A novel class of nonpeptidic biaryl inhibitors of human cathepsin K

J . Med. Chem. 2003, 46, 3709-3727

3709

A Novel Cla ss of Non p ep tid ic Bia r yl In h ibitor s of Hu m a n Ca th ep sin K

J oe¨l Robichaud,*^,†Renata Oballa,^†Peppi Prasit,^†J ean-Pierre Falgueyret,^‡M. David Percival,^‡

Gregg Wesolowski,^§Sevgi B. Rodan,^§Donald Kimmel,^§Colena J ohnson,^|Cliff Bryant, Shankar Venkatraman,

Eduardo Setti, Rohan Mendonca, and J ames T. Palmer

Department of Chemistry, Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway,

Kirkland, Quebec, Canada H9H 3L1, Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic

Research, 16711 TransCanada Highway, Kirkland, Quebec, Canada H9H 3L1, Department of Bone Biology & Osteoporosis,

Merck Research Laboratories, West Point, Pennsylvania 19486, Department of Laboratory Animal Resources, Merck Research

Laboratories, West Point, Pennsylvania 19486, and Department of Medicinal Chemistry, Celera, 180 Kimball Way,

South San Francisco, California 94080

Received March 5, 2003

A novel series of nonpeptidic biaryl compounds was identified as potent and reversible inhibitors

of cathepsin K. The P2-P3 amide bond of a known amino acetonitrile dipeptide 1 was replaced

with a phenyl ring, thereby giving rise to this biaryl series that retained potency vs cathepsin

K and showed an improved selectivity profile against other cathepsins. Structural modification

within this series resulted in the identification of compound (R)-2, a potent human cathepsin

K inhibitor (IC₅₀) 3 nM) that is selective versus cathepsins B (IC₅₀) 3950 nM), L (IC₅₀

)

3725 nM), and S (IC₅₀) 2010 nM). In an in vitro assay involving rabbit osteoclasts and bovine

bone, compound (R)-2 inhibited bone resorption with an IC₅₀of 95 nM. It was shown that,

unlike some peptidic nitrile inhibitors of cysteine proteases, the nitrile moiety of (R)-2 is not

converted to the corresponding amide 3 by cathepsin K. This indicates that this class of

nonpeptidic nitrile inhibitors is unlikely to be hydrolyzed by cysteine proteases. Furthermore,

the inhibition of cathepsin K by compound (R)-2 was shown to be fully reversible and not

observably time-dependent. To demonstrate the efficacy of compound (R)-2 in vivo, it was

administered to ovariectomized (OVX) rhesus monkeys at 20 mg/kg, po once daily for 8 days,

and a urinary marker of bone turnover, N-telopeptide of type I collagen (uNTx), was measured.

During the eight-day dosing period, the mean reduction by compound (R)-2 in uNTx was 80%

(p < 0.001). This demonstrates that inhibition of cathepsin K leads to an inhibition of this

bone resorption marker in OVX rhesus monkeys and strongly suggests that inhibition of

cathepsin K is a viable therapeutic approach for the treatment of osteoporosis.

In tr od u ction

dation of bone matrix. It is postulated that molecules

that inhibit the activity of cathepsin K could serve as

useful therapeutic agents against diseases such as

osteoporosis and other bone disorders displaying exces-

sive levels of resorption.

Osteoporosis is characterized by low bone mass and

accompanying microarchitectural deterioration that

results in skeletal fragility and an increased risk of

fracture. The bone loss associated with osteoporosis is

brought about by an imbalance between bone resorption

(performed by osteoclasts) and bone formation (per-

formed by osteoblasts).¹The extracellular matrix of bone

is a composite of hydroxyapatite and protein, 90% of

which is type I collagen. Bone resorption requires both

decalcification of the hydroxyapatite by an acidic mi-

croenvironment and degradation of the protein matrix.

These processes occur in the acidic lacunae beneath the

osteoclast. Cathepsin K is a member of the papain

superfamily of cysteine proteases that is abundantly and

selectively expressed in osteoclasts^2-4and has been

shown to play a key role in osteoclast-mediated degra-

Cathepsin K is one of a growing number of cysteinyl

cathepsins (B, H, L, S, C, K, O, F, V, X, and W),⁵all of

which are involved in intracellular protein degradation.

Some of these enzymes have been shown to have a broad

tissue distribution (i.e., cathepsins L and B) whereas

others possess a much more selective distribution (i.e.,

cathepsins K,⁶S,^7,8and W⁹). The design of therapeuti-

cally useful cathepsin K inhibitors would therefore

require a high degree of selectivity versus others

members of this class of cysteine proteases.

Most of the current approaches to cathepsin inhibition

involve molecules of a peptidic nature.^10,11While these

classes of compounds can achieve good potency and

selectivity, they are often plagued by high molecular

weights and poor pharmacokinetic profiles. Peptidic

molecules bearing a nitrile warhead have been reported

to be inhibitors of cathepsin K,^12-15B,¹⁶and S.¹⁷Our

present approach entailed the replacement of one pep-

tidic bond of the known nitrile dipeptide 1¹⁸with an aryl

or heteroaryl substituent (see Figure 1, dipeptide 1 to

biaryl 4). This strategy provided us with several potent

* To whom correspondence should be addressed. Tel.: (514) 428-

3665. Fax.: (514) 428-4900. E-mail: joel_robichaud@merck.com.

^†Department of Chemistry, Merck Frosst Centre for Therapeutic

Research.

^‡Department of Biochemistry and Molecular Biology, Merck Frosst

Centre for Therapeutic Research.

^§Department of Bone Biology & Osteoporosis, Merck Research

Laboratories.

^|Department of Laboratory Animal Resources, Merck Research

Laboratories.

Celera.

Published on Web 07/16/2003

3710 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

Sch em e 1^a

a

Conditions: (a) PdCl₂(dppf), Na₂CO₃, DMF or 2-propanol, 85-95 °C, 2-18 h.

Sch em e 2^a

a

Conditions: (a) LiHMDS, THF, 0 °C to rt; Me₂CHCH₂I; (b) (COCl)₂, DMF (cat.), MePh, rt; (c) EtNMe₂, 0 °C; (R)-pantolactone, MePh,

-78 °C to 0 °C; (d) HCl, AcOH, 85 °C, 18 h; (e) PyBOP, HCl‚H₂NCH₂CN, Et₃N, DMF, rt; (f) diboron pinacol ester, KOAc, PdCl₂(dppf),

DMF, 85-90 °C, 3 h.

strategy employed involved the deracemization of the

alkylated acid (R,S)-7 by the use of a chiral protonation

protocol.¹⁹According to this protocol, the alkylated acid

(R,S)-7 is first converted to its corresponding acid

chloride 8 with oxalyl chloride. Treatment of this

intermediate 8 with ethyldimethylamine generates the

ketene functionality in situ which is trapped at low

temperature with (R)-(-)-pantolactone to afford the

desired chiral ester 9. This methodology consistently

afforded under carefully controlled conditions, the de-

sired chiral pantolactone ester 9 in a 10:1 ratio in favor

of the desired diastereoisomer.

Hydrolytic cleavage of the chiral ester 9 under acidic

F igu r e 1. Replacement of one peptide bond of dipeptide 1

conditions yielded the required chiral acid (R)-7 without

with a phenyl ring affords biaryl compound 4.

compromising the integrity of the newly generated

stereocenter.¹⁹Following hydrolysis, the chiral acid

(R)-7 was converted to its aminoacetonitrile amide

derivative 10 by a standard PyBOP coupling. Finally,

a palladium-mediated cross-coupling reaction of this

chiral aryl bromide 10 with diboron pinacol ester

efficiently afforded the desired boronic ester (R)-5, now

and selective small biaryl molecules with good overall

profiles. The purpose of the present report is to report

some of the SAR and enzymology obtained in this series

and the in vivo efficacy in an OVX rhesus monkey model

of bone resorption.

ready for functionalization. The initial exploration of P3

SAR was carried out as described in Scheme 1, with

Suzuki cross-couplings between either the racemic or

chiral boronate esters (R,S)-5 or (R)-5 and various

halogenated aryl/heteroaryl bromides.

Ch em istr y

Our key step, a Suzuki cross-coupling between various

aryl/heteroaryl halides or triflates with the required

boronic ester intermediate 5, as outlined in Scheme 1,

provided us with access to a large number of compounds

that could be rapidly assembled in one final step. The

racemic boronic ester (R,S)-5 was constructed in three

steps (see Scheme 2). Deprotonation of 3-bromophenyl

acetic acid 6 with LiHMDS followed by alkylation with

1-iodo-2-methyl propane yielded the desired alkylated

acid (R,S)-7. Conversion of this racemic acid (R,S)-7 to

its aminoacetonitrile amide derivative was accomplished

by a standard PyBOP coupling. The palladium-mediated

cross-coupling reaction of the aryl bromide with diboron

pinacol ester proceeded smoothly to yield the desired

racemic boronic ester (R,S)-5.

P 3 Su bstitu tion . Extended P3 explorations were also

conducted by further substitution of the terminal ni-

trogen of the piperazine (R)-2 as depicted in Scheme 3.

Treatment of piperazine (R)-2 with chloroacetone and

triethylamine afforded the desired alkylated product 11.

Similarly, treatment of (R)-2 with 2-bromoethanol or

isobutylene oxide in the presence of K₂CO₃yielded the

desired alkylated products 12 and 13, respectively.

Reaction of piperazine (R)-2 with 2-amino-2-methylpro-

pyl hydrogen sulfate afforded the desired alkylated

product 14.

Preparation of the chiral boronic ester (R)-5 repre-

sented a more challenging synthetic approach. The

The synthesis of compound 15 (see Scheme 4) in

which the internal phenyl ring is replaced with a

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3711

Sch em e 3^a

a

Conditions: (a) chloroacetone, Et₃N, DMF, rt; (b) 2-bromoethanol, K₂CO₃, MeCN, ∆; (c) isobutylene oxide, K₂CO₃, DMF, 100 °C; (d)

2-amino-2-methylpropyl hydrogen sulfate, K₂CO₃, DMF, 100 °C.

Sch em e 4^a

a

Conditions: (a) PhB(OH)₂, PdCl₂(dppf), KOAc, DMF, 80 °C; (b) LDA, THF, 0 °C; 1-bromo-2-butene, -78 °C to rt; (c) H₂, 5% Pd/C*,

EtOH, 40 psi, 8 h; (d) NaOH, MeOH/H₂O (1:1), 18 h; (e) PyBOP, HCl‚H₂NCH₂CN, Et₃N, DMF, rt.

Sch em e 5^a

a

Conditions: (a) LDA, THF, 0 °C; 1-bromo-2-butene, -78 °C to rt; (b) H₂, 5% Pd/C*, EtOH, 40 psi, 8 h; (c) PyBOP, HCl‚H₂NCH₂CN,

Et₃N, DMF, rt.

Sch em e 6^a

a

Conditions: (a) pTsOH, HC(OCH₃)₃, MeOH, ∆; (b) (CF₃SO₂)₂O, Et₃N, CH₂Cl₂, 0 °C; (c) diboron pinacol ester, KOAc, PdCl₂(dppf),

DMF, 90 °C; (d) Na₂CO₃, PdCl₂(dppf), DMF, 100 °C; (e) KHMDS, THF, 0 °C to rt; RX, THF, 0 °C to rt; (f) LiOH, MeOH, H₂O; (g) PyBOP,

HCl‚H₂NCH₂CN, Et₃N, DMF, rt.

pyridine began with a Suzuki cross-coupling reaction

between commercially available methyl (5-bromopyri-

din-3-yl)acetate (16) and phenylboronic acid to afford

the desired biaryl compound 17. R-Alkylation of ester

17 with 1-bromo-2-butene, followed by hydrogenation

of the resulting double bond, yielded the desired func-

tionalized ester 18. This ester was saponified to its

corresponding carboxylic acid derivative using NaOH

and submitted to a standard PyBOP coupling reaction

with aminoacetonitrile hydrochloride and triethylamine

to afford the desired pyridine analogue 15. The synthe-

sis of the thiazole containing compound 19 (see Scheme

5) began with the R-alkylation of acid 20²⁰with LDA

and 1-bromo-2-butene to afford, after hydrogenation of

the generated double bond, the carboxylic acid 21.

Standard PyBOP coupling reaction of this intermediate

with aminoacetonitrile hydrochloride and triethylamine

afforded the desired thiazole analogue 19.

P 2 Su bstitu tion . Exploration of the P2 group in this

series was carried out to determine which substituents

would provide optimal cathepsin K activity. The syn-

thetic route began with the esterification of (3-hydroxy-

phenyl)acetic acid (22) with trimethylorthoformate and

tosic acid, followed by the treatment of the phenol with

triethylamine and trifluoromethanesulfonic anhydride

to yield the desired triflic ether 23 as indicated in

Scheme 6. The triflate 23 was then converted to the

corresponding boronic ester 24 by a palladium-mediated

reaction with diboron pinacol ester. Boronic ester 24 was

then cross-coupled under palladium-catalyzed Suzuki

3712 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

Ta ble 1. Inhibition of Human Cathepsins K, L, S and B by Biaryl Analogues

a

Each IC₅₀value corresponds to an average of at least two independent determinations except where noted with an asterisk.

conditions with 1-(4-bromophenyl)-4-methylpiperazine

(25) to afford the desired alkylation precursor 26. The

key intermediate 26 was then deprotonated with KH-

MDS and alkylated with a variety of electrophiles to

yield several P2 alkylated products 27b-f. All of these

ester intermediates 27b-f were then sequentially hy-

drolyzed using LiOH, and the corresponding carboxylic

acids were coupled with aminoacetonitrile hydrochloride

according to a standard PyBOP amide coupling protocol

to afford the desired amides 28b-f.

(40-fold), it was unselective against cathepsins L and

S. Other workers have previously reported that a P2-

P3 carbamate group could be replaced with a biaryl

group to afford cathepsin K inhibitors with improved

selectivity over their peptidic counterparts.²²The re-

placement of the P2-P3 amide bond of the dipeptide

nitrile 1 with a phenyl provided compound 4, which

retained activity against cathepsin K and showed a

significantly improved selectivity profile against the

other cathepsins (see Table 1). However, it was clear

that further improvements were required, especially

considering the lack of potency of the m-biphenyl 4 in

the in vitro bone resorption assay (IC₅₀) 3560 nM).

Bioch em istr y

Compounds were tested against human cathepsins K,

L, B, and S and a humanized form of rabbit cathepsin

K. Humanized rabbit cathepsin K was obtained by

mutation of two active site amino acid residues of the

rabbit enzyme to the corresponding human residues

(Tyr⁶¹f Asp⁶¹and Val¹⁵⁷f Leu¹⁵⁷). Both cathepsin K

enzymes also contain a Ser⁴⁹f Ala⁴⁹mutation to

remove a N-glycosylation site. Human cathepsins K, L,

and S and humanized rabbit cathepsin K were ex-

pressed and purified as described in the Experimental

Section while purified human liver cathepsin B was

obtained from Sigma. Inhibitory potency was deter-

mined after a 15 min preincubation with the various

enzymes prior to the addition of 2 µM of the substrate

Z-Leu-Arg-AMC. Assays were performed in 96-wells

plates, and IC₅₀values were determined by fitting

experimental values to a four-parameter logistic model.

Compounds were also tested in an in vitro bone resorp-

tion assay that evaluates degradation of bovine bone by

isolated rabbit osteoclasts as described previously.²¹

As previously observed,²²proper positioning of the

second aryl group appeared critical as the para-linked

biphenyl 29 lost considerable activity against cathepsin

K. Replacement of the internal phenyl group of 4 with

a either a pyridine (compound 15) or thiazole (compound

19) had a deleterious effect on cathepsin K potency. The

pyridine compound 15, however, showed increased

activity in the in vitro bone resorption assay. This

translated to a decrease in shift between the enzyme

inhibition and the ability to inhibit bone resorption in

vitro (60-fold shift for the biphenyl 4 vs 10-fold shift for

the phenyl pyridine 15).

Most of the remaining P3 SAR focused on modifica-

tions and substitutions to the terminal aryl ring. Para

substitution off the terminal phenyl group of 4 with

either a carboxamide, a methyl sulfone or a tetrazole

(30, 31, and 32, respectively) proved to be detrimental

to cathepsin K potency. Elaboration of the three posi-

tional isomers on the terminal phenyl group with a

morpholine group revealed surprisingly little SAR to

distinguish between these three spatially different

compounds. All three isomers, the para 33, the meta

34, and the ortho 35 showed equivalent potencies

against cathepsin K. When the terminal phenyl group

of 4 was replaced with the three isomeric pyridines, the

Resu lts a n d Discu ssion

The known dipeptide nitrile 1¹⁸and some of the biaryl

analogues synthesized are shown in Table 1. While the

original dipeptide 1 showed good potency against cathe-

psin K and modest selectivity against cathepsin B

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3713

Ta ble 2. Inhibition of Human Cathepsins K, L, S and B by Basic Biaryl Analogues

a

Each IC₅₀value corresponds to an average of at least two independent determinations except where noted with an asterisk.

potency of the compounds increased as the position of

the nitrogen moved away from the point of attachment

with the p-pyridine 39 being the most potent against

cathepsin K. Insertion of an extra 1,4-disubstituted

phenyl group between the pyridine and the internal

phenyl group of 39 (compound 40) did not significantly

alter potency against cathepsin K and roughly main-

tained the observed 10-fold shift between the enzyme

activity and the bone resorption assay.

While the selectivity profiles of several of these

analogues showed improvement over the original dipep-

tide 1, the potencies of these compounds were still

lacking, both in the cathepsin K enzyme assay, and in

the in vitro bone resorption assay. Encouraged by the

results obtained for the pyridine analogues 39 and 40,

we decided to focus our efforts on introducing other basic

functional groups in the P3 portion of the molecule. The

m-indole 41 (see Table 2) failed to give any improve-

ment, but there was a considerable increase in potency

against cathepsin K for the p-indole (R,S)-42. But while

this increase in potency only equaled that of the initial

biaryl 4, it was clear that the potency of this compound

in the bone resorption assay (IC₅₀) 249 nM) was a

dramatic improvement over the initial biaryl 4. The shift

between the enzyme assay and the bone resorption

assay was drastically reduced to 5-fold for the indole

(R,S)-42.

cathepsin K (IC₅₀) 79 nM and 8 nM, respectively). This

10-fold difference in potency between the enantiomers

prompted us to develop an efficient enantioselective

route to these compounds (see Scheme 2).

Although the substitution pattern around the termi-

nal phenyl ring afforded similar potencies with the

morpholine analogues (33, 34, and 35, see Table 1), it

was observed that this trend did not hold true for the

piperazine analogues. Ortho-branching of a piperazine

ring onto the biaryl core afforded 44 which showed no

improvement in cathepsin K activity over the previously

described compounds (2 and (R,S)-42). Shifting the

piperazine to the meta position (compound 43) resulted

in a loss of activity vs cathepsin K. We were pleased,

however, to discover that the para position was optimal

for the piperazine substitution, yielding compound

(R,S)-2, which showed a substantial increase in potency

against cathepsin K (IC₅₀) 9 nM). The chiral piperazine

(R)-2 exhibited not only a low single digit potency

against cathepsin K (IC₅₀) 3 nM) but also an improved

selectivity profile vs the other cathepsins (>1200-fold

for cathepsins L and B and >650-fold for cathepsin S).

The increase in cathepsin K potency also translated to

an increased potency in the bone resorption assay (IC₅₀

) 95 nM), although the shift remains somewhat high

(30-fold).

Interestingly, the racemic and chiral N-methylated

compounds (R,S)-46 and (R)-46 showed very similar

profiles (Cat K IC₅₀) 8 nM and 6 nM, respectively).

These results suggest that further elaboration of the

basic piperazine nitrogen of (R)-2 should be possible.

Replacement of the piperazine ring with a 1,2,3,6-

tetrahydropyridine ring (compound 47) afforded a mod-

At this point it was decided to separate the two

enantiomers of the racemic indole (R,S)-42 to determine

whether the potency of racemic (R,S)-42 resided pre-

dominantly in only one of the two enantiomers. Separa-

tion by chiral chromatography afforded enantiomers (S)-

42 and (R)-42 which showed very different potencies vs

3714 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

Ta ble 3. Inhibition of Human Cathepsins K, L, S and B by P3 Alkylated Analogues

a

Each IC₅₀value corresponds to an average of at least two independent determinations except where noted with an asterisk.

est loss in potency against cathepsin K; however,

hydrogenation of the double bond (compound 48) im-

proved the cathepsin K potency and also improved the

selectivity profile against the other cathepsins. In

conclusion, these efforts led to the discovery of the para-

oriented piperazine compound (R)-2 which displays a

range of highly desired properties (cathepsin K potency,

selectivity, and potency in the assay).

propyl, cyclopropyl methyl, 2-methylbutyl, and cyclobu-

tyl methyl) all resulted in a loss of cathepsin K potency

relative to the isobutyl group of (R,S)-46. We therefore

conclude that the isobutyl P2 substituent for this series

of compounds appears to be optimal for potency against

cathepsin K.

Mea su r em en t for P oten tia l Nitr ila se Activity of

ca th ep sin K w ith Com p ou n d (R)-2. It has been

previously described that peptide nitrile inhibitors of

papain family cysteine proteases can undergo slow

hydrolysis of the nitrile moiety to the corresponding

amide which is then rapidly hydrolyzed to the acid.^23,24

As shown in Scheme 7a, the nitrilase activity of the

enzyme can be favored by the addition of thiols. It was

hypothesized that the reversible thioimidate ester bond

formed between the enzyme and the inhibitor is at-

tacked by thiols to form a nonenzymic thioimidate that

is rapidly hydrolyzed to the amide. Because of the well-

known amidase activity of the papain cysteine protease

enzymes, the amide will be catalytically transformed to

the corresponding acid (Scheme 7b). To test whether the

present biaryl nitrile inhibitors could serve as substrates

for cathepsin K and be potentially inactivated by this

enzyme, 1 µM of compound (R)-2 was incubated with

two concentrations of cathepsin K in the presence of 1

mM â-mercaptoethanol. To identify the hydrolysis prod-

ucts of the reaction, the amide and the carboxylic acid

analogues 3 and 52 of compound (R)-2 were chemically

synthesized and used as standards for RP-HPLC analy-

sis. After a 30 min incubation, the assay mixture was

analyzed by RP-HPLC for the presence of the carboxylic

acid 52. As shown in Figure 2, no detectable amount of

acid 52 could be detected in the presence of either 10

nM (Figure 2B) or 1 µM (Figure 2C) enzyme. After 30

min, in the presence of 10 nM cathepsin K, 1 µM of the

synthetic amide 3 was extensively converted to the acid

52 (Figure 2D), consistent with the amidase activity of

cathepsin K. Amidase activity was also blocked by the

addition of 1 µM of the irreversible inhibitor E64 (Figure

2E). Incubation of compound (R)-2 with â-mercaptoet-

hanol up to 24 h did not lead to the formation of any

Exten d ed P 3 SAR. Following our observation that

the basic secondary nitrogen of the piperazine ring can

be substituted without adversely affecting potency and

selectivity (i.e., compound (R,S)-2 vs compound (R,S)-

46, see Table 2), we decided to conduct further SAR

studies on the terminal P3 portion of the molecule (see

Table 3). The Boc-protected compound 50 lost consider-

able activity against cathepsin K (IC₅₀) 922 nM),

possibly due to the loss in basicity of the terminal

piperazine nitrogen. The ketone-capped alkyl piperazine

11 recovered almost all of the potency against cathepsin

K and maintained a good overall selectivity profile

against the other cathepsins. Alkylation of the terminal

nitrogen of the piperazine ring with a bulky tert-butyl

group (compound 51) did not adversely affect the

potency against cathepsin K; the presumed equatorial

conformation of the tert-butyl substituent suggests a

hydrogen bond between the tertiary alkylamine and an

S3 residue via an axial vector. This result also supports

our initial hypothesis that high potency and selectivity

require the presence of a basic nitrogen in this P3

portion of the molecule. Other alkylated piperazines

(12-14) showed similar cathepsin K potencies but did

not have any substantial advantages over the lead

unsubstituted piperazine (R)-2.

P 2 Su bstitu tion . Having established some of the

SAR on the P3 portion of this series, we decided to

conduct SAR studies on the N-methylated version of our

optimized compound (R,S)-2 by varying the P2 substitu-

tion (see Table 4). It is interesting to note that compound

28a , devoid of any P2 substituent, is also devoid of

activity against any of the cathepsin enzymes. Introduc-

tion of a variety of groups in the P2 position (i.e., benzyl,

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3715

Ta ble 4. Inhibition of Human Cathepsins K, L, S, and B by P2 Alkylated Analogues

a

Each IC₅₀value corresponds to an average of at least two independent determinations.

Sch em e 7

hydrolysis products. Although cysteine proteases of the

papain family are known to possess nitrilase activity,

this enzyme-catalyzed reaction is extremely slow when

compared to the peptidase or amidase activity. It has

been previously demonstrated that the nitrilase activity

K_mof dipeptide nitrile inhibitors for papain corresponds

to their K_ifor protease inhibition.²⁴Our results show

that compound (R)-2 which possesses good affinity for

the enzyme (K_i) 5 nM) is clearly a poor substrate for

this reaction since no hydrolysis products were observed

at 100-fold K_iconcentration and 1 mM â-mercaptoet-

hanol. These results show that it is unlikely that the

nitrile moiety of this class of nonpeptidic inhibitors could

be hydrolyzed by cysteine proteases to cause the inac-

tivation of the inhibitor in vivo.

Tim e-Dep en d en cy of In h ibition . Previously de-

scribed cysteine protease inhibitors which form covalent

bonds with the cysteine active site of the enzyme often

inhibit in a time-dependent manner.^17,21,25That is, the

attainment of the equilibrium between the enzyme-

bound inhibitor and the free inhibitor is slow enough

to be experimentally observable (seconds time scale).

Figure 3 shows the time course of the reaction when

cathepsin K is incubated with the synthetic substrate

Z-Leu-Arg-AMC in the presence of various concen-

trations of compound (R)-2 (Figure 3A). The results

3716 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

F igu r e 3. Progress curves for the reaction of humanized

rabbit cathepsin K in the presence of compound (R)-2 (A) or

compound 45 (B). Progress curves were obtained after initia-

tion of the reaction by the addition of 0.2 nM of humanized

rabbit cathepsin K. Control reaction (b) without inhibitor; (A)

33 nM (3) or 100 nM (9) of compound (R)-2; (B) 100 nM (3) or

500 nM (9) of compound 45.

3 nM) (Figure 3B). The curves obtained in Figure 3B

(compound 45) were fitted to eq 1 (see Experimental

Methods) and the kinetic parameters k_offand k_onwere

calculated (see Experimental Methods) giving (n ) 2)

F igu r e 2. RP-HPLC profiles of the reaction mixture when

humanized rabbit cathepsin K is incubated with compound

(R)-2. (A) Coinjection of 1 µM of compound (R)-2 (CN) with 1

µM of the synthetic amide 3 (CONH₂) and 1 µM of the

synthetic acid 52 (COOH). (B) Reaction mixture when 1 µM

of compound (R)-2 is incubated with 10 nM humanized rabbit

cathepsin K and 1 mM â-mercaptoethanol for 30 min at room

temperature. (C) Same as (B) with 1 µM enzyme. (D) Reaction

mixture when 1 µM of the synthetic amide 3 is incubated with

10 nM enzyme and 1 mM â-mercaptoethanol for 30 min and

(E) in the presence of 1 µM E64.

values of 1.4 × 10^-3s^-1and 1.4 × 10⁵M^-1s^-1

,

respectively. The calculated K_i(10 nM) agrees with the

IC₅₀(10 nM) measured at sub K_mconcentrations of

substrate (where K_i∼ IC₅₀). A similar observation has

been reported for cathepsin S dipeptide nitrile inhibi-

tors.¹⁷There, the addition of a dimethylglycine residue

at P1 slows the on-rate and the off-rate of the compound

by 2 to 3 orders of magnitude when compared to the P1

monosubstituted compounds.

With both compounds ((R)-2 and 45), the kinetics of

the inhibition obtained were consistent with fully

reversible compounds. Experiments (data not shown) in

which the enzyme and the inhibitor were preincubated

together (100-fold concentrated) and then diluted into

obtained show that the rate of the reaction is linear for

two concentrations of compound (R)-2 assayed and

implies a relatively rapid attainment of the equilibrium.

However, the addition of a spiro-cyclopropyl ring at P1

gave a time-dependent inhibitor (compound 45) that had

a similar potency to that of compound (R)-2 (IC₅₀

)

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3717

associated with accelerated bone resorption and forma-

tion is established by surgical oophorectomy (OVX) at

least three months pretreatment. The same measure-

ment of bone resorption markers (bone collagen-specific

breakdown products), urinary N-telopeptides [uNTx]

used in humans taking antiresorptive therapy, is used

in the rhBMRA. A group of 21 OVX rhesus monkeys

was sequentially trained over a nine-week period to

chair restraint, chair restraint plus nasogastric (NG)

tubing, and NG tubing plus 5 mL saline dosing. These

monkeys, about three years post-OVX, aged 13.5 years,

and weighing 7.4 ( 1.1 kg were used and randomized

to groups (N ) 11 vehicle; N ) 10 compound (R)-2) by

baseline uNTx/Cre and body weight. Compound (R)-2

monkeys were treated po, qd for 2 days with 1%

carboxymethylcellulose (vehicle) and then treated with

compound (R)-2 po, qd for 8 days at 20 mg/kg. At the

end of drug treatment, monkeys were treated for three

additional days with vehicle. Vehicle monkeys received

vehicle throughout the 13-day period. Twenty-four hour

urine collections of both groups were made on the day

before the start of drug dosing, the second, fifth, and

eighth days of drug dosing, and the third day after the

end of drug dosing. One additional urine collection was

made 17 days after the last drug dose. The urine

samples were frozen at -70 °C within 4 h of collection

and on the day of assay, samples were thawed and

measured in duplicate using the N-telopeptides (NTx)

kit (Ostex International; Seattle, WA) using manufac-

turers’ instructions. Creatinine in each urine specimen

was also measured (Catalog 555-A, Sigma; St. Louis,

MO). The results are expressed as uNTx/Cre (nM/mM).

F igu r e 4. Mean plasma levels after intravenous administra-

tion of the HCl salt of compound (R)-2 in 5% dextrose and after

oral administration of the HCl salt of (R)-2 as a solution in

water in intact rhesus monkeys. Dosing volumes for both

intravenous and oral administration were 1 mL/kg. Compound

levels were determined in the plasma samples by LC/MS/MS

at the times indicated.

substrate-containing buffer showed a complete recovery

of the activity which is once again consistent with the

reversible nature of these nitrile inhibitors.

P h a r m a cok in etic P r ofile of In h ibitor (R)-2. To

determine if the compounds in this series possessed a

suitable pharmacokinetic profile, in vivo dosing studies

were performed with compound (R)-2. The results of this

study are presented in Figure 4. When the HCl salt of

compound (R)-2 was administered orally to intact rhesus

monkeys at a dose of 20 mg/kg in water, the compound

was well absorbed with a bioavailability of 32%. The

half-life of compound (R)-2 was determined after intra-

venous dosing in 5% dextrose and was found to be 9.6

h in rhesus monkeys. These results clearly demonstrate

that compound (R)-2 possesses good pharmacokinetic

properties (in contrast to most peptidic compounds) in

rhesus monkeys and could be used orally for in vivo

studies to test the effects of a cathepsin K inhibitor on

bone resorption.

As shown in Figure 5, the cathepsin K inhibitor (R)-2

dosed at 20 mg/kg po, qd decreased uNTx/Cre by 75-

87% relative to vehicle-treatment (p < 0.001). The

suppression was detected after the second dose, the first

sampling time. During the eight-day dosing period, the

mean reduction in uNTx/Cre by compound (R)-2 was

80% (p < 0.001). At 3 days after the last dose of

compound (R)-2, uNTx/Cre remained suppressed by 58%

(p < 0.004). However, by 17 days after the last dose,

uNTx/Cre in compound (R)-2-treated monkeys had

Rh esu s Mon k ey Bon e Resor p tion Ma r k er Assa y.

In the Rhesus Monkey Bone Resorption Marker Assay

(rhBRMA), estrogen-deficiency bone loss (i.e., low BMD)

F igu r e 5. Compound (R)-2 reduces bone resorption in OVX rhesus monkeys. Bone resorption in OVX monkeys treated with

either the HCl salt of compound (R)-2 (b) or vehicle (2) was evaluated using the urine biochemical marker NTx. Data are expressed

as uNTx/creatinine (nM/mM).

3718 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

flash chromatography over silica gel (33% ethyl acetate/

hexanes) to provide 2-(1,1′-biphenyl-3-yl)-4-methylpentenoic

acid (250 mg, 0.93 mmol). LCMS: 267.1 (M + H⁺). A solution

of 2-(1,1′-biphenyl-3-yl)-4-methylpentenoic acid (250 mg, 0.93

mmol) and Pd/C (10 mol %) in EtOH was subjected to

hydrogenation under pressure (40 psi) for 8 h. The reaction

mixture was filtered through Celite, and the filtrate was

concentrated in vacuo to yield 2-(1,1′-biphenyl-3-yl)-4-meth-

ylpentanoic acid. A solution comprised of 2-(1,1′-biphenyl-3-

yl)-4-methylpentanoic acid (251 mg, 0.91 mmol) in DMF (5 mL)

was treated with aminoacetonitrile (180 mg, 2.0 mmol), PyBOP

(520 mg 1.0 mmol) and triethylamine (500 µL, 3.0 mmol). The

mixture was stirred for 4 h and then diluted with water (50

mL) and ethyl acetate (20 mL). The organic layer was

separated, washed with an aq 1.0 M Na₂CO₃solution, aq 1.0

M hydrochloric acid solution, water, and brine, dried over

MgSO₄, and concentrated in vacuo. The residue was recrystal-

lized from 30% ethyl acetate/hexanes to provide the title

compound 4 (300 mg, 88% yield). LCMS: 307.3 (M + H⁺).

HRMS (+FAB): calcd for C₂₀H₂₃N₂O [MH⁺] 307.18104, found

307.18108. Anal. (C₂₀H₂₂N₂O) C: calcd, 78.40; found, 78.14;

H: calcd, 7.24; found, 7.24; N: calcd, 9.14; found, 8.99.

returned to control group levels. As indicated in the

pharmacokinetic studies in intact rhesus monkeys

(Figure 4), plasma levels of compound (R)-2 remained

at or above the bone resorption IC₅₀for a least 24 h

following a 20 mg/kg oral dose. The significant suppres-

sion of uNTx/Cre levels demonstrates that inhibition of

cathepsin K by compound (R)-2 leads to ∼80% inhibition

of bone resorption markers in OVX rhesus monkeys,

similar to what is observed in osteoporotic humans

treated with bisphosphonates.²⁶This finding suggests

that inhibition of cathepsin K may be a viable thera-

peutic approach for the treatment of osteoporosis and

other bone disorders displaying excessive levels of

resorption.

Con clu sion

In the present report we have shown that cathepsin

K can be inhibited by nonpeptidic nitrile compounds.

These biaryl compounds represent a new class of cys-

teine protease inhibitors. The SAR within this series

indicated that the R configuration of an isobutyl P2

group was optimal for cathepsin K potency as was the

p-piperazine biphenyl moiety in P3. These substitutions

resulted in the optimal nitrile compound (R)-2 which

was selective vs cathepsins L, S, and B and sub-100 nM

in an in vitro model of bone resorption. This series

inhibited cathepsin K in a non-time-dependent fashion,

and the potential nitrilase activity of cathepsin K (nitrile

to amide conversion) was shown not to occur with

compound (R)-2. Moreover, this inhibitor proved to have

good pharmacokinetic properties in rhesus monkeys and

showed excellent in vivo efficacy in the rhesus monkey

model for inhibition of bone resorption.

2-(1,1′-Bip h en yl-4-yl)-N-(cya n om et h yl)-4-m et h ylp en -

ta n a m id e (29). The title compound was synthesized according

to the same procedure used for compound 4 except 1,1′-

biphenyl-4-ylacetic acid was used instead of 1,1′-biphenyl-3-

ylacetic acid. LCMS: 307.1 (M + H⁺). HRMS (+FAB): calcd

for C₂₀H₂₃N₂O [MH⁺] 307.18104, found 307.18108.

N-(Cya n om et h yl)-4-m et h yl-2-(2-p h en yl-1,3-t h ia zol-4-

yl)p en ta n a m id e (19). To a cold (0 °C), stirred solution of (2-

phenyl-1,3-thiazol-4-yl)acetic acid²⁰(1.2 g, 5 mmol) in THF was

slowly added a solution of LDA (2.0 M in THF, 5.2 mL, 10.4

mmol). The reaction mixture was stirred at 0 °C for 1 h,

3-bromo-2-methylpropene (1.3 g, 10.0 mmol) was added, and

the 0 °C solution was stirred for an additional 1 h. HCl (1.0

N) was added, and the mixture was extracted with EtOAc,

washed with brine, and dried over Na₂SO₄. Upon concentration

in vacuo, the residue was purified by flash silica gel column

chromatography (1:1 EtOAc/hexanes) to afford 0.6 g (42%

yield) of 4-methyl-2-(2-phenyl-1,3-thiazol-4-yl)pent-4-enoic acid.

A solution of 4-methyl-2-(2-phenyl-1,3-thiazol-4-yl)pent-4-enoic

acid and 10% Pd/C (5 mol %) in EtOH was subjected to

hydrogenation under pressure (40 psi) for 8 h. The reaction

mixture was filtered through Celite, and the filtrate was

concentrated in vacuo to yield 4-methyl-2-(2-phenyl-1,3-thiazol-

4-yl)pentanoic acid. To a solution of 4-methyl-2-(2-phenyl-1,3-

thiazol-4-yl)pentanoic acid (300 mg, 1.1 mmol) in DMF (10 mL)

were added aminoacetonitrile HCl (184 mg, 2.0 mmol), PyBOP

(520 mg, 1.0 mmol), and TEA (0.5 mL, 3.0 mmol). The mixture

was stirred at room-temperature overnight. The DMF was

removed in vacuo, and to the residue was added EtOAc (20

mL) and a saturated aqueous solution of NaHCO₃(20 mL).

The organic layer was washed with brine and dried over Na₂-

SO₄. Upon concentration in vacuo, the residue was purified

by flash column chromatography (1/1, EtOAc/hexanes) to

afford 250 mg (72% yield) of the title compound (19). HRMS

Exp er im en ta l Section

Gen er a l. All substrates and reagents were obtained com-

mercially and used without further purification. Proton (¹H

NMR) magnetic resonance spectra were recorded on a Bruker

400 MHz instrument unless noted otherwise. All spectra were

recorded using residual solvent (CHCl₃, acetone, or DMSO)

as internal standard. Signal multiplicity was designated

according to the following abbreviations: s ) singlet, d )

doublet, app d ) apparent doublet, dd ) doublet of doublets,

t ) triplet, q ) quartet, dt ) doublet of triplets, dq ) doublet

of quartets, m ) multiplet, br s ) broad singlet, br t ) broad

doublet, br t ) broad triplet. Elemental analyses were provided

by Oneida Research Services Inc., Whitesboro, NY. High-

resolution mass spectra (HRMS-FAB⁺) were obtained at the

Biomedical Mass Spectrometry Unit, McGill University, Mon-

treal, Quebec, Canada. HPLC chromatograms were obtained

using a Waters Delta Prep 3000 HPLC with a Chiralpak AD

column (5 cm, I.D. × 50 cm length, 20 µm). Rotations were

measured on a Perkin-Elmer polarimeter. Reactions were

carried out with continuous stirring under a positive pressure

of nitrogen except where noted. Flash chromatography was

carried out with silica gel 60, 230-400 mesh.

(+FAB): calcd for

C

₁₇H₂₀N₃OS [MH⁺] 314.13271, found

314.13255. Anal. (C₁₇H₁₉N₃OS) C: calcd, 65.15; found, 65.07;

H: calcd, 6.11; found, 5.96; N: calcd, 13.41; found, 13.34.

N-(Cya n om et h yl)-4-m et h yl-2-(5-p h en ylp yr id in -3-yl)-

p en ta n a m id e Tr iflu or oa ceta te (15). To a solution of bro-

mobenzene (3.2 g, 20.0 mmol) in DMF (20 mL), 4,4,4′,4′,5,5,5′,5′-

octamethyl-2,2′-bi-1,3,2-dioxaborolane (5.0 g, 20.0 mmol), and

KOAc (3.5 g, 40.0 mmol) were added. The solution was

degassed with nitrogen for 5 min, PdCl₂(dppf) (0.5 g, 0.68

mmol) was added, and the mixture was heated at 80 °C for 2

h. After the mixture was cooled to room temperature, methyl

(5-bromopyridin-3-yl)acetate (16) (4.68 g, 20.0 mmol) and Na₂-

CO₃(10 mL) were added. The reaction mixture was degassed

for 5 min, PdCl₂(dppf) (0.5 g, 0.68 mmol) was added, and the

mixture was heated in a sealed vessel overnight at 85 °C. The

reaction mixture was filtered through a plug of Celite, and

the DMF was evaporated in vacuo. To the residue was added

a mixture of EtOAc/aq NaHCO₃(150 mL/150 mL), and the

N²-Ben zoyl-N¹-(cyan om eth yl)-L-leu cin am ide (1).¹⁸HRMS

(+FAB): calcd for

274.15553.

C

₁₅H₂₀N₃O₂[MH⁺] 274.15555, found

2-(1,1′-Bip h en yl-3-yl)-N-(cya n om et h yl)-4-m et h ylp en -

ta n a m id e (4). A solution of LDA (2.2 mL, 2.0 M in THF) in

THF (20 mL) was cooled to 0 °C and then treated with 1,1′-

biphenyl-3-ylacetic acid (0.212 g, 1.0 mmol). The mixture was

stirred for 40 min, cooled to -78 °C, and then treated with

3-bromo-2-methylpropene (135 µL, 1.3 mmol). The mixture was

stirred for 1 h, treated with 1.0 M hydrochloric acid (5 mL),

and then diluted with ethyl acetate (50 mL). The organic layer

was separated, washed with water, brine, dried over MgSO₄,

and concentrated in vacuo. The crude product was purified by

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3719

organic phase was separated and washed with brine. After

drying over Na₂SO₄and concentration in vacuo, the residue

was purified by silica gel, using EtOAc as eluent, to yield 3.0

g (66% yield) of methyl (5-phenylpyridin-3-yl)acetate (17).

Methyl (5-phenylpyridin-3-yl)acetate (17) was converted to

methyl 4-methyl-2-(5-phenylpyridin-3-yl)pent-4-enoate using

the same procedure used to generate 2-(1,1′-biphenyl-3-yl)-4-

methylpentanoic acid (intermediate in the synthesis of 38). A

solution of methyl 4-methyl-2-(5-phenylpyridin-3-yl)pent-4-

enoate and 10% Pd/C (10 mol %) in EtOH was subjected to

hydrogenation under pressure (40 psi) for 8 h. The reaction

mixture was filtered through Celite, and the filtrate was

concentrated in vacuo to yield methyl 4-methyl-2-(5-phenylpy-

ridin-3-yl)pentanoate (18). Methyl 4-methyl-2-(5-phenylpyri-

din-3-yl)pentanoate (18) was saponified following general

procedure 4 to yield 4-methyl-2-(5-phenylpyridin-3-yl)pen-

tanoic acid. 4-Methyl-2-(5-phenylpyridin-3-yl)pentanoic acid

(300 mg, 1.0 mmol) was converted to the title compound 15

using PyBOP (520 mg, 1.0 mol), aminoacetonitrile hydrochlo-

ride (184 mg, 2 mmol), and TEA (0.3 mL, 2.0 mmol) in DMF

(5 mL). The usual workup procedure afforded 250 mg (82%

yield) of N-(cyanomethyl)-4-methyl-2-(5-phenylpyridin-3-yl)-

pentanamide trifluoroacetate (15). HRMS (+FAB): calcd for

(2R)-N-(Cya n om et h yl)-4-m et h yl-2-[3-(4,4,5,5-t et r a m e-

th yl-1,3,2-d ioxa bor ola n -2-yl)p h en yl]p en ta n a m id e ((R)-5).

To the (2R)-2-(3-bromophenyl)-4-methylpentanoic acid ((R)-7)

(52.5 g, 193.7 mmol), PyBOP (105.83 g, 203.4 mmol), and

aminoacetonitrile hydrochloride (35.84 g, 287.5 mmol) was

added dry DMF (1.2 L) under an atmosphere of dry nitrogen

followed by Et₃N (84.0 mL, 600.5 mmol), and the reaction

mixture was stirred for 18 h at room temperature. The reaction

mixture was poured into aq sat. NaHCO₃, extracted with Et₂O,

and the organic layer washed with aq sat. NaHCO₃(3×), brine,

and dried over MgSO₄. The solvent was concentrated in vacuo

to afford a crude oil which was purified by flash chromatog-

raphy over silica gel (EtOAc/hexanes, 2/8, then 3/7) to yield

the desired (2R)-2-(3-bromophenyl)-N-(cyanomethyl)-4-meth-

ylpentanamide (10) as a pale yellow oil (59.9 g, quantitative

yield). [R]_D) -47.7° (c ) 1.49, MeOH). HRMS (+FAB): calcd

for C₁₄H₁₇BrN₂O [MH⁺] 309.0524, found 309.06034.

To (2R)-2-(3-bromophenyl)-N-(cyanomethyl)-4-methylpen-

tanamide (10) (21.7 g, 70.23 mmol), diboron pinacol ester (21.4

g, 84.27 mmol), and KOAc (20.68 g, 210.69 mmol) was added

dry DMF (400 mL) under an atmosphere of dry nitrogen,

followed by PdCl₂(dppf) (1.723 g, 2.11 mmol), and the reaction

was heated at 85 °C for 1 h. More PdCl₂(dppf) (0.03 equiv)

was added and the reaction mixture heated at 90 °C for

another 2 h. The reaction mixture was poured into water and

extracted with C₆H₆/Et₂O, and the organic phase was washed

with water, brine, and dried over Na₂SO₄. The solvent was

concentrated in vacuo to afford a crude oil which was purified

by flash chromatography over silica gel (EtOAc/hexanes, 3/7,

then 35/65) to yield the desired (2R)-N-(cyanomethyl)-4-

methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phe-

nyl]pentanamide ((R)-5) (20.0 g, 80% yield, 9:1 ratio of

enantiomers). Chiral HPLC: AD column; eluents 2-propanol/

hexanes, 5/95; 1.0 mL/min. UV detector 220 nm. t_R: (R)-5 10.12

min, (S)-5 8.11 min.

Gen er a l P r oced u r e 1 (Su zu k i Cr oss-Cou p lin g). To (2R)-

N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl]pentanamide ((R,S)-5 or (R)-5) (1 equiv),

aryl or heteroaryl bromide/iodide/triflate (1.1-1.5 equiv), and

PdCl₂(dppf) (0.03 equiv) was added DMF or 2-propanol (5-10

mL/mmol) under a dry nitrogen atmosphere followed by a

degassed aqueous 2.0 M solution of Na₂CO₃(2.5-3.0 equiv).

The reaction mixture was stirred at 80-85 °C for 0.5-1 h,

more PdCl₂(dppf) (0.03 equiv) was added, and the reaction was

heated at 80-95 °C until complete consumption of (R)-5 as

detected by TLC analysis (2-18 h). Upon completion of the

reaction, water was added, the product was extracted with

Et₂O (3×), and the combined organic phases washed with brine

and dried over Na₂SO₄or MgSO₄. The solvent was removed

in vacuo, and the product was purified by flash chromatogra-

phy over silica gel (EtOAc/hexanes, or CH₂Cl₂/MeOH/aq sat.

NH₄OH).

3′-(1-{[(Cya n om eth yl)a m in o]ca r bon yl}-3-m eth ylbu tyl)-

1,1′-bip h en yl-4-ca r boxa m id e (30). Following general pro-

cedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-5) was

submitted to a Suzuki cross-coupling reaction with 4-bro-

mobenzamide to afford title compound 3′-(1-{[(cyanomethyl)-

a m in o]ca r bon yl}-3-m et h ylbu t yl)-1,1′-biph en yl-4-ca r box-

amide (30) (145 mg, 59% yield, purified by flash chromatog-

C

₁₉H₂₂N₃O [MH⁺] 308.17629, found 308.17639. Anal. (C₂₁H₂₂

-

F₃N₃O₃) C: calcd, 59.85; found, 57.69; H: calcd, 5.26; found,

5.38; N: calcd, 13.52; found, 9.75.

2-(3-Br om op h en yl)-4-m eth ylp en ta n oic Acid ((R,S)-7).

To 3-bromophenyl acetic acid 6 (60.0 g, 279 mmol) dissolved

in dry THF (400 mL) at 0 °C under a dry nitrogen atmosphere

was added LiHMDS dropwise (1.06 M solution in THF, 552

mL, 586 mmol), and the slurry was stirred with a mechanical

stirrer for 2 h until it became a solution. 1-Iodo-2-methylpro-

pane (33.7 mL, 293 mmol) was added dropwise and the

reaction mixture stirred 2 days. The reaction was quenched

with ice followed by addition of citric acid (30 g), the product

was extracted with EtOAc (2×), and the organic phase washed

with brine and dried over Na₂SO₄. The solvent was removed

in vacuo and the product purified by trituration in hexanes

(250 mL) to afford 2-(3-bromophenyl)-4-methylpentanoic acid

((R,S)-7) (41.3 g, 55%). Anal. (C₁₂H₁₅BrO₂) C: calcd, 53.32;

found, 53.24; H: calcd, 5.59, found: 5.61.

(2R)-2-(3-Br om op h en yl)-4-m eth ylp en ta n oic Acid ((R)-

7). To a solution of 2-(3-bromophenyl)-4-methylpentanoic acid

((R,S)-7) (60.0 g, 221.4 mmol) in toluene (1.2 L) was added

DMF (350 µL, 4.43 mmol), followed by oxalyl chloride (23.2

mL, 265.7 mmol) gradually at room temperature, and the

reaction mixture was stirred for 2.25 h. The solution was cooled

to 0 °C, dimethylethylamine (60 mL, 553.5 mmol) was added,

and the reaction mixture stirred for 2.5 h. The reaction mixture

was cooled to -78 °C, and a solution of (R)-(-)-pantolactone

(34.6 g, 265.7 mmol) in toluene (1.0 L) was added gradually,

followed by warming to 0 °C and stirring for another 2.5 h.

Water was added and saturated with NaCl, and the organic

layer was separated, washed with aq 10% HCl, water, aq 50%

sat. solution of NaCO₃, and brine, and dried over MgSO₄. The

solvent was removed in vacuo to afford crude (3R)-4,4-

dimethyl-2-oxotetrahydrofuran-3-yl (2R)-2-(3-bromophenyl)-4-

methylpentanoate (9) (80.0 g, 10:1 diastereoisomeric ratio) that

was used as such in the next step. An analytically pure sample

of 9 was prepared by flash chromatography over silica gel

(EtOAc/hexanes, 2/8) for analysis. [R]_D) +7.9° (c ) 1.48,

MeOH). HRMS (+FAB): calcd for C₁₈H₂₃BrO₄[MH⁺] 383.07797,

found 383.08566. The (3R)-4,4-dimethyl-2-oxotetrahydrofuran-

3-yl (2R)-2-(3-bromophenyl)-4-methylpentanoate (9) (80.0 g,

221.4 mmol) was dissolved in acetic acid (640 mL), a solution

of HCl (aq, 2.0 M) was added, and the reaction mixture was

heated to 85 °C for 48 h. Approximately 50% of the solvent

was distilled off, and water was added along with seeding from

a previous reaction. Filtration afforded the desired (2R)-2-(3-

bromophenyl)-4-methylpentanoic acid ((R)-7) (47.0 g, 78%

yield) which retained the initial 10:1 diastereoisomeric ratio

of the starting ester 9. Anal. (C₂₀H₂₆BrNO₂) C: calcd, 61.23;

found, 61.48; H: calcd, 6.68; found, 6.42; N: calcd, 3.57; found,

3.85.

raphy, EtOAc/hexanes, 8/2). HRMS (+FAB): calcd for C₂₁H₂₄

-

N₃O₂[MH⁺] 350.18685, found 350.18695. Anal. (C₂₁H₂₃N₃O₂)

C: calcd, 72.18; found, 70.83; H: calcd, 6.63; found, 6.60; N:

calcd, 12.03; found, 11.68.

N-(Cya n om eth yl)-4-m eth yl-2-[4′-(m eth ylsu lfon yl)-1,1′-

bip h en yl-3-yl]p en ta n a m id e (31). Following general proce-

dure 1, (2R)-N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-5) was

submitted to a Suzuki cross-coupling reaction with 4-bromo-

phenyl methyl sulfone to afford title compound N-(cyano-

methyl)-4-methyl-2-[4′-(methylsulfonyl)-1,1′-biphenyl-3-yl]pentan-

amide (31) (265 mg, 98% yield, purified by flash chromatog-

raphy, EtOAc/hexanes, 1/1). HRMS (+FAB): calcd for C₂₁H₂₅

-

N₂O₃S [MH⁺] 385.15859, found 385.15870. Anal. (C₂₁H₂₄N₂O₃S)

3720 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

C: calcd, 65.60; found, 64.53; H: calcd, 6.29; found, 6.84; N:

calcd, 7.29; found, 6.65.

(C₁₉H₂₁N₃O) C: calcd, 74.24; found, 72.68; H: calcd, 6.89:

found, 7.39; N: calcd, 13.67; found, 13.72.

(2R)-N-(Cya n om eth yl)-4-m eth yl-2-[4′-(1H-tetr a a zol-5-

yl)-1,1′-bip h en yl-3-yl]p en ta n a m id e (32). Following general

procedure 1, (2R)-N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tet-

ramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R)-5)

was submitted to a Suzuki cross-coupling reaction with 5-(4-

bromophenyl)-1H-tetrazole to afford title compound (2R)-N-

(cyanomethyl)-4-methyl-2-[4′-(1H-tetraazol-5-yl)-1,1′-biphenyl-

3-yl]pentanamide (32) (130 mg, 26% yield, purified by flash

chromatography, CH₂Cl₂/MeOH/aq sat. NH₄OH, 80/18/2).

HRMS (+FAB): calcd for C₂₁H₂₃N₆O [MH⁺] 375.19333, found

375.19333. Anal. (C₂₁H₂₂N₆O) C: calcd, 67.36; found, 64.81;

H: calcd, 5.92; found, 6.59; N: calcd, 22.44; found, 17.79.

N-(Cya n om et h yl)-4-m et h yl-2-[4′-(4-m or p h olin yl)[1,1′-

bip h en yl]-3-yl]p en ta n a m id e (33). Following general pro-

cedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-5) was

submitted to a Suzuki cross-coupling reaction with 4-(4-

bromophenyl)morpholine (prepared by the bromination of

commercially available N-phenylmorpholine²⁷to afford the title

compound N-(cyanomethyl)-4-methyl-2-[4′-(4-morpholinyl)-

[1,1′-biphenyl]-3-yl]pentanamide (33) (172 mg, 63%, purified

by flash chromatography, EtOAc/hexanes, 4/6). MS (-ESI) m/z

390.3 (M - H)^-. HRMS (+FAB): calcd for C₂₄H₂₉KN₃O₂[M +

K⁺] 430.18968, found 430.18959. Anal. (C₂₄H₂₉N₃O₂) C: calcd,

73.63; found, 72.04; H: calcd, 7.47; found, 7.55; N: calcd, 10.73;

found, 9.52.

N-(Cya n om et h yl)-4-m et h yl-2-(3-p yr id in -3-ylp h en yl)-

p en ta n a m id e (38). To a cold (0 °C), stirred solution of (3-

pyridin-3-ylphenyl)acetic acid (1.2 g, 5.6 mmol.) in THF (50

mL) was slowly added a solution of LDA (1.6 M in THF, 8.0

mL, 13.0 mmol). The reaction mixture was stirred at 0 °C for

1 h. After cooling to -78 °C, 3-bromo-2-methylpropene (1.3 g,

10.0 mmol) was added, and the solution was warmed to room

temperature and stirred for an additional 1 h. The reaction

mixture was evaporated, dissolved in water (10 mL), and

acidified with 6.0 N HCl to pH 5. The crude product was

purified by preparative HPLC to give the desired compound

4-methyl-2-(3-pyridin-3-ylphenyl)pent-4-enoic acid (1.3 g, 60%

yield) as a white solid. This so-obtained compound was

dissolved in EtOH (20 mL), Pd/C (5 mol %) was added, and

the reaction mixture was subjected to hydrogenation under

pressure (40 psi) for 8 h. The reaction mixture was filtered

through Celite, and the filtrate was concentrated in vacuo to

yield the desired 4-methyl-2-(3-pyridin-3-ylphenyl)pentanoic

acid in quantitative yield (1.0 g). To a solution of this 4-methyl-

2-(3-pyridin-3-ylphenyl)pentanoic acid acid (1.0 g, 2.3 mmol)

in DMF (10 mL) at room temperature were added aminoac-

etonitrile hydrochloride (211 mg, 2.3 mmol), PyBOP (1.2 g, 2.3

mmol), and TEA (0.5 mL, 4.0 mmol). The reaction was left

stirring overnight. The DMF was evaporated in vacuo and the

residue was treated with a mixture of EtOAc/aq NaHCO₃(20

mL/20 mL). The organic phase was washed with brine and

dried over Na₂SO₄. The crude product was purified on pre-

parative HPLC to afford the desired product N-(cyanomethyl)-

4-methyl-2-(3-pyridin-3-ylphenyl)pentanamide (38) (600 mg,

61%) as a white solid. HRMS (+FAB): calcd for C₁₉H₂₂N₃O

[MH⁺] 308.17629, found 308.17639. Anal. (C₁₉H₂₁N₃O) C:

calcd, 74.24; found, 51.57; H: calcd, 6.89; found, 4.78; N: calcd,

13.67; found, 8.83.

N-(Cya n om et h yl)-4-m et h yl-2-(3-p yr id in -4-ylp h en yl)-

p en ta n a m id e tr iflu or oa ceta te (39). A solution of N-(cya-

nomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl]pentanamide((R,S)-5)(71mg,0.2mmol),4-bromopyri-

dine (47 mg, 0.3 mmol), PdCl₂(dppf) (13 mg, 0.018 mmol), and

Na₂CO₃(2.0 M, aqueous, 0.5 mL, 1.0 mmol) in dry DMF

(4 mL) was heated to 80-85 °C for 18 h. Water was added

and the product extracted with Et₂O (2×) and dried over

Na₂SO₄. The solvent was removed in vacuo, and the crude

product was purified by flash chromatography over silica gel

(EtOAc/hexanes, 1/1) to afford the desired compound (39).

HRMS (+FAB): calcd for C₁₉H₂₂N₃O [MH⁺] 308.17629, found

308.17639.

(2R)-N-(Cya n om eth yl)-4-m eth yl-2-[4′-(4-p yr id in yl)[1,1′-

bip h en yl]-3-yl]p en ta n a m id e (40). To 4-bromopyridine hy-

drochloride (6.61 g, 33.96 mmol), 4-methoxyphenylboronic acid

(5.16 g, 33.96 mmol), and PdCl₂(dppf) (835 mg, 1.02 mmol) was

added DMF (200 mL) under a dry nitrogen atmosphere

followed by an aq solution of Na₂CO₃(2.0 M, 67.9 mL). The

reaction mixture was stirred at 85 °C for 1.25 h, more

PdCl₂(dppf) (835 mg, 1.02 mmol) was added, and the reaction

was heated another 1 h. Water was added, the product

extracted with Et₂O, and the organic phase dried over Na₂-

SO₄. The solvent was removed in vacuo, and the crude product

was purified by flash chromatography over silica gel (EtOAc/

hexanes, 55/45) to afford the desired 4-(4-methoxyphenyl)-

pyridine (5.77 g, 92% yield). To 4-(4-methoxyphenyl)pyridine

(2.15 g, 11.62 mmol) in dry DMF (15 mL) was added NaSMe

(3.26 g, 46.48 mmol) at room temperature, and the reaction

mixture was heated at 130 °C for 5 h. After being cooled to

room temperature, the reaction was carefully poured into

water and the product extracted with Et₂O, and the organic

phase was dried over MgSO₄. The solvent was removed in

vacuo, and the product was purified by flash chromatography

over silica gel (EtOAc/hexanes, 8/2 then 100% EtOAc) to afford

the desired product 4-pyridin-4-ylphenol (875 mg, 44% yield).

A portion of the so obtained 4-pyridin-4-ylphenol (275 mg, 1.61

mmol) was converted to the triflate ether 4-pyridin-4-ylphenyl

trifluoromethanesulfonate (302 mg, 62% yield).²⁹Following

N-(Cya n om et h yl)-4-m et h yl-2-[3′-(4-m or p h olin yl)[1,1′-

bip h en yl]-3-yl]p en ta n a m id e (34). Following general pro-

cedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-5) was

submitted to a Suzuki cross-coupling reaction with 3-morpho-

lin-4-ylphenyl trifluoromethanesulfonate²⁸to afford the title

compound N-(cyanomethyl)-4-methyl-2-[3′-(4-morpholinyl)-

[1,1′-biphenyl]-3-yl]pentanamide (34) (155 mg, 51% yield,

purified by flash chromatography, EtOAc/hexanes, 4/6). MS

(-ESI) m/z 390.4 (M - H)^-. HRMS (+FAB): calcd for C₂₄H₂₉

-

KN₃O₂[M + K⁺] 430.18968, found 430.18980. Anal. (C₂₄H₂₉

N₃O₂) C: calcd, 73.63; found, 70.17; H: calcd, 7.47; found, 7.80;

N: calcd, 10.73; found, 9.82.

N-(Cya n om et h yl)-4-m et h yl-2-[2′-(4-m or p h olin yl)[1,1′-

bip h en yl]-3-yl]p en ta n a m id e (35). Following general pro-

cedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-5) was

submitted to a Suzuki cross-coupling reaction with 2-morpho-

lin-4-ylphenyl trifluoromethanesulfonate²⁹to afford the title

compound N-(cyanomethyl)-4-methyl-2-[2′-(4-morpholinyl)-

[1,1′-biphenyl]-3-yl]pentanamide (35) (180 mg, 59%, purified

by flash chromatography, EtOAc/hexanes, 4/6). MS (-ESI) m/z

390.4 (M - H)^-. HRMS (+FAB): calcd for C₂₄H₃₀N₃O₂[MH⁺]

392.23380, found 392.23362. Anal. (C₂₄H₂₉N₃O₂) C: calcd,

73.63; found, 72.44; H: calcd, 7.47; found, 7.36; N: calcd, 10.73;

found, 10.04.

N-(Cya n om eth yl)-4-m eth yl-2-(3-p yr im id in -5-ylp h en yl-

)p en t a n a m id e (36). Following general procedure 1, N-(cy-

anomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl]pentanamide ((R,S)-5) was submitted to a Suzuki

cross-coupling reaction with 5-bromopyrimidine to afford

the title compound N-(cyanomethyl)-4-methyl-2-(3-pyrimidin-

5-ylphenyl)pentanamide (36) (185 mg, 86% yield, purified

by flash chromatography, EtOAc/hexanes, 6/4, then 7/3).

HRMS (+FAB): calcd for C₁₈H₂₁N₄O [MH⁺] 309.17154, found

309.17150. Anal. (C₁₈H₂₀N₄O) C: calcd, 70.11; found, 69.95;

H: calcd, 6.54; found, 6.66; N: calcd, 18.17; found, 18.32.

N-(Cya n om et h yl)-4-m et h yl-2-(3-p yr id in -2-ylp h en yl)-

p en ta n a m id e (37). Following general procedure 1, N-(cya-

nomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl]pentanamide ((R,S)-5) was submitted to a Suzuki

cross-coupling reaction with 2-bromopyridine to afford the title

compound N-(cyanomethyl)-4-methyl-2-(3-pyridin-2-ylphenyl)-

pentanamide (37) (92 mg, 55% yield, purified by flash

chromatography, EtOAc/hexanes, 1/9). HRMS (+FAB): calcd

for

C

₁₉H₂₂N₃O [MH⁺] 308.17629, found 308.17639. Anal.

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3721

general procedure 1, (2R)-N-(cyanomethyl)-4-methyl-2-[3-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentana-

mide ((R)-5) was submitted to a Suzuki cross-coupling reaction

with 4-pyridin-4-ylphenyl trifluoromethanesulfonate to afford

title compound (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(4-pyridi-

nyl)[1,1′-biphenyl]-3-yl]pentanamide (40) (359 mg, 94% yield,

purified by flash chromatography, 100% EtOAc). MS (+ESI)

m/z 384.0 (M + H)⁺. HRMS (+FAB): calcd for C₂₅H₂₆N₃O

[MH⁺] 384.20759, found 384.20765. Anal. (C₂₅H₂₅N₃O) C:

calcd, 78.30; found, 77.94; H: calcd, 6.57; found, 6.95; N: calcd,

10.96; found, 11.00.

N-(Cya n om et h yl)-2-[3-(1H-in d ol-6-yl)p h en yl]-4-m et h -

ylp en ta n a m id e (41). Following general procedure 1, N-(cya-

nomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl]pentanamide ((R,S)-5) was submitted to a Suzuki

cross-coupling reaction with 6-bromoindole to afford title

compound N-(cyanomethyl)-2-[3-(1H-indol-6-yl)phenyl]-4-me-

thylpentanamide (41) (190 mg, 79% yield, purified by flash

chromatography, EtOAc/hexanes, 3/7). HRMS (+FAB): calcd

for C₂₂H₂₃KN₃O [M + K⁺] 384.14782, found 384.14799. Anal.

(C₂₂H₂₃N₃O) C: calcd, 76.49; found, 74.93; H: calcd, 6.71;

found, 7.51; N: calcd, 12.16; found, 11.17.

0.68 mmol) and methanesulfonic acid (0.18 mL, 2.73 mmol)

in 5 mL of anhydrous THF under nitrogen were stirred at room

temperature for 5 h. TLC analysis indicated complete con-

sumption of starting material. The reaction was diluted with

50 mL ether, and the solution was then decanted off. This was

repeated six times. The combined organics were concentrated

in vacuo, and the residue was then dried in vacuo to yield 407.7

mg of the title compound 43 as a brown solid. LC/MS (es):

391.07 (M + H)⁺.

N-(Cya n om eth yl)-4-m eth yl-2-[2′-(1-p ip er a zin yl)[1,1′-bi-

p h en yl]-3-yl]p en ta n a m id e (44). To N-(2-hydroxyphenyl)-

piperazine (2.00 g, 11.22 mmol) and DMAP (2.06 g, 16.83

mmol) in MeCN (40 mL) at 0 °C was added di-tert-butyl

dicarbonate (2.94 g, 13.17 mmol) dissolved in MeCN (20 mL),

and the reaction mixture was stirred at 0 °C for 3 h. The

reaction mixture was poured into water, the product extracted

with Et₂O and washed with brine, and the organic phase was

dried over MgSO₄. The solvent was removed in vacuo, and the

crude product was purified by flash chromatography over silica

gel (EtOAc/hexanes, 1/9, then 15/85) to afford the desired tert-

butyl 4-(2-hydroxyphenyl)piperazine-1-carboxylate (1.05 g,

34% yield).

N-(Cya n om et h yl)-2-[3-(1H-in d ol-5-yl)p h en yl]-4-m et h -

ylp en ta n a m id e ((R,S)-42). Following general procedure 1,

N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl]pentanamide ((R,S)-5) was submitted to

a Suzuki cross-coupling reaction with 5-bromoindole to afford

title compound N-(cyanomethyl)-2-[3-(1H-indol-5-yl)phenyl]-

4-methylpentanamide ((R,S)-42) (50 mg, 21% yield, purified

by flash chromatography, EtOAc/hexanes, 3/7). MS (-ESI) m/z

344.3 (M - H)^-. HRMS (+FAB): calcd for C₂₂H₂₄N₃O [MH⁺]

346.19194, found 346.19204. Anal. (C₂₂H₂₃N₃O) C: calcd, 76.49;

found, 72.62; H: calcd, 6.71; found, 7.62; N: calcd, 12.16;

found, 10.39.

A portion of the so obtained tert-butyl 4-(2-hydroxyphenyl)-

piperazine-1-carboxylate (405 mg, 1.46 mmol) was converted

to the triflate ether tert-butyl 4-(2-{[(trifluoromethyl)sulfonyl]-

oxy}phenyl)piperazine-1-carboxylate (550 mg, 100% yield).³⁰

Following general procedure 1, N-(cyanomethyl)-4-methyl-2-

[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentan-

amide ((R,S)-5) was submitted to a Suzuki cross-coupling

reaction with tert-butyl 4-(2-{[(trifluoromethyl)sulfonyl]oxy}-

phenyl)piperazine-1-carboxylate to afford the desired tert-butyl

4-[3′-(1-{[(cyanomethyl)amino]carbonyl}-3-methylbutyl)-1,1′-

biphenyl-2-yl]piperazine-1-carboxylate (562 mg, 70%, purified

by flash chromatography, EtOAc/hexanes, 3/7). Removal of the

Boc protecting group of 4-[3′-(1-{[(cyanomethyl)amino]carbo-

nyl}-3-methylbutyl)-1,1′-biphenyl-2-yl]piperazine-1-carboxy-

late (562 mg, 1.15 mmol) was accomplished following general

procedure 2 to afford the title compound N-(cyanomethyl)-4-

methyl-2-[2′-(1-piperazinyl)[1,1′-biphenyl]-3-yl]pentanamide (44)

(293 mg, 65%, purified by flash chromatography, CH₂Cl₂/

MeOH/aq sat. NH₄OH, 90/9/1). MS (+ESI) m/z 391.1 (M + H)⁺.

HRMS (+FAB): calcd for C₂₄H₃₁N₄O [MH⁺] 391.24196, found

391.24971. Anal. (C₂₄H₃₀N₄O) C: calcd, 73.81; found, 71.56;

H: calcd, 7.74; found, 7.42; N: calcd, 14.35; found, 13.37.

(2R)-N-(Cya n om et h yl)-2-[3-(1H -in d ol-5-yl)p h en yl]-4-

m eth ylp en ta n a m id e ((R)-42) a n d (2S)-N-(Cya n om eth yl)-

2-[3-(1H-in d ol-5-yl)p h en yl]-4-m et h ylp en t a n a m id e ((S)-

42). Racemic N-(cyanomethyl)-2-[3-(1H-indol-5-yl)phenyl]-4-

methylpentanamide ((R,S)-42) was separated by chiral HPLC

(Chiralpak AD column, mobile phase EtOH/hexanes, 2/8) to

afford at 8.44 min (2R)-N-(cyanomethyl)-2-[3-(1H-indol-5-yl)-

phenyl]-4-methylpentanamide ((R)-42) ([R]²³_D-54.6 [c ) 1.20,

CHCl₃]) and at 9.7 min (2S)-N-(cyanomethyl)-2-[3-(1H-indol-

5-yl)phenyl]-4-methylpentanamide ((S)-42) ([R]²³+41.0 [c )

D

1.00, CHCl₃]).

N-(Cya n om eth yl)-4-m eth yl-2-[4′-(1-p ip er a zin yl)[1,1′-bi-

p h en yl]-3-yl]p en ta n a m id e ((R,S)-2). Following general pro-

cedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-5) was

submitted to a Suzuki cross-coupling reaction with 1-(4-

bromophenyl)piperazine hydrochloride to afford title compound

N-(cyanomethyl)-4-methyl-2-[4′-(1-piperazinyl)[1,1′-biphenyl]-

3-yl]pentanamide ((R,S)-2) (155 mg, 57% yield, purified by

flash chromatography, CH₂Cl₂/MeOH/aq sat. NH₄OH, 90/9/1).

MS (+ESI) m/z 391.2 (M + H)⁺. HRMS (+FAB): calcd for

N-(Cya n om eth yl)-4-m eth yl-2-(3′-p ip er a zin -1-yl-1,1′-bi-

p h en yl-3-yl)p en ta n a m id e m eth a n esu lfon a te (43). Boc-

piperazine (7.89 g, 42.39 mmol), 1,3-dibromobenzene (13.0 g,

57.22 mmol), dicyclohexyl[3-(2-methyl-1,3-dioxolan-2-yl)phe-

nyl]phosphine (1.56 g, 2.54 mmol), Pd₂(dba)₃(0.8 g, 0.85 mmol),

and t-BuOK (5.71 g, 50.87 mmol) in 25 mL toluene were heated

to 105 °C overnight. The reaction was cooled to room temper-

ature, partitioned between ether and water, and extracted with

ether (2 × 75 mL). The combined ether layers were washed

with water (2 × 75 mL) and dried over MgSO₄. The organic

extracts were concentrated in vacuo, and the residue obtained

was purified by flash column chromatography (5-15% EtOAc/

hexanes) to yield 2.60 g of tert-butyl 4-(3-bromophenyl)-

C

₂₄H₃₁N₄O [MH⁺] 391.24979, found 391.24971. Anal. (C₂₄H₃₀

-

N₄O) C: calcd, 73.81; found, 70.89; H: calcd, 7.74; found, 7.44;

N: calcd, 14.35; found, 13.83.

(2R)-N-(Cya n om et h yl)-4-m et h yl-2-[4′-(1-p ip er a zin yl)-

[1,1′-bip h en yl]-3-yl]p en ta n a m id e ((R)-2). Following general

procedure 1, (2R)-N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tet-

ramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R)-5)

was submitted to a Suzuki cross-coupling reaction with 1-(4-

bromophenyl) piperazine hydrochloride to afford title com-

pound (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(1-piperazinyl)-

[1,1′-biphenyl]-3-yl]pentanamide ((R)-2) (773 mg, 55% yield,

purified by flash chromatography, CH₂Cl₂/MeOH/aq sat. NH₄-

OH, 90/9/1). MS (+ESI) m/z 391.2 (M + H)⁺. HRMS (+FAB):

calcd for C₂₄H₃₁N₄O [MH⁺] 391.24196, found 391.24196. Anal.

(C₂₄H₃₀N₄O) C: calcd, 73.81; found, 73.87; H: calcd, 7.74;

found, 7.64; N: calcd, 14.35; found, 14.28.

piperazine-1-carboxylate as

a yellow oil. tert-Butyl 4-(3-

bromophenyl)piperazine-1-carboxylate (1.0 g, 2.81 mmol),

N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl]pentanamide ((R,S)-5) (400 mg, 1.12

mmol), PdCl₂(dppf) (28 mg, 0.034 mmol), and Na₂CO₃(2.0 M

aqueous, 2.81 mL, 5.61 mmol) in 10 mL of DMF were heated

to 80 °C overnight. The reaction was cooled to room temper-

ature and partitioned between ethyl acetate and water and

extracted with EtOAc (3 × 75 mL). The combined ethyl acetate

layers were dried over MgSO₄and concentrated in vacuo. The

residue obtained was purified by flash chromatography (21-

37% EtOAc/hexanes) to yield 399 mg of tert-butyl 4-[3′-(1-

{[(cyanomethyl)amino]carbonyl}-3-methylbutyl)-1,1′-biphenyl-

3-yl]piperazine-1-carboxylate as a white foamy solid.

Gen er a l P r oced u r e 2 (Boc Dep r otection ). To a solution

of a Boc-protected amino derivative in dry THF (1-2 mL/

mmol) under dry nitrogen atmosphere was added 2 equiv of

MeSO₃H dropwise, and the reaction mixture was stirred for

tert-Butyl 4-[3′-(1-{[(Cyanomethyl)amino]carbonyl}-3-me-

thylbutyl)-1,1′-biphenyl-3-yl]piperazine-1-carboxylate (335 mg,

3722 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

approximately 1 h. Another 1 equiv of MeSO₃H was added,

and the reaction mixtures were stirred overnight (in certain

cases, another 1 or 2 equiv was required to drive the reaction

to completion). The reaction mixture was carefully quenched

using an aq sat. solution of NaHCO₃, the product was extracted

with EtOAc (2×), and the organic phase was dried over Na₂-

SO₄. The solvent was removed in vacuo, and the crude product

was purified by flash chromatography over silica gel (CH₂Cl₂/

MeOH/aq sat. NH₄OH, 90/9/1).

until a light yellowish color persisted. After stirring an

additional 15 min, acetic acid was added to quench any

remaining diazomethane, and the reaction mixture was con-

centrated in vacuo to afford a crude oil which was purified by

flash chromatography over silica gel (EtOAc/hexanes, 1/9) to

yield the desired methyl 2-(3-bromophenyl)-4-methylpen-

tanoate (3.32 g, 90% yield). To methyl 2-(3-bromophenyl)-4-

methylpentanoate (3.3 g, 11.58 mmol), diboron pinacol ester

(3.35 g, 13.90 mmol), and KOAc (3.31 g, 34.74 mmol) in dry

DMF (80 mL) under an atmosphere of dry nitrogen was added

PdCl₂(dppf) (285 mg, 0.347 mmol), and the reaction was heated

to 85 °C for 3 h. The reaction mixture was poured into water,

extracted with Et₂O, and washed with brine, and the organic

layer was dried over Na₂SO₄. The solvent was removed in

vacuo, and the crude product was purified by flash chroma-

tography over silica gel (EtOAc/hexanes, 1/9) to afford the

desired methyl 4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl]pentanoate (2.57 g, 67% yield). To 4-(4-

bromophenyl)-4-piperidinol (1.00 g, 3.90 mmol) was added TFA

(15 mL), and the reaction was stirred at room temperature

for 6 h. The solvent was concentrated in vacuo, aq sat. NaHCO₃

was added, the product was extracted with EtOAc, and the

organic layer was dried over Na₂SO₄. The solvent was removed

in vacuo and the crude product purified by flash chromatog-

raphy over silica gel (CH₂Cl₂/MeOH/aq sat. NH₄OH, 90/9/1)

to afford the desired product 4-(4-bromophenyl)-1,2,3,6-tet-

rahydropyridine (618 mg, 67% yield). To 4-(4-bromophenyl)-

1,2,3,6-tetrahydropyridine (3.40 g, 14.28 mmol) and DMAP

(3.48 g, 28.56 mmol) in MeCN (50 mL) at 0 °C under an

atmosphere of dry nitrogen was added di-tert-butyl dicarbonate

(4.68 g, 21.42 mmol), and the reaction mixture was stirred at

that temperature for 1 h. The reaction was poured into aq sat.

NaHCO₃and extracted with Et₂O, and the organic layer was

dried over Na₂SO₄. The solvent was removed in vacuo and the

product purified by flash chromatography over silica gel

(EtOAc/hexanes, 1/9) to afford the desired product tert-butyl

4-(4-bromophenyl)-3,6-dihydropyridine-1(2H)-carboxylate (3.84

g, 80% yield). To tert-butyl 4-(4-bromophenyl)-3,6-dihydropy-

ridine-1(2H)-carboxylate (2.62 g, 7.74 mmol), methyl 4-methyl-

2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pen-

tanoate (2.57 g, 7.74 mmol), and PdCl₂(dppf) (190 mg, 0.232

mmol) was added dry DMF (80 mL) under a nitrogen atmo-

sphere followed by a degassed aqueous 2.0 M solution of Na₂-

CO₃(11.6 mL, 23.2 mmol). The reaction mixture was stirred

at 85 °C for approximately 1 h, more PdCl₂(dppf) (190 mg,

0.232 mmol) was added, and the reaction was heated at 85 °C

for another 3 h. Water was added to the reaction, product

extracted with Et₂O (3×), and the combined organic layers

were washed with brine and dried over Na₂SO₄. The solvent

was removed in vacuo and the crude product purified by flash

chromatography over silica gel (EtOAc/hexanes, 2/8) to afford

the desired product tert-butyl 4-{3′-[1-(methoxycarbonyl)-3-

methylbutyl]-1,1′-biphenyl-4-yl}-3,6-dihydropyridine-1(2H)-

carboxylate (2.28 g, 63% yield). To tert-butyl 4-{3′-[1-(meth[

oxycarbonyl)-3-methylbutyl]-1,1′-biphenyl-4-yl}-3,6-dihydro-

pyridine-1(2H)-carboxylate (2.135 g, 4.61 mmol) in EtOH/

EtOAc (50 mL, 1:1) under an atmosphere of dry nitrogen was

added 10% Pd/C (250 mg), the reaction vessel was degassed

with hydrogen, and the reaction mixture stirred for 6.5 h. After

the reaction vessel was thoroughly degassed with dry nitrogen,

CH₂Cl₂was added, and the reaction mixture was filtered on a

pad of Celite. The solvent was removed in vacuo and the crude

product purified by flash chromatography over silica gel

(EtOAc/hexanes, 15/85) to afford the desired product tert-butyl

4-{3′-[1-(methoxycarbonyl)-3-methylbutyl]-1,1′-biphenyl-4-yl}-

piperidine-1-carboxylate (2.07 g, 97% yield). To tert-butyl 4-{3′-

[1-(methoxycarbonyl)-3-methylbutyl]-1,1′-biphenyl-4-yl}-

piperidine-1-carboxylate (1.22 mg, 2.63 mmol) in THF/MeOH

(40 mL, 1:1) was added an aqueous solution of NaOH (1.0 M,

2.9 mL, 2.9 mmol), and the reaction mixture was stirred for 6

h at room temperature. More NaOH (aq, 1.0 M, 2.9 mL, 2.9

mmol) was added, and the reaction mixture was stirred

another 18 h. The mixture was concentrated down, and aq 10%

HCl was added gradually until the solution reached pH ) 4.

(2R)-N-(1-Cya n ocyclop r op yl)-4-m eth yl-2-(4′-p ip er a zin -

1-yl-1,1′-bip h en yl-3-yl)p en ta n a m id e (45). Compound 45

was prepared according to the general PyBOP coupling

procedure 4 except 1-amino-1-cyclopropane-carbonitrile hy-

drochloride was used instead of aminoacetonitrile hydrochlo-

ride and coupled with (2R)-2-{4′-[4-(t-butoxycarbonyl)piperazin-

1-yl]-1,1′-biphenyl-3-yl}-4-methylpentanoic acid, followed by

the general Boc deprotection procedure 2 to afford the desired

(2R)-N-(1-cyanocyclopropyl)-4-methyl-2-(4′-piperazin-1-yl-1,1′-

biphenyl-3-yl)pentanamide (45). HRMS (+FAB): calcd for

C

₂₄H₃₃N₄O₂[MH⁺] 417.26544, found 417.26554.

1-(4-Br om oph en yl)-4-m eth ylpiper azin e (25). To a stirred

suspension of 1-(4-bromophenyl)piperazine (10 g, 1 equiv) in

DMF (120 mL) was added NaH (60% in oil, 4.3 g, 3 equiv) in

three portions. After being stirred at room temperature for 1

h, the mixture was cooled to -78 °C and a solution of methyl

iodide (2.24 mL, 1 equiv) in DMF (5 mL) was added dropwise

over 15 min. The mixture was stirred at -78 °C for 15 min

and then warmed to room temperature where stirring was

continued for 1 h. The reaction mixture was poured into H₂O

and extracted with EtOAc (4×). The combined organic extracts

were dried (MgSO₄) and concentrated under reduced pressure.

The residue was purified by flash chromatography (gradient

elution: 5% MeOH in CH₂Cl₂to 10% MeOH in CH₂Cl₂) to

afford the title compound 25 (6.5 g, 71% yield).

N-(Cya n om eth yl)-4-m eth yl-2-[4′-(4-m eth yl-1-p ip er a zi-

n yl)[1,1′-bip h en yl]-3-yl]p en ta n a m id e ((R,S)-46). Following

general procedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-

5) was submitted to a Suzuki cross-coupling reaction with 1-(4-

bromophenyl)-4-methylpiperazine (25) to afford title compound

N-(cyanomethyl)-4-methyl-2-[4′-(4-methyl-1-piperazinyl)[1,1′-

biphenyl]-3-yl]pentanamide ((R,S)-46) (85 mg, 30% yield,

purified by flash chromatography, CH₂Cl₂/MeOH/aq sat. NH₄-

OH, 90/9/1). MS (+ESI) m/z 405.1 (M + H)⁺. HRMS (+FAB):

calcd for C₂₅H₃₃N₄O [MH⁺] 405.26544, found 405.26535.

(2R)-N-(Cya n om eth yl)-4-m eth yl-2-[4′-(4-m eth yl-1-p ip -

er a zin yl)[1,1′-bip h en yl]-3-yl]p en ta n a m id e ((R)-46). Fol-

lowing general procedure 1, (2R)-N-(cyanomethyl)-4-methyl-2-[3-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentana-

mide ((R)-5) was submitted to a Suzuki cross-coupling reaction

with 1-(4-bromophenyl)-4-methylpiperazine to afford title

compound (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(4-methyl-1-

piperazinyl)[1,1′-biphenyl]-3-yl]pentanamide ((R)-46) (733 mg,

48%, purified by flash chromatography, CH₂Cl₂/MeOH/aq sat.

NH₄OH, 90/9/1). MS (+ESI) m/z 405.1 (M + H)⁺. HRMS

(+FAB): calcd for C₂₅H₃₂KN₄O [M + K⁺] 443.22132, found

443.22139. Anal. (C₂₅H₃₂N₄O) C: calcd, 74.22; found, 73.80;

H: calcd, 7.97; found, 7.94; N: calcd, 13.85; found, 13.70.

N-(Cya n om eth yl)-4-m eth yl-2-[4′-(1,2,3,6-tetr a h yd r o-4-

p yr id in yl)[1,1′-bip h en yl]-3-yl]p en ta n a m id e (47). Follow-

ing general procedure 1, N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R,S)-

5) was submitted to a Suzuki cross-coupling reaction with 4-(4-

bromophenyl)-1,2,3,6-tetrahydropyridine to afford the title

compound N-(cyanomethyl)-4-methyl-2-[4′-(1,2,3,6-tetrahydro-

4-pyridinyl)[1,1′-biphenyl]-3-yl]pentanamide (47) (70 mg, 36%

yield, purified by flash chromatography, CH₂Cl₂/MeOH/aq sat.

NH₄OH, 90/9/1). MS (+ESI) m/z 388.2 (M + H)⁺. HRMS

(+FAB): calcd for

388.23891.

C

₂₅H₃₀N₃O [MH⁺] 388.23889, found

N-(Cya n om eth yl)-4-m eth yl-2-[4′-(4-p ip er id in yl)[1,1′-bi-

p h en yl]-3-yl]p en ta n a m id e (48). To a solution of 2-(3-bro-

mophenyl)-4-methylpentanoic acid (R,S)-7 (3.15 g, 12.91 mmol)

in Et₂O (30 mL) at 0 °C was added diazomethane dropwise

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3723

The solvent was removed in vacuo and the crude product

purified by flash chromatography over silica gel (EtOAc/

hexanes, 3/7 + 1% AcOH) to afford the desired product 2-{4′-

[1-(tert-butoxycarbonyl)piperidin-4-yl]-1,1′-biphenyl-3-yl}-4-

methylpentanoic acid (1.02 g, 86% yield). To 2-{4′-[1-(tert-

bu t oxyca r bon yl)p ip er id in -4-yl]-1,1′-bip h en yl-3-yl}-4-

methylpentanoic acid (1.02 g, 2.22 mmol), PyBOP (1.22 g, 2.33

mmol), and aminoacetonitrile hydrochloride (411 mg, 4.44

mmol) was added dry DMF (50 mL) under an atmosphere of

dry nitrogen followed by Et₃N (0.96 mL, 6.88 mmol), and the

reaction mixture was stirred for 2 h at room temperature. The

reaction mixture was poured into aq sat. NaHCO₃and

extracted with Et₂O (3×), and the organic layer was washed

with brine and dried over MgSO₄. The solvent was concen-

trated in vacuo to afford a crude oil which was purified by flash

chromatography over silica gel (EtOAc/hexanes, 4/6) to yield

the desired product tert-butyl 4-[3′-(1-{[(cyanomethyl)amino]-

carbonyl}-3-methylbutyl)-1,1′-biphenyl-4-yl]piperidine-1-car-

boxylate (915 mg, 84% yield). Following general procedure 2,

tert-butyl 4-[3′-(1-{[(cyanomethyl)amino]carbonyl}-3-methyl-

butyl)-1,1′-biphenyl-4-yl]piperidine-1-carboxylate was depro-

tected to afford the title compound N-(cyanomethyl)-4-methyl-

2-[4′-(4-piperidinyl)[1,1′-biphenyl]-3-yl]pentanamide (48) (535

mg, 74% yield) following purification by flash chromatography

(CH₂Cl₂/MeOH/aq sat. NH₄OH, 90/9/1). MS (+ESI) m/z 390.9

(M + H)⁺. HRMS (+FAB): calcd for C₂₅H₃₂N₃O [MH⁺]

390.25454, found 390.25436.

1-yl-1,1′-biphenyl-3-yl)pentanamide hydrochloride ((R)-2) (100

mg, 0.234 mmol) in DMF (3 mL) was added Et₃N (82 µL, 0.589

mmol) followed by chloroacetone (22 µL, 0.281 mmol). The

reaction was stirred at room-temperature overnight. Et₂O and

H₂O were added, and the aqueous layer was extracted with

Et₂O (3×). The combined organic extracts were washed with

brine (1×), dried (MgSO₄), and concentrated in vacuo. The

residue was purified by flash chromatography (5% MeOH in

CH₂Cl₂) to afford the title compound (2R)-N-(cyanomethyl)-4-

methyl-2-{4′-[4-(2-oxopropyl)piperazin-1-yl]-1,1′-biphenyl-3-

yl}pentanamide (11) (92 mg, 88% yield). Anal. (C₂₇H₃₄N₄O₂)

C: calcd, 72.62; found, 70.64; H: calcd, 7.67; found, 7.54; N:

calcd, 12.55; found, 12.53.

(2R)-2-[4′-(4-ter t-Bu t ylp ip er a zin -1-yl)-1,1′-b ip h en yl-3-

yl]-N-(cya n om eth yl)-4-m eth ylp en ta n a m id e (51). A mix-

ture of 1,4-dibromobenzene (1.8 g, 50 mmol) and tert-

butylpiperazine dihydrobromide (3.04 g, 10 mmol)³⁰was

dissolved in 1:1 dioxane/toluene (60 mL). This solution was

degassed, charged with potassium tert-butoxide (5.61 g, 50

mmol), Pd₂(dba)₃(275 mg, 0.30 mmol), and (2′-dicyclohexy-

lphosphanylbiphenyl-2-yl)dimethylamine (DavePhos)³¹(210

mg, 0.60 mmol) and sealed. The reaction mixture was heated

at 80 °C overnight. 1:1 Ethyl acetate/THF (200 mL) was added.

The mixture was washed with 1:1 water/brine (200 mL) and

brine (100 mL), filtered through a plug of MgSO₄/DARCO/silica

gel, evaporated to dryness, and purified by flash chromatog-

raphy (0-50% ethyl acetate/dichloromethane, final elution of

product with 5% methanol/dichloromethane) to give 0.68 g

(23% yield) of 1-(4-bromophenyl)-4-tert-butylpiperazine. A

solution of (2R)-N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tet-

ramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pentanamide ((R)-5)

(0.356 g, 1.00 mmol), 1-(4-bromophenyl)-4-tert-butylpiperazine

(0.297 g, 1.00 mmol), and 2.5 M sodium carbonate (1.25 mL)

in DMF (5 mL) in a Schlenk tube was degassed and charged

with PdCl₂(dppf)‚(44 mg, 0.06 mmol). The tube was sealed and

heated at 85 °C overnight. After being cooled, the reaction

mixture was partitioned between 4:1 ethyl acetate/THF and

brine/water (2:3, 100 mL), the organic phase was washed with

saturated aqueous Na₂CO₃(50 mL) and brine (50 mL), filtered

through a plug of MgSO₄/DARCO/silica, concentrated in vacuo,

and purified by flash chromatography (0-70% ethyl acetate/

CH₂Cl₂followed by elution with 5-7% methanol/CH₂Cl₂). The

product was then precipitated from CH₂Cl₂/ether and hexanes,

yielding 55 mg (13% yield) of the title compound 51. HRMS

(2R)-2-[4′-(1-Aza bicyclo[2.2.2]oct-4-yl)-1,1′-bip h en yl-3-

yl]-N-(cya n om eth yl)-4-m eth ylp en ta n a m id e (49). To 4-phe-

nylquinuclidine (230 mg, 1.23 mmol) in AcOH (8 mL) was

added Br₂(1.25 mL, 4.6 mmol), and the reaction mixture was

refluxed for 3 h. The reaction mixture was poured into aq sat.

NaOH, the crude product extracted with EtOAc (3×), and the

organic layer dried over Na₂SO₄. The solvent was removed in

vacuo, and the crude product was purified by flash chroma-

tography over silica gel (CH₂Cl₂/MeOH/aq sat. NH₄OH, 90/9/

1) to afford the desired 4-(4-bromophenyl)quinuclidine (201 mg,

61% yield). Following general procedure 1, (2R)-N-(cyanom-

ethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl]pentanamide ((R)-5) was submitted to a Suzuki

cross-coupling reaction with 4-(4-bromophenyl)quinuclidine to

afford title compound (2R)-2-[4′-(1-azabicyclo[2.2.2]oct-4-yl)-

1,1′-biphenyl-3-yl]-N-(cyanomethyl)-4-methylpentanamide

(49) (127 mg, 41%, purified by flash chromatography,

CH₂Cl₂/MeOH/aq sat. NH₄OH, 90/9/1). HRMS (+FAB): calcd

(+FAB): calcd for

447.312387.

C

₂₈H₃₉N₄O [MH⁺] 447.31239, found

for

C

₂₇H₃₄N₃O [MH⁺] 416.27019, found 416.27005. Anal.

(C₂₇H₃₃N₃O) C: calcd, 78.03; found, 75.55; H: calcd, 8.00;

found, 7.73; N: calcd, 10.11; found, 9.62.

(2R)-N-(Cya n om eth yl)-2-{4′-[4-(2-h yd r oxyeth yl)-1-p ip -

er a zin yl][1,1′-bip h en yl]-3-yl}-4-m eth ylp en ta n a m id e (12).

To (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(1-piperazinyl)[1,1′-

biphenyl]-3-yl]pentanamide ((R)-2) (105 mg, 0.269 mmol) in

MeCN (10 mL) were added 2-bromoethanol (25 µL, 0.282

mmol) and K₂CO₃(40 mg, 0.289 mmol), and the reaction

mixture was heated to 80 °C for 3.5 h. More 2-bromoethanol

(10 mL, 0.141 mmol) and K₂CO₃(20 mg, 0.145 mmol) were

added, and the reaction mixture was refluxed for 3 h. Water

and brine were added, and the product was extracted with

EtOAc (2×) and dried over MgSO₄. The solvent was removed

in vacuo, and the crude product was purified by flash chro-

matography over silica gel (CH₂Cl₂/MeOH/aq sat. NH₄OH, 90/

9/1) to afford the desired (2R)-N-(cyanomethyl)-2-{4′-[4-(2-

hydroxyethyl)-1-piperazinyl][1,1′-biphenyl]-3-yl}-4-methyl-

pentanamide (12) (96 mg, 82% yield). MS (+ESI) m/z 435.1

(M + H)⁺. HRMS (+FAB): calcd for C₂₆H₃₅N₄O₂[MH⁺]

435.27600, found 435.27594. Anal. (C₂₆H₃₄N₄O₂) C: calcd,

71.86; found, 70.89; H: calcd, 7.89; found, 7.66; N: calcd, 12.89;

found, 12.46.

ter t-Bu tyl 4-[3′-(1-{[(Cya n om eth yl)a m in o]ca r bon yl}-3-

m et h ylb u t yl)-1,1′-b ip h en yl-4-yl]p ip er a zin e-1-ca r b oxy-

late (50). To 1-(4-bromophenyl)piperazine hydrochloride (103.15

g, 371.59 mmol), DMAP (4.54 g, 37.159 mmol), and Et₃N (155

mL, 1.115 mol) in MeCN (1.5 L) at room temperature was

gradually added di-tert-butyl dicarbonate (121.65 g, 557.39

mmol) dissolved in a minimal amount of MeCN, and the

reaction mixture was stirred for 5.5 h. The suspension was

filtered, EtOAc was added, and the organic phase was washed

with an aq 10% citric acid solution, water (2×), and brine and

dried over MgSO₄. The solvent was concentrated in vacuo to

afford the desired crude product tert-butyl 4-(4-bromophenyl)-

piperazine-1-carboxylate (118.75 g, 94% yield) that was used

as such in the following step. Following general procedure 1,

N-(cyanomethyl)-4-methyl-2-[3-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl]pentanamide ((R,S)-5) was submitted to

a Suzuki cross-coupling reaction with tert-butyl 4-(4-bro-

mophenyl)piperazine-1-carboxylate to afford the title com-

pound tert-butyl 4-[3′-(1-{[(cyanomethyl)amino]carbonyl}-3-

methylbutyl)-1,1′-biphenyl-4-yl]piperazine-1-carboxylate (50)

(231 mg, 74% yield, purified by flash chromatography, EtOAc/

hexanes, 35/65). HRMS (+FAB): calcd for C₂₉H₃₈KN₄O₃[M +

K⁺] 529.25810, found 529.25828.

(2R)-N-(Cyan om eth yl)-2-{4′-[4-(2-h ydr oxy-2-m eth ylpr o-

p yl)-1-p ip er a zin yl][1,1′-bip h en yl]-3-yl}-4-m eth ylp en ta n a -

m id e (13). To (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(1-piper-

azinyl)[1,1′-biphenyl]-3-yl]pentanamide ((R)-2) (135 mg, 0.346

mmol), isobutylene oxide (50 mg, 0.692 mmol), and K₂CO₃(48

mg, 0.346 mmol) was added DMF (8 mL), and the reaction

mixture was heated to 100 °C for 1 h. Additional aliquots of

isobutylene oxide (250 mg, 3.47 mmol) and K₂CO₃(96 mg,

(2R)-N-(Cya n om eth yl)-4-m eth yl-2-{4′-[4-(2-oxop r op yl)-

p ip er a zin -1-yl]-1,1′-bip h en yl-3-yl}p en ta n a m id e (11). To

a solution of (2R)-N-(cyanomethyl)-4-methyl-2-(4′-piperazin-

3724 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

0.692 mmol) were added along with MeOH to dissolve the

solids, and the reaction mixture was heated for another 2 h.

Water and brine were added, and the product was extracted

with EtOAc (3×) and dried over Na₂SO₄. The solvent was

removed in vacuo, and the crude product was purified by flash

chromatography over silica gel (100% EtOAc) to afford the

desired (2R)-N-(cyanomethyl)-2-{4′-[4-(2-hydroxy-2-methylpro-

pyl)-1-piperazinyl][1,1′-biphenyl]-3-yl}-4-methylpentana-

mide (13) (113 mg, 71% yield). MS (+ESI) m/z 384.0 (M + H)⁺.

HRMS (+FAB): calcd for C₂₈H₃₉N₄O₂[MH⁺] 463.30730, found

463.30723. Anal. (C₂₈H₃₈N₄O₂) C: calcd, 72.69; found, 71.77;

H: calcd, 8.28; found, 8.13; N: calcd, 12.11; found, 11.72.

mixture at 100 °C for 1 h, a further portion of Pd(Cl₂)dppf

catalyst (605 mg, 0.741 mmol) was then added, and the

mixture was heated at 100 °C for a further 1.5 h. The reaction

mixture was then cooled to room temperature, and H₂O was

added. The mixture was extracted with EtOAc (10×), and the

combined organic extracts were dried (MgSO₄) and concen-

trated under reduced pressure. The residue was purified by

flash chromatography (gradient elution: 3% MeOH in CH₂-

Cl₂to 5% MeOH in CH₂Cl₂) to afford the title compound (26)

(6 g, 75% yield).

Gen er a l P r oced u r e 3 (P 2 Alk yla tion ). To a cold (0 °C),

stirred solution of methyl [4′-(4-methylpiperazin-1-yl)-1,1′-

biphenyl-3-yl]acetate (26) (1 equiv) in THF (10 mL/mmol of

26) was added KHMDS (0.5 M in toluene, 1.2 equiv). The

resultant solution was stirred at 0 °C for 20 min, warmed to

room temperature, and stirred for an additional 2 h. The

solution was then cooled to 0 °C, and the alkyl bromide or

iodide (1.3 equiv) was added. After being stirred at 0 °C for

10 min, the reaction mixture was warmed to room temperature

and stirred until the reaction was complete by TLC analysis

(2 h to overnight). H₂O and EtOAc were added, and the

aqueous layer was extracted with EtOAc (3×). The combined

organic extracts were dried (MgSO₄), concentrated in vacuo,

and the resultant residue was purified by flash chromatogra-

phy to afford the desired alkylated compounds 27b-f.

Gen er a l P r oced u r e 4 (Meth yl Ester Hyd r olysis a n d

P yBOB Cou p lin g). To a stirred solution of alkylated methyl

[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]acetate (27a -f)

(1 equiv) in a 3:1 mixture of MeOH/H₂O (25-50 mL/mmol of

starting material) was added an aqueous solution of LiOH (2.0

M, 10 equiv). The reaction mixture was stirred at room

temperature until ester hydrolysis was complete by TLC

analysis (10 min to 48 h). The reaction mixture was concen-

trated under reduced pressure and neutralized to pH 7 with

10% aqueous HCl. Upon neutralization, the desired acid

precipitated out, filtered, and washed with H₂O. The solid was

placed in a round-bottom flask, and any excess H₂O was

removed by codistillation with toluene. The desired acid was

then used as such in the next peptide coupling reaction. To a

cold (0 °C), stirred solution of the acid (1 equiv), aminoaceto-

nitrile hydrochloride (3 equiv), and PyBOB (1.5 equiv) in DMF

(10 mL/mmol of acid) was added Et₃N (4 equiv). The resultant

suspension was stirred at room-temperature overnight. Satu-

rated aqueous NaHCO₃was added followed by EtOAc. The

aqueous layer was extracted with EtOAc (3×), and the

combined organic extracts were washed with brine (1×), dried

(MgSO₄), and concentrated under reduced pressure. The

resultant residue was purified by flash chromatography to

afford the desired amides (28a -f).

N-(Cya n om eth yl)-2-[4′-(4-m eth ylp ip er a zin -1-yl)-1,1′-bi-

p h en yl-3-yl]a ceta m id e (28a ). Following general procedure

4, methyl [4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]ac-

etate (26) (134 mg, 0.43 mmol) was converted to the title

compound 28a (150 mg, 100% yield, flash chromatography

using gradient elultion: 3% MeOH in CH₂Cl₂to 5% MeOH in

CH₂Cl₂). HRMS (+FAB): calcd for C₂₁H₂₅N₄O [MH⁺] 349.20284,

found 349.20289. Anal. (C₂₁H₂₄N₄O) C: calcd, 72.39; found,

68.93; H: calcd, 6.94; found, 6.82; N: calcd, 16.08; found, 14.92.

N-(Cya n om eth yl)-2-[4′-(4-m eth ylp ip er a zin -1-yl)-1,1′-bi-

p h en yl-3-yl]-3-p h en ylp r op a n a m id e (28b). Following gen-

eral procedure 3, methyl [4′-(4-methylpiperazin-1-yl)-1,1′-

biphenyl-3-yl]acetate (26) (300 mg, 0.93 mmol) was alkylated

with benzyl bromide to yield, after flash chromatography (5%

MeOH in CH₂Cl₂), methyl 2-[4′-(4-methylpiperazin-1-yl)-1,1′-

biphenyl-3-yl]-3-phenylpropanoate (27b) (88 mg, 23% yield).

Methyl 2-[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]-3-phe-

nylpropanoate (27b) (78 mg, 0.19 mmol) was converted to the

title compound 28b (40 mg, 49% yield) following general

procedure 4 (flash chromatography using 5% MeOH in CH₂-

Cl₂). HRMS (+FAB): calcd for C₂₈H₃₁N₄O [MH⁺] 439.24979,

found 439.24998.

(2R)-2-{4′-[1-(2-Am in o-2-m eth ylp r op yl)p ip er id in -4-yl]-

1,1′-b ip h en yl-3-yl}-N-(cya n om et h yl)-4-m et h ylp en t a n a -

m id e (14). To (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(1-piper-

azinyl)[1,1′-biphenyl]-3-yl]pentanamide ((R)-2) (284 mg, 0.666

mmol), 2-amino-2-methylpropyl hydrogen sulfate (451 mg, 2.67

mmol), and K₂CO₃(734 mg, 5.32) was added dry DMF (6 mL),

and the reaction mixture was heated to 70 °C for 18 h.

Additional aliquots of 2-amino-2-methylpropyl hydrogen sul-

fate (450 mg, 2.66 mmol) and K₂CO₃(730 mg, 5.29 mmol) were

added, and the reaction mixture was heated for another 48 h.

The reaction mixture was poured into water, extracted with

EtOAc (3×), dried over MgSO₄, concentrated in vacuo, and

purified by flash chromatography over silica gel (CH₂Cl₂/

MeOH/saturated aqueous NH₄OH, 90/9/1) to afford (2R)-2-{4′-

[1-(2-amino-2-methylpropyl)piperidin-4-yl]-1,1′-biphenyl-3-yl}-

N-(cyanomethyl)-4-methylpentanamide (14) as a foamy solid

(11.5 mg, 4%). HRMS (+FAB): calcd for C₂₈H₄₀N₅O [MH⁺]

462.32329, found 462.32340.

Meth yl [4′-(4-m eth ylp ip er a zin -1-yl)-1,1′-bip h en yl-3-yl]-

a ceta te (26). To a solution of (3-hydroxyphenyl)acetic acid (22)

(20.0 g, 131.6 mmol) and p-TsOH (2.5 g, 14.5 mmol) in MeOH

(100 mL) was added trimethylorthoformate (29 mL, 263.2). The

solution was refluxed for 4 h, cooled to room temperature, and

stirred overnight. The reaction mixture was concentrated in

vacuo, and the residue obtained was dissolved in Et₂O and

washed with saturated aqueous NaHCO₃(4×). The organic

layer was dried (MgSO₄) and concentrated in vacuo to yield

methyl (3-hydroxyphenyl)acetate (23.0 g, quantitative yield)

which was used as such in the next reaction. To a solution of

methyl (3-hydroxyphenyl)acetate (23.0 g, 138.6 mmol) in CH₂-

Cl₂(700 mL) was added Et₃N (31 mL, 221.7). The resultant

yellow solution was cooled to 0 °C followed by the dropwise

addition of trifluoromethanesulfonic anhydride (35 mL, 207.9

mmol) over 20 min. The reaction mixture was stirred at 0 °C

for 1 h and then diluted with a 1:1 mixture of Et₂O/EtOAc

(700 mL). The mixture was washed with H₂O (2 × 300 mL)

and brine (1 × 300 mL), dried (MgSO₄), and concentrated in

vacuo. The residue was purified by flash chromatography

(gradient elution: 7% EtOAc/hexanes to 20% EtOAc/hexanes)

to afford methyl (3-{[(trifluoromethyl)sulfonyl]oxy}phenyl)-

acetate (23) (33.7 g, 82% yield). A solution of methyl (3-{-

[(trifluoromethyl)sulfonyl]oxy}phenyl)acetate (23) (33.0 g, 110.7

mmol), diboron pinacol ester (33.7 g, 132.9 mmol), KOAc (32.5

g, 332.1 mmol), and Pd(Cl₂)dppf (1.8 g, 2.20 mmol) in DMF

(500 mL) was heated at 70 °C for 1.5 h. A further portion of

Pd(Cl₂)dppf catalyst (1.8 g, 2.20 mmol) was then added, and

the mixture was heated at 90 °C for 1.5 h. The reaction was

cooled to room temperature, diluted with a 1:1 mixture of C₆H₆/

Et₂O (1 L), and washed with H₂O (1 × 500 mL) and brine (2

× 300 mL). The combined aqueous layers were extracted with

Et₂O (2 × 400 mL), and the combined organic extracts were

dried (MgSO₄) and concentrated in vacuo. The residue was

purified by flash chromatography (gradient elution: 5% EtOAc/

hexanes to 30% EtOAc/hexanes) to afford methyl [3-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]acetate (24) (27.5

g, 90% yield). To a solution of methyl [3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)phenyl]acetate (24) (10.2 g, 36.96 mmol)

and 1-(4-bromophenyl)-4-methylpiperazine (25) (6.3 g, 36.96

mmol) in DMF (200 mL) was added an aqueous solution of

Na₂CO₃(2.0 M, 37 mL, 110.88 mmol). The solution was

degassed with N₂for 30 min followed by the addition of Pd-

(Cl₂)dppf (605 mg, 0.741 mmol). After heating the reaction

N-(Cya n om eth yl)-2-[4′-(4-m eth ylp ip er a zin -1-yl)-1,1′-bi-

p h en yl-3-yl]p en ta n a m id e (28c). Following general proce-

dure 3, methyl [4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3725

yl]acetate (26) (300 mg, 0.93 mmol) was alkylated with

1-iodopropane to yield, after flash chromatography (5% MeOH

in CH₂Cl₂), methyl 2-[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-

3-yl]pentanoate (27c) (137 mg, 40% yield). Methyl 2-[4′-(4-

methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]pentanoate (27c) (137

mg, 0.37 mmol) was converted to the title compound N-(cya-

nomethyl)-2-[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]-

pentanamide (28c) (56 mg, 38% yield) following general

procedure 4 (flash chromatography using gradient elultion: 3%

MeOH in CH₂Cl₂to 10% MeOH in CH₂Cl₂). HRMS (+FAB):

calcd for C₂₄H₃₁N₄O [MH⁺] 391.24979, found 391.24994. Anal.

(C₂₄H₃₀N₄O) C: calcd, 73.81; found, 50.65; H: calcd, 7.74;

found, 6.24; N: calcd, 14.35; found, 9.91.

N-(Cya n om eth yl)-3-cyclop r op yl-2-[4′-(4-m eth ylp ip er -

a zin -1-yl)-1,1′-bip h en yl-3-yl]p r op a n a m id e (28d ). Follow-

ing general procedure 3, methyl [4′-(4-methylpiperazin-1-yl)-

1,1′-biphenyl-3-yl]acetate (26) (300 mg, 0.93 mmol) was

alkylated with (bromomethyl)cyclopropane to yield, after flash

chromatography (5% MeOH in CH₂Cl₂), methyl 3-cyclopropyl-

2-[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]propanoate (27d)

(198 mg, 57% yield). Methyl 3-cyclopropyl-2-[4′-(4-methylpip-

erazin-1-yl)-1,1′-biphenyl-3-yl]propanoate (27d ) (198 mg, 0.52

mmol) was converted to the title compound N-(cyanomethyl)-

3-cyclopropyl-2-[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]-

propanamide (28d ) (79 mg, 38% yield) following general

procedure 4 (flash chromatography using gradient elultion: 5%

MeOH in CH₂Cl₂to 10% MeOH in CH₂Cl₂). MS (+ESI) m/z

404.2 (M + H)⁺. HRMS (+FAB): calcd for C₂₅H₃₀KN₄O [M +

K⁺] 441.20567, found 441.20585. Anal. (C₂₅H₃₀N₄O) C: calcd,

74.59; found, 73.72; H: calcd, 7.51; found, 7.46; N: calcd, 13.92;

found, 13.72.

N-[(2R)-4-Met h yl-2-(4′-p ip er a zin -1-yl-1,1′-b ip h en yl-3-

yl)p en ta n oyl]glycin e (52). To the hydrochloride salt of (2R)-

N-(cyanomethyl)-4-methyl-2-[4′-(1-piperazinyl)[1,1′-biphenyl]-

3-yl]pentanamide ((R)-2) (1.37 g, 3.2 mmol) in 1,4-dioxane (8

mL) and MeOH (2 mL) was added dropwise a solution of HCl

(4.0 M, in 1,4-dioxane, 2.4 mL, 9.6 mmol) under an atmosphere

of dry nitrogen, and the reaction was stirred at room temper-

ature for 1.5 h. A further aliquot of HCl (4.0 M, in 1,4-dioxane,

2.4 mL, 9.6 mmol) was added, and the reaction mixture was

stirred for a total of 6 h. The acid was carefully quenched with

a solution of aq sat. NaHCO₃, brine was added, the product

was extracted with EtOAc (2×) and dried over Na₂SO₄, and

the solvent was removed in vacuo. Purification of the crude

product by flash chromatography (CH₂Cl₂/MeOH/aq sat. NH₄-

OH, 90/9/1) afforded the desired intermediate methyl N-[(2R)-

4-methyl-2-(4′-piperazin-1-yl-1,1′-biphenyl-3-yl)pentanoyl]gly-

cinate (750 mg, 55% yield). To methyl N-[(2R)-4-methyl-2-(4′-

piperazin-1-yl-1,1′-biphenyl-3-yl)pentanoyl]glycinate (750 mg,

1.77 mmol) in MeOH (20 mL) was added dropwise an aq

solution of LiOH (2.0 M, 4.4 mL, 8.8 mmol), and the reaction

was stirred for 3.5 h. The reaction mixture was carefully

neutralized with an aq solution of 10% HCl, and the solvent

was removed in vacuo. The crude product was rinsed with

water and then EtOAc and dissolved in MeOH, and the solvent

was removed in vacuo to afford the desired product N-[(2R)-

4-methyl-2-(4′-piperazin-1-yl-1,1′-biphenyl-3-yl)pentanoyl]gly-

cine (52) (520 mg, 72% yield). HRMS (+FAB): calcd for

C

₂₄H₃₃N₄O₂[MH⁺] 410.24437, found 410.24418.

En zym e Exp r ession a n d P u r ifica tion . Recombinant

human cathepsin K was expressed in P. pastoris essentially

as described,³²activated by diafiltration at pH 4.5, and purified

by ion exchange chromatography on a 20 mL Pharmacia

Source 15S column equilibrated with 50 mM sodium acetate,

pH 4.5, 1 mM EDTA, 1 mM dithiothreitol. Cathepsin K was

eluted from the column with a linear gradient of 0 to 1.0 M

NaCl in the same buffer. Humanized rabbit cathepsin K

(rabbit cathepsin K with S163A, Y175D, and V274L mutations

introduced; numbering from the initiation methionine) was

expressed in P. pastoris and purified following the same

protocol used for human cathepsin K. A human cathepsin L

cDNA was isolated by PCR, cloned into the expression vector

pPIC9, and transformed into P. pastoris strain KM71. Human

cathepsin S, cloned as described,³³was subcloned into the

expression vector pPIC9 and transformed into P. pastoris

strain KM71. Fermentation of high expressing clones for

cathepsins L and S was carried out according to the supplier’s

protocol (Invitrogen). Recombinant cathepsin L was activated

by diafiltration and purified on a Source 15S column as

described for cathepsin K. Recombinant cathepsin S was

activated by diafiltration at pH 4.5 and purification was

carried out as described for cathepsin K except that a 0-0.5

M linear NaCl gradient was used.

En zym e In h ibition . To measure enzyme activity, assays

were carried out in 50 mM MES pH 5.5 containing 2.5 mM

DTT, 2.5 mM EDTA, and 10% DMSO. IC₅₀values of com-

pounds were determined for human cathepsins K, L, B, and S

and for humanized rabbit cathepsin K using 2 µM of Z-Leu-

Arg-AMC as substrate. Prior to the addition of substrate,

different concentrations of the inhibitor ranging from 100 µM

to 0.2 nM were preincubated for 15 min with each enzyme

(0.2-1 nM) to allow the establishment of the enzyme-inhibitor

complex. Substrate was then added and the enzyme activity

(4S)-N-(Cya n om et h yl)-4-m et h yl-2-[4′-(4-m et h ylp ip er -

a zin -1-yl)-1,1′-bip h en yl-3-yl]h exa n a m id e (28e, m ixtu r e of

2 d ia ster eoisom er s). Following general procedure 3, methyl

[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]acetate (26) (300

mg, 0.93 mmol) was alkylated with (S)-(+)-1-bromo-2-meth-

ylbutane to yield, after flash chromatography (5% MeOH in

CH₂Cl₂), methyl (4S)-4-methyl-2-[4′-(4-methylpiperazin-1-yl)-

1,1′-biphenyl-3-yl]hexanoate (27e) (105 mg, 29% yield). Methyl

(4S)-4-methyl-2-[4′-(4-methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]-

hexanoate (27e) (75 mg, 0.19 mmol) was converted to the title

compound 28e (36 mg, 767% yield) following general procedure

4 (flash chromatography using gradient elultion: 3% MeOH

in CH₂Cl₂to 5% MeOH in CH₂Cl₂). HRMS (+FAB): calcd for

C

₂₆H₃₅N₄O [MH⁺] 419.28109, found 419.28094. Anal. (C₂₆H₃₄

-

N₄O) C: calcd, 74.61; found, 65.80; H: calcd, 8.19; found, 8.48;

N: calcd, 13.39; found, 14.32.

N-(Cyan om eth yl)-3-cyclobu tyl-2-[4′-(4-m eth ylpiper azin -

1-yl)-1,1′-bip h en yl-3-yl]p r op a n a m id e (28f). Following gen-

eral procedure 3, methyl [4′-(4-methylpiperazin-1-yl)-1,1′-

biphenyl-3-yl]acetate (26) (300 mg, 0.93 mmol) was alkylated

with (bromomethyl)cyclobutane to yield, after flash chroma-

tography (5% MeOH in CH₂Cl₂), methyl 3-cyclobutyl-2-[4′-(4-

methylpiperazin-1-yl)-1,1′-biphenyl-3-yl]propanoate (27f) (22

mg, 6% yield). Methyl 3-cyclobutyl-2-[4′-(4-methylpiperazin-

1-yl)-1,1′-biphenyl-3-yl]propanoate (27f) (21 mg, 0.05 mmol)

was converted to the title compound 28f (20 mg, 91% yield)

following general procedure 4 (flash chromatography using 5%

MeOH in CH₂Cl₂). HRMS (+FAB): calcd for C₂₆H₃₃N₄O [MH⁺]

417.26544, found 417.26554.

4-Met h yl-2-(4′-p ip er a zin -1-yl-b ip h en yl-3-yl)p en t a n o-

ic Acid Ca r ba m oylm eth yla m id e (3). To the hydrochloride

salt of (2R)-N-(cyanomethyl)-4-methyl-2-[4′-(1-piperazinyl)-

[1,1′-biphenyl]-3-yl]pentanamide ((R)-2) (1.04 g, 2.44 mmol) in

CH₂Cl₂(50 mL) was added TFA (380 µL, 4.88 mmol) dropwise,

and the reaction was stirred at room temperature for 5 h. The

acid was carefully quenched with a solution of aq sat. NaHCO₃,

the product extracted with EtOAc (3×) and dried over Na₂-

SO₄, and solvent was removed in vacuo. Purification of the

crude product by flash chromatography (CH₂Cl₂/MeOH/aq sat.

NH₄OH, 90/9/1) afforded the desired compound 4-methyl-2-

(4′-piperazin-1-yl-biphenyl-3-yl)pentanoic acid carbamolmethy-

lamide (3) (502 mg, 51% yield). HRMS (+FAB): calcd for

measured from the increase of fluorescence at 460 nm (λ_ex.

)

355 nm). The final volume of the reaction was 100 uL. Assays

were performed in 96-well plate format and the plate read

using a Spectramax (Molecular Devices) plate reader. The

percent inhibition of the reaction was calculated from a control

reaction containing only vehicle. IC₅₀curves were generated

by fitting percent inhibition values to a four parameter logistic

model (SOFTMAX PRO, Molecular Devices).

Nitr ila se Assa y. Detection for nitrile hydrolysis was evalu-

ated by RP-HPLC analysis. The reaction was carried out in

50 mM MES pH 5.5 containing 2.5 mM DTT, 2.5 mM EDTA,

and 1 mM â-mercaptoethanol. Reaction products were ana-

C

₂₄H₃₃N₄O₂[MH⁺] 409.26035, found 409.26050.

3726 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17

Robichaud et al.

(9) Linnevers, C.; Smeekens, S. P.; Bromme, D. Human cathepsin

W, a putative cysteine protease predominantly expressed in

CD8+ T-lymphocytes. FEBS Lett. 1997, 405 (3), 253-259.

(10) Hernandez, A. A.; Roush, W. Recent advances in the synthesis,

design and selection of cysteine inhibitors. Curr. Opin. Chem.

Biol. 2002, 6, 459-465.

(11) Lecaille, F.; Kaleta, J .; Bromme, D. Human and parasitic papain-

like cysteine proteases: their role in physiology and pathology

and recent developments in inhibitor design. Chem. Rev. 2002,

102 (12), 4459-4488.

(12) Bryant, C. M.; Palmer, J . T.; Rydzewski, R. M.; Setti, E. L.; Tian,

Z.-Q.; Venkatraman, S. Novel compounds and compositions as

protease inhibitors. World Patent 2000/55126, Chem. Abstr.

2000, 133, 252050.

(13) Gabriel, T.; Pech, M.; Rodriguez Sarmiento, R. M. â-amino acid

nitrile derivatives useful for the treatment of diseases which are

associated with cysteine proteases. World Patent 2001/96285,

Chem. Abstr. 2001, 136, 53544.

lyzed by RP-HPLC using a linear gradient from 25% acetoni-

trile to 60% acetonitrile 0.1% TFA on a C18 column (Zorbax

RX-C18 4.6 × 150 mm). The chromatogram was developed in

6 min at a flow rate of 2 mL/min. Under those conditions, the

retention times for compound (R)-2, the synthetic acid 52, and

the amide 3 were 3.9, 3.2, and 2.7 min, respectively.

Kin etic An a lysis. The measurement of enzyme activity to

determine kinetic parameters was performed in 50 mM MES

pH 5.5 containing 2.5 mM DTT, 2.5 mM EDTA, and 10%

DMSO. Z-Leu-Arg-AMC (2 µM) was used as substrate, and

enzymatic activity was measured at room temperature in 1

mL stirred cells using a Quantamaster spectrofluorometer

(Photon Technology International) with excitation and emis-

sion wavelengths of 355 nm of 460 nm, respectively. Product

formation was measured at different inhibitor concentrations

following initiation of the reaction by the addition of the

enzyme (0.2 nM final concentration). Formation of product (P)

with time for an enzyme inhibited by a time-dependent

inhibitor is described by the following equation³⁴

(14) Missbach, M. Preparation of dipeptide cathepsin K inhibitors.

World Patent 2001/58886, Chem. Abstr. 2001, 135, 180786.

(15) Altmann, E.; Betschart, C.; Gohda, K.; Horiuchi, M.; Lattmann,

R.; Missbach, M.; Sakaki, J .; Takai, M.; Teno, N.; Cowen, S. D.;

Greenspan, P. D.; McQuire, L. W.; Tommasi, R. A.; Van Duzer,

J . H. Dipeptide nitriles. World Patent 9924460, Chem. Abstr.

1999, 130, 352553.

P ) v_st - (v_s- v₀) (1 - e^-k)/k_obsd

(1)

obs

(16) Greenspan, P. D.; Clark, K. L.; Tommasi, R. A.; Cowen, S. D.;

McQuire, L. W.; Farley, D. L.; van Duzer, J . H.; Goldberg, R. L.;

Zhou, H.; Du, Z.; Fitt, J . J .; Coppa, D. E.; Fang, Z.; Macchia, W.;

Zhu, L.; Capparelli, M. P.; Goldstein, R.; Wigg, A. M.; Doughty,

J . R.; Bohacek, R. S.; Knap, A. K. Identification of dipeptidyl

nitriles as potent and selective inhibitors of cathepsin B through

structure-based drug design. J . Med. Chem. 2001, 44, 4524-

4534.

(17) Ward, Y. D.; Thomas, D. S.; Frye, L. L.; Cywin, C. L.; Morwick,

T.; Emmanuel, M. J .; Zindell, R.; McNeil, D.; Bekkali, Y.;

Giradot, M.; Hrapchak, M.; DeTuri, M.; Crane, K.; White, D.;

Pav, S.; Wang, Y.; Hao, M.-H.; Grygon, C. A.; Labadia, M. E.;

Freeman, D. M.; Davidson, W.; Hopkins, J . L.; Brown, M. L.;

Spero, D. M. Design and synthesis of dipeptide nitriles as

reversible and potent cathepsin S inhibitors. J . Med. Chem.

2002, 45, 5471-5482.

where v_sis the rate of the reaction at steady-state, v₀is the

initial velocity of the reaction, and k_obsdis the apparent first-

order rate constant characterizing the establishment of the

steady-state velocity. Experimental values were fitted by

nonlinear regression to eq 1, and fitted parameters v_s, v_0,and

k

_obsdwere used to estimate pre-steady-state kinetic parameters

k_onand k_off(eqs 3 and 4) and the dissociation constant K_i(eq

2) using the following relationships.³⁴

K_i) [I]/((v_o/v_s) - 1)

k_on) k_obsd/([I] + K_i)

k_off) k_onK_i

(2)

(3)

(4)

(18) Suzue, S.; Irikura, T. Studies on hepatic agents. I. Synthesis of

aminoacyl (and hydroxyacyl) aminoacetonitriles. Chem. Pharm.

Bull. 1968, 16, 1417-1432.

(19) Larsen, R. D.; Corley, E. G.; Davis, P.; Reider, P. J .; Grabowski,

E. J . J . R-Hydroxy esters as chiral reagents: asymmetric

synthesis of 2-arylpropionic acids. J . Am. Chem. Soc. 1989, 111,

7650-7651.

(20) Huntress, E. H.; Pfister, K., III. The conversion of 2-phenyl-4-

chloromethylthiazole to 2-phenyl-4-hydroxymethyl-5-chlorothia-

zole. J . Am. Chem. Soc. 1943, 65, 1667-1669.

Su p p or tin g In for m a tion Ava ila ble: ¹H NMR spectral

data. This material is available free of charge via the Internet

at http://pubs.acs.org.

Refer en ces

(21) Falgueyret, J . P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.;

Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S.

B.; Percival, M. D. Novel, nonpeptidic cyanamides as potent and

reversible inhibitors of human cathepsins K and L. J . Med.

Chem. 2001, 44 (1), 94-104.

(22) DesJ arlais, R. L.; Yamashita, D. S.; Oh, H.-J .; Uzinskas, I. N.;

Erhard, K. F.; Allen, A. C.; Haltiwanger, R. C.; Zhao, B.; Smith,

W. W.; Abdel-Meguid, S. A.; D′Alessio, K.; J anson, C. A.;

McQueney, M. S.; Tomaszek, T. A.; Levy, M. A.; Veber, D. F.

Use of X-ray cocrystal structures and molecular modeling to

design potent and selective non-peptide inhibitors of cathepsin

K. J . Am. Chem. Soc. 1998, 120, 9114-9115.

(23) Gour-Salin, B. J .; Storer, A. C.; Castelhano, A.; Krantz, A.;

Robinson, V. Inhibition of papain by peptide nitriles: Reactions

of external nucleophiles with the thioimidate ester adduct.

Enzyme Microb, Technol. 1991, 13, 408-411.

(24) Dufour, E.; Storer, A. C.; Menard, R. Engineering nitrile

hydratase activity into a cysteine protease by a single mutation.

Biochemistry 1995, 34, 4 (50), 16382-16388.

(1) Peck, W. A.; Burckhardt, P.; Christiansen, C.; Fleisch, H. A.;

Genant, H. K.; Gennari, C.; Martin, T. J .; Martini, L.; Morita,

R.; Ogata, E.; Rapado, A.; Shulman, L. E.; Stern, P. H.; Young,

R. T. T.; Barrett-Connor, E.; Brandi, M. L.; Chesnut, C. H.;

Delmas, P. D.; Heaney, R. P.; J ohnston, C. C.; Kanis, J . A.;

Lindsay, R.; Meunier, P. J .; Papapoulos, S. E.; Reginster, J . Y.;

Riggs, B. L.; Riis, B. J .; Seeman, E.; Wasnich, R. D. Consensus

development conference: diagnosis prophylaxis and treatment

of osteoporosis. Am. J . Med. 1993, 94, 646-650.

(2) Bromme, D.; Okamoto, K.; Wang, B. B.; Biroc, S. Human

Cathepsin O2, a matrix protein-degrading cysteine protease

expressed in osteoclasts. J . Biol. Chem. 1996, 271, 2126-2132.

(3) Bossard, M. J .; Tomaszek, T. A.; Thompson, S. K.; Amegadzie,

B. Y.; Hanning, C. R.; J ones, C.; Kurdyla, J . T.; McNulty, D. E.;

Drake, F. H.; Gowen, M.; Levy, M. A. Proteolytic activity of

human osteoclast cathepsin K. Expression, purification, activa-

tion, and substrate identification. J . Biol. Chem. 1996, 271,

12517-12524.

(4) Tezuka, K.; Tezuka, Y.; Maejima, A.; Sato, T.; Nemoto, K.;

Kamioka, H.; Hakeda, Y.; Kumegawa, M. Molecular cloning of

(25) Bossard, M. J .; Tomaszek, T. A.; Levy, M. A.; Ijames, C. F.;

Huddleston, M. J .; Briand, J .; Thompson, S.; Halpert, S.; Veber,

D. F.; Carr, S. A.; Meek, T. D.; Tew, D. G. Mechanism of

inhibition of cathepsin K by potent, selective 1,5-diacylcarbohy-

drazides: a new class of mechanism-based inhibitors of thiol

proteases. Biochemistry 1999, 38, 8 (48), 15893-15902.

(26) Christgau, S.; Bitsch-J ensen, O.; Hanover-Bjarnason, N.; Gam-

well-Henriksen, E.; Qvist, P.; Alexandersen, P.; Bang-Henriksen,

D. Serum CrossLaps for monitoring the response in individuals

undergoing antiresorptive therapy. Bone 2000, 26, 505-511.

(28) Prepared from commercially available 3-morpholinophenol ac-

cording to Kotsuki, H.; Kobayashi, S.; Suenaga, H.; Nishizawa,

H. High-pressure organic chemistry; XI. A new convenient

synthesis of aromatic amines from activated phenols. Synthesis

1990, 12, 1145-1147.

a

possible cysteine proteinase predominantly expressed in

osteoclasts. J . Biol. Chem. 1994, 269, 1106-1109.

(5) Turk, B.; Turk, D.; Turk, V Lysosomal cysteine proteases: more

than scavengers. Biochim. Biophys. Acta 2000, 1477, 98-111.

(6) Drake, F. H.; Dodds, R. A.; J ames, I. E.; Connor, J . R.; Debouck,

C.; Richardson, S.; Lee-Rykaczewski, E.; Coleman, L.; Rieman,

D.; Barthlow, R.; Hastings, G.; Gowen, M. Cathepsin K, but not

Cathepsin B, L, or S, is abundantly expressed in human

osteoclasts. J . Biol. Chem. 1996, 271, 12511-12516.

(7) Kirschke, H.; Bro¨mme, D.; Wiederanders, B. Cathepsin S, a

lysosomal cysteine protease. In Proteolysis and Protein Turnover;

Bond, J . S., Barret, A. J ., Eds.; Portland Press: London and

Chapel Hill, 1993; pp 33-37.

(8) Shi, G. P.; Villadangos, J . A.; Dranoff, G.; Small, C.; Gu, L.;

Haley, K. J .; Riese, R.; Ploegh, H. L.; Chapman, H. A.; Cathepsin

S required for normal MHC class II peptide loading and germinal

center development. Immunity 1999, 10 (2), 197-206.

(29) Prepared from commercially available 2-morpholinophenol ac-

cording to ref 28.

Nonpeptidic Biaryl Inhibitors of Human Cathepsin K

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3727

(30) Poindexter, G. S.; Bruce, M. A.; LeBoulluec, K. L.; Monkovic, I.

Use of 2-oxazolidinones as latent aziridine equivalents. III.

Preparation of N-substituted piperazines. Tetrahedron Lett.

1994, 35, 7331-7334.

(31) Old, D. W.; Wolfe, J . P.; Buchwald, S. L. A Highly Active Catalyst

for Palladium-Catalyzed Cross-Coupling Reactions: Room-Tem-

perature Suzuki Couplings and Amination of Unactivated Acyl

Chlorides. J . Am. Chem. Soc. 1998, 120, 9722-9723.

D. Expression of human cathepsin K in Pichia pastoris and

preliminary crystallographic studies of an inhibitor complex.

Protein Sci. 1997, 6 (4), 919-921.

(33) Bromme, D.; McGrath, M. E. High level expression and crystal-

lization of recombinant human cathepsin S. Protein Sci. 1996,

5 (4), 789-791.

(34) Izquierdo-Martin, M.; Stein, R. L. Transition-state structural

features for the association of metalloproteases with phosphorus-

containing inhibitors. J . Am. Chem. Soc. 1992, 114, 1527-1528.

(32) Linnevers, C. J .; McGrath, M. E.; Armstrong, R.; Mistry, F. R.;

Barnes, M. G.; Klaus, J . L.; Palmer, J . T.; Katz, B. A.; Bromme,

J M0301078

Article Doi

A novel class of nonpeptidic biaryl inhibitors of human cathepsin K

DOI: 10.1021/jm0301078

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm0301078

Full text of DOI:10.1021/jm0301078

Products guided by the article

R&D Labs maybe for 597584-50-4

Relevant to this article

Hot Product