CA2197306A1 - Cysteine protease and serine protease inhibitors - Google Patents

Abstract

The present invention is directed to irreversible inhibitors of serine and cysteine proteases which most preferably contain a 1-oxytriazole or 1oxyimidazole functionality. Methods for the use of the protease inhibitors are also described.

Description

WO9~14067 PcT~s9s/l4794 PROTEASE AND SERINE PROTEASE lN~ OKS

FIELD OP T~E ~v~-~ L _ We have discovered and in this patent ~s t we disclose novel inhibitors of cysteine or serine proteases, methods ~or making our novel compounds, and methods for using our novel _ '~. - We reier to our compounds as "heterocyclic-~-hetero atom methyl~ketones.;' BA~KUI OF T~E l~V

~umerous cysteine and serine proteases have been i~nt;fj~ in human tissues. A "protease" is an enzyme which degrades proteins or peptides into smaller ~ neLI~8. The terms "cysteine protease" and "serine protease" refer to proteases which are distinguished by the presence of a cysteine or serine residue which plays a critical role in the catalytic process. ~ n systems,~incïuding humans, normally degrade and process proteins via a variety of ~ Anirmc including the actions of cysteine and serine proteases. ~owever, when present at elevated levels or when Ahr~rr lly activated, ~ cysteine and serine proteases are involved in pathophysiological processes.
For example, calcium-activated neutral proteases ~ncalpains") comprises a family of intrAc~llnlAr cysteine proteases which are ubiquitously expressed in I liAn tissues. Two major rAlrA;n~ have been i~nt;f;ed: calpain I
and calpain II_ While calpain II is the pr~ nAnt form in - . ~ SUBSrlllJTE SHEET (RU E 261 .. .

W096114067 2 1 ~ ~ 3 ~ 6 PCT~S9~14794 many tissues, calpain I i3 thought to be the pr~ ncnt form in pi~th~logical conditions of nerve tissues. The calpain family of cysteine proteases has been implicated in many diseases and disorders, including neurodeg~nPrat;~n, stroke, ~7h~ r~s diseage, amyotrophy, motor neuron damage, acute central nervous system injury, qr~ r dygtrophy, bone resorption, platelet aggregation, cataracts and ;nfli tion.
Calpain I has been implicated in excitatory amino-acid induced neurotoxicity disorders including ischemia, hypoglycemia and epilepsy. The lysosomal cysteine protease cathepsin B has been implicated in the following disorders: arthritis, inflammation, myocardial infarction, tumor metastasis, and muscular dystrophy. Other ly~c~_ 1 cysteine proteases include cathepsins C, ~, L and S. Interleukin-l~ converting enzyme ("I OE") is a cysteine protease which catalyzes the formation of interleukin-l~. Interleukin-l~ is an immunoregulatory protein implicated in the following disorders and diseases:
;nfli tion, diabetes, septic schock, rhPn~tQid arthritis, and ~17hP;r---'s disease. IOE has also been linked to the 2C apoptotic cell death of neurons which i8 implicated in a variety of neurodegenerative disorders including Parkinson's disease, ischemia and amyotrophic lateral sclerosis (ALS~ .
Cysteine proteases are also produced by various p~th~g~nc. The cysteine protease clostripain i5 produced by Clostridium histolvticum. Other proteases are produced by ~rvoanossma cruzi, malaria parasites P~ ';um falci~arum and p vinckei and, stre~tococcus strains. Hepatitis A viral protease (HAV 3~ C is a cysteine protease ~cc~nt;iRl for proc~cc;ng of picornavirus structural proteins and enzymes.
Exemplary serineproteases implicatedin degenerative disorders include thrombin, human leukocyte elastase, pancreatic elastase, chymase and cathepsin G. Specifically, thrombin is produced in the blood coagulation cascade1 cleaves fibrinogen to form fibrin and activates Factor VIII; thrombin is implicated in thL~ ' ~hlebitis, thrombosis and asthma.
Human leukocyte elastase is implicated in tissue degenerative disorders such as rheumatoid arthritis, osteoarthritis, SUBS~ITUTE SHEET (RULE 26~

o96~14~67 PCT~S9~14794 ~- 3 -atherosclerosis, bronchitis, cystic fibrosis, and emphysema.
Pancreatic elastase is implicated in pancreatitis. Chymase, an enzyme important in angiotensin synthesis, is implicated in hypertension, myocardial infarction, and coronary heart disease. Cathepsin G i8 implicated in abnormal connective tissue degradation, particularly in the lung.
Given the link between cysteine or serine proteases and various debilitating disorders, , , ~ which inhibit these proteases would be useful and would provide an advance in both research and clinical settings.

- SUMMARY OF TH3 iNv We have developed novel cysteine and serine protease ;n~ih;t~rg which we refer to as ~heterocyclic-N-hetero atom methyl ketones." They are represented by the following formula:
R5 R3 Xt R2 : G-C-C-~-C--C-C-Q-A
R5 X2 R4 i 1 Constituent m~mbers are defined infra. Preferred embodiments are heterocyclic-N-oxy methyl ketones represented by the following formula:
I
'~ O
'~ NH~r'~~~X3 .

Constituent members are defined infra.
20Our ~ '~ are useful for the irreversible inhibition of cysteine and serine proteases. Beneficially, the - compounds find utility in a variety of settings. For example, - ,;, SUBSTITUTE SHEET (RULE 26) . ::~. . . ..
" .
, ., ~ .

WO96/14067 2 t ~ 7 3 ~ ~ PCT~S95/14794 in a research arena, the claimed compounds can be used, for example, as standards to screen ior natural and synthetic cysteine protease and serine protease inhibitors which have the same or similar functional characteristics as the disclosed compounds. In a clinical arena, our compounds can be used to alleviate, mediate, reduce and/or prevent disorders which are associated with abnormal and/or aberrant activity of cysteine proteases and/or serine proteases.
We also disclose methodologies for making our heterocyclic-N-hetero atom methyl ketones.
These and other features of our compounds will be set forth in P~r~P~ form as our disclosure cnnt;n-1PR.

n~ATT-~n Vr~K~ UN OF T~E ~K~rrKK~U r~ D
We have discovered novel cysteine and serine protease inhibitors which are represented by the general formula:

R~ R3 X' R2 G--C--C-M-C--C--C--Q--A
R~ X~ R4 R~

wherein:
M is O, NR7 or CR1R7, and most preferably NR7;
Xl is O, S or NR7, and preferably 0;
-- X~ is O, S, NR7 or two hydr~ atoms, and preferably 0;
Q is O, S or NRl, and preferably O;
R1 and R2 are ;n~PpPn~Pntly H, allyl having from l to lC carbons, heteroaryl having from l to lO carbons, alkalnoyl having from l to lO carbons, or aroyl, wherein the alkyl, heteroaryl, alkanoyl and aroyl groups are optionally substituted with J;
R3, R4, Rs and R6 are ;n~PpPn~Pn~ly H, alkyl having from l to l-O carbons, aryl, or heteroaryl, wherein the alkyl, aryl and heteroaryl groups are optionally substituted with J;
Preferably, R1, R2 and R4 are H; and R3 is H, n-butyl, SUBSTITUTE SHEET (RULE 26~

096/14067 2 ~ 9 7 3 ~ G PCT~S95/14794 isobutyl or benzyl;
R7 and R8 are ;n~ tly H, alkyl having from 1 to ~ 10 carbons, aryl, or heteroaryl, wherein the alkyl, aryl and heteroaryl groups are optionally substituted with J;
J is halogen, CooR7, R70Co, R70CoNH, OH, CN, NO2, NR7R7, N=C(R7)RB, N=C(NR7R8)z, SR7, oR7, phenyl, naphthyl, heteroaryl, or a cycloalkyl group having from 3 to 8 carbons;
G is N~7.~, NHRl, CH,R1, CH~C(O)B, carbobenzyloxy-NH, succinylNH,R70-succinyl-NH,R70C(O)NH,-CH,C(O)-(xanthen-9-yl), CH,COR' where R' i8 an alkyl, aryl, or arylalkyl group of up to 13 carbons; or AA'NHC(O)OCH,C~Hs where AA is one of the 20 natural amino acids or its opposite AntiroA~;
-- B is alkyl having from 1 to 10 carbons, aralkyl havi~g from 1 to 10 carbons, aryl having 1 to 3 carbocyclic rings, or heteroaryl having 1 to 3 rings, wherein the alkyl, aralkyl, aryl and heteroaryl groups are optionally substituted with J; and A has the structure:

~N / ~N
D
~W~
E F

wherein Y is N or CRl;
W is a double bond or a single bond;
D is C=O or a single bond;
E and F are ;n~r~n~rtly Rl, R', J, or when taken together E and F comprise an aliphatic carbocyclic ring having from 5 to 7 carbons, an aromatic carbocyclic ring having from 5 to 7 carbons, an Ai;phAt;c heterocyclic ring having from 5 to 7 atoms, or an aromatic heterocyclic ring having from 5 to ? atoms; wherein.. the Ai ;rhAt;C heterocyclic ring and the - aromatic heteroryclic ring each have from 1 to 4 heteroatoms;

; - 'SUBSTITUTE 5HEET (RULE 26) ....

~Iq7~
W09C114067 PCT~S9~14794 and the ~lirh~tiC carbocyclic ring, the aromatic carbocyclic ring, the ~liph~t;c heterocyclic ring, and the aromatic heterocyclic ring are each optionally substituted with J.
Preferred embodiments of the invention have the formula:
J~ O
,~ NH~X3 O P

wherein:
R is NHC~O)OCH2C6H6, -CH2ClO)-(xanthen-9-yl) or -CH2C~O)CHlC6Hs)C2Hs.
P~ i6 isobutyl, isopropyl, benzyl, ethyl or carboxyalkyl of 2-9 carbons; and X3 has the formula:

D

X'~X~

X X

wherein:
D is C=O or a single bond;
X' is CH, CCl, CCH3, CF or N;
~ Xs is H, CH1, Cl, OCH3 or F;
X6 is H, CH3, Cl, F, OCH3, CF3, ethyl or phenyl;
X7 is N, CCl, CH, COCH3 or CF; and Y is N or CH.
In some preferred embodiments, K is -CH2C(O)-SUBSmUTE SHEE~ (RULE 26) 096114067 PCT~S95114794 ~xanthen-9-yl) or ~,Lub~l.zyloxy-NH, and ln other preferred ~; c, P3 is benzyl, igobutyl or ethyl.
In further preferred ~; ~R Y is N. Preferably, X3 i8 0-l-oxybenzotriazole, or X7 is N, or Y is CH, or Y is N
and D is C=0.
I~ some ~ '_'; c Q i8 NR3, or R3 and R are both not ~. In other ~mho~; ~c one of R3 or R2 is a group other than H. In further rmh~ c, X1 ig S or NR', or M is 0 or CRlR2 _ ' In other ~ tC X2 i8 S, NR1, or two hydrogen atoms, or R has the formula:

O ~
--CH2-C~ 0 :
:
As used herein, the term "alkyl" is meant to include straight-chain, branched and cyclic hydrocarbon groups such as, for example, ethyl, isopropyl and cyclopropyl groups.
Preferred alkyl groups have l to about lO carbon atoms.
"Cycloalkyl" groups are cyclic alkyl groups. "Aryl" groups are aromatic cyclic ~ _ lc including but not limited to phenyl, tolyl, benzyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl The term "carbocyclic, n as used herein, refers to cyclic groups in which the ring portion is composed solely of carbon atoms. The term "heterocyclic" refers to cyclic groups in which the ring portion ;nr~ Pc at least one heteroatom such as 0, N or S. ~Heteroalkyl~ groupg are heterocycles crn~in;ng ~olely single bonds within their ring portions, i.e. saturated heteroatomic ring systems. "Alkanoyln groups are those which contain an alkyl portion linked through a carbonyl group.
nAroyl" groups are those which contain an aryl portion linked - through a carbonyl group. "Aralkyln groups have both aryl and ,.
SUBSmUTE SHEET (RULE 26) W096/14067 Z~ PCT~S9~/14794 alkyl portions, and are ~tt~rhPd through their alkyl portions.
Because the disclosed compounds are useful in lnhibiting the activity of serine and cysteine proteases, and because the usPfl~lnp~3-3 of such compounds can be applied to both research and therapeutic settings, methodologies for inhibiting the activity of cysteine and serine proteases by contacting the protease with a compound of the invention include providing the compound to a mammal, including a human, as a I ';~ or pharmaceutical agent.
As used herein, the term "contacting" means directly or indirectly causing at least two moieties to come into physical association with each other. Contacting thus includes physical acts such as placing the moieties together in a ~nnt3;n~r, or administering moieties to a patient. Thus, for example, administering a compound of the invention to a human patient ev;~3Pn~;ng a disease or disorder associated with abnormal and/or aberrant activity of such proteases in a method for ;nh;h;t;ng the enzymatic activity of such protease which are associated with disease or disorder, falls within the scope of the definition of the term ~contacting.
As used herein, the terms "inhibit~ and "inhibition~
mean having an adverse effect on enzymatic activity. The term "irreversible," when used to modify "inhibit~ and "inhibition,"
means that such adverse effect on catalytic activity can not be reversed once it is initiated. Inhibition of cysteine or serine protease activity can be ~ptp~n;npd using a variety of methodologies. Two convenient -- h~l3~10gies are preferred.
The first involves determining the rate of inactivation of a protease using a ~ ~ ' of the invention; the second involves determining the percent inhibition of a defined amount of the protease by a 3 of the invention. With respect to the cysteine protease calpain I, a whole cell assay, which measures inhibition of calpain I activity via a decrease in the amount of cleavage of a preferred calpain I substrate, ~-spectrin, is also useful in determining the inhibition of catalytic activity.
In a research environment, preferred ou...~uu-lds having SUBSTITUTE SHEET (RULE 26) 21 ~730i~
~ Wo96/liO67 PCT~S9~14794 _ 9 _ defined attributes can be used to screen for natural and synthetic . ul-ds which evidence similar characteristics of n~; hit; ng protease activity. The uilds can also be used in the r~f;n of in vitro and ir, vi w models for determining the effects of inhibition of particular proteases on particular cell types or bi~lsg;ci~l conditions.
Pharmaceutically acceptable salts of the cysteine and serine protease inhibitors also fali within the scope of the c _~uullds a6 disclosed herein. The term "phar~-~e~lt;ci~lly acceptable salts" as used herein means an inorganic acid addition salt such as hydrorhl~r~ sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, islki~l;n~ earth metal salts such as magnesium~salt ànd calcium salt, aluminum salt, and zinc salt.
Examples of pharmaceutically acceptable ammonium salts are ammonium salt and tetramethyli ; nm salt. Examples of pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of pharmaceutically acceptable amino a~ ;tion salts are salts with lysine, glycine, and pheny~ qnin~.
Compounds provided herein can be formulated into pharr~ceut; riil compositions by admixture with pharr-~eutiri~lly acceptable n~nt~ excipients and carriers. The compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions;
or oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, via, for example, trans-derm-al patches; or prepared in other suitable ~ n~ for these and other forms of administration as will be d~ enL to those skilled in the art.
The composition may conveniently be administered in unit dssage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in ~ Remington~s ph~r~-cpl~tical Sciences (Mack Pub. Co., Easton, PA, SUES~ITUTE SHEET (RULE 26i Wo 96/14V67 ~ PCrlUS95/14794 1980). Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydLugPI1ated n~rhthAlenes and the like. In particular, 5 bio~ _-t;hle, biodegradablelactidepolymer, lactide/glycolide copolymer, or polyoxyethylene-poly~y~L~.~ylene copolymers may be useful excipients to control the release of the active compounds. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl 10 acetate copolymer particles, osmotic pumps, ;~ nt~hle infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be a~ueous solutions r~rt~;ninS, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, 15 or oily solutions for administrationln the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may al80 include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration. For~nnl~t;rnq for 20 transdermal patches are preferably lipophilic: 1 qirnq.
The materials of this invention can be employed a6 the sole active agent in a pharmaceutical or can be used in combination with other active ingredients.
The c~nrPntrations of the ~ described herein 25 in a therapeutic composition will vary rlPrPnr~in~ upon a number of fac~:ors, ;nrln~;ng the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the c v~ Ju.ld8 employed, and the route of administration In general terms, the compounds of this 30 invention may be provided in an aqueous physiological buffer solution crnt~;n~ng about 0.1 to 10~6 w/v. compound for parenteral administration. Typical dose ranges are from about 1 ~g/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight 35 per day. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or ~ P~, the overall health SUBSTITUTE SHEET(RULE 26) WO96114067 ~ PCT~S9Y14794 atatus of the particular patient, the relative biological e~icacy of the c~ ulld selected, and form~ n of the c _ ~ excipient, and its route of administration.
~ The ~ of the invéntion are r -h~n; rT-ba8ed i-.~v~Y;hle ;nhih;t~rs of cysteine and serine proteases which we believe, although not wisking to be bound thereby, provide a novel -h~nir~ of proteage inactivation. The inkibitors most preferably contain a l-oxytriazole, 3-oxytriazin-4-one, or l-oxy;m~7~1e functionality. It has been found that the ,: _~onn~r of the invention are irreversible inhibitors. While not wishing to be bound by any specific theory, it is believed that linkage through the N-hetero, preferably N-oxy bond, makes the l-oxytriazole, 3-oxytriazin-4-one, or l-oxy;m;~7O1e moiety a superior leaving group, thus facilitating inactivation upon lS interaction with the target protease. Scheme l depicts a proposea 1 ~-h~n; r~ of inactivation of a cysteine protease by an inhibitor of the invention:

Scheme l G~ NZ

G ~

B5 01~ ~NZ B5 ~
~- G~ G~NZ

.
.
. , , SUBSnTUrE SHEET (RULE 26 -~ ' - . ' .. .. ~.t~

W096/14067 ~ Q' ~ PCT~S9~14794 The invention is further illustrated by way of the following examples which are ;nt~n~d to ~1l7ri~te the invention. These ~ q are not intended, nor are they to be construed, as limiting the scope of the disclosure nor the appended claims.

Example lA:
Inhibition and Rate of Cysteine Protease Activity To evaluate inhibitory activity of our compounds, stocks (40 times rnnc~ntrated) of each compound to be tested were prepared in 100~ anhydrous dimethyl sulfoxide (DMSO) and 5~1 of each inhibitor preparation was ali~uoted into each of three wells of a 96 well plate. Calpain I was purified from human red blood cells using a modification of the method described by Lee, W.J. et al. (Biochem.
Internatl. (1990)22(1): 163-171). Briefly, 3 units of packed outdated red blood cells were washed 3x by repeated centrifugation at 500rpm for 10 minutes through 900mls of 0.9~ NaCl. Cells were lysed in 20mM Tris/lmM EDTA/lmM
EGTA/5mM mercaptoethanol (Buffer A) and centrifuged for 1 hour at 12,000 rpm in a GSA rotor using a Sorval centrifuge.
The supernatant was collected and applied to a DEAE-sepharose FF column ecl-;l;hn~t~d in Buffer A + 25mM NaCl.
After washing the column in Buffer A + 25mM NaCl, the bound protein was eluted at 117ml/hr with a 25mM to 150mM NaC1 linear gradient in Buffer A rnllent;n3 200, lOml fractions.
Two ~l of every fifth fraction was applied to nitrocelluose using a dot blot apparatus and calpain cnnt~;n;ng fractions i~nt;fied by Western analysis. For detection of calpain, nitrocellulose sheets were first blocked in 5~ Blotto (5~
Carnation instant milk/lOmM Tris/150mM NaC1) for 30 minutes, followed by ;ncllhation for 1 hour in antibody directed against Calpain I (rabbit anti-human calpain I polyclonal sera, 1:1000 dilution in 5~ Blotto). After three 5 minutes washes in lOmM Tris/150mM NaC1/.05~ Tween 20 the nitror~llnlnse was incubated for 1 hour in secondary ~1 k~l ;n~ phn5rh~t~qe conjugated antibody (Biorad Cat#170-SIIBSTITUTE SHEET ~RULE 26) 2 ~ 973~
. .
96/14067 PCT~89~l4?94 ~ - ~. 13 -6518 1:2000 in 5~ Blotto). After three 5 minutes washes in lOmm Tris/i50mM NaClj.05~ Tween 20 and 1 wash in lOmM
Tris/150mM NaCl the nitrocellulose was ;nnl~h~t~ for up to 2 hours in a n~l nr; triC substrate solution (Biorad Alakaline Phosphatase Substrate conjugate kit, cat#l70-6432). The reaction was stopped by washing in water. After fr~rt; nn~
rnnt~;n;ng calpain, and those in between, were pooled, Eolid ammonium sulfate was added to achleve a 30~ solution and the mixture stirred for 1 hour at 4~C. Following collection of the precipitate by centrifugation for 1 hour at 4~C at 12K
rpm in a GSA rotor, the supPrn~tAnt was collected and brought to 45~ in ammonium sulfate. After stirring for 1 hour at 4~C, centrifugation was repeated. The supPrn~tAnt was discarded and the precipate resuspended in approximately 7mls and dialyzed overnight at 4~C against Buffer A + 50mM
NaCl. The sample was then applied to a S300 gel filtration column preequilibrated in Buffer A + 50mM NaCl, washed and eluted at a flow rate of 20ml/hr, rollpnt;ng 200, 4 ml fr~ct;nnq. The peak of calpain was determined by assaying aliquots of every fifth Cr~rt;nn for enzyme activity monitored by the hydrolysis of a fluorogenic dipeptide substrate as described below. The peak fractions were pooled and the enzyme prepa~ation used to test ~ ~ for inhibitory activity. As an alternate to enzyme isolated from tissue sources, reF ~ ~ ;n~nt human calpain I has also been used to monitor _ A~ for inhibitory activity.
The foregoing enzyme preparation was diluted into assay buffer (i-e-, 50mM Tris, 50mM NaCl, lmM EDTA, lmM
EGTA, and 5mM ~mercaptoethanol, pH 7.5 including 0.2mM Succ-Leu-Tyr-MNA) and 175~1 aliquoted into the same wells ~nnt~;n;ng the ;n~pPn~Pnt inhibitor stocks as well as to positive control wells nnnt~;n;n~ 5~1 DMSO, but no ~.
To start the rP~nt;~n, 20~1 of 50mM CaCl2 in assay buffer was added to ail wells of the piate, excepting three, which were used as background signal baseline controls. Substrate hydrolysis was monitored every 5 minutes for a total of 30 - minutes using Flouroskan Il (ex=340nM; em=430nM). Substrate SlITUTE SHEE~ (RULE 26) ~ ~ ~7~
WO96/14067 ~ PCTrUS9~rl4794 hydrolysis in the absence of inhibitor was linear for up to 15 minutes.
To demonstrate activity agalnst two other cysteine protease3, cathepsin B ~Calbiochem, cat#219364) and cathepsin L ~Calbiochem, cat#219402), assays were performed subst~nt;Ally the same as o~ltl;nPd above except that the cathepsin B and ~thPp~in L were diluted into a different assay buffer consisting of 50mM sodium acetate (p~ 6.0)/lmM
EDTA/lmM ditheothreitol and 2 the substrate used was Cbz-Phe-Arg-AMC (Bachem cat# I-1160; O.lmM for cathepsin s;
.006mM for ~thPp~in L). ~;t;nn~lly, the order of reagents added to the plate was altered because both enzymes are constitutively active. Following inhibitor addition to the plates appropriate 2x ~nncPntrated stock dilutions of the enzyme preparations were made in assay buffer and lOCul added to each well. The assay was initiated by addition of lCOul of 2x cnn~pntrated stock dilution of substrate in assay buffer. Substrate hydrolysis was monitored using a Fluofoskan II lex=390nm; em=460nM).
Inhibition of enzyme activity was calculated as the percent decrease in the rate of sub3trate hydrolysis in the presence of inhibitor (ui) relative to the rate in its absence (uO). Compari30n between uO and ul was made within the linear range-for substrate hydrolysis. For screening, ,- _ '~ were tested at 10 ~M. ~ , ul-ds having 2 50~
inhibition at 10 ~M were rnn~i~Pred active. Apparent second order rate constants were determined from analysis of reaction progress curves under pseudo-first order conditions. Each determin~t;nn represents the mean of three or more ;nA~rPn~Pnt single cuvette analyges ront;n~ ly monitored via a Perkin-Elmer LS50B spectrofluorimeter. The rate of inhibition of hydrolysis was obtained by fitting the curve to the ~pnnPnt;~l equation ~

y = Ae~~ob~-t + B ~1) where y ~Pt) is the product formed at time t. A and B are SUE;SrlTUTE StlEET tRuLE 261 96114067 2 1 ~ 7 3 ~ 6 PCT~S9~/14794 ~ ~ - 15 -constants. A, the amplitude of the reaction, is given by [P~-P~] and B (= P~) is the maximal product formed when the reaction ~i8 determined as k~,/[I]. This was corrected for the presence of equation (2):

k2~k.~l+[S]/~) (2) Values for k2 are provided in Table IA.

- ' ' ! , .
;
'.

~ SUBSlITUT~ SHEET ~ULE 26) . ~ , .

WO 96/1406~ ? 7 ~ Q ~ PCT/US95/14794 ;~

Table IA
J~ o K f ~~ _ O
K~ZNH,LorW
w. ~ L, \~0 z~ J~o ~

Compound P,iC i~,xlO' oi E~atmplo: X3 ~M 's-'l A'B'C' 3 1: ~: BereyiZNH150 100117 4 1 ~ BulyiZNHI?5 19 1. ~: EthylZlYH633000443 6 3-oxy~3H}tri~1O[4.5-i~]pyridine BenzylZNH184 40107 7 6~ ' ' BenzylZNH160 18 1 0 8 6 o i: ~ BenzyiZNH 101 9 6-methoxy 1 . ~: BeruyiZNH125 52 ti - I : Bem~yiZliH 50 Il 6-chloro-5: J' I . ~: BenzylZNH 98 28 12 1 . ,: I VA~ ' BeruylZNH 46 13 5-: ts: /11: ~: BenzylZNH11629 14 1. 1~: BemylZNH157 6-phenyl-1: ,: BenzyiZNH 77 16 16 4,5,6,7 . I ,: BeluyiZNH 93 17 5: 1: ,: BenzyiZNH 164 18 5,ts l~ ' BesuylZNH 65115 19 1 oxy-(lHA~-triluolo[4,5blpyridine BenzylZNH 57 4,5,6,7. 1. ~I BenzylZNH 1.3 21 4,6,7-i 1: ~L BenzyiZNH 1.8 10 22 1 ~ Bem~ylZNH 4 23 g~5: I VA~8 ' ' BenuyiZNH 64 24 5~ ti ~ BereylZNH 84 30 4.54ifluorr~ : BenzylZNH 67300 26 6-methyl-1 c~: BeruylZNH112 27 5: Jl I c~: BenzylZNH230 3 0 28 3 . ~ BenzylZNH122 70 7 29 5,7-dimethoxy-3: ~8 1: BenzyiZNH 171 6~hloro-3. ~: 1. BeruyiZliH180 , SUBSTITUTE SHEET (RULE 26) 2 jl 9730~

Table IA (continucd) - Compound P,i; k~10 of En;lmple: X3 (M~s ') 'C
31 3. ~ BuylZNH 46 39 1 c.~' 'Ben~ylL 46 '5 1~ BuylL 2.7 48 1 0 46 l: ~' ' BenylW 3 ~ A - Calpain 1: B - Cathepsin L: C - Cathcpsin B
~- Examples 32-38 and 41-45 are synthetic intenmedi-tes in the pneparation of the compounds of examples 39. 40 and 46. n~spectively.
Ex~mple ls: Inhlbltlon of 8-rln- Prot-a~- Activlty To demonstrate activity against the serine .: .
protease a-chymotry-psin (Sigma Chem. Co. cat# C-3142) the protocol of Example lA was followed except that the enzyme was diluted into assay buffer consisting of 50mM Hepes (pH
7.5)!0.5M NaCL and the final substrate mnn~ntrAtinn used was o.03mM Succ-Ala-Ala-Pro-Phe-A~C ~Bachem, Inc. Cat# 1-1465). Additionally, because a-ch-ymotrypsin is not a calcium sensitive enzyme and is constituitively active, followlng addition of inh;hitnr stocks to the 96 well plates, lOOul of a 2 fold cn-llm~ ed stock of enzyme in ~;3~lt;nn buffer was first added and the reaction started by addition of lOOul of a 2 fold cnn~ntrated stock of substrate in assay buffer. Substràte hydrolysis was monitored every 5 minùtes up to 30 minutes using a .
Fluoroskan II (em=390nM ex-460nM). Results, expressed as ~

;nh;h;t;nn of ~-~y, LLy~gin at lO~M, are presented in Table IB.

- - . , iSUBSTlTUTE SHEET (RULE 26) .. j .

W 096/14067 ~ 3 ~~6~ P~rrUS9SI14794 Tahle IB

Compound of Example:~ Inhibition of ~-Chymotrypsin at lO~M

Ex~mple lC: Inhibition of Calpain Activation in Intact Cells Sodium Dodecy/Sulfate-Polyacrylamide Gal Electrophoresis (SDS/PAGE)/Coomas6ie stainjDensitometric analysis of calpain cleavage of endogenous substrates has Berved a8 a standard method for measuring inhibition of calpain activation in intact cell systems following exposure to calcium and ionophore ~Mehdi et al., 1988, Biochem.
Biophys. Res. Conunum., 157:1117-1123; McGowan et al., 1989, Biochem. Biophys. Res. Co~runun. 158:432-435; Hayashi et al., 1991, Biochem. Biophys. Acta. 1094:249-256) . In our analysis, we monitored the degradation of a preferred calpain substrate, the ~-subunit of non-erythrocyte spectrin, using two ;n~p~n~nt ~ntiho~;es which recognize the two 150 kDa cleavage products specifically generated by calpain proteolysis ~Roberts-l.ewis et al., 1994, IJ.
Neurosci. 14~3934-3944) . The use of these ~ntihQr~;es has greatly far;l;tat~d the evaluation of calpain inhibition in intact cell systems. For intact cell assays, the human SUBS~lIU~ SHEET (RULE 26) 2l 9~30f~
~ WO 96/14067 ~ . e PCT/US95/14794 lymphoid celL~line Molt-4, in which the calpain I isozyme ~L~ ~ ;n~tPq (DPqhr~n~p et al., 1993, Neurochem. Res. 18:
767-773) was chosen for inhibitor screening. The ef_ectiveness of our ~ q in intact ~olt-4 cells is measured as a decrease in the amount of calpain-generated spectrin breakdown products compared to the amount gpnpr~ted in the presence of calcium and ionophore alone. Molt-4 cells were first washed and subsequently resuspended in ~epes-Buffered Saline ~5.4mM KCl, 120mM NaCl, 25mM glucose, 1.5mM MgSO~, lmM sodium pyruvate, 20 mM Hepes p~ '.7.0) to lx107 cells/ml. Test compounds were _irst solubilized in DMSO to 50mM and subsequently diluted into ~epes-Buf~ered saline to a final concentration of 200~M --;nt~;n;ng 8~ DMSO
final r~nCpntr~ti~n~ Five microliters of inhibitor stock solutions (200~m) were then aliquoted into each of three wells of a 96 well microliter plate, followed by 100~1 of cell suspension. R~ut;nPly, celis were prP;nr~hatP~ with 40~M inhibitor for 10 minutes. Subsequently, 100~1 of ~epes-buffered saline solution rrnt~;n;ng 20~m calcium - 20 ;~nrFhnre (ionomycin (Sigma, St. Louis, MO, I-0634)) and 5mM
CaCl2 was added to the cells and allowed to incubate for up to 30 minutes. The calcium was then chelated by addition of 2~1 of lM FDTA and the cells were~_arvested by cPntr;fugation in a Beckman table top rPntr;f-~ge. The ~nrPrn~t~nt was removed and the cells lysed by addition of 20mM Tris-~C1 p~ 3.0jl~ NP-40/.137M NaC1/13mM EDTA/iO~g/ml aprotinin/lO~g/ml leupeptin/.lM PMSF . Tnqol llh] e material is - removed by c~ntr;fug~t;on and the protein c~nrpntration of -~ SUBSTITUTE SHEET (RULE 26) , WO96114067 2 1 ~7306 PCTIUS95114794 ~

the lysates ~t~r~;nPti by a BCA micro protein assay (Pierce, Inc., Rockford, I~). Twenty mi~L~y~ .s of each sample was then applied to a 6~ SDS-PAGE gel and electrophoresed for 45min at 200V (T.AI 1;, U.K. 227 Nature 6B0, 1970).
Electrophoresed protein is then transferred to nitrocellulose (Towbin, H et al. 76 PNAS 4350, 1979). For dètection of spectrin breakdown products, nitrocellulose sheets were first blocked in 5~ Blotto (5~ Carnation instant milk~lOmM Tris/150mM NaCl, pH 8.0) for 30 minutes, followed by ;nrllhstion for 1 hour in antibody directed against spectrin breakdown products (Ab 38 and/or 41; Roberts Lewis et al., J. Neurosci 14, 3934-3944, 1994, 1:500 dilution in 5~ Blotto). After three 5 minute washes in 10mM Tris/150mM
NaCl/.05~ Tween 20 the nitroc~ellulose is incubated for 1 hour in secondary alkaline phosphatase conjugated antibody (Biorad, Hercules CA Cat# 170-6518 1:2000 in 5% Blotto).
After three=5 minutes washes in 10mM Tris/150mM NaCl/.05 Tween 20 and 1 wash in 10mM Tris/150mM NaC1 the nitrocellulose iB incubated for up to 2 hours in a - 20 colorimetric substrate solllr;rn (Biorad ~12k2~;n~
ph~srh2t2qe Substrate conjugate kit cat#170-6432). The reaction is stopped by washing in water. After the nitror~ 1rse sheets are drird the amount of spectrin breakdown products detected is guantified using a BioQuant-Osk image analysis system (R&M Biometrics, Inc., Nashville, TN). The amount of breakdown products in c ~ d treated cells is compared relative to the amount in non-compound treated cells and expressed as the ~ inhibition of breakdown SUBS~ITUT~ SHEET ~RULE 26) W096114~67 PCT~S9~/14794 ~ 21 - ~
products ("~DPs"~. Result~ are presented Ln Table IC.

: .. .

.
. .

,~ .

, ,"
- -; . :

~i .

, - SUBSTITUT~ SHEET (RU-E 26) ~. ; . . -, -- -~096114067 2 i ~ 7 3 ~ ~ PC~US95/147g4 TA8~E IC
Compound of Example:~ Inhibition of 8DPs llO~M) Synthe~is of r 1~ry C _- ~
lS HPLC analysis and purification of final products and int~L, ';i~t~q was conducted under the conditions described in each example using a VyDac reverse-phase C-18 lO micron column (1.0 x 25 cm) at a flow rate of 3.5 ml/min.
coupled to a ~V detector.
The use of Ag20 in the alkylation of alcohols is described in T.W Creene et al., Protective Grollp~ in Org~nic Synthesis, New York, N.Y. John Wiley ~i Sons, Dec.
1991, pp. 15 and 48.

Starting ~aterials:
1-Xydroxybenzotriazole can be purchased from various commercial sources (e.g , Aldrich Chemical Company) and was used as recei~ed. All other benzo~r; ~7~ were prepared according to the procedures described in 8rady, 0.
L. et al., ~. Chem. Soc. 37, 2258-2267 (1960) ; Ronig, W. et al , Chem. ~er. 103, 788-798 (1970), and Carpino, L. A., ~.
Am. Chem. Soc. 115, 4397-4398 ~1993). 1-~ydL~yb~7imi~7Qle was prepared according to Seng, F. et SUBSTITUTE SHEET (RULE 26) 2 ~ ~73~
096/i4067 ~ ~ L ~ , PCT~S9~l4794 ~ ~- 23 - ~, al,, Syn~he~is~:1975, page 703. Leucine chloromethylketone and phenyl~l ~n~ n~ chloromethylketone can be purchased ~rom various commercial sources (e.g., BACH8M Bioscience, Inc.) - and were used as recelved, Amino acid or N-t~rmin~l protecte-d dipeptide b,romomethyl ketones were prepared ~rom - the COL1 ~ ;ng diazomethylk~t~n~ by treatment with E'13r/AcOH or ~3r (gas~ according to the standard proc~nres cited and described in Harbeson, S.~. et al., ~. Med. Chem 32, 1378-1392 (1989).

.
Exnmple 2 .. ~
Methods A and s are general methods ~or preparing compounds o~ ~he invention ~rom halomethylk~nP~ 1 and 2.
.

Dipeptide halomethylketone 1: m.p. 135.5-136.5~C , ~ ~ r and dipeptide h~l om~hylketone 2: m.p. 103-104~C

, I

o3bH

SUBS~ITIJTF SHEET ~RULE 26~

W096114067 2 ~ 973a6 PCT~S95/14794 Method A: To a solution of the appropriate bromo or iod~k~nn~ (0-05-0.1 mmol) and an N-hydroxyheterocycle ~1.1 eq.) in 1 m~ of dimethylformamide was added Ag20 (1.1 -2.2 eq.) under inert a -~lh~re The reaction mixture was stirred at room temperature m;~imiz;ng exposure to light for 0.5- 72h. The mixture was then diluted with ethyl acetate and filtere through a pad of ~;~t~ ~~nus earth. The filter pad was thoroughly washed with ethyl acetate and the c~ ~ n~ filtrateg were waghed twice with 1 volume of H20 and 1 volume of brine. After drying over ~nhydrous magnesium sulfate, and filtration, the solvent was removed under reduced pressure. The desired product was isQlated and purified by HPLC as described for each example.
Method B: To a solution of the appropriate brn~k~tnn~ (0.1-0.15 mmol) in dry dimethylformamide was added anhydrous potassium fluoride (3.5 eq.), and the mixture was stirred at room temperature for approximately ~
min under an inert ~ ~h~re. An N-hydroxyheterocycle (1.2 eq.) was added, and the resulting mixture was stirred for 24h. The reaction mixture was diluted with ethyl acetate, washed su~c~RR;vely with 1 volume each of water, saturated aqueous NaHC03, 10% aqueous citric acid, water and finally with brine. After drying over magnesium 8ulfate, and filtration, the solvent was removed under reduced pressure.
~ 25 The desired product was isolated and purified by flash chromatography and/or HPLC.

SUBSTITUTE SHEET (RULE 26) ~ WO96/14067 ~;' PCT~S95/14794 .
s ~ 25 -Exucple 3 l-[N-[N-Benzyl~Ay~LL~l~yl-L-leucyl]-3s-4-phenyl-3-~mlno-2 oxobutyloxy]benzotriazole.

J~ ~ c Z~N J ~

Method A; reaction time l9h; purification: flash chromatography (hexane:ethyl acetate 1:1) followed by HPLC
(reverse phase, acetonitrile:water (~nnti~;n;ng 0.1~
trifluoroacetic acid) 10~-100~ over 40 min) R~ 30.10 min;
yield 66~; mp 128.5-i30C; lH NMR (300 MHz, CDCl3): ~ 8.00 (d, IH), 7.83 (d, lH), 7.56 (t, lH), 7.43 (t, lH), 7.38-6.98 (m, lOH), 6.57 (m, lHj, 5.29 (bd, lH), 5.06-4.9 (8 overlapping with m, 4H), 4.76 (q, lH), 4.12 (m, lH), 3.00 (m, 2H), 1.7-1 3 (series of m, 3H), 0.98 (m, 6H); FA~3MS m/z 544 (MH);
Anal. C (66.23), E (6-07), N (12.83); calc. c (66.29), H
(6.07), N (12.89).

Exumple 4 l-[N-[N-Benzyloxycarbonyl-L-leucyl]-3S-5-~ethyl-3-~ino-2-~ . -. oxohexyloxy]b -L~lazole.
- , , ,, ~

~SUBSllTUTE SHEET tRULE 261 WO96114067 2 ~ PCT/US951147g4 - 2Ç -~ ~ o Il ~ ~N

Method B; reaction time 24h; purification: flash chromatography (hexane:ethyl acetate 1:1) followed by HP~C
(reverse phase, acetonitrile:water (~nt~;nin~ 0.1~
trifluoroacetic acid) 10~-100~ over 40 min) Rt 29.31 min;
yield 43~; lH NMR (300 MHz, CDCl3): ~ 7.96 (m, lH), 7.81 (bd, lH), 7.53, m, lH), 7.44-7.16 (m, 6H), 6.7 (bd, lH), 5.41 (m, 2H~, 5.24-5.00 (g overlapping with m, 3H), 4.62 (m, lH), 4.18 (m, lH), 1.72-1.35 (m, 6H~, 0.92 (m, 12H); FABMS m/z (510, MH+).

~x~mpl~ 5 1-[N-[N-Benzyloxycarbonyl-L-leucyl]-3S-3-amino-2-oxopentyloxy] benzotriazole.

- 15 Z~N~

SUBSTITUTE SHEET ~RULE 26) 21 ~7~
WO96114067 ~CT~S95li4794 . - - 27 -Method A; reaction time l9h; purification flash ~llr. tn~rapy (hexane:ethyl acetate 1:1) followed by HPLC
(revèrse phase, acetonitrile:water~(rnnt~;n;nrJ 0.1~
trifluoroacetic acid) 10~-100~ over 40 min) Rt 30.10 min;
yield 24t; IH NMR (300 MHz, CDCl3): b 7.98 (d, lH), 7.83 (bd, lH), 7.S3 (m, lH), 7.38 (m, lH), 7.30 ~s, 5H), 6.69 (b d, lH), 5.40 ~s, 2H), 5.15 (d, lH), 5.07 (8, 2H), 4.58 (m, lH), 4.15 (m, lH), 1.90 (m, lH), 1.75-1.4 (m, 4H), 0.92 (m, 6H), 0.83 (bt, 3H); FA;3MS m/z (482, MH+).

Example 6 3-[N-[N-Benzyl~y~ b~yl-L-leucyl]-3S-4-phenyl-3- ~ino-2-oxobutyloxy]-3H-triazolo[4,5-b]pyridine.

Method A; reaction time: 18h; yield 36~;
pur;~ir~t;rn: HPLC (reverse phase, acetonitrile:water (rrnt~ining 0.1~ trifluoroacetic acid) 10~-100~ over 40 min) Rt: 28.25 min' lH ~MR (300 MHz, CDCl~ 8.78 (m, lH), 8.44 ~ (bd, lH), 7.49 (m, lH), ~7.44-7.12 (m, lOH), 6.83 (m, lH), 5.43-5.00 (m, 6H), 4.16 (m, lH), 3.35-3.05 (m, 2H), 1.69-1.35 (m, 3H), 0.92 (m, 6H); FA;3MS m/z (545, MH+) SUBS~I~UTE SHEET (RULE 26) - ~

- L

WO96114067 2~ t 3 ~ $ PCT~S9~14794 Example 7 l-[N-[N-Benzyl~y~Lv~yl-L-leucyll-3s-4-pheny-3-amino-2 oxobutyloxy]-6'-trlf 1UOL, ~ hY1benZOtriaZO1e .

Z'i~

Method A; reaction time: 15h; yield 12%;
purification: HPLC Irever~e pha~e, acetonitrile:water (cnntA;n;ng 0.1~ trifluoroacetic acid) 10%-100~ over 40 min) Rt: 32.90 min; ~H NMR (300 MHz, CDCl3): ~ 8.23 (s, lH), 8.10 (d, lH~, 7.63 (d, lH), 7.43-6.86 (m, lOH), 6.52 (m, lH), 5.33 (m, lH), 5.15-4.86 (~ overlapping with m, 4H), 4.69 (m, lH), 4.09 (m, lH), 2.95 (m, 2H), 1.66-1.26 (m, 3H), 0.89 (m, 6H); FABMS m/z (612, MH+).

Ex~mple 8 l-[N-~N-Benzyl~y~ L~yl-L-leuayl]-3s-4-phenyl-3-amino-2 oxobutyloxy]-6'-chloL~L_ zo~iazole.

-- ~ Cl .

SU8S~ITUT~ SHEET (RULE 26) 3 ~ 6 WO9~14067 PCT~S95114794 ~ - 29 -Method Ai reaction time: 15h; yield 30~;
purification: HPLC (reverse phase, acetonitrile:water (C~nt~;n;n~ 0.1~ trifluoroacetic acid) 10~-100~ over 40 min) ~: 32.21 min; 3H NMR (300 MHz, CDCl3): ~ 7.90 (m, 2H), 7.46-6.95 (m, llH), 6~.60 (m, lH), 5.29 (m, lH), 5.18-4.89 (m, 4H), 4.72 (m, lH), 4.09 (m, lH), 2.97 (m, 2H), 1.67-1.3 (m, I ! ' 3H), 0.89 (m, 6H); FA;3MS m/z (578, MH+).

Exnmple 9 l-[N-IN-Benzyloxycarbonyl-L-leucyl]-3S-4-phenyi-3-a~ino-2-10 oxobutyloxy]-6i-methu~yL_.~zutri~zole.

N

~c1~30J~

Method A; reaction time: 49h; yield 7.5~;

purification:~HPLC (reverse phase, acetonitrile:water ~r~nt~;n;ng 0.1~ trifluoroacetic acid) 10%-100~ over 40 min) ~: 31.55 min, 3H NMR ~300 MHz, CDCl3): ~ 7.82 ~dd, lH), 7.46-6.85 ~m, 12H), 6.60 ~m, lH), 5.29 (m, lH), 5.15-4.9 (m, 4H), 4.80 (m, lH), 4.12 (m, lH), 3.93 (S, 3H), 3.03 (m, 2H), 1.67-1.32 (m, 3H), 0.86 (m, 6H); FA;3HS m/z (574.5, MH+).

. ' Example io 1-~N-[N-Benzyloxycnrbonyl-L-leucyl~-3S-4-phenyl-3-amino-2-SUBSTlTllTE SHEET (RULE 26) .
W096/~4067 21 973Q~ PCT~S9~la94 oxobutyloxy~-6'-fluoL~L-~zotLiazole.
z ~

Method A; reaction time: 15h; yield 48%;
purification: HP~C (reverse pha~3e, acetonitrile:water (rnnt~;nin~ 0.1~ trifluoroacetic acid) lO~-100~ over 40 min) ~: 31.33 min; IH NMR (300 MHz, CDCl3): ~ 7.98 (m, 2H), 7.52 (bd, lH~, 7.43-7.09 (m, lOH), 6.69 (m, lH), 5.29 (m, lH), 5.18-4.89 (m, 4H), 4.72 (m, lH), 4.13 (m, lH), 3.00 (m, 2H), 1.66-1 29 (m, 3H), 0.89 (m, 6H); FASMS m/z (562, MH+) SUBSTITUTE SHEET (RULE 26) 2 1 973~

7 PCT~S9~14794 Example 11 l-[N-[N-Benzylo~y~L~yl-L-leucyl]-38-4-phenyl-3-amino-2 oxobutyloxy]-6'-chloro-5'-methylbenzotriazole.

~' Cl)~
C~3 . .

Method A; reaction time: 15.5h; yield 37~;
pl~;f;c~t;on: HPDC (reverse phase, acetonitrile:water ~r~n~;n;n~ 0,1~ trifluoroacetic acid) 10%-100~ over 40 min) ~: 32.53 min; IH NMR (300 MHz, CDC13): ~ 7.87 ~5, lH), 7.83 (9, lH), 7.43-6.92 ~m, lOH), 6.58 ~m, lH), 5.26 ~m, lH), 5 15-4.89 (m, 4H), 4.72 ~m, lH), 4.12 ~m, lH), 2.98 ~m, 2H), 2.53 (s, 3H), 1.63-1.32 (m, 3H), 0.89 (m, 6H), FABMS m/z (592, MH+).

Ex~mple 12 l-[N-[N-Benzyloxycarbony]-L-leucyl]-3S-4-phenyl-3-amlno-a-oxobutyloxy]-4'-methylb~..zoL..azole.

~ 3 SUBSTITUTE SHEET (RULE 26~

WO96/14067 2 1 q ~ PCT~S9~14794 Method A; reaction time: 15.5h; yield 15~r;

purification: HPLC (reverse phaae, acetonitrile:water (~nnt~ln1ns O.l~r trifluoroacetic acid) 10~-100~ over 40 min) Rt: 32.63 min; lH NMR (300 MHz, CDCl3): ~ 7.61 (d, lH), 7.52-6.92 (m, 12H), 6.55 ~m, lH), 5.26 (m, lH), 5.15-4.92 (m, 4H), 4.80 (m, lH), 4.10 (m, lH), 3.03 (m, 2H), 2.76 (9, 3H), 1.8-1.34 (m, 3H), 0.89 (m, 6H), FABMS m/z (558, MH+).

Example 13 l-[N-[N-Benzyloxycnrbonyl-L-leucyl]-3S-4-phenyl-3-amino-2-oxobutyloxy]-5'-chloro-6'-methylbenzotriazole.

J'~ rl o ~~rr~

~ ,[~
~ H3C
Cl Method A; reaction time: 15h; yield 25~;
purification: HPLC (reverse phase, acetonitrile:water (c~n~;n;n~ 0.1~ trifluoroacetic acid) 10~-100~ over ~0 min) Rt: 34.06 min; lH NMR (300 MHz, CDCl3): ~ 8.00 (s, lH), 7.70 (9, lH), 7.43-6.93 (m, lOH), 6.58 (m, lH), 5.29 (m, lH), 5.15-4 90 (m, 4H), 4.73 (m, lH), 4.10 (m, lH), 2.97 (m, 2H), 2.58 (9, 3H)~ 1.66-1.30 (m, 3H), 0.89 (m, 6H); FABMS m/z (592, MH+).

SUBSTITUT~ SHEET (RULE 26) 2l ~73a6 WO96114067 PCT~S9~14794 -~33 -~xample 14 1-[N-[N-Benzyl~a L~~ L-leucyl]-3S-4-phenyl-3-nmino-2-oxobutyloxy]-4'-chiorobenzotriazole.

~ ~~--N ~ZN

Method A; reaction time: 3h; yield l9~;
purification: HPLC keverse phase, acetonitrile:water (r~nt~;n;n~ 0.1~ trifluoroacetic acid) lO~-lO0~ over 40 min) R~: 32.13 min; lH NMR (300 MHz, CDC1,): ~ 7.75 (d, lH), 7:49-6.86 (series of m, 12H), 6.59 (m, lH), 5.29 (m, lH), 5.12-4.89 (m, 4H), 4.66 (m, lH), 4.09 (m, lH), 2.96 (m, 2H), 1.66-1.29 (m, 3H), 0.89 (m, 6H); FA3MS m/z (578, M+).

~x~pl~ 15 1-[N-[N-Benzyloxycarbonyl-L-l-ucyl]-3S-4-phonyl-3-amino-a-oxobutyloxyl-6'-phenylbenzotriazole.

--~ ~ O
y ~ ~~ ~N

~ ~ Ph~
, ... . : .

.' . ': ,, , SUBSrlTUTE SHEET (RULE 26) ;~ .
,y WO96114067 2 1 9 7 3 0 6 PCT~S95/~4794 Method A; reaction time: 15h; yield 3~;
purification: HPLC ~reverse phase, acetonitrile:water (cnnt~;n;ng 0.1~ trifluoroacetic acid) 10~-100~ over 40 min) R~: 33.23 min; lH NMR ~300 MHz, CDCl3): ~ 8.02 (m, 2H), 7.79-6.94 (series of m, 16H~), 6.63 (m, lH), 5.30 (bd, lH), 5.15-4.9 (m, 4H), 4.77 (m, lH), 4.12 (m, lH), 3.00 (m, 2H), 1.7-1.3 (series of m, 3H), 0.87 (m, 6H) ; FABMS m/z (620, mH+).

Example 16 -- l-[N-[N-Benzyloxycarbonyl-L-leucyl]-3S-4-phenyl-3-~ino-2-oxobutyloxyl-4',5',6',7'-tetrafluoI~b~zotriazole.

~~~r~_N~

~ F~=

Method A; reaction time: 15h; yield 4~;
purification: HPLC (reverse phase, acetonitrile:water (cnnt~;n;n~ 0.1~ trifluoroacetic acid) 10~-100~ over 40 min) R~: 33.08 min; lH NMR (300 MHz, CDC13): ~ 7.43-7.00 (m, lOH), 6.57 (m, lH), 5.35 (bd, lH), 5.1-4.89 (8 overlapping with m, 4H), 4.70 (m, lH), 4.12 (m, lH), 3.03 (m, 2H), 1.7-1.2. (m, 3H), 0.92 (ml 6H); FA;3MS m/z (616, MH+).

.

SUBSTITUT~ SHEET (RUL~ 26~

~ WO96/14067 2 t 9 7 3 0 6 PCT~S9~14794 Example 17 l-[N-[N-Benzyloxycarbonyl-L-leucyli-36-4-phenyl-3-amino-2-oxobutyloxy]-5'-chlorobenzotrinzole.

~ ~ o 'N ~ ~0 ~ N ~
~,, " &~ Cl Method ~; reaction time: 3h; yield 58~;
pur;~;r~t;~n- MPLC (reverse phase, acetonitrile:water (~nt~;n;ng 0.1~ trifluoroacetic acid) 10~-100~ over 40 min) Rt: 30.36 min; lH NMR (300 MHz, CDC1,): ~ 8.00 (s, lH), 7.83 (d, lH), 7.52 (d, 1H), i.44-6.9 (m, lOH), 6.63 (m, lH), 5.30 (bd, lH), 5.15-5 00 (s and m, 4H~, 4.90 (m, lH), 4.12 (m, lH), 2.97 (m, 2H), 1.66-1.35 (m, 3H), 0.90 (m, 6H); FABMS
m/z (578, MH+).
The following Examples of; the s_ ~_u~lds listed in Table IA were prepared in a manner similar to that for Example 17.

Example 18 1-[N-[N-Benzyi~y~rLv~yl-~-leucyl]-3S-4-phenyl-3-nmino-2-oxobutyloxy]-5',6'-dichlolob ~zoL~lazole.
Rt:31.85 min; ~H NMR (300 MHz, CDC13): ~ 8.12 (s, 1~), 8.04 (s, lH), 7.40-6.88 (m, lOH), 6.68 (m, lH), 5.30 SUBS~ SHEET (RULE 26) ' ' .'''' . ~

WO96/14067 2 ~ q 7 3 ~ ~ PCT~S9~14794 (m, lH), 5.16-4.88 (8 and m, 4H), 4.64 (m, lH), 4.12 (m, lH), 3.00 (m, 2H), 1.66-1.3 (m, 3H), 0.92 (m, 6H); F~3MS M/Z
(614, mh+).

Example 19 1-[N-[N-Benzyloxycarbonyl-L-leucyl]-3S-4-phenyl-3-amino-2-oxobutyloxy]-(lH)-triazolo[4,5-b]pyridine.
Rt:27.24 min; IH NMR (300 MHz, CDCl3): ~ 8.8 (m, lH), 8.37 (m, lH), 7.54 (m, lH), 7.43-6.89 (m, lOH), 6.80 (m, lH), 5.40 (m, lH), 5.23-4.97 (s and m, 4H), 4.66 (m, lH), 4.14 (m, lH), 2.97 (m, 2H), 166-1.32 (m, 3H), 0.88 (m, 6H); FABMS m/z (545, MH+).

Example 20 l-[N-[N-Benzyloxycarbonyl-L-leucyl]-3S-4-phenyi-3-amino-2-oxobutyloxy]-4~,5,6',7~-tetrachlorobenzotriazole.
Rt:34.67 min; 3H NMR (300 NHz, CDCl3): ~ 7.40-7.06 (m, lOH), 6.63 (m, lH), 5.32 (m, lH), 5.14-4.91 (s and m, 4H), 4.86 (m, lH), 4.10 (m, lH), 3.08 (m, 2H), 1.49-1.2 (m, 3H), 0.89 (m, 6H); FABMS m/z (682, MH+).

SUESIITUTE SHEET (RULE 26) WO961140C7 t / I ~ PCTr~S95rl4794 Ex~mple 21 l-tN-[N-Benzyl~y~L~l~yl-L-leucyl]-3s-i-phenyl-3-ucino-2 oxobutyloxy]-4',6',7'-trichlorotriazole.
Rt:33.56 min; 3H NMR (300 MHz, CDCl3): ~ 7.52 (s, 1~), 7.437.00 (m, lOH), 6.57 (m, lH), 5.28 (bd, lH), 5.17-4.83 (m, 5H), 4.1~ (m, lH), 3.08 (m, 2H), 1.63-1.2 (m, 3H), 0.85 (m, 6H); FABMS m/z (648, MH+).

Example 22 l-[N-[N-Benzyloxycarbonyl-L-leucyl]-(3S)-4-phenyl-3-amino-2-oxobutyloxy]b~n~;m;~ole.

~ ~ o ,~r~

Method B, reaction time 40 min; purification: HPLC(reverse phase, acetonitrile:watér (r~nt~in;n~ 0.1~
tri ~ln~roacetic acid) 10~-100~ over 4D min.) Rt: 24.64 min;
I5 yield 41%; 3H NMR (300 MHz, CDCl,): ~ 9.14 (br, lH), 7.81 (m, lX), 7.6-7.0 (m, 16H), 5.5-4.9 (m, 3H), 4.59 (m, lH), 4.10 (m, lH), 3.00 (m, 2H), 1.53 (m, lH), 1.40 (m, 2H), 0.83 (2d, 6~); FABMS m/z 543 (MH'); mp 56-60 C.

.
~xa~ple 23 1-[N-[N-Benzyl~y~lL~l.yl-L-leucyl]-36-4-phenyl-3-amino-2-SUBSTITUTE SHEET ~RULE 26) - ~ t:

; ~ ~
WO96/14067 2 ~ q 7 ~ ~ ~ PCT~S95/14794 oxobutyloxy~-4 ,5 -dichl~-~L~zot nzole.
Rt:32.70 min; lH NMR ~300 MHz, CDCl3): ~ 7.63 (d, lH), 7.46 (d, lH), 7.32-6.75 (m, lOH), 6.53 (m, lH), 5.20 (m, lH), 5.03-4.8 (8 and m, 4H), 4.50 (m, lH), 3.97 (m, lH), 2.80 (m, 2H), 1.45-1.14 (m, 3H), 0.80 (m, 6H); FA5MS m/z (614, MH+).

Exsmple 24 l-tN-[N-Benzyloxycarbonyl-L-leucyl~-3S-4-phenyl-3-amino-2-oxobutyloxy~-5~-chloro-6 -ethylbenzotriazole.
R~: 33.90; lH NMR (300 MHz, CDCl3): ~ 7.91 (~, lH~, 7.63 (s, lH), 7.32-6.85 (m, lOH), 6.57 (m, lH), 5.23 (m, lH), 5.06-4.86 (8 and m, 4H), 4.66 (m, lH), 4.03 (m, lH), 3.03-2.8 (m, 4H), 1.57-1.2 (m and t, 6H), 0.80 (m, 6H);
FA;3MS m/z (606, MH+).

Ex~ple 25 l-[N-[N-Benzyl~y.~L~l-L-leucyl~-3S-4-phenyl-3-smino-2-oxobutyloxy]-4 ,5 -difluu ~b~zo~Liazole.
Rt:31.54 min; lH NMR (300 MHz, CDCl3): ~ 7.63 (m, lH), 7.51-6.90 (m, llH), 6.68 (m, 3.H), 5.34 (m, lH), 5.2-4.92 (~ and m, 4H), 4.67 (m, lH), 4.09 (m, lH), 2.97 (m, 2H), 1.66-1.31 (m, 3H), 0.92 (m, 6H), FABMS m/z (580, MH+).

Exnmple 26 l-[N-EN-Benzylo~y~rL~yl-L-leucyl~-3S-4-phenyl-3-amino-2-oxobutyloxy~-6 -methylb_ zoL- szole.
Rt: 31.57 min; lH NMR (300 MHz, CDCl3) ~ 7.88 (d, SUBSrllUTE SHEET (RULE 26) WO96/14067 PCT~S95/14794 lH~, i.57 (s, lH~, 7.43-6.94 (m, llH), 6.67 (m, lH), 5.25 (d, lH), 5.11-i.92 (s and m, 4H), 4.80 (m, lH), 4.11 (m, lH), 3.03 (m, 2H), 2.59 (8, 3H), 1.66-1.3 (m, 3H), 0.90 (m, 6H); FABMS m/z (558, MH+).

, Ex~mple 27 l-[N-[N-Benzyl~y~ L~,-yl-L-leucyl]-3S-4-ihenyl-3-~mino-2-oxobutyloxy]-5i-methylbenzotrinzole.
Rc: 31;59 min; lH MMR (300 MHz, CDCl3): ~ 7.74 (m, 2H), 7.436.94 (m, ilX), 6.63 (m, lH), 5.27 (bd, lH), 5.14-4.91 (s and m, 4H), 4.74 (m, lH), 4.il (m, lX), 3.00 (m, 2H), 2.52 (s, 3H), 1.68-1.3 (m, 3H), 0.86 (m, 6H); FABMS m/z (558, MH+).

Example 28 l-[N-[N-Benzyl~y.~.L~l.yl-L-leucyl]-3S-4-phenyl-3-am~no-2-oxobutyloxy]-3-benzotriazin-4-one.
Method B reaction time 4h; pur;f;r~t;r,~, recryst~lli7~tl~n (EtOAc/Hexanes); yield 76~1; mp 155-157~C;
3H-NMR (3bo MHz, CDCl~) ~ 8.38 (d, 5 Hz, lH), 8.22 (d, 5 Hz, lH), 8.01 (d, 5 Hz, lH), 7.85 (d, 5 Hz, lH), 7.35-7.15 (m, 10H), 6.95 (m, lH), 5.20-4.90 (m, 6H), 4.20-4.10 (m, lH), 3.40-3.30 (m, lH), 3.20-3.05 (m, lH), 1.65-1.35 (m 3H), ~ 0.95-0.85 (m, 6H); MS (ESI): 572 (M+H)i; Anal, calcd _or C3lH33NsO~- C (65.12), H (5.83), N (12.25); Fd: C (65.06), H
(5.74), N (12.39).

. .
Example i9 ~BSll~UrE SHEET (RULE 2~) WO96/14067 2 1 9 7 } ~ 6 PCT~S9~14794 I-[N-[N-Benzyloxyc~rbonyl-L-leucyl]-3S-4-phenyl-3-~mino-2-oxobutyloxy]-3-~6',7'-dimethoxy~benzotriazin-4-one.
Method B. reaction time Sh; purification, recrystallization (EtOAc/Hexanes); yield 73~; mp 180-185~C(dec); 'H-NMR (300 MHz, CDCl3): ~ 7.61 (s, lH), 7.52 (s, lH), 7.35-7.15 (m, 10H), 6.90 (m, lH), 5.20-4.80 (m, 6H), 4.22-4.10 (m, lH), 4.05 (2d, 6H), 3.40-3.30 (m, lH), 3.18-3.05 (m, lH), 1.65-1.35 (m, 3H), 0.95-0.85 (m, 6H); MS
(ESI): 632 (M+H)'; Anal, calcd for C33H3~NsOC: C(62.74), H(5.92), N(11.09); Fd: C(62.51), H(5.76), N(11_03).

Example 30 l-[N-[N-Benzyloxycarbonyl-L-leucyl]-3S-4-phenyl-3-amino-2-oxobutyloxy]-3-(6~-chloro)benzotriazin-4-one.
Method B. reaction time 4h; purification, recrystallization (EtOAc/Hexanes); yield 57~; mp 150-153~C
(dec); 3H-NMR (300 MHz, CDCl3): ~ 8.35 (s, lH), 8.15 (d, 5 Hz, lH), 7.92 (d, 5 Hz, lH), 7.35-7.15 (m, 10H), 6.90 (m, lH), 5.15-4.85 (m, 6H), 4.20-4.10 (m, lH), 3.35-3.25 (m, lH), 3.15-3.05 (m, lH), 1.65-1.35 (m 3H), 0.95-0.85 (m, 6H);
MS (ESI): 607/609 (M+H)'; mono-chloro isotope pattern; Anal, calcd for C3lH32NsO6~: C(61.42), H(5.33), N(11.56), CI(5.85);
Fd: C(61.34), H(5.33), N(11.54), CI(6.20).

Exampl- 31 l-[N-[N-Benzyl~Ay~rL~.yl-L-leucyl]-3S-5-methyl-3-amino-2-oxohexyloxy]-3-b_~zot.lazin-4-one.
Method B. reaction time 21h; purification, SUBS;rlTUTE SHEET ~RULE 261 2 1 973~6 WO96/14067 PCT~S95114794 ~ 41,- :
rec~yst~ ti~n (EtOAc/Hexanès); yield si~; mp 147-148.5~C; IH-NMR (300 MHz, CDCl~ 8.i8 ~d, 5 Hz, lH), 8.22 (d, 5 Hz, lH~, 8.01 (d, 5 Hz, lH), 7.85 (d, 5 Hz, lH), 7.35 (m, 5H), 6.80 (m, lH), 5.20-4.g5 (m, 6H), 4.30-4.20 (m, lH), 1.90-1.50 (m, 6H?, i.oo-o.so (m, 12H); MS (ESI): 538 (M+H)-;
Anal, calcd for C2,H,5NsO~: C (62-54j, H(6.58), N(13.03); Fd:
C(62-43), H(6.52), N(12.96).

synthe~l~G ol~ n~-;n;n~ Y---th~n~-9-yl and l-phenyipropyl Functlonallty Synthesi~ of c ,_ ' r~nt~;n~n~ the xanthen-9-yl ~nd lphenylpropyl functionality are depicted in Scheme 2.
-I Ra ~ oc~3 o ,1 o ,1 o~, Ra ~ ~~ Ra ~ - a ___~, 4 R ~ C~2Ph 5 IL- C112CI~(CI~ ) R ~ ~ ~ N~N

8 ~--C~2Ph R ~ ~ Ph 2 ( 3)2 ~ - 2 ( 3)2 ~,SUBSTITUTE SHEET(RULE 26) Wo96fl4067 2 ' 9 7 ~ ~ ~ PCT~Sg5/147g4 Example 32 Synthesis of Tnt~ te 1 (8cheme 2):

To a cooled (0~C) solution of Y~nthPnP-9_ carboxylic acid (9.05g, 0.04 mole) in anhydrous THF (40mL) was added 1,1' carbonyldiimidazole ~6.81g, 0 042 mole). The mixture was stirred at 0~C for 0.5h and~then at room temperature overnight. The next day, this solution was added slowly/ over lh, to a cooled (-7a~C) solution of tert-butyl lithioacetate (0.088 mole, generated, in situ fromtert-butyl acetate and lithium diisopropylamide~ in TEF (40 m~) hexane (35 m~). The mixture was stirred for an additional 0.5h, ~lpnrhpd with lN HC1 ~88 mL), brought to OoC and acidified wit_ lN HC1 to pH 3-4. The resulting a~ueous solution was extracted with ethyl acetate ~2x 100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Purification by flash chromatography ~silica gel, 6~ ethyl acetate-hexane) gave 8.7g of the desired product: lH-NMR (300 MH~, CDC13) ~ 7.40-7.00 Im, 8H), 5.00 (s, lH), 3.20 (8, lH) 1.40 (8, 9H). A general description of this ~luceduL~ can be found in Harris, 8.D.
et al., Tetrahedro~ ~ett. 28(25) 2837 (1987), and in Hamada, SUBSrITUTE SHEET ~RULE 26~

96/14067 ICT~S9S/14794 . - L 43 - ~

Y_ et al., ~. Am. Chem. SOC. 111, 669 (1989).

, Exumple 33 ~ynthesi~ of Tnt~te 2 (Scheme 2):
.

~i~3 To a stirred slurry of 60~ sodium hydride in oil (0.860g, 0.021S mol) in anhydrous T~E (10 m~), was added 810wly the keto ester Intermediate 1 (6.63g, 0.02 mol) in anhydrous T~F (20 mL) . After the evolution o~ hydrogen gas cea8ed, the solution was treated with 6.82g of the leucine-triflate methyl ester tgenerated from the corresponding (D)-hyd~u~y~Ler (4.00g) and triflic anhydride (8.05g) in the presence of 2,6-l~ ;n~ (3.06g)) adapted from the pLucedu,e described in ~offman, R.V. et al., Tetrahedron ~ett. 34(13), 2051 (1993). The resulting mixture was stirred overnight, 1~ diluted with ether (100 mL) washed with water (30 m~) and ~nnc~ntrated under reducea pressure to give-7.00g of crude diester ; nt~ te. This material was then dissolved in trifluoroacetlc acid (TFA, 7 m~) and stirred at room temperature for lhr_ The TEA was removed and the residue dissolved in benzene (30 mL) and heated at reflux for lh.
The solvent was removed under reduced pressure and ' . ' SUBSrlTUrE S~IEET (RULE 26) ~ i s '~
WO96114067 2 ~ ~ 7 3 3 6 PCT~S951147~4 purification by flash ChL~ tography (silica gel, 4~ ethyl acetate-hexane) gave 2.34g of the product, Intermediate 2: lH
NMR (300MHz, CDCl3) ~ 7.40-7.0 (m, 8H), 4.90 (8, lH), 3.55 (8, 3H), 2.80-2.60 (m, 2H), 2.30 (dd, J=8Hz and 2Hz, lH), 1.30 (m, 2H), 1.00 (m, lH), 0.80 (d, J=8Hz, 3H)~ 0.70 (d, ~-8Hz, 3H).

3xamp1e 34 Tnt~ te 3 (Scheme 2):

~H
0~ 0 A mixture of InteL ~ te 2 (2.33g, 6.6 mmol) lithium hydroxide-monohydrate (0.360g~, methanol (27 mL), and water (9 mL), was heated at 70-75~C for 1.5h. The methanol was removed under=reduced ~Le~uL~. The resulting aqueous solution was washed with diethyl ether (20 mL), acidified at 0~C with lN HCl and then extracted with diethyl ether ~3xlO mL). The organic layer was washed once with brine and dried over anhydrous sodium sulfate. Filtration - followed by removal of the solvent under reduced pressure produced 1_83g of the product, Intermediate 3. lH-NMR (300 - 20 .~Hz, CDCl~) ~ 7.40-7.00 (m, 8H), 4.95 (8, lH), 2.80-2-60 (m, 2H~, 2.30 (dd, J=8Hz and 2Hz, lH), 1.35 (m, lH), 1.00 (m, SUESrl~UrE SHEET (Rl)LE 26~

W096ll4067 PCT~S9~/14794 -~45 - -lH), 0.80 (d, J=aHz, 3H), 0.70 (d, J=8Hz, 3H).

Fx~mple 35 Tnt~ t~ 4 (8chome 2~:

, ~Cl ~ ~ ~ ~

To a cooled (-60~C) solution of Intermediate 3 [0.148g, 0.4373 mmol] in anhydrous THF (3 m~) was added N-methylmorpholine (0.142g) followed by isobutyl chloroformate (0.066g). The mixture was stirred for 0.5h and the cooling bath replaced by an iceiwater bath. To the reaction mixture was added 0.012g of phenyl~l~n;n~ chloromethyl ketone hydrochloride in DMF (3 mL). The resulting mixture was stirred at 0~C for lh then at room temperature overnight.
The mixture was then diluted with ethyl acetate (20 m~), washed with 2~ acueous citric acid (2 x lOm~), 2~ agueous NaHCO, (2 x lOmL), brine (l x lOm~), and dried over anhydrous sodium sulfate. Filtration and removal of the solvent under reduced pressure gave crude Tntp ~ te g. Purification by flash chromatography (silica gel, 15% ethyl ~~ptat~ n~) afforded o.lOsg of Tnt~ te 4. l~-NMR (300MHz, CDC13), 7.10-7.30 (m, 13H), 6.15 (d, J=6Hz, lH), 4.90 (s, lH), 4.70 (q, J=6Hz, lH), 4.05 (d, J=16Hz, lH), 3.85 (d, J=16Hz, lH), StlBSrITUTF Sl IEET (RULE 26~
-, -~

2 ' ~7~06 WO96/14067 PCT~S95/14794 3.00 (m, lH), 2.50 (m, 2H), 2.30 (dd, J=8Hz and 2Hz, lH), 1.30 (m, 2H), 0.90 (m, lH), 0.75 (d, J=6Hz, 3H), 0.65 (d, J=6Hz, 3H).

Example 36 Tnt~ te 5 (Scheme 2):

Following the same procedure described for the 3ynthesis o~ Tnt~ ~;Ate 4, Int~ te 3 [0.408g, 1.205 mmol] was coupled with leucine chloromethyl ketone hydrochloride 10.241g) to yield 5 (0.146g): 1H-NMR (300MHz, CDCl3) ~ 7.40-7.00 (m, 8H), 6.00 (d, J=8HZ, lH), 4.90 (s, lH), 4.60 (m, lH), 4.20 (s, 2H), 2.70-2.50 (m, 2H), 2.35 (dd, J=8HZ and 2Hz, lH), 1.60-1-20 (m, 4H), 0.95 (d, J=8Hz, 3H), 0.90 (m, 2H), 0.85 (d, J=8Hz, 3H), 0.80 (d, J=8Hz, 3H), 0.70 (d, J=8HZ, 3H).

Ex~mple 37 Tnt~ te 6 (Scheme 2) A mixture o~ Tn~ te 4 (0.030g, 0.058mmol), sodium iodide (0.022g) and acetone (3 m~) was stirred at room temperature ~or lh. The solvent was removed under SU85TllUTE SHEE ,T (RULE 26) 2t973a6 WO96/14067 PCT~S9~14794 - ~7 -reduced préssure and the residue was partitioned between H20 (5m~) and CH~Cl~ (2 x 5m~). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent removed under reduced pressure, to give 0.036g of Int~L, ';~te 6: lH-NMR (300MHz, CDCll) ~ 7.40-7.00 (m, 13H), 6.15 (d, J=6Hz, lE), 4.90 (s, lH); 4.85 (q, J56Hz, lH), 3.70 (d, J=8Hz, lH), 3.60 (d, J=8Hz, lH), 3.00 (m, 2H), 2.50 (m, 2H), 2.30 (dd, J=8Hz and 2Hz, lH), 1;30 (m, 2H), 0.85 (m, lH), 0.75 (d, J=6Hz, 3H), 0.65 (d, J=6Hz, 3H).
.

Ex~rple 38 Tntl ~; nte 7 ~Scheme 2):
Following the same ~r~cedur~ described for the synthesis of Intermediate 6, Intermediate 5 (0.105g, 0.217 mmol) was converted to Intermediate 7 (0.120g): ~H-NMR
(300MHz, CDCl3) ~ 7.40-7.00 (m, 8H), 6.00 (d, J=8Hz, lH), 4.90 (s, lH), 4.70 (m, lH), 3.90 (d, J=6Hz, lH), 3.85 (d, J=6Hz, lH), 2 70-2.50 (m, 2H), 2.35 (dd, J=8Hz and 2Hz, lH), 1.60-1_20 (m, 4H), 0.95 (d, J=8Hz, 3H), 0.90 (m, 2H), 0.85 (d, J=8HZ, 3H), 0.80 (d, J=SHz, 3H), 0.75 (d, J=8Hz, 3H).

Ex ple 39 l-[N-[2-(2-Methylpropyl)-1,4-dioxo-4-(xanthen-9-yl)butyl]-3S-3amino-2-oxo-4-phenylbutyloxy]benzotriazole.

~su~snTuTE SHEET (RULE 26) ..

WO96114067 2 1 9 7 3 ~ 5 PCT~S9~114794 Using method A, InteL, ~;~t~ 6 (0.046g, 0.075 mmol) was coupled with l-hydL~yb~lzotriazole 10.014g) to give the product (0.046g) as a white solid after puri~ication by cryst~lli7at~n from ethyl acetate-hexane:
mp 99-101 ~C; FABMS 618 m/z (M'); lHNMR (300MHz, CDCl3) ~ 8.00 (d, J=6Hz, lH), 7.80 (d, J=6Hz, lH), 7.70 (t, J-6Hz, lH), 7.00 (t, J=6Hz, lH~, 7.30-6.90 (m, 13H), 6.10 (d, J=8Hz, lH), 5.15 (d, J=16Hz, lH), 4.90 (d, J=16Hz, lH), 4.85 (8, lH), 4.55 (m, lH), 2.90 ~m, 2H), 2.50 (m, 2H), 2.30 (dd, J=8Hz and 2Hz, lH), 1.30 (m, 2H), 0.85 (m, lH), 0;75 (d, J=6Hz, 3H), 0.65 (d, J=6Hz, 3H).

Example 40 l-[N[-[2-(2-Nethylpropyl)-1,4-dioxo-4-(xanthen-9-yl)butyl]-38-3a~ino-5-methyl-2-oxohexyloxy]~_ zotllazole.

~ ~ 7 ~

Using method A, Int,-L ~~te 7 (0.115g, 0.2 mmol) was coupled with l-hydLo~yb~zorr;~7~ (o.o34g) to give the ~ ~ ~ (O.051g) as a white solid: m.p. 92-94 ~C; lH-NMR
(300MHz, CDC13); ~ 8-00 (d, J=6Hz, lH), 7.80 (d, J=6Hz, lH), 7.65 (t, J=7Hz, lH), 7.40 (t, J=7Hz, 1~), 7.35-7.00 (m, 8H), 6.00 (d, J=8Hz, lH), 5.40, (8, 2H), 4.90 (8, lH), 4.50 (m, SUBSTlTUrE SHEET ~RlJLE 261 - 2 1 973~6 ~ ~096/l4067 ~ PCT~S95/14794 , 1~) ~ 2.70-2.50 (m, 2Hj, 2.30 (dd, J=8Hz and 2H, lH), 1.60-1.20 (m, 4H), 0.95 (d, J-8Hz, 3H), 0.90 (m, 2H), 0.85 (t, J-8Hz, 3H~, 0.80 (d, J-8Hz, 3X), 0.75 (d, J=8Hz, 3H~.

Example 41 Sy3thesis of Int~ te la (Scheme 2) , . i ~ ' .

Following the same method for the synthesis of the InteL -';~te 1 of Example 32 (RF g-xanthenyl), (S)-(+)-2-phenylbutyric acid (3.93g, 0.024 mole) was converted to Tnt~ te 1ar R= 1-phenylpropyl (4.13g): lH~MR (300 MHz, lo CDC13) ~: 7.38-7.18 (m, 5H), 3.70 (t, J=6Hz, lH), 3.35 (d, J=16Hz, lH), 3.20 (d, J=16Hz, lH), 2.10 (m, lH), 2.70 (m, lH), i.45 (8, 9H), 0.85 (t, J-17Hz, 3H).

Ex~3ple 42 Sy3thesis of I3t~ 'tAte 2a (Sche~e 2) :
- ' ~oc~.

~ .

Follo~ing the game method for the synthesis of the Intermediate 2 of Example 33, (R= 9-xanthenyl), Intermediate - . .
1'. SUBSTITUT~ SHEET (RULE 26j :,. . ..

WO96114067 2 i q 7 3 ~ 6 PCT~S9~/14794 la ~3.25g, 0.0124 mole) was converted to Tnt~ te 2a (2.85g): lHNMR (300 MHz, CDCl3) ~ 7.40-7.18 (m, 5H), 3.60 (S, 3H), 3.50 ~t, J=6Hz, lH), 2.85 ~m, lH), 2.75 ~m, lH), 2.45 ~dd, J=18Hz and 2Hz, lH), 2.05 (m, lH), 1.70 ~m, lH), 1.45 ~m, 2H), 1.15 ~m, lH), 0.90-0.70 (m, 9H).

ExDmple 43 Sy~thesis o~ Tnt~ te 3a (Scheme 2) ~CH , InteL ~ te 2a (0.570g, 1.963 mmol) was hydrolyzed to Int~r~ 3a ~R- I-phenylpropyl? ~0.507g), following the same procedure for the synthesis of Tn~ -A;?te 3 of Example 34 ~R- 9-xanthenyl): 3HNMR (300 MHz, CDCl3) ~. 7.40-7.10 (m, 5H), 3.60 (m, lH) 2.90 (m, lH), 2.75 (m, lH), 2.50 (m, lH), 2.05 (m, lH), 1.70 (m, lH), 1.50 (m, 2H), 1.15 (m, lH), 0.90-0.70 (m, 9H).

15 Exa~ple 44, Sy~the~i~ of Tnt~ te 4a (Scheme 2) -Following the same procedure for the synthesis of Tnt~ te 4 of Example 35 ~R=9-xanthenyl~ R=benzyl), SUBSnTUTE SHEET IRULE 26) 2 1 97336~
WO96/14067 ' PCT~S9~14794 Intermediàte 3a (0.386g, 1.40 nmol) was converted to . . .
Tnt~ te 4a (R~ phenylpropyl, Rsbenzyl) (0.382g, 72.28 diastereomeric mixture): IH~MR (300 MHz, CDCl3) ~ 7.40-7.10 (m, lOH), 6.30 ~d, J=6Hz, lH), 4.85 and 4.75 (2 set of q, . .
72:28, J=6Hz, lH), 4.10 and 4.05 (2 sets of doublet, 72:28, J=18Hz, lH), 3.90 and 3.85 (2 sets of doublet, 72:28, J=18Hz, lH), 3.50 (m, lH), 3.10 (m, lH), 2.95 (m, lH), 2.65 (m, 2H), 2.40 (m, lH), 2.00 (m, lH), 1.70 (m, lH), 1.35 (m, 2H), 1.00 (m, lH), 0.90-0.65 (m, 9H).

..
Example 45 8ynthesis of Tnt~ te 5a (8che~e 2) 0.087g (0.1907 mmol) of Intermediate 4a, R~=l-phenylpropyl, R=benzyl, was converted to I~termediate 5a (R=l-phenylpro~yl, R=benzyl) (0.094g, 72:28 diastereomeric mixture) following the same procedure for the synthesis of TntP ~ te 6 of Example 36, (R~=9-xanthenyl, R=benzyl):
~HNMR (300 MHz, CDCl3) ~ 7.40-7.10 (m, lOH), 6.30 (mixture of fl~l~hlPt~ lH), 5.00 and 4.85 (i sets of quartets, 72:28, J=6Hz, lH), 3.75 and 3.65 (2 sets of doublets, 72:28, .
J=16Hz, lH), 3.70 and 3.60 (2 sets of doublets, 72-28, J=16Hz, lH), 3.50 (m, lH), 3.10 (d, J=6Hz, lH), 3.00 (m, lH), 2.65 (m, 2H), 2.40 (m, lH), 2.00 (m, lH), 1.70 (m, lH), 1.40 (m, 2X), 1.00 (m, lH), 0.85-0.70 (m, 9H).

~.

. .

ITUlE SHEET lRUL~ 26) .. . .

WO96/14067 2 ~ ~ 7 ~ ~ ~ PCT~S9~14794 Ex~mple 46 l-[N-~2R-(2-methylpropyl)-1,4-dioxo-5S-phenylheptan-l-yl]-3S-3amino-2-oxo-4-phenylbutyloxy]benzotriazole Following method A, Inte~ te 5a, (0.094g, 0.1716 mmol) was coupled with l-hyd~yb~--zotriazole (0.030g) to give 1-[N-[2R-(2-methylpropyl)-1,4-dioxo-5S-phenylheptan-l-yl]-3S-3-amino-2-oxo-4-phenylbutyloxy]benzotriazole (0.080g) as a mixture (72:28) of diastereomers: lHNMR (300 MHz, CDCl3) ~ 8.00 (d, J=6Hz, lH), 7.85 and 7.80 (2 sets of doublets, 72:28, J=6Hz, lH), 7.55 (t, J=6Hz, lH), 7.40 (t, J-6Hz, lH), 7.30-6.20 (m, lOH), 6.25 (mix. of doublets, lH), 5.30 and 5.20 (2 sets of ~nnhletc~ 72 28, J=16Hz, lH), 4.95 and 4.90 ~2 sets of doublets, 72:28. J=6HZ, lH), 4.70 and 4.50 (2 sets of quartet, 72:28, J=7Hz, lH), 3.50 (m, lH), 3.00 and 2.90 (2 sets of ~n~lhlet~l 72:28, J=8Hz, lH), 2.60 (m, 2H), 2.40 (m, lH), 2.00 (m, lH), 1.65 (m, 2H), 1.30 (m, lH), 0.90 (m, lH), 0.85-0.60 (m, 9H).

Each of the p..hl;~h~d ~n~ t~ --n~;on~d in this spen;~;~ation is hereby incorporated by reference in their entirety.
Those skilled in the art will appreciate that SUBSTIME SHEET (RULE 26) ~ WO96/14067 2 1 9 7 3 ~ PC~US9D14794 numerous changes and modifications may be made to the preferred embodiments of the invention and that such changes and ';f;r~tions may be made without departing from the spirit of the invention. It is therefore intended that the appended claims cover all eguivalent variations as fall within the true spirit and scope of the invention.

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SUBSrllU~E SHEET /RULE 26)

Claims (18)

WHAT IS CLAIMED IS:

1. A compound represented by the formula:

wherein:
M is selected from the group consisting of O, NR7 and CR1R2, X1 is selected from the group consisting of O, S
and NR7;
X2 is selected from the group consisting of O, S, N7 and two hydrogen atoms;
Q is selected from the group consisting of O, S
and NR1;
R1 and R2 are each independently selected from the group consisting of H, alkyl having from 1 to 10 carbons, heteroaryl having from 1 to 10 carbons, alkanoyl having from 1 to 10 carbons, and aroyl, wherein said alkyl, heteroaryl, alkanoyl and aroyl groups are optionally substituted with J;
R3, R4, R5 and R6 are each independently selected from the group consisting of H, alkyl having from 1 to 10 carbons, aryl, and heteroaryl, wherein said alkyl, aryl and heteroaryl groups are optionally substituted with J;
R7 and R8 are each independently selected from the group consisting of H, alkyl having from 1 to 10 carbons, aryl, and heteroaryl, wherein said alkyl, aryl and heteroaryl groups are optionally substituted with J;

J is selected from the group consisting of halogen, COOR7, R7OCO, R7OCONH, OH, CN, NO2, NR7R8, N=C(R7)R8, N=C(NR7R8)2, SR7, OR7, phenyl, naphthyl, heteroaryl, and a cycloalkyl group having from 3 to 8 carbons;
G is selected from the group consisting of NH2, NHR1, CH2R1, CH2C(O)B, carbobenzyloxy-NH, succinyl-NH, R7O-succinyl-NH, R7OC(O)NH, CH2C(O)-(xanthen-9-yl), CH~COR' wherein R' is selected from the group consisting of alkyl, aryl, and arylalkyl group of up to 13 carbons, and AA'NHC(O)OCH~C~H~ wherein AA~ is selected from the group consisting of one of the 20 natural amino acids and an opposite antipode of said amino acid;
B is selected from the group consisting of alkyl having from 1 to 10 carbons, aralkyl having from 1 to 10 carbons, aryl having 1 to 3 carbocyclic rings, and heteroaryl having 1 to 3 rings, wherein said alkyl, aralkyl, aryl and heteroaryl groups are optionally substituted with J; and A is represented by the structure:

wherein:
Y is selected from the group consisting of N and CR1;

W is selected from the group consisting of a double bond and a single bond;
D is selected from the group consisting of C=O and a single bond;
E and F are each independently selected from the group consisting of R1, R2, J, and when E and F comprise a joined moiety, said moiety is selected from the group consisting of an aliphatic carbocyclic ring having from 5 to 7 carbons, an aromatic carbocyclic ring having from 5 to 7 carbons, an aliphatic heterocyclic ring having from 5 to 7 atoms, and an aromatic heterocyclic ring having 5 to 7 atoms; wherein:
said aliphatic heterocyclic ring and said aromatic heterocyclic ring each have from 1 to 4 heteroatoms; and said aliphatic carbocyclic ring, said aromatic carbocyclic ring, said aliphatic heterocyclic ring, and said aromatic heterocyclic ring are each optionally substituted with J.

2. The compound of formula 1 represented by the formula:

wherein:
K is selected from the group consisting of NHC(O)OCH2C6H5, -CH2C(O)-(xanthen-9-yl) and -C2C(O)CH(C6H5)C2H5;
P1 is selected from the group consisting of isobutyl, isopropyl, benzyl, carboxyalkyl of 2-9 carbons and ethyl; and X3 is represented by the structure:

wherein:
D is selected from the group consisting of C=O and a single bond;
X4 is selected from the group consisting of CH, CC1, CCH3, CF and N;

X5 is selected from the group consisting of X, CH3, C1, OCH3 and F;
X6 is selected from the group consisting of H, CH3, C1, F, OCH3, CF3, ethyl and phenyl;
X7 is selected from the group consisting of N, CC1, CH, COCH3 and CF; and Y is selected from the group consisting of N and CH.

3. The compound of claim 2 wherein K is -CH2C(O)-(xanthen-9yl).

4. The compound of claim 2 wherein P1 is benzyl.

5. The compound of claim 4 wherein Y is N.

6. The compound of claim 4 wherein X3 is O-1-oxybenzotriazole.

7. The compound of claim 2 wherein X7 is N.

8. The compound of claim 2 wherein Y is CH.

9. The compound of claim 1 wherein Q is NR1.

10. The compound of claim 1 wherein one of R1 or R2 is other than H.

11. The compound of claim 1 wherein X1 is selected from the group consisting of S and NR7.

12. The compound of claim 1 wherein neither of R3 and R4 are H.

13. The compound of claim 1 wherein M is selected from the group consisting of O and CR1R2.

14. The compound of claim 1 wherein X2 is selected from the group consisting of S, NR7, and two hydrogen atoms.

15. The compound of claim 2 wherein K has the formula:

16. A composition for inhibiting the enzymatic activity of a serine protease or a cysteine protease comprising a compound of claim 1.

17. A method for inhibiting the enzymatic activity of a serine protease or cysteine protease comprising contacting a protease selected from the group consisting of serine protease and cysteine protease with an inhibitory amount of a compound of claim 1.

18. A method for inhibiting the enzymatic activity of a serine protease or cysteine protease comprising contacting a protease selected from the group consisting of serine protease and cysteine protease with an inhibiting amount of a compound of claim 2.