecting Groups in Organic Synthesis; John Wiley & Sons, New York, 1981. For example to prepare compound where R7 is 4-hydroxybenzyl, a ~-butyldimethylsilyl group is used as protecting group and removed with HF in the last step of the scheme.

W 097/30080 PCTrUS97~2S75 SCHEME IV
H O
CBZ (CH2J~oH H2N~ J~N~'~
~ (CH2)5 ~j IVc H COz-~Bu ~ ~~2 IVb IVd O (CH2)s V ~U~NH

~ ',',CHo2)5 SEMO~NH2 ~--C02-~-B~

HN~ IVf ~ NH H~N ~NH
NH (CH2)5 ~ H 1--O

HN~NH ~g ~ o NH (CH2)5 o~Nb~

HN
H2N~NH

WO 97130080 PCT/US97/02~75 Another method of synthesis is illustrated by Scheme V and may be used to prepare a compound of Formula I where m is 5;
A is where R3 is methoxy;
n is 1 ;

B is R6~

R4~

where R4 is 4-chlorobenzyl and Rs is taken together with R6 to form a carbonyl; and G is Rg E~Rg ~N

20 where E is carbon, R8 and Rg are hydrogen and R7 is hydrogen.
A known Fmoc-protected amino aldehyde Va, is reductively aminated at room temperature to a known t-butoxy-protected amino acid Vb, using NaB(02CCH3)H in an inert solvent, such as CH2CI2, over 5-24 h. The nitrogen of the resulting intermediate is protected with CBZ and the O- ~-25 butoxy group is cleaved to give the acid Vc. This intermediate is coupled toa ~-protected dipeptide Vd using HOBT/DCC followed by the removal of the FMoc protection with an anhydrous base such as diethylamine to give Ve.
Intermediate Ve is coupled to 6-[[imino[4-methylbenzenesulfonyl)-ami no]methyl]amino]-2-(R,S)-[[2-(trimethylsilyl)ethoxy] methoxy]-3(S)-30 [9-fluorenyllmethoxycarbonyl)-amino]hexanoic acid, ( Maryanoff et al.

Journal of the Amer~can Chemical Society 1995, 1 17, 1225-39) using HOBT/DCC at room temperature for 4-24 h in an inert solvent and deprotected with diethylamine to give the acyclic arginine derivative ~~. The ~-butoxycarbonyl and SEM protecting groups are removed with TFA and the 5 resulting intermediate is coupled at room temperature with BOP-CI and DMAP in an inert solvent such as CH2Cl2 to give the hydroxy macrocyclic derivative ~g. Compound Vg is oxidized using periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger to give a compound of Formula I.
This Scheme V may be used to form the compounds of the invention where m is 2-12, Ais (CH2)n~ ~
~ ~N~

15 Bis 0~
N-H
R4~

where R4 is hydrogen, C1 5alkyl, phenyl, substituted phenyl (where the 20 phenyl substituents are C1 5alkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 5alkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted phenylC1 5alkyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C
25 salkylcarbonylamino, C1 5alkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 5alkyl naphthyl or diphenylC1 2alkyl, and G is H ,H
,N%

W 097/30080 PCTAUS97~2S75 where R7 is hydrogen, C1 salkyl, phenyl, substituted phenyl (where the phenyl substitoents are C1 5alkoxy, fluorine or chlorine), phenylC~ 5alkyl, substituted phenylC1 salkyl (where the phenyl substituents are C1 salkoxy, fluorine or chlorine), 3-pyridylC1 ~alkyl, 4-pyridylC1 salkyl naphthyl or 5 diphenylC1 2alkyl and E is C(CH2)q, where q is 0-12. For example to prepare compounds where m is 2-12, the illustrated reactant Vb, is repl~oed with an analog of ~m~ methylenes such as 8-aminooctanoic acid -~-butyl ester. To prepare a compound whcre A is ~h o CN~

and B is ~ 0~
NJ~H

replace the illustrated reactant Vd with 3-pyridylalanine-D-pipecolinic acid -~-butyl ester. A compound where G is ,Nc,s and R7 is hydrogen can be prepared by replacing Va W;th 6-(N-9-fluorenylmethoxycarbonyl)aminobutylraldehyde. To prepare compounds where R7 is other than hydrogen, start with an N-protected a-amino acid, reduce the carboxy to an aldehyde. Any of the standard reagents and 25 conditions may be used including 1,1'-carbonyldiimidazole in THF at 0-10~
C, followed by treatment with DlBAlJhexane at -42 ~C. This a-substituted aldehyde is used in place of Va and the remaining steps of the synthesis are carried through with only minor modifications.

SCHEME V

FmocHN~H ÇBZ
Va ~ ~ FmocNH(cH2)~N-(cH2)5co2H
o Vc H2N~o~ / C~
Vb ~ ~N~Ç",0,2-~Bu Cl NH2 1_~
~CH3 Vd CH30 0~ ~ N~
(CH2)4 0 C02~-Bu CBZ
/ N--(CH2)s ~~ r H2N~ CBZ Ve ~~ Q~NH NH ~I
HO~ t BuO2(~>~eo rN--(CH2)s NH
~H ~H~ Ts'~,//

HN~NH ,H
Ts V~ ~ ~N--(CH2)s ~H~

W O 97~0080 PCTrUS97/02S75 Yet another method of synli,esis is illustrated by Scheme VI. This scheme is used to prepare a compound where m is 5 and G is ~ ,N~

and R7 is 4-chlorobenzyl.
A known ~-protected amino acid ~a. is coupled at room temperature to a known C-protected amino acid VIb, using HOBT/DCC in an inert solvent, 10 such as DMF, CH3CN or THF, over 5-24 h to give VI~. . Although HOBT/DCC is the preferred coupling agent other agents be used and include: BOP, BOP-CI and PyBrOP. The preferred protecting groups are CBZ for nitrogen and l-butoxycarbonyl for carboxy; however, other protecting groups may be substituted as ~lisclJsse~l previously. The protecting groups 15 are removed by sequentiaJ treatment with TFA and Pd/(OH)2/H2 to give VIc lnterrnediate VIc is coupled to 6-[[imino~4-methylbenzenesulfonyl~-amino]methyl]amino]-2-(R,S)-[~2-(trimethylsilyl)ethoxylmethoxy]-3(S)-[9-phenylmethoxycarbonyl)-amino]hexanoic acid, ( Maryanoff ~. Joumal of the American Chemical Society 199~, 117, 1225-39) using HOBT/DCC
20 at room temperature for 4-24 h in an inert solvent to give y~. The CBZ, ~-butoxycarbonyl and SEM protecting groups are removed by sequential treatment with TFA and Pd(OH)2/H2 to give VIe. This intermediate is coupled at room tempersturQ with BOP-CI and DMAP in an inert solvent such as - CH2Cl2 to give the hydroxy macrocyclic derivative VIf. Compound VIf is 25 oxidized using periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carboc~tion scavenger such as anisole, thioanisole, pentamethylbenzene, dimethylsulfide or cresol to give a compound of Formula I.
This Scheme Vl may be used to form the compounds of the invention 30 where m is 2-12, and G is ~N5 ..

W 097~0080 rCT~USg7~2~75 where R7 is hydrogen, C1 salkyl, phenyl, substituted phenyl Iwhere the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C1 salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), phenylC1 salkyl, substituted 5 phenylC1 salkyl (where the phenyl substituents are C1 salkyl, carboxy C1 salkoxycarbonyl, carboxamido, amino, C1 salkylamino, hydroxy, C
salkylcarbonylamino, C1 salkoxy, fluorine bromine or chlorine), 3-pyridylC1 salkyl, 4-pyridylC1 salkyl naphthyl or diphenylC1 2alkyl. For example to prepare compounds where m is 2-12, the illustrated reactant VIb 10 is repl-ced with an analog of ~.mu methylenes such as 8-aminoooctanoic acid l-butYl ester. A compound where G is o ,N%

15 and R7 is butyl can be prepared by replacing VIb with N-BOC-D-norleucine W 097/30080 PCTnUS97/02575 SCHEME VI
o ~~OH
CBZ' Cl~
~ - H
H2N\~CO2-~-Bu o (CH2) 5 Cl~ f 'NH

O NH '~=HO2)s ~NHCBZ C~-~Bu C~'~.~NH

J-- VId '(CH2)C
HN o~ ,~NH ,Lo HTN~NH SEMOlr 2 ~ VIe HNJ
HTN~NH

, WO97/3NU~ PCTrUS97~2S75 Cl~ ~NH
VIe o~NH ~;>
H
HO~N~
~ O
HNJ
HTN~NH

Cl~ f 'NH VIf o~NH ~>
o~HN_~

HN
H2N~NH

Scheme VII may be used to prepare the compounds of Formula III
where m is 4, W is sulfur, 5 Ais (CH2)n-~ ~
J ~N~

where R3 is hydrogen and n is 1;
B is R6~

R4~

15 where R4 is benzyl and Rs is taken together with R6 to form a carbonyl; and W O 97~0080 PCT~USg7~2~7 G is Rg~ E~Rg - ~ ~NSS

where E R8 and Rg are taken together to form a carbonyl and R7 is benzyl.
N-a-Fmoc~ r-tosyl-L-arginine is treated with carbonyldiimidazole at 0 ~C
in THF and reduced with DIBAL at about -48 ~C to give ths corresponding aldehyde. The cyanohydrin VIIb is produced by treating the aldehyde with KCN
10 and H2O at room temperature over several days in ethyl acetate. Treatment of VIIb with gaseous HCI in an alcohol such as MeOH at about -40 to -15 ~C over several hours gives the imidate VIIc. Treatment of the imidate with VIId (cysteine methyl ester hydrochloride) in an inert solvent such as CH2CI2 at room temperature for 1-5 h gives the Fmoc protected amino alcohol VlIe. The 15 hydroxy of VIle can be converted to the trisilylalkyl ether with standard silylating agents such as t butyldimethylsilyltriflate and an organic base such as 2 6-lutidine at 0 ~C. The Fmoc group is removed by treatment with an organic base such as diethyl amine at room temperature over 2-5 h. The free amine can be protected as the N-Boc derivative by treatment with di-t-butyl dicarbonate in an20 inert solvent at 0 ~C over 16 h to give the 4 5-dihydrothiazole intermediate VIIf.
Intermediate V~If can be oxidized to the thiazole by treatment with with an oxidizing agent such as MnO2 in an inert solvent such as CH2CI2 at room temperature over several hours. The isolated 5-carboalkoxy thiazole intermediate is saponified at room temperature with LiOH in dioxane/water to 25 give the 5-carboxy thiazole derivative ~g.
Derivative VIIg is coupled using HOBT/DCC to the ~-protected tripeptide VIlh, where the peptide is prepared using any of the methods r~;scussed in the previous schemes. Treatment with TFA removes the Boc group to give the coupled inler",ediate VIIi. This intermediate is treated at room temperature with 30 BOP-CI and DMAP in an inert solvent foliowed by removal of the silyl protecting group with Bu4NF/THF at room temperature to give the macrocycle VIIj. This intermediate is oxidized using Dess-Martin periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger to give a compound of Formula III.

To produce compounds where W is nitrogen or oxygen, VIId iS replaced with 2,3~iamino propionic acid methyl sster or serine ethyl ester respectively.
Intermediate ~'Ilc is used to produce all of the compounds of Formula III.
This intermediate can be used in place of 6-[[imino[4-methylbenzenesulfonyl)-5 aminoJmethyl]amino]-2-(R,S)-[[2-(trimethylsilyl)ethoxy]methoxy]-3(S)-[9-phenylmethoxycarbonyl)-amino]hexanoic acid or 6-[[imino[4-methylbenzenesulfonyl)-amino]methyl]amino]-2-(R,S)-[~2-(trimethylsilyl)ethoxy]methoxy]-3(S)-[9-fluorenylmethoxycarbonyl)-amino]hexanoic acid in Schemes I through ~ with only minor modification to 1 o giYe the desired compounds.

WO 97130080 PCr/US97/02S75 SCHEME VII

H_,~ Fmoc-N_~H

Ts-HN~NH Ts-HN~NH

VIIa ~ VIIb ~=NH ~ HCI S~rC02Me Fmoc-HN )=N
~' OH NH2 Fmoc-HN

~? ~CO2Me Ts-HN--6NH VIId Ts-HN~NH

Vllc ~ ~

S~rC02Me S~CO2H
)=N )=N
Boc-HN~ Boc-HN ~"
OSi(Me)2t-Bu ~' OSi(Me)zt-Bu NH I~H
Ts-HN~NH Ts-HN~NH

vnf ~g W 097~0080 PCTAUS97~S75 ~3-- S~CO2H
. H )=N
H2N~ (~H2)4 1 ~OSi(Me)2t-Bu HN ~ >

H02C~ Ts-HN~6NH VIIg \~ ~ H
'~. N_ ~ N~ (CH2~o S N O~ '' t-Bu-(Me)2S~NH2 ~N~C02H
HN
HN~
Ts NH VIIi ~. H ~

S N HN ~ ~~. H

t ~

HN~NH

W O 97~0080 , rcTrusg7/02s7s The compounds of the invention were tested for their ability to inhibit thrombin mediated hydrolysis. Two in vitro enzyme assays were performed to give both Michaelis-Menten kinetics or slow, tight-bir~ding kinetics In addition, the compounds were tested in vitro for their ability to inhibit trypsin, 5 as an indication of their selectivity Thrombin-cataJyzed hydrolysis rates were measured spectrophotometrically using commercial human alpha-thrombin (American Diagnostica), a chromogenic substrate (Spectozyme~ TH (H-D-HHT-Ala-Arg-pNA 2AcOH), American Diagnostica) in aqueous buffer (10 mM Tris, 10 10 mM Hepes, 150 mM NaCI, 0.1% PEG; pH 7.4), and a microplate reader (Molecular Devices). Changes in absorbance at 405 nm were monitored (Softmax, Molecular Devices), upon addition of enzyme, both with and without inhibitor present at 37~C over 30 minutes. Inhibition constants (Ki) were determined by fixing the enzyme and inhibitor concentrations and 15 varying the substrate concenl,~lion (1 nM thrombin, 5-100 ~M Spectozyme~
TH). Michaelis-Menton kinetics were applied to the initial reaction slopes using the program K Cat (Bio Metallics Inc.).
Trypsin-catalyzed hydrolysis rates were measured using the same method as the thrombin procedure. Bovine type 1 trypsin (Sigma) and 20 Spectrozyme ~ TRY (Cbo-Gly-D-Ala-Arg-pNA-AcOH. American Diagnostics) rerl-~ed their thrombin equivalents in a concentration range of 3.2 U/ml trypsin and 0.1-0.3 mM Spectrozyme.
Compounds of the invention showed slow binding inhibition with thrombin which was demonstrated in the following assay. Serial dilutions of 25 compounds and human alpha-thrombin (0.1 nM, American Diagnostica) were incubated at 25 ~C for 4h. A chromogenic substrate was added (50 IlM
Spectozyme~D TH (H-D-HHT-Ala-Arg-pNA02AcOH), American Diagnostica) and the increase in absorbance at 405 nM was measured with a microplate reader (Molecular Devices) at 25 ~C using an aqueous buffer (10 mM Tris, 30 10 mM Hepes, 150 mM NaCI, 0.1%PEG; pH 7.4). Data was collected over 4h and plotted (MOD v. Time). A compound was determined to be slow binding if the plot for any compound at any concentration was concave.
Ki-slow was determined by measuring enzyme-catalyzed hydrolysis rates. A mixture of human alpha-thrombin (0.1 nM, American Diagnostica), 35 substrate (50 ~M Spectozyme~9 TH (H-D-HHT-Ala-Arg-pNA02AcOH), American Diagnostica), compound and aqueous buffer (10 mM Tris, 10 mM
Hepes, 150 mM NaCI, 0.1%PEG; pH 7.4) was monitored over 4h for changes in absorbance at 405 nM at 25 ~C. A vehicle mixture was n~n , W O 97~0080 PCTrUS97~2S7S

und~r the same condition and inhibition constan~s were determined by applying the data to the following equation: P=vst-(1/kl(vs-vz)(1-exp(-kt)).
Plotting k' Vs. I giv~s Ki from the equation k'=k6(1 +S/Km)+k61Ki(l). (Ref:
Tight-Binding Inhibitors- I. Sungman Cha, Biochemical Pharmacolo~y 1995.
Vol24 pp2177-2185). The Kjs and Kj-slow (~M) for representative compounds are listed in Table A. Cyclotheonamide, N-Me PPACK aldehyde (~GYKI-14766/LY-294468, Anticoagulant Thrombin Inhibitor", Drugs Future 1993,18,1159-1160) and argatroban (~Argaroban/Novastan/Slonnon, AnRco~glJl~nt Thrombin Inhibitor., Dmgs Future 1990, 15,1115-1116) were 10 used as reference standards and their values are listed below. The compound numbers in the table correspond to the examples described hereinafter.
Table A
CDd ~ Thr lcl Tr~ Kl Thr lCl-~low 2 0.35iO.1 (N=8) 0.45iO.37(N=6) 0.15iO.1 (N=10) 0.026iO.018 (N=8) 14 1.61i1.32 (N=10) 0.011iO.003 (N=5) 3 0.021iO.012 (N=6) 0.015+0.0038 (N=6) 11 0.0031iO.0008 (N=5) 0.004+0.0018 (N=6) 18 3.8iO.2(N=3) 0.31iO.01 (N=3) 16 85.9i16.3 (N-3) 0.62iO.48(N=8) 7 0.2_0.072 (N=7) 0.039_0.025 (N=5) 6 0.021iO.005 (N=6) 0.0068+0.005 (N=8) O.O9iO.0086 (N=6) 0.085+0.029 (N=5) 1 0.014iO.001 (N=5) 0.045iO.036 (N=6) 0.015+0.004 (N=6) 0.025+0.02 (N=4) 8 1.8i1 (N=6) 0.28iO.02(N=6) 491.996i268.809( N~
17 0.018iO.004 (N=6) 0.0029~0.0015 (N=5) 21 0.092iO.046 (N=5) 0.022iO.014 (N=6) 4.705+1.509( N=2) 9 O.OO99iO.0017 (N57) 0.0021+0.0011 (N=5) 4 0.16iO.06 (N=9) 0.013+0.005 (N=6) 22 no activity at 50 ~M
19 0.0053iO.0026 (N56) 0.0025+0.00075 (N=5) 12 1iO.12 (N=6) 0.017iO.0073 (N=6) 0.019+0.0044 (N=6) 0.0053+0.0026 (N=5) 1 362i 0.7(N=3) 23 1+0.4(N=6) 0.066+0.026 (N=6) 13 0.0023iO.0005( N=6) 0.0015iO.OOO9 (N=3) CtA .170 + 0.08 23.0 40 + 1.9(N=4) WOg71300B0 rCTAUS97~257 N-Me-PPAC 0.01~ 0.0039 Argatroban 0.01~ 2.9 As indicated by Table A, the compounds of Formula I may be used in 5 pharmaceutical composiliGns to treat patients (humans and other primates) with thrombotic disorders in a similar manner as known heparins and coumarins. The compounds can be administered by any parenteral route (intravenous, intraperitoneal, subc~lt~neous, dermal patch), where the prefer,ed route is intravenous infusion. Infusion doses can range from about 10 0.1-300 ~lg/kglmin of inhibitor, admixed with a pharmaceutical carrier over a period ranging from several minutes to several days.
The pharmaceutical compositions can be prepared using conventional pharmaceutical excipients and compounding techniques. Oral dos~ge forms may be elixers, syrups, capsules tablets and the like. Where 15 the typical solid carrier is an inert subst~rlce such as lactose, starch, glucose, methyl cellulose, magnesium sterate, dicalcium phosphate, mannitol and the like; and typical liquid oral excipients include ethanol, glycerol, water and the like. All excipients may be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like 20 using conventional techniques known to those skilled in the art of preparing dosage forms. Parenteral dosage 1Orms may be prepared using water or another sterile carrier.
Typically the compounds of Formula I are isolated and used as their pharmaceutically acceptable salts. Examples of such salts include 25 hydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric, maleic, fumaric, malic, tartatic, citric, benzoic, mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, ~Q-toluenesulfonic, cyclohexanesulfamic and saccharic.
In addition to the treatment of thrombotic disorders, the compounds of 30 Formula I may be used to prevent coagulation of stored blood samples and as coatings on medical devices such as stents and orthopedic devices.
Generally they may be used in any circumstance where one seeks to inhibit coagulation by placing the compounds in contact with the medium containing thrombin. Those experienced in the use of anticoagulant agents, 35 may find a variety of other uses for the thrombin inhibitors of this invsntion.
These uses are considered to be within the scope of this invention, for this invsntion contemplates the use of compounds of Formula I as antithrombotic agents.

W 097/30080 PCT~US97~2S7 Yet another use for the compounds of the invention is as trypsin inhibitors. Inhibitors of trypsin have been used clinically in the treatment of pancreatic disorders, such as pancreatitis. The ICso values for the compounds of the invention compare favorably with the pancreatic agents 5 camostat mesilate and nafamostat (ICso s, 1 x 10-8 and 1.3 x 10-8 respectively). The compounds of Formula I may be used in the same manner as those therapeutic agents.
Although all of the claimed compounds are useful as thrombin or trypsin inhibitors, the preferred compounds of Formula I include ~~_ N--(CH2) 5 ~ ~~_ N _ (CH2)6 ~;~ H ~~ ~ ~j H ~~ ~0 o~N~

HN HN
H2N~NH H2N~NH
(CH )~ (CHZ)2 ~ 0,~_ HN _ (CHZ)4 ~3 ~_H~ N~

HN HN
H2N~NH H2N~NH

W 097~80 PCTAUS97/02S7S

~3--- H

-(CHz)3 5 N HN o~3 ~CH H ~ =~ ~, 0~ 0 0 o~N~ 1~1 ~S N~

HN HNq~NH
H2N~NH NH2 ~ C1~3_ 5 O s o O N (CHz)7 N NH (CH2)6 ~5~ N~ o~ N~
O ~ O

HN~NH HN~NH

NH2 and NH2 With respect to compounds of Formulas I and II, the particularly preferred substituents are as follows. The particularly preferred "A"s are ~ O
_~* _~' (CH2)n 10 where n is 1, R3 is hydrogen and the preferred stereochemistry of the starred carbon is S.
The particularly preferred"B"s are , WO 97/30080 . PCT/US97/02575 R6~

R4~

where Rs and R6 are taken with the carbon to which they are each attached to form a carbonyl; and R4 is naphthylmethyl, diphenylmethyl, 5 phenylC1 salkyl or substituted phenylC1 salkyl where the phenyl substituents are chlorine or fluorine. The preferred stereochemistry of the starred (~) carbon is R.

The particularly preferred "Gns are R8 ~Rg ~ ~,N~

where R8 and Rg are taken with the carbon to which each is attached to form a carbonyl; and R7 is naphthylmethyl, diphenylmethyl, phenylC1 salkyl or substituted phenylC1 salkyl where the phenyl substituents are chlorine or fluorine. The ring size of the macrocycle is determinsd by A, B, G and m, where 20 a ring size of 15 to 25 is particularly preferred.
With respect to the compounds of Formuta nI~ the particularly preferred "W~ is sulfur. All other particularly preferred substituents are as described for Formulas I and II.
In order to illustrate the invention the following examples are included.
2~ These examples do not limit the invention. They are only meant to suggest a method of practicing the invention. Those skilled in the art may find other methods of practicing the invention, which are obvious to them. However, those methods are deemed to be within the scope of this invention~

WO 9~/~0080 PCTnUS97/02575 5R, 1 8S, 21S-N-13-(4, 7, 16, 17, 20-PENTAOXO-5-PHENETHYLElCOSAHYDRO-3a, 6,1 5,1 9 -TETRAA~ACYCLOPENTACYCLONONADECENE-1 8-YL) PROPYL] GUANIDINE
Step 1 a ~,~,N~
H ~ C02~-Bu A solution of CBZ-D-homophenylalanine 5.6 9 (18 mmol), ProO-~Bu (3.4 g, 19.8 mmol) and HOBT, (3.65 g, 27 mmol) in CH3CN (70 mL) was stirred for 20 min, 10 treated with a solution of DCC (4 9, 19.8 mmol) in CH3CN (30 mL) and stirred overnight. This mixture was filtered, the filtrate was concentrated and dissolved in CHCI3 (400 mL). This solution was washed with successive portions of 2%
Na2CO3(aq) (100 mL) and brine (100 mL). The resulting organic layer was dried (Na2SO4), concentrated in v~llo and purified by fiash column 15 chromatography (silica, CHCI3) to afford 1a as an oil: (8.4g).

Step 1 b ~ I~
i~,N~.

0 C0~-Bu A mixture ~ (6.0 g) and Pd(OH)2-C in MeOH (100 mL) was shaken under 20 psig for 1.5 h. The catalyst was filtered, and the filtrate concentrated to give the free amine ~, as an oil (61.6 g).

Step 1c W 097~80 PCTrUS97~2S7S
Q~

~ N~
(CH2)7H ~ C02~-Bu I

NHCBZ

A solution of DCC (3.8 g, 18.5 mmol) in CH3CN (40 mL) was added to a stirred mixture of 1 b (6.2 9, 18.5 mmol) 8-carbobenzoxyaminooctanoic acid ( 5 4.94 9, 16.8 mmol) and HOBT (3.4 g, 25.2 mmol) in CH3CN (300 mL~. The resulting mixture was stirred for 16 h, filtered and concentrated in v~cuo . Theresidue was dissolved in CHCI3, washed sequentially with 5% Na2CO3(aq) and brine, dried (Na2SO4), and concentrated in vacuo . The residue was purified by flash column chromatography (ether-MeOH; 100%->95:5) to give ester 1c as 10 an oil: (7.8 9, 70%); mle = 607 (MH+).

Step 1d -, r 09~ N~

(CH2)7H ~ CO~-Bu 1d A mixture of 1c, (7.8 g, 12.8 mmol), 20% Pd(OH)21C (5.0 9) and 150 ml of MeOH was shaken under 20 psig of H2 for 3 h. The mixture was filtered and concentrated to give the amine 1d as an oil: (5.5 9, 91%), m/e = 474 (MH+).

Step 1e W0971~U~ PCT~US97~2575 (CH2~)~LN r~
- ~f ~JHCBZ~=O SEMO ~\ ~ C02~-Bu HNJ
HN ~NH
Ts 1e A solution of DCC (0.91, 4.4 mmol) in DMF (15 mL) was added to a solution of 6-~[imino[4-methylbenzenesulfonyl)amino)methyl]amino]-2-(R,S)-[[2-5 (trimethylsilyl)ethoxy]methoxyl-3(S)-~9-phenylmethoxycarbonyl)-amino]hexanoic acid, (2.5 9 ,4.0 mmol: Maryanoff et al. Joumal of the American Ch~mical Socioty 1995, 117, 1225-39), amine 1 d (2.0 g, 4.4 rnmol) and HOBT
(0.8 9) in DMF (150 mL). This mixture was stirred overnight, and filtered. The filter cake was washed with CH3CN, and the filtrate was concentrated in v~ o 10 . The residue was purified by flash column chromatography (silica gel, 100%
CHCI3, then 2% MeOH-CHCI3) to give ester 1 e as a foam: (3.4 9 78%); m/e =
1078 (MH+).

Step f (CH2~N' r~
NH \ ~' f NH2 )=o SEM~ C02~-Bu HN
HTN ~NH

1f A solution of ~ (3.3 g) in MeOH (50 mL) was treated with 20%
Pd(OH)2/C (2.0 9) and shaken under 20 psig of H2 for 3 h The mixture was filtered and co-,centl~led to the ester 1f as a foam: (2.7 9); m/e = 944 (MH+).

W 097/30080 PCTAUSg7~2575 Step g (CH2)~LN' NH \ ~
~=l' ~IH2 )'~
HO~\ ~_c02H
I~
HNJ
HTN ~NH

A solution lf (2.6 g) in CH2CI2 (10 mL) was added to a solution of 1:1 CH2CI2:trifluoroacetic acid (40 mL) at 0 ~C and stirred for 2.5 h. Volatiles were removed under a stream of N2, and the resulting gum was triturated three times with ether to give the acid 19 as a white solid: (2.5 9); m/e = 758 (MH+).

Step h NH
H~H_~ ~

HN
HT ~NH

1h A mixture of 19 (1.2 9, 1.38 mmol) in 1.4 L of CH2CI2 was treated with DMAP (0.93 9, 7.6 mmol), and stirred for 20 min. BOP-CI (0.1, 2.76 mmol) was WO97~0080 PCT~US97~2~75 added and the mixture was stirred for another 2 h and concentrated in v~ o .
The residue waS dissolved in CH2CI2 (500 mL) and washed twice with 10%
citric acid (aq)(2x250 mL). The organic layer was washed With brine, dried (Na2SO4), fittered, and concenl,dl6.1 in V~ . The residue was purified by 5 flash column chromatography (CHCI3-MeOH; 100% -> 90%; silica gel) to give the coupled intermediate ~: (600 mg, 81 %); m/e = 740 (MH+).

Step i NH ~;;
~N~

HN
HT ~NH

1i Dess-Martin periodinane (499 mg, 1.2 mmol) was added to a stirred solution of 1 h (580 mg, 0.78 mmol) in CH2CI2 (50 mL) at room temperature.
ThiS mixture waS stirred for 1.5 h, treated with an excess Of 25% Na2S2O4 (aq) in NaHCO3 (sat'd. aq) and stirred for another 5 min. The aqueous layer was 15 extracted with several portions of CH2CI2 and the combined organic extracts were washed twice with NaCI (sat'd, aq), dried (Na2SO4), filtered and concentrated to give the diketone 1 i as a white solid: (495 mg); m/e = 738 (MH+).

(ctl2)~LN' NH ~' ~N~J~'O

HN
H2N~NH

5R, 18S, 21 S-N-[3-(4, 7, 16, 17, 20-PENTAOXO-5-PHENETHYLElCOSAHYDRO-3a, 6, 15, 19 -TETRAAZACYCLOPENTACYCLONONADECENE-1 8-YL) , W O 97/30080 PCTAUS97~2575 PROPYL] GUANIDINE

A suspension of 1 i (480 mg, 0.65 mmol) in anisole (3 mL) was cooled to -78~C and treated with anhydrous HF ( ca. 10 mL) using a standard HF
5 apparatus. This mixture was stirred at 0~C for 3.5 h, concentrated in v~llo and triturated twice with 25 mL portions of ether. A solid was collected, washed with ether, and purified by reverse-phase HPLC (MeCN-water-TFA, 35:65:0.2). The resulting solid was Iyophilized to give the title compound as a white solid: 272 mg; mp 50 ~C; FAB-MS m/e 584 (MH+);
10 Anal Calcd. for C30H4sN705 ~ 2.5 CF3C02H ~ 1.25 H20:
Calcd.:C, 47.16; H, 5.65; N, 11.00; H20, 2.53.
Found:C, 46.90; H, 5.23; N, 11.12; H20 2.60.

(CH2)!~N
~H_~ O

HN
1 5 H2N~NH
5R, 16S, 19S-N-~3-(5-BENZYL-4, 7, 14, 15, 18-PENTAOXOOCTADECAHYDRO) -3a, 6, 13, 17-(TETRAAZACYCLOPENTACYCLOHEPTADECEN-1 6-YL)PROPYL]-GUANIDINE Dl-TRIFLUOROACETIC ACID
SESQU IHYDRATE.

Compound 2 was prepared using the general method of Example 1.
CBZ-D-PheOH replaced CBZ-D-homophenylalanine in Step 1a and 5-carbobenzoxyaminopentanoic acid replaced 8-carbobenzoxyaminooctanoic 25 acid in Step 1c to give the title compound as a solid. FAB-MS rn/e 541 (MH+);Anal. Calc'd for C~l 13~N705-2tC2HF302)-1.5 H20:
Calc'd: C, 46.73; H, 5.67; N, 12.31, H20, 3.39.
Found: C, 46.71; H, 5.73; N, 12.78; H20, 3.52.

W O 97/~W~0 PCTnUS97~2~75 (CH2)~N

~N~ ~

HN
H2N~NH
2S, 5S, 1 8R-N-[3-(1 8-BENZYL~-3,6, 7,16,19-(PENTAOXOEICOSAHYDRO)-1A, 4, 8,17-6 (TETRAAZACYCLOPENTACYCLONONODECEN-5-YL)PROPYL]
GUANIDINE TRIFLUOROACETIC ACID HYDRATE.

Compound 3 was prepared following the method of Example 1 with only slight modifications. CBZ-D-PheOH repl?cs-l CBZ-D-homophenylalanine in 10 Step 1a to give the title compound aS a solid. FAB-MS m/z 570, (MH+) Anal. Calc'd for C29H43N70s-2.5C2HF302-H20;
Calc'd: C, 46.79; H, 5.49; N,11.23; H2O, 2.06.
Found: C, 47.05; H, 5.43; N, 11.29; H2O, 2.25.

(CH2)~N' NH
~N~=O

HN
H2N~NH
2S, 5R, 18S, 21S-[3-(5-BENZYL-2METHOXY-4, 7, 16, 17, 20-PENTAOXOElCOSAHYDRO-3a, 6, 15,19-TETRMZACYCLOPENTACYCLONONADECEN-1 8-YL) PROPPYL] GUANIDINE TRIFLUOROACETIC ACID HYDRATE

W 097~0080 rCTrUS97/0257S

Compound 4 was prepared following the method of Example 1 with only slight modifications. CBZ-D-PheOH replaced CBZ-D-homophenylalanine in Step 1a and ~i~- methoxyproline (prepared according to Barlos, K., et al. Tetrahedron Lett. 1983, 39, 475) ~epl~~e~J ProO-I Bu in the same step to give the title 5 compound as a solid: FAB-MS mlz 600 (MH+);
Anal. Calc'd for C30H4sN7O6-2.25 C2HF3O2-1.5 H2O
Calc'd:C, 46.91; H, 5.73; N, 11.10; H2O, 3.05 Found:C, 46.90; H, 6.03; N, 11.44; H2O, 3.00.

O H~
(CH2)~LN' )~
NH
~H_4~,~O

HN
H2N~NH
6R, 19S, 22S-N-[3-(6-BENZYL-5, 8, 17, 18, 21-PENTAOXODOCOSAHYDRO-4a, 7,16, 20-TETRAAZABENCBZOCYCLONONADECEN-19-YL) PROPYL]
t S GUANIDINE TRIFLUOROACETIC ACID HYDRATE

Compound 5 was prepared following the method of Example 1 with only slight modifications. CBZ-D-PheOH replaced CBZ-D-homophenylalanine in Step 1a and L-pipecolinic acid replaced ProO-t-Bu in the same step to give the title compound as a solid: FAB-MS m/z 584 (MH+); Anal. Calcd tor C30H4sN7os-2 0 C2HF302-1.0 H2O:
Calc'd: C, 49.21; H, 5.95; N, 11.82; H2O, 2.17 Found: C, 49.28; H, 5.66; N, 11.67, H2O, 2.56.

W O 97/30080 PCTAUS97~2575 H ~0 ~H_~ O

HN
H2N~NH
5R, 2S, 18S-N-13-(5-NAPTHALEN-2-YLMETHYL-4, 17, 16, 17, 20-PENTAOXOElCOSAHYDRO-3a, 6,1 5, 1 9-TETRMZACYCLOPENTACYCLONONADECEN-1 8-YL)PROPYL]
GUANIDINE TRIFLUOROACETATE HYDRATE

Compound 6 was prepared following the method o~ Example 1 with only slight modifications. CBZ-D-2-napthylalanine replaced 10 CBZ-D-homophenylalanine in Step 1 a to give the title compound as a solid:
FAB-MS m/z: FAB-MS mlz 620 (MH+); Anal. Calc'd for C33H45N70s-1.5 C2HF3O2-1 75 H20:
Cacl'd:C, 51.80; H, 5.93; N, 11.59; H20, 3.19 Found:C, 51.44; H, 5.95; N, 11.44; H20, 3.23.

~L H O

HN~ O

HN
H2N~NH
3S, 1 6R, 1 9S-N-[3-(1, 4, 5, 14, 1 7-PENTAOXO-1 6-PHENYL-2, 6, 15, 18-TETRAAZACYCLOPENTACYCLONONADECAN-3-YL)PROPYL] GUANIDINE TRIFLUOROACETIC ACID HYDRATF

WO 97/~W~0 PCTrUS97/0257S

Compound 7 was prepared following the method of Example 1 with only slight modifications. CBZ-D-phenylglycine replaced CBZ-D-homophenylalanine in Step 1a to give the title cornpound as a solid: FAB-MS mlz 556 (MH+); Anal.
Calcd for C28H41N705-1.75 C2HF302-1.5 H20:
Calc'd:C, 48.37; H, 5.90; N, 12.53; H20 3.45 Found:C, 48.40; H, 5.95; N, 12.52; H20, 3.64.

(CHZ)6~LN' NH ~"' O~N~O
IJ
HNJ
H2N~N~
5R, 17S, 19AS-N-[3-(5-BENZYL-4, 7, 15, 16, 19-PENTAOXOOCTADECAHYDRO-3a, 6,14, 18-TETRAAZACYCLOPENTACYCLOOCTADECFN-1 7-YL)PROPYL]
GUANIDINE TRIFLUOROACETIC ACID HYDRATE

Compound 8 was prepared using the general method of Example 1.
CBZ-D-PheOH repl~ed CBZ-D-homophenylalanine in Step 1a and 7-carbobenzyloxyaminoheptanoic acid rQplaced 8-carbobenzoxyaminooctanoic acid in Step 1c to give the title compound as a solid: FAB-MS m/z 556 (MH+);
Anal. Calc'd for C2BH41N70s-1 .5C2HF302-2.0 H20:
Calc'd:C, 48.82; H, 6.15; N, 12.85, H20, 4.72 Found:C, 48.68; H, 6.07; N, 12.74, H20, 4.83.

glL H Ç3 ~5~H_~ O
HN
H2N~NH

.

5R, 20S, 23S-[3-(5-BENZYL-4, 7, 18, 19, 22-PENTAOXOCYCLOEICOSAHYDRO-3A, 6,17, 21-YL)PROPYL]GUANIDINE TRIFLUOROACETIC ACID HYDRATE

A slurry of 9-cyanopelargonic acid (8.6 g, 48 mmol), a catalytic amount 5%Rh/AI203, and 2û0 mL of 2.0 N NH3-EtOH was shaken under 50 psig of H2 pressure for 6 h, then filtered through dicalite. The filter pad was washed with100 mL of hot 1 :1 MeOH-H2O, and the combined filtrates were concentrated to give 6.1 g of 10-aminGcle~noic acid. The crude product (5.7 9) was dissolved in 15.5 mL of 2N NaOH and treated simultaneously with 23 mL of 2N NaOH and 5.8 9 of carbobenzoxychloride at 0 ~C with vigorous stirring over 0.5 h. Water and 2N NaOH were added as needed to maintain stirring and a pH between 10-14. After stirring for 2.5 h, the reaction was diluted with 400 mL of H2O andfilter~d through dicalite. The filtrate was acidified (pH 2) with H2SO4, then extracted with ether. The combined ether extracts were dried (Na2S04), filtered and concenl~aled. The residue was dissolved in CH3CN, filtered, and the filtrate concsntrated to afford 5.3 9 of 1 0-(N-carbobenzoxy)-aminodecanoic acid which was used without further pu~fication: FAB-MS mlz 322 (MH+).
Compound 9 was prepared using the general method of Example 1.
CBZ-D-PheOH replaced CBZ-D-homophenylalanine in Step 1a and 10-(N-carbobenzoxy)-aminodecanoic acid replaced 8-carbobenzoxyaminooctanoic acid in Step 1c to give the title compound as a solid: FAB-MS mlz 598 (MH+);
Anal Calc'd for C31H47N7Os-1.75 C2HF3O2.1.75 H2O;
Calc'd: C, 50.00; H, 6.35; N, 11.83; H2O, 3.80 Found: C, 49.62; H, 6.23; N, 11.93; H2O, 3.46.

(cH2)1~LN' Ç3 ~H_~ O

HN
HzN~NH

, W O 97~C80 PCTAUS97~2S75 21 S, 24S, 27R-N-[3-(5-BENCBZYL-4, 7, 19, 20, 23-PENTAOXOTETRACOSAHYDRO-3a, 6,18, 22-YL)PROPYL]GUANIDINE

Compound 10 was prepared using the general method of Example 1.
CBZ-D-PhsOH replaced CBZ-D-homophenylalanine in Step 1a and 11-carbobenzoxyaminoundecanoic acid replaced 8-carbobenzoxyaminooctanoic acid in Step 1 c to give the title compound as a 10 solid: FAB-MS m/z 612 (MH~); Anal Calc'd for C32H4gN7Os-1.75 H2O-1.5 C2HF302:
Calc'd:C, 51.62; H, 6.68; N, 12.05; H2O, 3.93 Found:C, 51.83; H, 6.12; N, 11.98; H2O, 3.93 Preparation of Compound 11 Step 11a H2N~ ~CO2-t-Bu (CHzh Carbobenzoxy chloride (271 mL, 1.9 mol) and 4N NaOH (475 mL) were added simultaneously to a solution of 6-aminopentanoic acid (1009, 0.76 mol) in 4 N NaOH (aq.) (190 mL) at such a rate as to maintain the temperature c 10~C. The reaction was stirred an additional 2 h at 0-5~C while the pH was maintained between 10 and 12. The mixture was then diluted with H2O (250 mL) and extracted 10ur portions of ether (250 mL). The aqueous extract was acidified with 3N H2SO4 (pH = 3), and extracted repeatedly with CH2CI2 . The organic extracts were combined and washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo to yield 196 g of 6-carbobenzyloxyaminopentanoic acid as an oil which solidified upon standing:
FAB-MS m/z 266 (MH+).
A solution of 6-carbobenzyloxyaminopentanoic acid (50 9) in CH2CI2 (500 mL) was treated with 2.2 mL of H2SO4 (conc.), saturated with isobutylene and stirred for 4 h. The resulting mixture was treated with 5% KOH (aq) (200 mL) and the layers were separated. The organic layer was washed twice with brine (2x100 mL), dried (Na2SO4) and concentrated. in vacuo. The residue was purified by flash column chromatography (silica gel, hexanes-ether) to - afford 38 g of 6-carbobenzyloxyaminopentanoic acid t-butyl ester: MS mlz 322 (MH+).
A mixture of 6-carbobenzyloxyaminohexanoic acid ~-butyl ester (9 g, ??
mmol), Pd(OH)21C (4.5 9) and 50 mL of ethanol was shaken under 15 psig for 2 5 h, filtered and concentrated in v~ o to give the amine 11~. as an oit: MS m/z 188 (MH+). The material was used without further purification.

Step 11b CBZNH~N~ ~CO2-t-Bu o (CH2)5 11b A solution of HOBT (4.0 9, 30.2 mmol), DCC (4.6 9, 22.3 mmol) in DMF
(35 mL) was added to a solution of amine lla (4.2 9, 22.2 mmol) and CBZ-D-Phe (6.0 g, 20.0 mmol) in DMF (35 mL). This mixture was stirred overnight, filtered and concentrated in v~ n. The residue was dissolved in CHCI3, washed sequentially with 10% NaHCO3 (aq) and brine, dried (Na2SO4) and concentrated in v~ r~. A 2.0 g portion of the crude residue was purified by flash column chromatography (silica gel, CHCI3) to afford 1.8 9 of the coupled ester 11b: MS m/z 469 (MH+).

Step 11c ~H
CBZNH N~ ~C02H
o (CH2)5 A solution of 11 b (2.2 9, 4.7 mmol) in CH2CI2 (5 mL) was added to a solution of 1:1 TFA-CH2CI2 (25 mL) at 0 ~C. This solution was gradually 25 warmed to RT and stirred for an addtional 1.5 h. The volatiles were removed under a stream of N2, and the residue was triturated with ether to give the acid11c as a white solid: (1.8 9); MS m/z 413 (MH+).

W 097~ PCTrUS97~257S

Step 11d (C,H2)~LN'H ~3 HN \..~

~ NH2 ~ CO2-t-Bu A solution of DCC (1.1 g, 5.5 mmol) in CH3CN (5 mL) was added to a 5 stirred solution of 11 c (2.0 g, 5.0 mmol), D-Phe-Pro-O-~-Bu (1.8 g, 5.~ mmol) and HOBT (1.07 g, 7.5 mmol ) in CH3CN (35 mL). This mixture was stirrsd for 16 h, filtered and concentrated in v~çllo . The residue was dissolved in CHCI3, washed sequentially with 10% NaHCO3 and brine, dried (Na2SO4) and concentrated in v~ o . This residue was purifisd by flash column 10 chromatography (silica gel, CHCI3->CHCI3-MeOH) to give the coupled product as a foam: MS mlz 542 (M - Pro-O-t-Bu)+. This material was combined with MeOH (50 mL) and Pd(OH)2 (1.2 g) and shaken under H2 (20 psig) for 2.5 h.
The reaction was filtererd through dicalite and the filter pad thoroughly washedwith MeOH. The filtrate was concentrated to give intermediate .L~ as a foam:
(1.8g,); MS 579 (MH+).

Step 11e o H (CH~)s o~,~NH O~) ~
SEMO ~ NHCBZ ~ C02-t-Bu HNJ
HN ~NH

1 1 e A solution of 6-[~imino[4-methylbenzenesulfonyl) amino]methyl]amino]-2-(R,S)-[~2-(trimethylsilyl)ethoxy]methoxy]-3(S)-[9-phenylmethoxycarbonyl)-amino]hexanoic add, (1.0 g, 1.6 mmol) of 1 ld (1.0 g, 1.8 mmol) and HOBT (0.3 g, 2.2 mmol) in CH3CN (35 mL) was treated with a solution of DCC (0.4 9, 1.8 mmol) in CH3CN (5 mL) and stirred overnight. The mixture was filtered and the filtrate was concentrated in vacuo . The residue was dissolved in CHCI3, ,~

WO 97130080 PCT~US97102575 washed successivety with 10% Na2CO3 and H2O, dried (Na2SO4) and concentrated in v~ . The residue was purified by flash chromatography (siica gel, CHCI3 ->CHCI3.MeOH) to give the arginine derivative 11 e as a solid:(1.5 9); MS m/z 1184 (MH+~.

Step 11f o H (CHz)s ~ =~"''13 HNJ
HT ~NH

11f A mixture of 11 e (1.5 9, 1.26 mmol), Pd(OH)2, (0.8 g) and MeOH (50 10 mL)was shaken under of H2 at 20 psig for 2.~ h. The mixture was filtered and the filtrate was concentraled in v~ o to give the deprotected intermediate 1 1 f1.2 ~ as an off white solid: MS m/z 1049 (MH+).

Step 119 H (CH2)s o ~ Oz~) '13 HN
HN ~NH
Ts To a solution of 1:1 TFA-CH2CI2 (15 mL) was added to a solution of llh (1.24 9, 1.2 mmol) in CH2C12 (5 mL) at 0 ~C. This mixture was stirred for 2.~ h at room temperature, and the volatiles were removed under a stream of N2. The 20 residue was triturated with ether and collected to afford llQ, 1.1 9 as a white solid: MS 863 (MH+).

W 097/30080 PCTfUS97~2S7S

Step 1 1 h ~~--N--(CH

~ ~
HN

HN ~NH

A mixture of llg (1.1 g, 1.1 mmol) in CH2CI2 (1.1 L) was treated with DMAP (0.7 g, 5.7 mmol) followed by BOP-CI (0.6 g, 2.3 mmol). This mixture was stirred for 24 h, then concentrated in v~ o. The residue was dissolved in CH2CI2, washed with 10% citric acid (aq), dried (Na2SO4) and concentrated.
in VR~llo The residue was purified by flash column chromatography (silica gel, CH2CI2-> 95% CH2CI2-MeOH to yield ~b., ~0.3 g )as a solid: MS 845 (MH+).
Step 11i _(CH

~ 0 HN

HN ~NH
1 1 i Intermediate 11 h (0.3 g, 0.3 mmol) was added to a mixture of o~ Dess-15 Martin periodinane (2.0 g, 0.5 mmol) in CH2CI2 (10 mL~. The mixture was stirred for 1.5 h, and treated with an excess of 10% Na2S2O3 (aq) in NaHCO3 (satd. aq.). The layers were separated, and the aqueous layer was extracted three times with CHCI3. The combined organic extracts were washed with H2O, dried (Na2SO4), and concentrated in VR~UO to give ~i (0.3 g) as a white solid 20 which was used without purification.

WO 97130080 PCTtUS97102S7S

~_ H _ (CH 2 ~=

~N~

HN
H2N~NH
5R, 15R, 19S, 21AS-N-[3-(5, 1~DIBENZYL-4, 7, 14, 17, 18, 21, HEXAOXOElCOSAHYDRO-3a, 6,16, 20-YL)PROPYL]GUANIDINE TRIFLUOROACETATE

A mixture ot intermediate 11 i (0.3 g) and anisole (6 mL) was treated with HF (ca 10 mL) at -78~C, and warmed to 0~C. This mixture was stirred for 4.5 h and the HF was removed in v~ o. The residue was triturated with ether and 10 the resulting solid was purified by reverse phsse HPLC (1:1 0.2 CH3CN-H2O-TFA) to afford the title compound (0.1 9 ) as a white powder: MS 620 (MH+);
Anal. Calc'd for C23H4sN7Os-1.75 C2HF3O2-1.5 H2O:
Calc'd:C, 51.14; H, 5.63; N, 11.93; H2O, 2.40.
Found:C, 51.19; H, 5.70; N, 12.08; H2O, 2.59.

o N_(CH2)4 ~~,~;'13 HN
H2N_~NH
5R, 15S, 19S, 21a-S-N-[3-(5, 15-DIBENZYL-4, 7, 14, 17, 18, 21-HEXAOXODOCOSAHYDRO-3a, 6, 13, 16, 20-YL)PROPYL)GUANIDINE TRIFLUOROACETATE

W097/~W~0 PCT~US97~2S75 Compound 12 was prepared following the method of Example 11. ~a-CBZ-L-Phe was used in place of N-a-CBZ-D-Phe in Step 11 b and all other steps were carried out with only minor modifications: Anal calc'd for C36H48N8O6-1.35 C2HF3O2-2-0 H2O:
Calc'd:C, 52.89; H, 6.12; N, 12.75; H2O, 4.10 Found:C, 53.02; H, 5.92; N, 12.82; H2O, 3.97 5R, 18S, 20a-S - N - ~3-(5-BENZYL-4, 16, 17, 20-TETRAOXOElCOSAHYDRO-3a, 6, 15, 19-TETRAAZACYCLOPENTACYCLONONADECEN-18-YL) PROPYLl GUANIDINE TRIFLUOROACETATE
Step 13a FM (CH2?7 Fluorenylmethoxycarbonyl chloride (4.87 g, 0.02 mol) to a mixture of aminooctanoic acid (3.0 g ,0.02 mol) in 200 mL of 10% Na2CO3 (aq) and dioxane (150 mL) at 0~C. The mixture was stirred for 2.5 h at 0~C, acidified to pH 5 with acetic acid and extracted thres times withCHCI3. The combined 20 organic extracts were washed with H2O, dried (Na2SO4~ and concentrated in v~cuo . The residue was purified by flash chromatography (silica gel, CHCI3-> 95:5 CHCI3-MeOH) to afford 8-fluorenylmethoxycarbonylaminooctanoic acid ~6.4 9) as a solid: MS 382 (MH+).
The product was dissolved in CH2CI2 (40 mL), cooled to 0~C, and treated with 25 methoxymethyl amine hydrochloride (2.05 9, 21.1 mmol), triethylamine (8.1 mL)and BOP reagent (8.0 9). This mixture was stirred for 12 h at 0~C, washed sequentially with 3N HCI, NaHC03 (sat"d. aq.) and brine. The organic phase was dried (Na2SO4) and concentrated. in v~cllo. The residue was purified by flash column cllro,--atoyraphy (silica gel; CHCI3-~98:2 CHCI3-MeOH) to afford 30 8-fluorenylmethoxycarbonyl-aminooctanoic acid N,N-methoxymethyl amide (7.1 9): MS mlz 425 (MH+). A solution of ths product (6.8 g) in THF ~80 mL) was cooled to -40~C and treated dropwise with 1.0 M DIBAL/THF (48.5 mL). The mixture was stirred an additional 15 min, quenched with 3N HCI (50 mL) and warmed to room temperature. The resulting aqueous layer was extracted 35 repeatedly with CHCI3 and the combined organic extracts were washed with brine, dried (Na2SO4) and concentrated in v~uo. The residue was purified by WO97/3N~ PCT~US9710257 chromatography (silica gel; CH2CI2->2% MeOH-CH2CI2) to afford aldehyde 1~ as a solid: 5.2 g; MS m/z 366 (MH+~.
Step 13b H (CH2)8 FMoc- N
HN

0 =~>N " ~[3 ~ C02-t-8u 13b A solution of ~ (4.2 9,11.6 mmol) and D-Phe-Pro-O-t-Bu (4.1 9,12.7 mmol) in CH2CI2 (100 mL) was treated with sodium triacetoxy borohydride (3.7 g,17.4 mmol), followed by glacial acetic acid (0.7 9). This mixture was stirred for 3.5 h, 10 treated with excess NaHCO3 (sat'd) and the resulting aqueous layer was extracted repeatedly with CH2CI2. The combined organic layers were washed with brine, dried (Na2S04), and concentrated in v~ o to give the coupled product 13b as a semi-solid: 7.8 9; MS m/z 668 (MH+).

Step 13c H (CH2)s CBZ-N
~ "~
N ~
~ C02-t-Bu 13c A mixture of 13~ ~7.8 9, in 1 :1 CH2CI2 -H2O (30 mL) was cooled to 0 ~C and llea~ed with NaHCO3 (1.1 9,12.7 mmol) of followed by dropwise addition of benzylchloroformate ~1.8 mL,12.7 mmol). This mixture was stirred for 2h at O ~C, and the a~lueous phase was extracted repeatedly with CH2C12. The combined organic layers wers washed sequentially with H2O and brine, dried (Na2SO4) and conce,~tr~ J in v~ . The residue was purfied by flash chromatography (silica gel, CHCI3) to give 13c as a semi-solid: 7.3 9; MS m/z 802 (MH+).

W 097/30080 PCT~USg7~2S75 Step 13d (CH2)s H2N-- \
CBZ-N
o~ '13 ~ CO2-t-Bu A soiution of 1 3c (2.4 9) in DMF (20 mL) was treated with piperidine 4 mL and 5 stirred for 25 min. The resulting mixture was concentrated in vacuo and triturated with hexane to give 13d as an oil: 1.5 9; MS m/z 580 (MH+).

Step 13e (cH2)8--N-CBZ

~ ~IHFMoc SEMO ~ C02~-Bu l_ HNJ
HN ~NH

13e A solution of 6-~limino[4-methylbenzenesulfonyl) amino]methyl]amino]-2-(R,S)-[[2-(trimethylsilyl)ethoxy]methoxy]-3(S)-~9-fluorenylmethoxycarbonyl)-amino]hexanoic acid (1.6 9, 2.3 mmol), ~ (1.4 g, 2.5 mmol), and HOBT
(0.46 g, 3.4 mmol) in CH3CN (35 mL) was treated with a solution of DCC
15 (0.51 g, 2.5 mmol) in CH3CN (5 mL). This mixture was stirred for 12 h, filtered, and the filtrate was concentrated in v~cuo . The residue was purified by flash column chromatography (silica gel, CHCI3-~5% MeOH-CHCI3) to afford 13e as a semi solid: 1.8 9; MS m/z 1273 (MH+) .. . ..

W 097/30080 PCTnUS97/02S75 -Step 1 3f (C,H2)8_N'CBZ

~ ~JH2 ~; ;~\
SEM0~ .~ C02t-Bu HNJ
HNT ~NH
~f Piperidine (4 mL) was added to a solution of 1 3e (1.7 9) in DMF (20 mL) and 5 stirred for 30 min. This mixture was concentrated in vacuo and the residue was washed with hexanes to yield 13f as an oil: 1.4 9; MS m/z 1050 (MH+) Step 139 (CH2)8_N-CBZ
0 ~NH
H0~.~ C02t-Bu HNJ
HTN ~NH
1 0 .~g A solution of TFA (15 mL) and CH2C12 (10 mL) was added a solution of 13f (1.4 g) in CH2CI2 (5 mL) at 0 ~C. This mixture was warmed to room temperature and stirred for 2 h. The volatiles were removed under a stream of N2 and the residue was triturated with ether to afford ~g as a solid 1.1 9; MS m/z 864 1 5 (MH+) Step 13h (C72)8.,N-H~H~ o HN

HTNs~NH
L~
BOP-CI (0.5 9, 2.0 mmol) was added to a solution of .~, (1.1 g, 1.0 mmol) and 5 DMAP (0.63 9, 5.2 mmol) in CH2CI2 (1.0 L). This mixture was stirred for 6 h and concentrated in V~CI~O. The residue was purified by flash column chromatography (silca gel; 5%MeOH-CH2CI2 ) to afford 1 3h as a white foam:
0.31 9; MS m/z 846 (MH+).

(C~2)8_N-H
NH
~H~ '0 HN
1 0 H2N~N~
~R, 18S, 20a-S - N - [3-(5-BENZYL-4, 16, 17, 20-TETRAOXOElCOSAHYDRO-3a, 6,1 5, 1 9-TETRMZACYCLOPENTACYCLONONADECEN-18-YL) PROPYL]
GUANIDINE TRI~LUOROACETATE

A suspension of 13e (0.2 9, 0.4 mmol) in CH2CI2 (10 mL) was treated with (0.3 9, 0.6 mmol) of the Dess-Martin periodinane and stirred for 2 h. The mixture was treated with excess solution of 25% NaS2O3 in NaHCO3 (sat'd aq.) and the the aqueous layer was extracted repeatedly with CH2CI2. The combined 20 organic extracts were washed with water, dried (Na2SO4) and concentrated in v~uo to yield the keto-amide product (0.3 g)which was used in the next step without purification: MS m/z 844 (MH+). A stirred mixture of the keto-amide and W097/30080 PCT~US97~2S75 anisole (3 mL) was treated with HF (ca. 15 mL) at -78~C. This mixture was stirred an additional 3.5 h at 0 ~C, and the HF was removed in v~ o at 0 ~C.
The residue was triturated with ether, and the residue was purified by reverse-phase HPLC (30:70:0.2 CH3CN-H2O-TFA). The desired fractions were 5 Iyophilized to give the title compound as a white solid: 0.1 9; MS m/z 556.5 (MH+); Anal. Calcd for C2gH4sN7O4-2.75 C2HF3Oz-1.25 H2O
Calc'd: C, 46.46; H, 5.68; N, 10.99; H2O, 2.47 Found: C, 46.53; H, 5.72; N, 11.15; H2O, 2.77.

2S, 5S, 9R-N[3-(9-BENZYL-3, 6, 7,10,19-(PENTAOXOEICOSAHYDRO)-1a, 4, 8, 11-(TETRAAZACYCLOPENTACYCLONONADECEN-5-YL)PROPYL] GUANIDINE TRIFLUOROACETIC ACID.
Step 14a H O CO2-t-Bu ,N~ J~N~ ~;

14a A solution of N-a-Cbz-aminooctanoic acid (2.5 g, 8.5 mmol), Pro-O-~-Bu (1.6 9, 9.4 mmol) and HOBT (1.7 9, 12.8 mmol) in CH3CN (80 mL) was added a 20 solution of DCC (1.4 g, 9.4 mmol) in CH3CN. (15 mL). The reaction was stirredovernight, filtered and the resulting filtrate concentrated in vacuo. The residue was purified by flash column chromatography CH2CI2 -~ 2% MeOH-CH2CI2 ) to afford 1~ :3.8 9; MS m/z 447 (MH+).

Step 14b ~ CO2-t-Bu H2N~ J~NJ;
(cH2)7 l_j 14b A mixture of ~ (3.6 g), Pd(OH)2 (1.8 9) in MeOH (75 mL) was shaken under H2 (20 psig) for 2.5 h. This mixture was filtered, and the filtrate was 30 concentrated in vacuo to give the free amine 1 4b (2.5 g) which was used without further purification.

Step 14c ZBCNH~ H ~ CO2-~-Bu O~r (CH2)7 N~

A solution of N-a-Cbz-D-Phe (2.2 9, 7.3 mmol) of, 1 4c ~2.5 9, 8.1 mmol) and HOBT (1.4 9, 11.0 mmol) in CH3CN (10 mL) was treated with DCC (1.7 9, 8.1 mmol)in CH3CN (10 mL~. The mixture was stirred overnight, filtered and the filtrate was concentrated in v~cuo. The residue was dissolved in CHCI3, washed with NaHCO3 (sat'd. aq), dried (Na2SO4) and concentrated in v~ o .
This residue was purified by flash column chromatography (silica gel, 97:3 CHCI3 MeOH) to afford 14c: 3.7 g; MS mlz 594 (MH~).

Step 14d ¢~
NH2 H ~ CO2-t-Bu (CH2)7 N~
14d A mixture of 14c (3.7 g), MeOH ( 75 mL) and PdOH2 (1.4 g)was shaken under H2 (20 psig) for 2.5 h and filtered. The filtrate was concentrated in vacuo to afford 1 4d (2.5 9) which was used in the next step without purification.

Step 14e ~ NH
O NH 'LHo2'7 SEMO~ 'CBZ ~~ CO2-t-Bu l_ HNJ
HN ~NH
Ts 14e A mixture of 6-[~imino~4-methylbenzenesulfonyl) amino]methyl]amino]-2-(R,S)-1[2-(trimethylsilyl)ethoxylmethoxy]-3(S)-[9-phenylrnethoxycarbonyl)-amino]hexanoic acid (1.0 9, 1.6 mmol), .1 4d (0.81 g, 1.8 mmol) and HOBT
(0.32 9, 2.4 mmol) of HOBT in CH3CN (60 mL) was treated with DCC (0.36 9, 5 1.8 mmol) in CH3CN (60 mL). This mixture was stirred overnight, filtered and the resulting filtrate was concentrated in v~ o . The residue was dissolved in CHCI3, washed with 10% aqusous Na2CO3, dried (Na2SO4) and concentrated in vP~uo. This residuc was purified by flash column chromatography (silica gel, CHCI3-~98% CHCI3-MeOH) to afford 1 4e (1.42 9) as a white solid: MS m/z 1064 (MH+).

Step 14f ~ O
\=/ '~ NH
o~ Lo SEMO~NH2 ~ COz-t-Bu HN

HTNs~NH
14f 15 A mixture of 14e (1.42 9) Pd(OH)2 (0.8 9) and MeOH (50 mL) under H2 (20 psig)for 2.5 h. This mixture was filtered, and the filtrate concentrated in v~uo to yield amine ~f (1.18 9): MS m/z 930 (MH+).

Step 149 ~ O
\=/ '~ NH
O~ NH '~Ho2)7 . C02H

HN
HN ~NH
Ts WO 971300B0 PCT~US9710257 A solution of ~9, (1.18 g) in CH2CI2 (5 mL) was added to a solution of 1:1 TFA-CH2CI2 (20 mL) at 0 ~C. This mixture was stirred for 2 h at room temperature and the solvent was removed under a stream, of N2. The residue was triturated with ether to afford 149 (1.02 9) which wasd used without additional purification.

Step 14h H
O~ NH ( ~CL2) 7 r o HNJ

TH,N NH
14h A solution of 14h (1.01 9, 1.18 mmol) in CH2CI2 was treated with DMAP (0.73 9, 10 6.0 mmol) of DMAP followed by BOP-CI (0.60 g, 2.35 mmol). This mixture was stirred for 24 h and the total volume was reduced to 100 mL in vacuo. This solution was washed with 10% aqueous citric acid, dried (Na2SO4) and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, CH2CI2 ->10% MeOH-CH2CI2 ) to yield ~h (0.30 g) S~ep 14i ~ I\~H
0~ NH ( ,CL2)7 o~ N~
, - o HNJ

HN ~NH
14i 20 A solution of .~L (0.30 g, 0.42 mmol) in CH2CI2 (10 mL) was added to a suspension of Dess-Martin periodinane (0.26 9, 0.62 mmol) in CH2CI2 (10 mL) After 2.~ h, the mi\xture was treated with an excess of 25% Na2S2O4 (aq) in ,.

WO 97t30080 PCT/US97/02575 NaHC03 (sat'd. aq) and stirred for 5 min. The aqueous layer was sxtracted with Cl 12CI2 and the combined organic extracts were washed with H20, dried (Na2SO4) and concentrated under in v~ n to afford 1 4i (0.30 g) which was used in the next step without purification: MS m/z 724 (MH+).

H
o~NH (CH2)7 o~ N~7 r o HNJ
H2N~NH
2S, 5S, 9R-Nl3-(9-BENZYL-3, 6, 7, 10, 19-(PENTAOXOEICOSAHYDRO)-1 a, 4, 8, 11-(TETRAAZACYCLOPENTACYCLONONADECEN-5-YL)PROPYL] GUANIDINE TRIFLUOROACETIC ACID

A suspension of 1 4i (0.30 9) in anisole (2 mL) was cooled to -78~C and treated with anhydrous HF (ca. 10 mL) using a standard HF apparatus. This mixture was stirred for 4h at 0 ~C and the HF was removed in vacuo at 0~C. This 15 residue was tnturated twice with ether (2 X 25 mL) and the resulting solid was purified by reverse-phase HPLC (MeCN-water-TFA, 40:60:0.2). Lyophillization of the eluate provided the title compound (0.44 9) as a white solid: FAB-MS m/z 570 (MH+) Anal. Calc'd for C29H43N7Os-1.25 C2HF3O2-2.0 H2O
20 Calc'd: C, 50.56; H, 6.50; N, 13.10; H2O, 4.82.
Found: C, 50.31, H, 6.25; N, 12.70; H2O; 4.33.

, . . .

~ ~H
o~NH (CH2)5 o~ N~

HN
H2N~NH
1 2R, 1 6S ,1 9S-N-[3-(1 2-BENZYL-4, 1 1 ,14,1 5,1 8-PENTAOXOOCTADECAHYDRO)-3a, 10.1 3 ,1 7-(TETRMZACYCLOPENTACYCLOH EPTADECEN-1 6-YL)PROPYL]-GUANIDINE TRIFLUOROACETIC ACID

The preparation of compound 15 is analogous to Example 14. The hexanoyl analog of ~, namely 1 (S)-[6-(2(R)-amino-3-phenylpropionylamino)hexanoyllpyrrolidine-2-carboxylic acid I-butyl ester, was 10 prepared by coupling CBZ-D-Phe with 6-aminohexanoic acid -I-butyl ester, saponi~ying the ester and coupling the resulting acid with Pro-O-I-Bu. The remaining steps of Example 14 were carried out to give the title compound as a solid: FAB-MS m/z 542 (MH~; Anal Calc'd. ~or C27H3gN70s-1.75 CF3CO2H-1.5 H20:
Calc'd: C, 47.69; H, 5.74; N, 12.76; H20, 3.53 Fo~lnd: C, 47.81; H, 5.74; N, 13.04; H20, 3.57.

~ O
~ H
o ~ NH (CH2)s o~N~

HN
H2N~NH
1 2S, 1 6S, 1 gS-3-[1 6-(3-GUAN ID INOPROPYL)-4 ,1 1 ,14 ,15,18-PENTAOXOOCTADECAHYDRO-3,10,1 3,1 7-TETRAAZACYCLOPENTACYCLOHFPTADECEN-1,2-YL]PROPIONIC ACID
TRIFLUOIROACETATE

..

WO g7/30080 PCTAUS97~2S75 The preparation of compound 16 is analogous to Example 14. The hexanoyl analog of 1~Lg. namely 1 (S)-[6-(2(R)-amino-3-(carboxymethyl)propionylamino)hexanoyl]pyrrolidine-2-carboxylic acid ~-butyl 5 ester, was prepared by coupling L-N-a-Fmoc-Glu(OBzl)-OH with 6-aminohexanoic acid -~-butyl ester, saponifying the ester and coupling the resulting acid with Pro-O-~-Bu. The remaining steps of Example 14 were carried out to give the title compound as a solid: FAB-MS m/z 523 (MH+); Anal Calc'd. for C23H37N7Or2.0CF3CO2H-1 5 H2O:
10 Calc'd: C, 41.65; H, 5.44; N, 12.59; H2O, 3.47 Found: C, 41.83; H, 5.30; N, 12.64; H2O, 3.57.

5R,1 8S,21 S-N-[3-(4,7,16,17,20-PENTAOXO-5-PHENETHYLEICOSAHYDRO-3a, 6, 15, 19-TETRAACBZACYCLOPENTACYCLONONADECEN-18-YL)PROPYL]GUANINDINE TRIFLUOROACETICACID
Step 17a Fmoc' ~ co2~Bu H

20 Sodium Trriacetoxyborohydride (6.1 g, 29 mmol) and acetic acid (1 mL) were added to a solution of N-a-(fluorenylmethyloxycarbonyl)glycinal (5.4 g, 19 mmol: prepared by the method of Ho, et al. Jovrnal Of Organic Chemistry 1983, 58, 2313-16) and 5-aminopentanoic acid ~-butyl ester (3.6 g, 20 mmol) in CH2CI2 (30 mL). This mixture was stirred overnight and concentrated in vacuo 25 to give ~ which was used without purification Step 17b Fmoc' ~i N ~ C02~Bu CBZ
17b 30 To a solution of 11 g of intermediate 3 and 5.6 mL of triethylamine in 150 mL of CH2CI2 at 0 ~C Carbobenzoxychloride (3.1 mL) was added to a stirred solution of 14a (11g) andtriethylamine (5.6 mL) in CH2CI2 (150 mL). After2 h, the reaction was quenched with H2O, and the resulting organic iayer was extracted twice with CH2CI2. The combined organic layers were dried (Na2SO4) and 35 concentrated. The residue was purified by flash chromatography (silica gel; 3:1 W O 97~0080 PCTrUS97~2S75 hexanes-ether~> 3:2 hexanes-ether) to afford 2.6 9 of 1 7b as a white solid: MS
m/z 573 (MH+).

Step 17c H2N N--,C02tBu CBZ

A solution of intermediate 17b ~2.42 9) in CH2CI2 (5 mL) was added to a 1:1 solution of TFA-CH2CI2 (20 mL) at 0 ~C. The reaction was stirred for 1 h at room temperature, then volatiles were removed under a stream of N2 at room temperaature. The residue was purified by chromatography (flash column, silica gel; 9:1 CHCI3-MeOH) to afford 1.8 g of ~: MS mlz 517 (MH+).

Step 17d r\
0~ ~N~
(CH2)4' H O CO2t-Bu N~ CBZ
Fmoc 17d A solution of 17c (1.72 9, 3.33 mmol) and D-Phe-Pro-O-~-Bu (1.58 9, 4.99 mmol) and HOBT (0.67 g, 4.99 mmol) was treated with a solution of DCC (1.03 g, 5.00 mmol) in CH3CN (3 mL) and stirred overnight. The mixture was filtered and concentrated, and the residue was purified by flash chromatography (silica gel, 100 % CHCI3 -> 98% CHCI3-MeOH) to give 2.28 9 of intermediate 17d: MS m/z 817 (MH+) Step 17e r\
0~ ~N~
(CH2)4/ H O C02t-Bu _rN~CBZ

W 097/30080 PCTrUS97~2S75 A solution of ~ (2.24 g) in CH3CN (25 mL) was treated with diethylamine (6 mL) and the mixture was stirred for 2.6 h. The solution was concentrated in v~ o, and the residue was purified by flash chromatography (silica gel, 100 5 % CHCI3 -> 90% CHCI3-MeOH) to give 1.27 g of intermediate 1 7e:MS m/z 595 (MH+).

Step 17t N--(CH2)4 ~O

O~,NH
SEMO_( N
TsHN~ NHFmoc ~ CO2-tBu NH
1 7f To a stirred solution of [[imino[4-methylbenzenesulfonyl)amino]methyl]amino]-2-(R,S)-[[2-(trimethylsilyl)ethoxy]methoxy]-3(S)-[9-fluorenyl methoxycarbonyl)-amino]hexanoic acid, (2.5 g ,4.0 mmol: prepared analogous to the CBZ
derivative in Maryanoff et al. Joumal of the American Chem~cal Society 1995, 117, 1225-39) and intermediate 17f (1.27 9, 2.13 mmol) and HOBT (0.39 9, 2.91 mmol) in CH3CN (45 mL) was added DCC (0.44 g, 2.13 mmol) and this mixture was stirred overnight. The reaction was filtered, the filtrate was concentrated,and the residue was purified by flash chromatography (silica gel, 100 % CHCI3 -~ 95% CHCI3-MeOH) to yield 1.96 g of intermediate 17f: MS m/z 1287 (MH+).
Step 17g N- (CH2)4 ~~
O ~/ NH ~3 SEMO _( N
TsHN_6 NH2 ~ CO2-tBu NH
17q A solution of intermediate lZg (1.93 9) in CH3CN (20 mL) was treated with 25 diethylamine (5 mL) and stirred for 2h. The solution was concentrated, and the residue was triturated repeatedly with ether to afford 1.31 g of intermedlate l~g:
MS m/z 1065 (MH+).

Step 17h N- (CH2)4 ~~
O /J ~H~3 SEMO _~ O
H _~~~ NH2 ~ C02H
TsHN~
NH
17h To a solution of 1:1 TFA -Cl 12CI2 (20 mL) at 0 ~C was added a solution of intermediate 179 (1.31 g, 1.23 mmol) in CH2CI2 (5 mL). After stirring at room temperature for 1 h, the solution was concentrated under a stream of N2, and the10 residue was triturated with ether to afford 1 .1 g of intermediate 1 7h as a white solid: MS m/z 879 (MH+).

Step 17i C~Z
~N- (CH2)4 ~ 'H NH~3 HOI O (~

TsHN_ NH
17i A mixture of intermediate ~h (1.1 g, 1.0 mmol) and DMAP (0.63 g, 5.1 mmol) in CH2CI2 (1 L) was treated with BOP-CI (0.5 1 g, 2.0 mmol) and stirred for 4 h.
The mixture was reduced ca. 75%t washed twice with 10% aqueous citric acid, dried (Na2SO4) and concentrated. The residue was purified by flash column 20 chromatography (silica gel, CH2CI2-> 10% MeOH-CH2CI2) to give 0.53 g of intermediate 17i: MS m/z 861 (MH+).

WO 97130080 rCT~US97/02S7S

rN- (CH2)4 Oq'N'H N\~H~3 NHZ~N~~
NH
5R,18S,21 S-N-[3-(4,7,16,17,20-PENTAOXO-5-PHENETHYLEICOSAHYDRO-3a, 6,1 5, 1 9-TET~AAZACYCLOPENTACYCLONONADECEN-1 8-YL)PROPYL]GUANINDINE TRIFLUOROACETICACID

To a mixture of Dess-Mar~in periodinane (0.39 9, 0.89 mmoi) in CH2CI2 (10 mL) was added to a solution of intermediate1:Zi (0.51 9) in CH2CI2 (10 mL).
After stirring for 1 h, the reaction was treated with excess 25% Na2S203(aq) in NaHC03 (sat'd., aq.) and the layers were separated. The aqueous layer was extracted three times with CHCI3, and the combined organic layers were washed with water, dried (Na2SO4), and concentrated in v~uo to afford 0.46 9 of the corresponding keto-amide: MS mlz 859 (MH+). A mixture of the keto-amide ~0.44 g) in anisole (3 mL) was treated with ca. 10 mL of HF at -78 ~C
and stirred at 0 ~C tor 3.5 h. FYcess HF was removed under in vacuo at 0 ~C, and the residue was triturated twice with ether. The residue was purified by reverse-phase HPLC (80:20:0.2 H2O-CH3CN-TFA) to afford 0.052 g of the title compound as a white solid: MS mlz 571.5 (MH+); mp t02-106 ~C; Anal. Calcd.
for C2~H42N8O5-2.5 C2HF3O2-3.5 H2O:
Calculated C, 43.14; H, 5.65; N, 12.20; H2O, 6.86;
Found: C, 42.91; H, 5.38; N, 12.42; H2O, 6.44.

N-[3-(3-BENZYL-2,5,6,9-TETFIAOXO-1 ,4,8-TRIAZACYCLOTETRADEC-7-YL)PROPYLlGUANlDtNE TRIFLUOROACETIC ACID
Step 1 8a ~H
CBZNH N~ ~C02-t-Bu O (CH2) 5 W097/30080 PCT~US97102S7S

Intermediate ~ was prepared following steps 11 a and 11 b of example by using 6-aminohexanoic acid instead of aminopentanoic acid in step 11a.

Step 18b '~ NH
O NH '~L~Ho2)s ~NHCBZ O-~Bu HNJ
HN ~N~-I
Ts 18b A mixture of ~ (0.72 9, 2.15 mmol), 6-[[imino[4-methylbenzenesulfonyl) amino~methyl]amino}~2-(R,S)-[[2-(trimethylsilyl)ethoxylmethoxyl-3(S)-[9-phsnylmethoxycarbonyl)-amino]heKanoic acid (1.47 9, 2.36 mmol) and HOBT
10 (0.44 g, 3.22 mmol) dissolv0d in CH3CN (30 mL) was treated with a solution ofDCC (0.49 g, 2.36 mmol) in CH3CN (30 mL)and stirred overnight. The mixture was filtered, concentrated in v~tlo and the residue was dissolved in mL ethyl acetate (50 mL) and washed with saturated aqueous NaHCO3 (5 mL). The organic extract was dried (Na2SO4) and concentrated. The residue was 15 purified via flash column chromatography (9:1 CH2CI2-MeOH) to yield 1.72 9 of intermediate 1~

Step 18c NH
(CH2)5 0 ~,~NH ,~o OH
SEMO ~ NHCBz HNJ

Ts 1~
A solution of intermediate 1 8b (1.71 9, 1.82 mmol) in CH2CI2 (5 mL) was added to 1:1 TFA-CH2CI2 (28 mL) solution at 0 ~C. The mixture was stirred for 1 h at room temperature and concenlr~led under a stream of N2. The residue was triturated with ether to afford 1.6 9 of intermediate ~ as a white solid.

Step 18d ~ ~
\=/ " ~ NH
(CH2)5 0~ NH ,~o OH
SEMO~ NH2 HNJ
HN ~NH
Ts A mixture of intermediate ~ (500 mg, 0.5 mmol), Pd(OH)2 (600 mg) and metl,anol (30 mL) was shaken under H2 at 21 psig for 5 h. The mixture was filtered, and the filtrats was concentrated to afford 390 mg of inte,-"ecJiale 10 as a white solid: m/z 619 (MH+).

Step 18e ~ O
~ NH
O NH <~

HO~ N

HN

HTNs~NH
18e 15 A solution of intermediate ~ (340 mg, 0.464mmol) and DMAP (283 mg, 2.32 mmol) in 450 mL of CH2CI2 was treated with BOP-CI (236 mg, 0.928 mmol) and stirred for 5 h. Solvent was removed under reduced pressure, and the residue was purified via flash column chromatography (silica gel, 95:5 CH2CI2 -MeOH-~90:10 CH2CI2 -MeOH) to yield 78 mg of intermediate 1~ m/z 601 (MH+~.

W 097130080 PCTAUSg7/02S75 Step 18f ~- o ~ NH

Oo~NH

HN

HTNs~NH
1 8f A solution of intermediate ~ (78 mg, 0.13 mmol) in CH2CI2 (15 mL) was 5 treated with (82 mg, 0.19 mmol) of the Dess-Martin periodinane. The mixture was stirred for 3.5 h, treated with of aqueous 4:1 NaHCO3-Na2S2O3 (20mL).
This mixture was extracted tour times with 25 mL portions of CH2CI2 . The combined CH2CI2 extracts were washed with brine, dried (Na2SO4), and conc~nllale~l to provide 49 mg of inl~r,l,~Jiale l~lf which was used in the 10 fa"3wing step without further pu-i1ication: m/z 599 (MH+).

~ Nl I
o~N~

HN
H2N~NH
N-[3-(3-BENZYL-2,5,6,9-TETRAOXO-1 ,4,8-TRIAZACYCLOTETRADEC-7-YL)PROPYL]GUANIDINE TRIFLUOROACETIC ACID

A suspension of intermediate ~ (49 mg) in anisole (1 mL) was treated with ca.
5 mL of anhydrous HF at -78 ~C. The mixture was stirred at 0 ~C for 3.5 h and the excess HF was removed under vacuum. The residue was triturated with ether and purified by reverse-phase HPLC (MeCN-water-TFA, 30:70:0.2) to give 20 10.4 mg of the title compound as a white solid: mlz 445 (MH+); Anal. Calcd for CZ2H32N6o4-1 .75C2HF30z-1.1 8H20:

WO 97t30080 PCT/US97/02S75 Calculated: C, 46.03; H, 5.47; N, 12.63; H20, 3.19 Found:C, 46.03; H, 4.95; N, 12.99; H20, 3.49.

~ 11 ~~
~N~

HN
H2N~NH
5R,15R,19S,21AS-N-[3-(15-BENZHYDRYL-5-BENZYL-4,7,14,17,18,21-HEXAOXODOCOSAHYDRO-3a,6, 13,1 6,20-PENTMZACYCI OPENTACYCLOEICOSEN-19-YL)PROPYL]GUANIDINE
TRIFLUOROACETIC ACID

Compound 19 was prepared usin~ the method of Example 1 1 with the following modifications. 8-Aminohexanoic acid is used in place of 5-aminoheptanoic acid in step 11 a and CBz-D-phe is r~ eJ with CBZ-D-diPhe (prepared via the method of US Pat 5,198,548) in step 11 b to give the title compound as a solid:
15 FAB-MS m/z 766 (MH+); Anal. Calc'd tor C42Hs2NgO6-1.75 C2HF302-2.25 H20:
CaJc'd:C, 54.38; H, 5.84; N, 11.15; H20, 4.03.
Found:C, 54.57; H, 5.71; N, 11.29; H20, 4.09.

W097130080 PCTnUS97~2S7S

o H_(CH2)5 ~3 ~$H =~ '13 ~ O
HN
H2N~NH
5R,15S,19S,21aS-N-~3-(5,15-DlBENZYL-4,7,14,17,18,21-HEXAOXODOCOSAHYDRO-3a,6, 13, 16,20-PENTAAZACYCLOPENTACYCLOEICOSEN-19-YL)PROPYLlGUANIDINE TRIFLUOROACETICACID

Compound 20 was prepared using the method of Example 11 with the following modifications. 6-Aminohexanoic acid is used in place of 5-aminoheptanoic acid 10 in step 1 1a and CBZ-D-diPhe-ProO-~-Bu replaced with D-Phe-ProO-I-Bu in step 11d to give the title cG",pound as a solid: FAB-MS m/z 766 (MH+); Anal. Calc'd for C42Hs2NgO6-2.0 C2HF302-2~75 H2O:
Calc'd: C, 53.00; H, 5.75; N, 10.75; H20, 4.75.
Found: C, 53.30; H, 5.51; N, 10.70; H2O, 4.96.

o H
H3C~N- (CH2)4 ~ 'H NH~0 0~ 0 ., NH2_6N--~LN~ ~~

NH
5R,14R,18S,20aS-N-[3-(5-BENZYL-14-METHYL-4,7,13,16,17,20-HEXAOXOElCOSAHYDRO-3a,6l12,15,1 9-PENTAAZACYCLOPENTA-CYCLONONADECEN-18-YL)PROPYL]GUANIDINETRIFLUOROACETIC ACID

Compound 21 was prepared using the method of Example 11 with the following modifications. 6-Aminohexanoic acid is used in place of 5-aminoheptanoic acid W O 97~ PCT~US97/02S75 in step 11a and CBZ-D-Phe is rer~laced with CBZ-D-Ala in step 11b to give the title compound as a solid: FAB-MS m/z 600 (MH+~; Anal. Calc'd for C29H42N806-2.15 C2HF302-2.50 H20:
- - Calc'd:C, 45.00; H, 5.57; N, ~2.16; H2O, 5.07.
Found:C, 45.01; H, 5.46; N, 12.82; H2O, 5.31.

(CH2~)~N~

H~;~H_~ ~
HN
H2N~NH
[20aR-(20aR, 5R, 1 8S)l-N-[3-(5-PHENYLETHYL-1 7-HYDROXY-4, 7,16, 20-TETRAOXOElCOSAHYDRO-3a, 6, 15, 19-TETRA-AZACYCLONONADECANE-18-YL) PROPYL] GUANIDINE

A suspension of Intermediate 1 h (0.245 g, 0.372 mmol) anisole (2 mL) 15 was cooled to -78~C and lreate~J anhydrous HF (ca. 1 OmL) using a standard HFapparatus. After stirring for 4 h, HF was removed under re~lo~ed pressure at 0~C, and the residue was triturated twice with 25 mL portions of ether. The solid was collected, washed with ether, then purified by reverse-phase HPLC
(MeCN-water-TFA, 30:70:0.2). Lyophillization of the eluate provided the title 20 cG-"pound as a white solid: FAB-MS m/z 586 (MH~); Anal. Calcd for C30H47N7O5~2C2HF302-O.75 H20:
Calc'd: C, 49.36; H, 6.15; N, 11.85; H20, 1.63.
Found: C, 49.10, H, 6.07; N, 12.15; H20; 1.42.

W O 97/~W~0 PCTAJS97/02S7S

Step a H O
- Fmoc-N

~H
Ts-HN~NH

1,1-Carbonyldiimidazole (1.8g, 11.0 mmol~ was added to a solution of N-a-Fmoc-NG-tosyl-L-arginine (6.0 9, 10.0 mmol) in anhydrous THF (30 mL) at 0 ~C under argon and stirred at 0 ~C for 1.5 h. The reaction mixture was cooled to -48 ~C and 1 M DIBAL (28 mL, 28 mmol) was added dropwise over 20 min.
The resulting mixture was stirred for another 1.5 h and 1.2 N HCL (67 mL) was 10 added with stirring. The mixture was allowed to warm up to room temperature and partitioned between 0.6N HCI (65 mL) and chloroform. The resulting eous layer was washed with several portions of chloroform. The combined organic extracts were washed withsu¢cessive portions of water and brine, dried (Na2SO4) and concentrated in v~cuo to affordthe aldehyde ~ as a white 15 flakey solid.

Step b H OH
Fmoc N~,~H
~' CN

~IH
Ts-HN~NH
2~
20 A solution of KCN (1.44 9, 22 mmol) and H2O (125 ml ) was added to a solutionof aldehyde 2~. (5.9 9, 11.0 mmol) in ethyl acetate (250 mL) and the resulting mixture was stirred for 40 h at room temperature under argon. The organic layer was separated and the aqueous layer was washed with three portions of ethyl acetate. The combined ethyl acetate extracts were washed with brine, 25 dried (Na2SO4), concenlr~ed in v~uo and stored in the refrigerator under argon. The residue was partitioned between ethyl acetate (100 mL) and saturated aqueous NaHCO3 (200 mL) and the pH was maintained at 7.0 by the WO 9~30080 PCT/US97/02S7S

addition of solid NaHCO3. The solid NaHCO3 was removed by filtration and the resulting aqueous layer was washed with several portions of ethyl acetate.
The combined organic layer was washed twice with brine, dried (MgSO4) and concentrated in v~cuo to give the cyanohydrin 23b as a white solid; FAB-MS
5 rn~z562 (MH)+.

Step c ~H
Ts-HN~NH
2~
10 HCI (21 g) was b~bbler~ into a solution of nitrile ~ (3.0 ~, 5.34 mmol) and mell)anol (53 mL) under argon at a temperature of less than -40 ~C over 20 min.
The reaction vessel was closed under nitrogen and placed in a freezer at -15 ~C for 46 h and con~ thle~ in v~cuo at room temperature. The residue was partitioned betwesn saturated aqueous NaHCO3 solution (250 mL) and 15 ethyl acetate. The organic layer was washed with two portions of brine, dried (MgSO4) and concenl,at~-J in v~ o to give the imidate 2~ as a solid.

Step d ~~r CO2Me Fmoc-HN
OH

i~lH
Ts-HN~NH
~
Cysteine methyl ester hydrochloride (2.7 9, 15.9 mmol) was added to a solution of imidate ~ (5.0 9, 7.9 mmol) and CH2C12 ~100 mL) and the resulting mixture was stirred under argon at room t~mperature for 2 d. The mixture was washed sequentially with brine and water, then dried (Na2S04) and concentrated. The W 097130080 PCTrUS97~2575 -residue was purified by flash chromatography (silica gei, 95:~ CH2C12-MeOtl) to ~ive 3.8 9 Of 2~ as a white foam: MS m/z 680 (MH+).

s ~r Co2Me )=N
Boc-HN_~
OSi(Me)2t-Bu ~IH
Ts-HN~NH
2~
t-Butyldimethylsilyltriflate (3.3 g, 12.6 mmol) was added dropwise to a solutionof ~ (1.9 g, 2.8 mmol) and 2,6-lutidine and cooled to 0 ~C. The mixture was stirred for 1 h at 0 ~C, then quenched with ice. The CH2CI2 layer was washed with water, dried (Na2SO4) and concentrated to give the corresponding si~yl 10 ether which was used without purification: rn/z 794 (MH+). The silyl ether was dissolved in 50 mL of 20 % diethylamine -CH3CN and stirred for 2.5 h. The solution was concentrated, and the residue was purified via flash chromatography (silica gel, CH2C12->10% MeOH-CH2C12) to yield 1 g (1.85 mmol) of the corresponding a-amino derivatiYe as an oil. This material (1.0 9, 15 1.85 mmol) was dissolved in CH2CI2 (25 mL) and treated with di-t-butyl dicarbonate (0.49 9, 2.25 mmol) at 0 ~C. After stirring overnight at room ternperature the reaction was v~ashel~ with water and the CH2CI2 layer was dried (Na2SO4) and concentrated. The residue was purified by flash chromatography (silica gel, 95:5 CH2CI2-MeOH) to afford 1.1 9 of ~ as a 20 semi-solid: m/z 558 (MH+).

Step f S~ COzH
~=N
Boc-HN ~
OSi(Me)2t-Bu ~hH
Ts-HN~NH
2~f WO 9'7/30080 PCT/US97/02575 -Activated MnO2 (3.5 g) was added to a solution of 2~ (1.1 g, 1.7 mmol) in CH2CI2 (100 mL). The mixture was stirred for 6.5 h and fUtered through dicalite.The filtrate was concen~,ated, and the residue was purified by flash column chromatography (silica gel; 95:5 CH2CI2-MeOH) to afford 800 mg (1.3 mmol) of 5 the corresponding thi~ole derivative as a white foam. The material was combined with LiOH (94 mg, 3.9 mmol) and 9:1 dioxane-water solution (12 mL).
The mixture was stirred for 4 h, diluted with water and acidified to pH 5 with acetic acid. The mixture was exlla~ecl three times with ethyl acetate, dried (Na2SO4) and concentrated to give 2~f as a white semi-solid: m/z 656 (MH+).
Step g r\
0~ ~N~
(CH2)~ H O C02~u ~ g Intermediate 2~Q was pr~par~-~ from D-Phe~ ProO-~-Bu and 15 4-(N-car~obenzoxy)a",inobutanoic acid, using steps a-d of Example 1.

Step h H (CH2)4 0~_N-- ~0 5-Bu-(Me)25~ NH~ ~C13 H_~N
Ts NH
~h 20 A solution of intermediate 2~ (0.4 9, 0.60 mmol), intermediate 2~Q (0.26 9, 0.63 mmol), and HOBt (121 mg, 0.90 mmol) in CH3CN (10 mL) was treated with DCC
~130 mg, 0.63 mmol) in CH3CN (2 mL). The mixture was stirred overnight, filtered and concentr~ted. The r~si~ue was dissolved in CH2CI2, washed sequentially with saturated ~qlleol)s NaHC03, dried (Na2SO4) and 25 concentrated. The residue was purified via flash column chromatography (silica gel, 95:5 CH2CI2-MeOH) to afford 350 mg of the coupled product as a white WO 971~0080 PCTIUS97102S7S

toam: m/z 1055 (MH+). The material was dissolved in CH2CI2 (10 mL) and treated with a solution of 1:1 TFA-CH2CI2 (10 mL) at 0 ~C. The mixture was stirred for 1 h at room temperature and concentrated under a stream of N2 at room temperature. The residue was triturated with ether to give 413 mg of 5 intermediate 2;~h as a white solid: MS m/z 899 (M~t+).

Step i H N ~ /
N~
Ts NH
23i 10 A solution of int~rmediatc 2~. (1.0 9, 0.90 mmol) in CH2CI2 (900 mL) was tr~&l~d with DMAP (560 mg, 4.6 mmol) followed by BOP-CI (450 mg, 1.8 mmol).
The mixture was stirred for 2 h and concentrated in vacuo. The residue was purified via flash column chromdlogr~phy (silica gel, 95:5 CH2CI2-MeOH) to 9iY8 500 mg of an off-white solid: mtz 882 (MH+). This solid was stirred for 1 h15 in 1 M Bu4NFlTHF and concentrated under reduced pressure. The residue dissolved in CH2CI2 and washed repe~tedly with H20. The organic layer was dried (Na2SO4) and concenl-dted under reduced pressure to yield 27~ mg of the corresponding alcohol ~Lwhich was used in the following step without further purification: mlz 767 (MH+).

N--H2N~
NH
3S,6S, 1 2R-N-[3-(1 2-BENZYL-2, ~,1 1, t 4,20-PENTAOXO-23-THIA-4,10,13,19,24-PENTAAZATRICYCLO[19.2.1 .3]-6, 10-TETRACOSA-1(2H), 21-DIEN-3-YL)PROPYL]GUANIDINE

W097/30080 . PCTrUS97~2S7S

The alcohol (275 mg) was suspended in 30 mL of CH2CI2 and added to a suspel-sion of the Dess-Martin periodinane (228 mg, 0.54 mmol) in 1 mL of CH2CI2. The mixture was stirred for 3h, quenched with 25% Na2S203 (aq) in 5 NaHCO3 (sat'd., aq.) and stirred for 0.5 h. The layers were separated, and theC~2CI2 layer was washed twice with water, dried (Na2SO4) and concentrated under rer~lced pressure to yield 273 mg of the corresponding keto-amide derivative as a foam: mlz 6t 1 (MH+). A mixture of 270 mg of the keto-amide derivative was suspended in 2 m- of anisole and treated with excess (ca. 10 mL) 10 anhydrous HF at -78 ~C. The mixture was stirred at 0 ~C for 4.5 h, then HF was r~moved at 0 ~C under red~ce~ pressure. The residue was triturated with ether and purified by reverse-phase HPLC (70:30:0.2 H20-CH3CN-TFA).
Lyophill;Lalion of the eluate provided 120 mg of the title compound as a white solid: FAB-MS rn/z 556 (MH+); Anal. Calc'd for C29H38N8OsS-1.75C2HF3O2-1.5 15 ~:
C~lc'd: C, 46.62; H, 5.15; N, 13.38, H20, 3.23.
Found: C, 46.40; H, 5.03; N, 13.57; H20; 3.33.

Claims

What is claimed is:

1. A compound of the Formula I

wherein:
m is 2 to 12;
A is where the amido carbonyl is bound to B and the .alpha.
aminomethine is bound to the depicted ring carbonyl, R3 is hydrogen, hydroxy or C1-5alkoxy, n is 1 or 2, and a is 0 or 1;
B is where the amido carbonyl of B is bound to the depicted ring methylene and the methine is bound to A, R4 is independently selected from the group consisting of hydrogen, C1-5alkyl, carboxy C1-5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), 3-pyridylC1-5alkyl, 4-pyridylC1-5alkyl, diphenylC1-2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1-5alkyl, carboxy C1-5alkoxycsrbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), R5 and R6 are hydrogen or taken together with the carbon of attachment to form a carbonyl, and b is 0 or 1;

G is where the amine of G is bound to the ring methylene and the methine is bound to the depicted amide, R7 is independently selected from the group consisting of hydrogen, C1-5alkyl, carboxyC1-5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), 3-pyridylC1-5alkyl, 4-pyridylC1-5alkyl, diphenylC1-2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), E is carbon or C(CH2)q-, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25, R8 and R9 are hydrogen or taken together with the carbon of E
to form a carbonyl, and g is 0 or 1;
or the pharmaceutically acceptable salt thereof.

2. The compound of Claim 1 where a is 1, b is 0 and g is 0.

3. The compound of Claim 2 where n is 1.

4. The compound of Claim 1 where a is 0, b is 1 and g is 0.

5. The compound of Claim 4 where R5 and R6 are taken together with the carbon to which each is attached to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substttuents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

6. The compound of Claim 1 where a is 0, b is 0 and g is 1.

7. The compound of Claim 6 where E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylsmino, C1-5alkoxy, fluorine bromine or chlorine).

8. The compound of Claim 6 where R8 and R9 are hydrogen, E is C(CH2)q and q is 0-6.

9. The compound of Claim 1 where a is 1, b is 1 and g is 0.

10. The compound of Claim 9 where n is 1, R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where ths phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fiuorine bromine or chlorine), ciiphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

11. The compound of Claim 1 where a is 1, b is 1 and g is 1.

12. The compound of Claim 11 where n is 1, R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

13. The compound of Claim 11 where n is 1, R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl. carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2atkyl. naphthyl and substitutsd naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), R8 and R9 are hydrogen, E is C(CH2)q, and q is 0-6.

14. The compound of Claim 1 where a is 1, b is 0 and g is 1.

15. The compound of Claim 14 where n is 1, E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

16. The compound of Claim 14 where n is 1, R8 and R9 are hydrogen, E is C(CH2)q and q is 0-6.

17. The compound of Claim 1 where a is 0, b is 1 and g is 1.

18. The compound of Claim 17 where R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylaminot hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine). diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine);
E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

19. The compound of Claim 11 where R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl. substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonyiamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine) R8 and R9 are hydrogen, E is C(CH2)q, and q is 0-6

20. The compounds of Claim 1 selected from the group consisting of and

21. The compounds of Claim 1 selected from the group consisting of and

22. The compounds of Claim 1 selected from the group consisting of and

23. The compounds of Claim 1 selected from the group consisting of and

24. The compounds of Claim 1 selected from the group consisting of and

25. A compound of the Formula II
wherein:
R1 is hydroxy;
R2 is hydrogen;
m is 2 to 12;
A is where the amido carbonyl is bound to B and the a aminomethine is bound to the depicted ring carbonyl, R3 is hydrogen or C1-5alkoxy, n is 1 or 2, and a is 0 or 1 ;
B is where the amido carbonyl of B is bound to the depicted ring methlylenes and the methine is bound to A, R4 is independently selected from the group consisting of hydrogen, C1-5alkyl, carboxyC1-5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonyiamino, C1-5alkoxy, fluorine bromine or chlorine), 3-pyridylC1-5alkyl, 4-pyridylC1-5alkyl, diphenylC1-2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), R5 and R6 are hydrogen or taken together with the carbon of attachment to form a carbonyl, and b is 0 or 1 ;

G is where the amine of G is bound to the ring methylenes and the methine is bound to the depicted amide, R7 is independently selected from the group consisting of hydrogen, C1-5alkyl, carboxyC1-5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), 3-pyridylC1-5alkyl, 4-pyridylC1-5alkyl, diphenylC1-2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

E is carbon or C(CH2)q-, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25, R8 and R9 are hydrogen or taken together with the carbon of E
to form a carbonyl, and g is 0 or 1;
and pharmaceutically acceptable salts thereof.

26. A compound of the Formula III.

wherein:
m is 2 to 12;
W is nitrogen, sulfur or oxygen;
A is where the amido carbonyl is bound to B and the .alpha.
aminomethine is bound to the depicted ring carbonyl, R3 is hydrogen, hydroxy or C1-5alkoxy, n is 1 or 2, and a is 0 or 1 ;

B is where the amido carbonyl of B is bound to the depicted ring methylene and the methine is bound to A, R4 is independently selected from the group consisting of hydrogen, C1-5alkyl, carboxyC1-5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), 3-pyridylC1-5alkyl, 4-pyridylC1-5alkyl, diphenylC1-2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine);

R5 and R6 are hydrogen or taken together with the carbon of attachment to form a carbonyl, and b is 0 or 1;

G is where the amine of G is bound to the ring methylene and the methine is bound to the depicted amide, R7 is independently selected from the group consisting of hydrogen, C1-5alkyl, carboxyC1-5alkyl, phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), 3-pyridylC1-5alkyl, 4-pyridylC1-5alkyl, diphenylC1-2alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), E is carbon or C(CH2)q-, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25.

R8 and R9 are hydrogen or taken together with the carbon of E
to form a carbonyl, and g is 0 or 1;
and pharmaceutically acceptable salts thereof.

27. The compound of Claim 26 where a is 1, b is 0 and g is 0.

28. The compound of Claim 27 where n is 1.

29. The compound of Claim 26 where a is 0, b is 1 and g is 0.

30. The compound of Claim 29 where R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyf, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyt, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

31. The compound of Claim 26 where a is 0, b is 0 and g is 1.

32. The compound of Claim 31 where E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

33. The compound of Claim 31 where R8 and R9 are hydrogen, E is C(CH2)q and q is 0-6.

34. The compound of Claim 26 where a is 1, b is 1 and g is 0.

35. The compound of Claim 34 where n is 1, R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

36. The compound of Claim 26 where a is 1, b is 1 and g is 1.

37. The compound of Claim 36 where n is 1, R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkyicarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl.
carboxy C1-5alkoxycarbonyl, carboxamido, amino. C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

38. The compound of Claim 36 where n is 1, R5 and R6 are taken together with the carbon of attachment to form a carbonyl; and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), R8 and R9 are hydrogen, E is C(CH2)q, and q is 0-6.

39. The compound of Claim 26 where a is 1, b is 0 and g is 1.

40. The compound of Claim 39 where n is 1, E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylarnino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

41. The compound of Claim 39 where n is 1, R8 and R9 are hydrogen, E is C(CH2)q and q is 0-6.

42. The compound of Claim 26 where a is 0, b is 1 and g is 1.

43. The compound of Claim 42 where R5 and R6 are taken together with the carbon of attachment to form a carbonyl, and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl. carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), E is carbon, R8 and R9 are taken with the carbon of attachment for form a carbonyl, and R7 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine).

44. The compound of Claim 42 where R5 and R6 are taken together with the carbon of attachment to form a carbonyl and R4 is selected form the group consisting of phenyl, substituted phenyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), phenylC1-5alkyl, substituted phenylC1-5alkyl (where the phenyl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5alkoxy, fluorine bromine or chlorine), diphenylC1-2alkyl, naphthyl and substituted naphthyl (where the aryl substituents are C1-5alkyl, carboxy C1-5alkoxycarbonyl, carboxamido, amino, C1-5alkylamino, hydroxy, C1-5alkylcarbonylamino, C1-5atkoxy, fluorine bromine or chlorine);
R8 and R9 are hydrogen, E is C(CH2)q and q is 0-6

45. The compounds of Claim 26 selected from the group consisting of and

46. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound of Claim 1 or of Claim 26 for treating thrombin mediated diseases in a mammal.

47. A method for inhibiting thrombin comprising contacting a compound of Claim 1 or of Claim 26 with a medium containing thrombin.

48. The method of Claim 47 where the compound contacts the medium via an orthopedic or a surgical device.

49. The method of claim 47 where the medium is mammailian blood.

50. A method of claim 49 where the mammal is a human.

51. A method treating a thrombin mediated disease in a mammal comprising administering an effective amount of a compound of Claim 1 or of Claim 26.

52. A method for inhibiting trypsin comprising contacting a compound of Claim 1 or of Claim 26 with a medium containing trypsin.

53. A method of treating a trypsin related disorder in a mammal comprising administering an effective amount of a compound of Claim 1 or of Claim 26.