Azepanone-based inhibitors of human and rat cathepsin K

1380

J . Med. Chem. 2001, 44, 1380-1395

Azep a n on e-Ba sed In h ibitor s of Hu m a n a n d Ra t Ca th ep sin K

Robert W. Marquis,*^,†Yu Ru,^†Steven M. LoCastro,^†J in Zeng,^†Dennis S. Yamashita,^†Hye-J a Oh,^†

Karl F. Erhard,^†Larry D. Davis,^†Thaddeus A. Tomaszek,^‡David Tew,^‡Kevin Salyers,^§J oel Proksch,^§

Keith Ward,^§Brian Smith,^§Mark Levy,^§Maxwell D. Cummings,^|,1R. Curtis Haltiwanger,^|Gudrun Trescher,^|

Baoguang Zhao, Michael S. McQueney,^#Karla D’Alessio,^#Chao-Pin Lee,^@Antonia Marzulli,^§

Robert A. Dodds,^∧,2Simon Blake,^∧Shing-Mei Hwang,^∧Ian E. J ames,^∧Catherine J . Gress,^∧Brian R. Bradley,^∧

Michael W. Lark,^∧Maxine Gowen,^∧and Daniel F. Veber*^,†

Departments of Medicinal Chemistry, Mechanistic Enzymology, Drug Metabolism and Pharmacokinetics, Physical and

Structural Chemistry, Structural Biology, Protein Biochemistry, Life-Cycle Management and Drug Delivery Systems,

and Bone and Cartilage Biology, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406

Received November 10, 2000

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors

of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show

improved configurational stability of the C-4 diastereomeric center relative to the previously

published five- and six-membered ring ketone-based inhibitor series. Studies in this series

have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K_i)

0.16 nM) as well as 24, a potent inhibitor of both human (K_i) 0.0048 nM) and rat (K_i,app) 4.8

nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S

stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected

equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher

energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature

subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20

to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues

through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by

a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a

conformational constraint has served the dual purpose of increasing inhibitor potency by locking

in a bioactive conformation as well as locking out available conformations which may serve as

substrates for enzyme systems that limit oral bioavailability.

In tr od u ction

to expand.⁵This expansion is based upon several studies

which have shown that members of this class of protease

are expressed selectively and that they play a critical

role in the pathology of disease. The design and activity

of a new class of reversible, 1,3-bis(acylamino)-2-pro-

panone inhibitors of cathepsin K, exemplified by 1, have

been reported (Figure 1).⁶Despite potent inhibition of

cathepsin K, the low oral bioavailability, short duration

of action, and low potency in cell-based assays of bone

resorption limited the utility of this class of compounds.

Initial attempts to address these issues focused on the

incorporation of peptidomimetic elements designed to

reduce the peptide nature of 1. This has led to the

discovery of compounds 2 and 3 which are potent

inhibitors of human cathepsin K displaying potent

inhibition in cell-based assays of bone resorption.⁷

However, because both 2 and 3 displayed poor oral

bioavailability in the rat, their utility was limited to

subcutaneous and intraperitoneal routes of administra-

tion. A particularly successful modification of 1, involv-

ing the introduction of a conformational constraint, has

led to the previously reported 3-amido-pyrrolidin-4-one

4 and the 4-amido-piperidin-3-one 5, both of which are

potent, reversible inhibitors of cathepsin K.^8,9Several

analogues within these cyclic 1,3-diaminoketone series

displayed promising preliminary pharmacokinetics as

Cathepsin K (EC 3.4.22.38), a 24-kDa cysteine pro-

tease of the papain superfamily, has been shown to be

selectively expressed within osteoclasts as a proen-

zyme.¹Upon activation, mature cathepsin K is secreted

from the osteoclast into the acidic resorption lacuna

where it plays a critical role in the degradation of the

protein matrix of bone. Several studies have reinforced

the role of cathepsin K in bone resorption and high-

lighted the attractiveness of this cysteine protease as a

target for inhibition in diseases characterized by el-

evated levels of bone turnover such as osteoporosis.^2-4

Recently the evolution of a variety of templates

targeting the inhibition of cysteine proteases has begun

* Corresponding authors: Dept. of Medicinal Chemistry, Glaxo-

SmithKline, 709 Swedeland Rd., P.O. Box 1539, UW 24-1055, King of

Prussia, PA 19406. Phone: 610-270-6570/6561. Fax: 610-270-4451.

E-mail: robert_w_marquis@sbphrd.com or daniel_f_veber@sbphrd.com.

^†Department of Medicinal Chemistry.

^‡Department of Mechanistic Enzymology.

^§Department of Drug Metabolism and Pharmacokinetics.

^|Department of Physical and Structural Chemistry.

Department of Structural Biology.

#

Department of Protein Biochemistry.

^@Department of Life-Cycle Management and Drug Delivery Sys-

tems.

^∧Department of Bone and Cartilage Biology.

¹Current address: 3-Dimensional Pharmaceuticals Inc., 665 Stock-

ton Dr., Eagleview Corporate Center, Exton, PA 19341.

2

Current address: Osiris Therapeutics Inc., 2001 Aliceanne St.,

Baltimore, MD 21207.

Published on Web 03/24/2001

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1381

F igu r e 1. Templates leading to azepanone inhibitors.

well as adequate potency versus rat cathepsin K.¹⁰Due

to rapid epimerization of the C-4 chiral center contained

within these inhibitors, they were unsuitable for more

detailed analyses of their pharmacokinetics in either

rodents or primates. The rapid rate of epimerization of

the C-4 diastereomeric center associated with these

analogues represents a significant pharmacokinetic

issue since it is difficult to determine whether the more

potent of the two diastereomers has been absorbed and

is actually present in the blood. The timelines of the

analytical methods employed for the pharmacokinetic

analysis of compounds is normally long relative to the

epimerization rates of these inhibitors. On the other

hand, inhibitor epimerization is of a time scale such that

equilibration may not occur in blood prior to excretion.

As such, the chiral instability present in the five- and

six-membered ring cyclic diaminoketones would lead to

racemization prior to serum analysis. The reality of this

situation is fully recognized by the stereoselective oral

bioavailability of the chirally stable azepanones de-

scribed in this paper.

The combination of potent enzyme inhibition and

promising, but inconclusive, pharmacokinetics of the

five- and six-membered ring diaminoketones prompted

the investigation of several strategies designed to

eliminate the epimerization of the labile C-4 chiral

center of these analogues. Initial attempts to address

this issue included blocking the offending epimerization

by incorporation of a methyl group at C-4 or by elimina-

tion of the ketone moiety. Although both of these

strategies produced compounds capable of inhibiting

cathepsin K, their potencies were substantially reduced

relative to the unsubstituted or ketone analogues.¹¹An

ultimately successful approach to this epimerization

issue was based on data which had shown that the rate

of ketone enolization in the cyclic 1,3-diaminoketone

series became slower as the heterocyclic ring was

expanded from five to six members. Additionally, epimer-

ization rates in the acyclic 1,3-diaminoketone series (see

structure 2, Figure 1) had also been shown to be

negligible. These data suggested that further ring

expansion could slow the rate of ketone enolization to a

degree sufficient to address the pharmacokinetic issues

described above.

In this paper we describe a series of configurationally

stable, azepanone-based inhibitors of human cathepsin

K. Ring expansion of the five- and six-membered ring

cyclic 1,3-diaminoketone templates to a seven-mem-

bered ring azepanone such as 6 (Figure 1) has proven

to be highly effective in improving the C-4 chiral

stability of these inhibitors. This modification has

simultaneously provided compounds with improved oral

bioavailability in rats. Additionally, incorporation of

functional groups derived from structure-activity stud-

ies (SARs) in the acyclic 1,3-diaminoketone series into

this azepanone template has led to the identification of

20, a potent, reversible inhibitor of human cathepsin K

which displays good oral bioavailability in the rat, as

well as inhibitor 24, a potent inhibitor of both human

and rat cathepsin K.

Syn th esis Ch em istr y

The syntheses of the azepanone and acyclic diami-

noketone inhibitors described in this paper are detailed

in Schemes 1-7. As shown in Scheme 1, N-alkylation

of benzyl N-allylcarbamate (7)¹²with 5-bromo-1-pentene

in the presence of sodium hydride provided the olefin

metathesis substrate 8. Treatment of diene 8 with

catalytic bis(tricyclohexylphosphine)benzylidineruthen-

ium(IV) dichloride according to the procedure developed

by Grubbs and co-workers¹³cleanly effected the ring-

closing olefin metathesis to provide azepine 9. Epoxi-

dation of 9 with m-CPBA provided oxirane 10 which was

treated with sodium azide under standard conditions

to cleanly afford azido alcohols 11 and 12 in a ratio of

9:1 (as determined by HPLC analysis) favoring the

desired regioisomer 11. The regioselectivity of this

epoxide opening is likely the result of the preferred chair

conformation of 10 which places the bulky carbonylben-

zyloxy nitrogen protecting group in a pseudo-axial

orientation. This conformation effectively blocks the C-3

carbon leading to the preferred trans-diaxial nucleo-

philic opening of the epoxide at C-4.¹⁴The undesired

azido alcohol 12 was carried through the synthesis

sequence and was removed at a later stage.

Elaboration of the azepane core to 15 and 16 is

outlined in Scheme 2. The mixture of azides 11 and 12

was reduced with 1,3-propanedithiol in methanol and

1382 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9

Marquis et al.

Sch em e 1. Synthesis of 4-Azido-3-hydroxyazapanes 11 and 12^a

a

Reagents and conditions: (a) 5-bromo-1-pentene, NaH, DMF; (b) bis(tricyclohexylphosphine)benzylidineruthenium(IV) dichloride,

CH₂Cl₂, reflux; (c) m-CPBA, CH₂Cl₂; (d) NaN₃, NH₄Cl, H₂O, CH₃OH, 70 °C.

Sch em e 2. Synthesis of Azepanones 15 and 16^a

a

Reagents and conditions: (a) 1,3-propanedithiol, TEA, CH₃OH; (b) 10% Pd/C, H₂, CH₃OH; (c) Cbz-leucine, EDC, HOBt, CH₂Cl₂; (d)

Dess-Martin periodinane, CH₂Cl₂; (e) HPLC separation.

triethylamine¹⁵to provide the intermediate amino

alcohol 13. Hydrogenolysis of the benzyloxycarbonyl

protecting group, followed by bis-acylation of the result-

ing 4-aminoazepan-3-ol (not shown) with 2 equiv of Cbz-

leucine in the presence of EDC, provided the diastere-

omeric mixture of alcohols 14. Oxidation of alcohols 14

followed by HPLC separation of the diastereomeric

ketones provided azepanones 15 and 16.

The syntheses of analogues 20 and 21 are detailed in

Scheme 3. Acylation of amino alcohol 13 with N-Boc-L-

leucine in the presence of EDC followed by hydrogenoly-

sis of the carbonylbenzyloxy protecting group provided

amine 17. Treatment of 17 with freshly prepared

2-pyridinesulfonyl chloride in the presence of triethyl-

amine followed by removal of the tert-butoxycarbonyl

protecting group and acylation of the resulting amine

salt with benzofuran-2-carboxylic acid gave 18 as a

mixture of two diastereomers. Application of the 2-py-

ridylsulfonamide moiety to the azepanone template had

been derived from earlier work in the acyclic 1,3-

diaminoketone series where this functionality was

shown to be a potency-enhancing, Cbz-leucine peptido-

mimetic. Oxidation of the mixture 18 with pyridine

sulfur trioxide complex¹⁶gave the mixture of diastere-

omeric ketones 19 which were separated by HPLC to

provide azepanones 20 and 21. Diastereomer 21 could

be recycled to 20 by epimerization with triethylamine

in methanol and water followed by a second HPLC

separation. The small-molecule X-ray crystal structure

of 20 unequivocally established the stereochemistry of

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1383

Sch em e 3. Synthesis of Azepanones 20 and 21^a

a

Reagents and conditions: (a) N-Boc-leucine, EDC, HOBt, CH₂Cl₂; (b) 10% Pd/C, H₂, CH₃OH; (c) 2-pyridylsulfonyl chloride, TEA, CH₂Cl₂;

(d) HCl, EtOAc, CH₃OH; (e) benzofuran-2-carboxylic acid, EDC; (f) SO₃-pyridine complex, TEA, DMSO; (g) HPLC separation; (h) TEA,

CH₃OH, H₂O.

the C-4 chiral center as S (Figure 2). In this structure

the azepanone ring is in a pseudo-boat conformation

with the C-4 leucinamide group in an equatorial orien-

tation.

ridinesulfonyl chloride gave intermediate 30. Removal

of the N-Boc protecting group followed by acylation with

N-Boc-L-leucine, deprotection, and acylation with ben-

zofuran-2-carboxylic acid gave intermediate 32 which

was oxidized with the Dess-Martin periodinane to

provide ketone 2.

As shown in Scheme 7, treatment of the known

epoxide 33¹⁹with N-methylamine followed by sulfony-

lation with 2-pyridinesulfonyl chloride provided alcohol

intermediate 34. Removal of the N-Boc protecting group

and acylation of the subsequent amine salt with N-Cbz-

L-leucine gave alcohol 35. Deprotection of 35 followed

by acylation with benzofuran-2-carboxylic acid and

oxidation provided ketone 36.

As outlined in Scheme 4, the synthesis of 24 and 25

began with acylation of amine 17 with 2-(3-pyridin-2-

ylphenyl)acetic acid¹⁷in the presence of EDC. Removal

of the tert-butoxycarbonyl protecting group followed by

coupling of the resulting amine with 5-[2-(4-morpholi-

nyl)ethoxy]-2-benzofuran-2-carboxylic acid provided 22

as a mixture of two diastereomers. Oxidation of the

mixture 22 gave azepanones 23 as a 1:1 mixture of

diastereomers which were separated by HPLC to pro-

vide 24 and 25. Triethylamine-induced epimerization

of 25 to the diastereomeric mixture of ketones 23

followed by a second HPLC separation allowed for a

recycling of this less desired diastereomer.

The syntheses of acyclic inhibitors 2, 28, and 36 are

outlined in Schemes 5-7. As shown in Scheme 5,

acylation of the commercially available 1,3-diamino-

2-hydroxypropane (26) with N-Boc-L-leucine in the

presence of EDC followed by sulfonylation with 2-py-

ridinesulfonyl chloride provided the alcohol intermedi-

ate 27. Removal of the N-Boc protecting group and

acylation of the resulting amine salt with benzofuran-

2-carboxylic acid and oxidation provided ketone inhibi-

tor 28.

Resu lts a n d Discu ssion

P oten cies a n d Selectivities. Studies of cyclic five-

and six-membered ring ketone-derived cathepsin K

inhibitors had highlighted the possibility of improved

pharmacokinetics but had also shown that these ring

systems were configurationally unstable at the C-4

diastereomeric center. As shown in Table 1, ring expan-

sion to the azepanone 6 improved the stability of the

C-4 chiral center to the point that separation of the

individual diastereomers 15 and 16 was now possible.

Azepanone 15 was shown to be a 2.0 nM inhibitor of

cathepsin K, while diastereomer 16 was at least 7-fold

less potent with a K_i,app) 15 nM. Inhibitor 15 was

Scheme 6 details the synthesis of inhibitor 2. Sulfo-

nylation of the known amino alcohol 29¹⁸with 2-py-

selective for cathepsin K versus cathepsins L (K_i,app

)

1384 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9

Sch em e 4. Synthesis of Inhibitors 24 and 25^a

Marquis et al.

a

Reagents and conditions: (a) 2-(3-pyridin-2-ylphenyl)acetic acid, EDC, HOBt; (b) HCl, EtOAc, CH₃OH; (c) 5-[2-(4-morpholinyl)ethoxy]-

2-benzofuran-2-carboxylic acid, EDC, HOBt; (d) SO₃-pyridine complex, TEA, DMSO; (e) HPLC separation; (f) TEA, CH₃OH, H₂O.

Sch em e 5. Synthesis of Acyclic Inhibitor 28^a

a

Reagents and conditions: (a) N-Boc-L-leucine, EDC, HOBt; (b) 2-pyridylsulfonyl chloride, NMM, CH₂Cl₂; (c) TFA; (d) benzofuran-2-

carboxylic acid, HBTU, DMF; (e) Dess-Martin periodinane, CH₂Cl₂.

47 nM; cathepsins K/L ) 23) and S (K_i,app) 26 nM;

cathepsins K/S ) 13). Azepanone 15 is 11-fold more

potent than its acyclic counterpart 1 (cathepsin K K_i,app

) 22 nM) highlighting the utility of a conformational

constraint in locking in a bioactive conformation of this

inhibitor.²⁰Having established both the viability of the

ring expansion concept for the potent inhibition of

cathepsin K as well as the improved chiral stability of

the C-4 diastereomeric center, this class of seven-

membered ketones became the focus of a medicinal

chemistry effort to optimize selectivity and oral bio-

availability. Functional groups which had displayed

potent inhibition of human and rat cathepsin K and had

led to effective inhibition of native cathepsin K in cell-

and tissue-based in vitro assays systems in the acyclic

1,3-diaminopropanone series were applied to this

azepanone template.

As shown in Table 1, the incorporation of the 2-py-

ridinesulfonamide group, which had been shown in the

acyclic 1,3-diaminopropanone series to be an effective

potency-enhancing replacement for the Cbz-leucine

moiety, combined with replacement of the carbonylben-

zyloxy group with the benzofuran-2-carboxamide, pro-

vided 20 which is a potent inhibitor of human cathepsin

K with a K_i) 0.16 nM. Analogue 20 is 13-fold selective

for cathepsin K versus cathepsin L, 25-fold selective

versus cathepsin S, and greater than 3000-fold selective

versus cathepsin B. The C-4 R diastereomer 21 was

approximately 1/6000th as active as 20 with a K_i,app

980 nM, highlighting the critical nature of this center

)

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1385

Sch em e 6. Synthesis of Inhibitor 2^a

a

Reagents and conditions: (a) 2-pyridinesulfonyl chloride, NMM, DMF; (b) 4 M HCl/dioxane, CH₂Cl₂; (c) N-Boc-L-leucine, NMM, HBTU;

(d) 4 M HCl/dioxane, CH₃OH; (e) benzofuran-2-carboxylic acid, HBTU, NMM; (f) Dess-Martin periodinane, CH₂Cl₂.

Sch em e 7. Synthesis of Inhibitor 36^a

a

Reagents and conditions: (a) N-methylamine, 2-propanol; (b) 2-pyridinesulfonyl chloride, NaHCO₃, CH₂Cl₂; (c) 10% Pd/C, H₂, EtOH,

10% HCl; (d) N-Boc-leucine, DIPEA, EDC, HOBt; (e) 4 M HCl/dioxane, CH₃OH; (f) benzofuran-2-carboxylic acid, DIPEA, HOBt, EDC; (g)

Dess-Martin periodinane.

Ta ble 1. Inhibitory Potencies versus Human Cathepsins K, L,

S, B and Rat Cathepsin K and in Vitro Cell-Based Assays

rat^c

IC₅₀(nM)

human^bK_i(nM)

K_i(nM)

osteoclast in situ

Cat

K

Cat

L

Cat

S

Cat

B

Cat

K

resorption cytochem

compd

assay

1

2

4

5

6

22

340

890

20

1,300

59

>1000

440

7.5

2.3

2.6

30

39

16

15

16

2.0

15

47

290

26

97

F igu r e 2. ORTEP representation of the small-molecule X-ray

crystal structure of azepanone 20. The C-4 chiral center is of

the S configuration with the leucinamide group in an equato-

rial orientation off of the azepanone ring.

18^a>1000

20

21

22^a

24

25

28

36

0.16

980

54

0.0048

4.7

1.6

2.2

4.0

500 60

70

30

80

30

for potent inhibition of cathepsin K. (We are unable to

exclude the possibility that even this low level of activity

may be the result of contamination (<0.01%) by 20.) The

mixture of diastereomeric alcohols 18 was not active

when tested up to a concentration of 1 µM. The

incorporation of a conformational constraint has in-

creased the inhibitor potency of 20 at least 10-fold

relative to acyclic analogues 28, 2, and 36 which are

1.6, 7.5, and 670 nM inhibitors of cathepsin K, respec-

tively. Compound 20 has been characterized as a

competitive, reversible inhibitor of cathepsin K. As

shown in Figure 3 the Lineweaver-Burk plot of 20

displays a 1/v axis intercept which is constant with

increasing inhibitor concentration, indicative of com-

petitive inhibition. There was no time-dependent inhibi-

0.49 14

100

4.8

670

a

Inhibitory potencies for alcohols 18 and 22 are reported as

mixtures of diastereomers. ^bInhibitory potencies versus cathepsins

K, B, L and S were determined as describe in ref 6b. ^cAssay versus

rat cathepsin K was performed as described in ref 25.

tion observed over a 30-min period. The reversible

nature of inhibition of 20 was established by mass

spectral analysis which showed that upon incubation

of 20 with cathepsin K for 2 h there had been no

irreversible modification of the protein by the inhibitor.

Incubation of 20 with excess cysteine in D₂O/DMSO

1

followed by H NMR analysis showed 20 to be stable to

1386 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9

Marquis et al.

membered ring ketones to be the result of the elimina-

tion of eclipsing interactions within these ring systems

upon enol formation. For the conformationally more

flexible seven-membered ring ketone system, enol for-

mation results in greater ring strain thereby leading to

a higher energy transition state.

As a 60 nM inhibitor of rat cathepsin K, 20 is

approximately 375-fold less potent an inhibitor of this

enzyme than of human cathepsin K. The characteriza-

tion of both cynomolgus monkey²⁴and rat²⁵cathepsin

K have recently been reported from these laboratories.

These studies have shown that mature cathepsin K from

cynomolgus monkey is identical to the mature human

enzyme. This has permitted the in vivo evaluation of

potent inhibitors of human cathepsin K, such as 20, to

determine their ability to inhibit bone resorption in a

recently developed medically ovariectomized monkey

model of bone turnover. In this model, 20 significantly

inhibited bone resorption with once daily subcutaneous

dosing at 12 mg/kg as measured by the decrease of the

serum bone markers NTx and CTx.²⁶

F igu r e 3. Lineweaver-Burk plot of 20 at increasing inhibitor

concentrations of 0 nM (O), 100 nM (b), 200 nM (0), 350 nM

(9), 500 nM (4).

Ta ble 2. First-Order Rate Constants of Epimerization at pH

11 and 12.5

k (s^-1

)

compd

pH 11

pH 12.5

Alternatively, mature rat cathepsin K has been shown

to be 88% identical to the human enzyme.²⁵Despite the

relatively high sequence identity of the rat and human

enzymes, their kinetic behavior toward the fluorogenic

substrate Cbz-Leu-Arg-aminomethylcoumarin varies

considerably (human cathepsin K K_m) 6 µM; rat

cathepsin K K_m) 99 µM) with a k_cat/K_m12-35-fold

lower for the rat versus the human enzyme. This

difference in substrate specificities is also reflected in

the significant difference in the potencies of inhibitors

such as 20 against the human and rat enzymes. This

has precluded the evaluation of many potent inhibitors

of human cathepsin K in standard rodent models of bone

turnover.⁷Furthermore, this difference has necessitated

the development of separate SARs in order to identify

suitably potent inhibitors of rat cathepsin K for evalu-

ation in these models.

20 to 21

21 to 20

24 to 25

25 to 24

6.2 × 10^-5

6.0 × 10^-5

1.2 × 10^-5

1.0 × 10^-5

1.7 × 10^-3

2.3 × 10^-3

2.2 × 10^-4

2.9 × 10^-4

epimerization. Within the level of detection at 500 MHz,

this experiment also showed no hemithioketal formation

highlighting the unreactive nature of the azepanone

carbonyl moiety. Azepanone 20 is a potent inhibitor of

native cathepsin K in an in vitro, cell-based assay of

bone resorption²¹(IC₅₀) 70 nM) as well as a potent

inhibitor in an in situ cytochemical assay (IC₅₀) 80 nM)

which measures cathepsin activity in osteoclasts in

sections of human tissue.²²

An HPLC method allowing for the baseline separation

of 1:1 diastereomeric mixtures of azepanones was

developed in order to more precisely elucidate the rate

of epimerization of the C-4 chiral center of this class of

compound. Under acidic to neutral pH conditions (pH

2-7 phosphate buffer) the C-4 centers of the diastere-

omeric pair 20 and 21 were each stable in aqueous

buffer for days with no detectable change by HPLC

analysis. At pH > 12 these compounds epimerized

rapidly to reach a 1:1 ratio within a few hours. Similar

behaviors were observed for azepanones 24 and 25 and

for the acyclic derivative 2. To further elucidate the

mechanism of enolization, kinetic studies in pH 11 and

12.5 buffers, in which the epimerization could be

monitored in a reasonable experimental time frame,

were carried out on 20, 21, and their corresponding

2,2′,4-trideuterated analogues 37 and 38.²³The first-

order rate constants of epimerization are shown in Table

2. A deuterium isotope effect of approximately 5 was

observed for the deuterated derivatives 37 and 38. The

rate of epimerization increases with pH at about a 10-

fold increase per pH unit. The kinetic result is consistent

with a hypothesized mechanism of base-catalyzed for-

mation of the achiral enol intermediate leading to

racemization of the C-4 chiral center of 20 as well as

21. By extrapolation, epimers 20 and 21 would have a

half-life of approximately 1 year in pH 7.4 buffer. We

understand the faster rate of enol formation in the five-

and six-membered ring ketones compared to the seven-

Azepanone 24 was identified as an exceptionally

potent inhibitor of human cathepsin K (K_i) 0.0048 nM)

and a sufficiently potent inhibitor of rat cathepsin K to

permit in vivo evaluation (K_i,app) 4.8 nM). The incor-

poration of the 2-(3-pyridin-2-ylphenyl)acetamide as

well as the 5-[2-(4-morpholinyl)ethoxy]-2-benzofuran-

2-carboxamide was derived from earlier work in the

acyclic 1,3-diaminopropanone series which had shown

these functional groups to be favorable for potent

inhibition of rat cathepsin K. The potency of 24 versus

rat cathepsin K has afforded the opportunity to examine

the ability of this inhibitor to attenuate bone resorption

in the thyroparathyroidectomized (TPTX) rat as well as

bone loss in the ovariectomized (OVX) rat. Continuous

intravenous infusion of 24 reduced PTH-induced hy-

percalcemia in the TPTX rat in a dose-dependent

manner. Compound 24 also inhibited bone matrix

degradation in the ovariectomized rat.²⁷

Analogue 24 is selective for human cathepsin K

versus human cathepsins B, L, and S with K_i,app) 100,

0.49, and 14 nM, respectively. As with inhibitor 20,

compound 24 has been characterized as a competitive,

reversible inhibitor of cathepsin K. Azepanone 24 is a

potent inhibitor of native cathepsin K in the osteoclast-

mediated bone resorption assay (IC₅₀) 30 nM) and in

the in situ cytochemical assay, a measure of osteoclast

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1387

cathepsin activity (IC₅₀) 30 nM). Diastereomer 25 is

approximately 1/1000th as potent as 24 versus human

cathepsin K with a K_i,app) 4.7 nM. The precursor, two-

component mixture of trans diastereomeric alcohols, 22

inhibited cathepsin K with a K_i,app) 54 nM confirming

the importance of thiol addition to the ketone of the

inhibitor as a critical component to the very high

potency of 24. The potency of alcohols 22 is likely the

result of the excellent noncovalent interactions at P₃,

P₂, and P₃′ of the inhibitor with critical residues within

the active site of the enzyme. In addition, the expected

active isomer of alcohols 22 with the S configuration at

C-4 of the azepanone ring when presenting the amino

group as axial would position the C-3 hydroxyl group

also in an axial orientation. This relationship has the

potential to place the C-3 hydroxyl moiety in the same

position as the hydroxyl group of the hemithioketal of

the bound 24 (vide infra). The only difference between

the ketone and the alcohol would then be the lack of a

covalent bond and the possible introduction of non-

bonded sulfur-CH interactions accounting for the 3

orders of magnitude loss of potency between 24 and 22.

Molecu la r Mod elin g a n d X-r a y Cr ysta llogr a p h y.

1. P r ed icted Str u ctu r e of 20 Bou n d to Ca th ep sin

K. On the basis of conformational analysis of both

diastereomers of the representative model hemithio-

ketal 39²⁸(see also Experimental Section), we developed

a representative set of seven azepanone conformers to

use in predicting the cathepsin K binding mode of the

azepanone scaffold, as part of a structure-based design

effort around this structural class. Since this work was

initiated prior to separation of the diastereomers, both

the R and S configurations at C-4 were considered. The

representative set comprised three conformers of the R

diastereomer and four conformers of the S diastereomer

with both axial and equatorial orientations of the C-4

substituent for each diastereomer. Conformational analy-

sis indicates that the equatorial C-4 conformer is favored

over the axial conformer with a calculated energy

difference of approximately 2 kcal mol^-1(see Experi-

mental Section). This is consistent with the small-

molecule crystal structures of both 20 (Figure 2) and

21 (the R diastereomer; data not shown).

F igu r e 4. Schematic representations of the 2.0 Å resolution

X-ray co-crystal structure of inhibitor 20 bound within the

active site of cathepsin K. The inhibitor spans the S₃to S₂′

pockets of the active site of the protein with the thiol of Cys

25 adding to the sterically more congested Re face of the

carbonyl group of the inhibitor. The C-4 leucinamide group is

in an axial orientation off of the azepanone ring which is in a

pseudo-chair conformation. The methylene groups of the

azepanone ring (highlighted in red) are oriented away from

the enzyme toward solvent.

active site during refinement of the crystal structure

proved impossible. Inspection of the active site density

led to speculation that the bound compound might in

fact be the active diastereomer 20. We tested this

hypothesis in an unbiased manner by automated fitting/

refinement of our representative set of seven azepanone

conformers to the observed active site density. On the

basis of visual selection of the conformer that best fit

the active site density, an axial conformer of the S

diastereomer 20 was unambiguously selected as the

bound inhibitor.

Preferred binding of the active S diastereomer 20

versus the inactive R diastereomer 21 is dramatically

highlighted by this exclusive crystallographic selection

of 20 from a solution of predominantly (96%) 21 during

crystal formation and growth. As predicted by the

modeling studies, the C-4 leucinamide substituent

adopts an axial orientation upon binding to cathepsin

K (Figure 6).

Each conformer in the representative set was used

as a starting point for iterative modeling within the

protein active site involving superposition onto related

cathepsin K-inhibitor crystal structures.⁶Despite the

preferences seen in small-molecule crystal structures

and conformational analysis, modeling within the pro-

tein active site led to the prediction of a cathepsin K

binding preference for a pseudo-chair conformation of

the S diastereomer with the C-4 substituent in the

higher energy axial orientation (Figure 2).

2. Cr ysta l Str u ctu r es of 20 a n d 24 Bou n d to

Ca th ep sin K. Initial difficulty in obtaining a cathepsin

K co-crystal structure with an azepanone led to the

screening of many analogues and both active and

inactive diastereomers for co-crystal formation. Ulti-

mately, we obtained crystals of the complex prepared

from cathepsin K and the relatively inactive R diaste-

reomer 21. In previous modeling studies it was ex-

tremely challenging to develop a plausible binding

model for the R diastereomer; consistent with these

modeling studies, fitting of the R diastereomer 21 to the

The 2.0 Å resolution X-ray co-crystal structure of the

inhibitor 20-cathepsin K complex shows the inhibitor

spanning the S₃-S₂′ pockets of the active site of the

enzyme (Figure 4). The C-4 leucinamide of 20 is oriented

on the unprimed side of the active site with the isobutyl

group bound within the hydrophobic S₂pocket formed

by residues Leu 160, Ala 134, and Met 68. The terminal

1388 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9

Marquis et al.

F igu r e 6. Comparison of the predicted and observed struc-

tures of 20 bound to cathepsin K. The conformations and

relative positions of several critical active site residues are

particularly well-conserved in cathepsin K crystal structures

(Gln 19, Cys 25, Trp 26, Tyr 67, His 162, Trp 184; see

structures described in ref 6). Heavy-atom superposition of

these residues of the enzyme used in modeling onto that

observed in the crystal structure yields a close overlap between

the predicted binding model (purple) and that observed in the

co-crystal structure (color-by-atom). The overlap is especially

close for the azepanone rings and proximal parts of the

pendant groups. CR, Câ, and Sγ of Cys 25 and the atoms of

20 are shown.

active site Cys 25 and the ketone carbonyl of the

inhibitor. In contrast to 20, inhibitor 24 spans the S₃-

S₃′ binding region of the cathepsin K active site with

the morpholinoethoxy group extending beyond S₃, ori-

ented toward solvent. The pyridyl moiety of the 2-(3-

pyridin-2-ylphenyl)acetamide extends into the S₃′ pocket,

forming a face-to-face π-π stacking interaction with Trp

184. The amide oxygen forms a hydrogen bond with the

indole N-H of Trp 184, similar to that observed for the

sulfonamide oxygen of 20.

F igu r e 5. Schematic representations of the 2.8 Å resolution

X-ray co-crystal structure of inhibitor 24 bound within the

active site of cathepsin K.

benzofuran carboxamide group is bound in the S₃

binding pocket forming an edge-to-face π-π stacking

interaction with Tyr 67.²⁹Two hydrogen bonds are

observed between Gly 66 of the enzyme and the leuci-

namide portion of the inhibitor similar to the antipar-

allel â-sheet interactions observed in other inhibitor-

protease structures.³⁰As observed in an earlier co-

crystal structure of acyclic inhibitor 2 bound within the

active site of cathepsin K,^6athe 2-pyridyl moiety of 20

occupies the S₂′ binding pocket with one of the sulfona-

mide oxygens forming a hydrogen bond with the indole

N-H of Trp 184. The methylene groups of the azepanone

ring of 20 are oriented toward solvent (highlighted in

red in Figure 4) making no contacts with the protein.

The X-ray co-crystal structure is consistent with the

formation of a hemithioketal between Cys 25 of cathe-

psin K and the carbonyl group of the azepanone,

confirming our initial hypothesis for the design of

reversible, transition-state inhibitors of cysteine pro-

teases. The thiol of the active site cysteine has added

to the diastereotopically more hindered Re face of the

ketone, syn to the C-4 leucinamide moiety. The oxygen

of the resulting hemithioketal is within hydrogen bond-

ing distance of the C(O)NH₂group of Gln 19 and the

N-H of Cys 25.

P h a r m a cok in etic An a lysis. Parameters which re-

late the physiochemical properties of a compound to

absorption play a critical role in determining the phar-

macokinetics of orally administered drugs.^31-33It has

been recognized that the oral bioavailability of com-

pounds may also, in part, be a function of enzymatic

processes such as metabolism by cytochrome P-450

enzymes and/or transporter efflux mechanisms medi-

ated by P-glycoproteins.³⁴The configurational stability

of the seven-membered ring ketones has allowed us to

delineate the effects of inhibitor cyclization as well as

the importance of the stereochemistry of the C-4 chiral

center in determining the factors which influence the

oral bioavailability of these compounds. The pharma-

cokinetic profiles and efflux properties³⁵of cyclic ana-

logues 20, 21, and 24 as well as the acyclic analogues

2, 28, and 36 are presented in Tables 3 and 4. Cyclic

analogue 20 showed good oral bioavailability in the rat

of 42% with a T_1/2of 30 min and a volume of distribution

approximately twice that of total body water when dosed

orally at 6.3 mg/kg as a solution in 6% aqueous encapsin

and 0.5% DMSO. Azepanone 20 was cleared from the

systemic circulation at a rate of 50 mL/min/kg. As shown

in Table 4, the m-to-s and s-to-m in vitro permeabilities

in rat distal colon show 20 to have good membrane

permeability and that it is not a substrate for P-

glycoprotein transporters as indicated by the similar

rates of flux in both the serosal-to-mucosal and mucosal-

to-serosal directions. The less potent, C-4 R diastere-

omer 21 was 9.7% orally bioavailable with a T_1/2) 33

min and a volume of distribution 1.7 times that of total

The 2.8 Å resolution X-ray co-crystal structure of

ketone 24 bound within the active site of human

cathepsin K (Figure 5) shares most of the features

described for the X-ray co-crystal structure of 20. The

co-crystal structure of 24 is consistent with both the S

stereochemistry and an axial orientation of the leuci-

namide substituent at the C-4 chiral center as well as

hemithioketal formation between the thiol group of the

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1389

Ta ble 3. Pharmacokinetic Properties in the Male Sprague-Dawley Rat

a

Pharmacokinetic properties of analogue 28 were determined in a four-component mixture study format.

Ta ble 4. Oral Bioavailability of Cyclic and Acyclic Analogues

which now has the same hydrogen bond donor/acceptor

as a Function of Physiochemical and Efflux Properties

flux (cm/h)

count as 20, did not lead to an improvement in oral

bioavailability.³⁷The potent rat cathepsin K inhibitor

24 showed poor oral bioavailability with a %F < 2.0%.

The low permeability of 24 in the m-to-s direction (<0.01

cm/h) and moderate permeability in the s-to-m direction

(0.05 cm/h) suggest that the bioavailability of 24 is likely

limited by an apical recycling mechanism and not by

poor permeability across membranes.

MW

clog H bond

H bond

%F

compd (g/mol)

P

donors acceptors m-to-s s-to-m (rat)

2

20

21

24

28

36

500.6 3.19

526.6 3.08

709.8 5.40

486.5 2.88

514.6 2.79

3

2

3

2

6

9

6

0.011 0.158

0.030 0.084 42.1

0.046 0.069

3.2

9.7

2.1

3.3

2.5

<0.01

ND

0.050

0.159

ND

The s-to-m transport of 2 and 28 is 10-100-fold

greater than their m-to-s transport. These results

suggest involvement of specific transport mechanism-

(s) present in the apical membrane of the intestinal

epithelial cells which recycle 2 and 28 from the intra-

cellular compartment back into the lumen of the intes-

tine. Therefore, when these cathepsin K inhibitors were

added to the mucosal bathing solution, their transepi-

thelial transport appears to be limited by apical recy-

cling. Addition of these cathepsin K inhibitors to the

serosal bathing solution results in an apparently greater

permeability due to transcellular movement through the

cell aided by this apical membrane transporter. Apical

recycling has been suggested to be responsible for low

bioavailability, increased variability, drug interactions,

and the potential for food effects.³⁸

body water. Diastereomer 21 was cleared at a rate of

52 mL/min/kg. Like 20, azepanone 21 showed good cell

permeability and was not a substrate for apical recy-

cling. The lower oral bioavailability of diastereomer 21

relative to that of diastereomer 20 is therefore likely to

be the result of first-pass hepatic and/or intestinal

metabolism, perhaps by cytochome P-450 enzymes. The

different inhibitory and pharmacokinetic profiles of

diastereomers 20 and 21 highlight the importance of a

configurationally stable C-4 chiral center in these

analogues.³⁶The low m-to-s flux but high s-to-m flux of

the corresponding acyclic derivatives 2 and 28 (Table

4) shows these analogues to have good cell permeability

which are subject to P-glycoprotein-mediated apical

recycling, resulting in poor oral bioavailability relative

to azepanone 20. The acyclic N-methyl analogue 36,

1390 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9

Marquis et al.

A comparison of the physiochemical properties of

azepanone 20 to those of the acyclic derivatives 2, 28,

and 36 reveals all four analogues to have similar

molecular weights (mean MW ) 500 g/mol), calculated

lipophilicities (mean clog P ) 2.95), and hydrogen

bonding capacities. All three of these parameters fall

within the empirically derived values developed by

Lipinski and co-workers for estimating solubilities and

permeabilities of new chemical entities and suggests

that other factors are responsible for determining rela-

tive oral bioavailabilities within this set of compounds.

As shown in Table 4 and discussed above, the relative

oral bioavailabilities of acyclic analogues 2 and 28

compared to the cyclic azepanone 20 appear to be a

function of apical recycling from intestinal epithelial

cells. One mechanism for apical recycling is the P-

glycoprotein (P-gp) efflux pump which is expressed on

the luminal surface of intestinal epithelial cells.³⁹P-

Glycoproteins are membrane-bound proteins which are

capable of binding a variety of disparate substrates. The

drug-binding domains of these enzymes have not yet

been fully elucidated, and there has been speculation

that several of the postulated transmembrane domains

may be involved in substrate recognition. On the basis

of the oral bioavailability and efflux properties of these

compounds, we suggest that the improved oral bioavail-

ability of azepanone 20 may be the result of the

conformational constraint imparted by ring formation

effectively locking the rotational freedom of this mol-

ecule thereby limiting the number of conformations

available for binding to enzymes. From the data pre-

sented above it appears that the conformational limita-

tions placed by this specific ring system have locked out

those conformations that interact well with those en-

zyme systems which serve to limit oral bioavailability

(i.e. P-gp’s) while retaining a bioactive conformation that

leads to potent inhibition of the targeted enzyme,

cathepsin K. Alternatively, the conformationally flexible

acyclic derivatives 2, 28, and 36 are postulated to allow

access to conformations which are more likely to be

substrates for enzyme systems thereby reducing their

relative bioavailability.

erties which are key factors in determining oral bio-

availability. These studies have suggested that limiting

the rotational freedom of an inhibitor by the introduc-

tion of a conformational constraint may serve the dual

purpose of locking in a bioactive conformation (thereby

leading to potent enzyme inhibition) as well as locking

out P-glycoprotein, cytochrome P-450, and other me-

tabolizing enzyme substrate conformations (thereby

leading to improved oral bioavailability).

We expect that incorporation of alternative specificity

elements into this azepanone template should allow for

the selective inhibition of other cysteine proteases while

retaining the desirable pharmacokinetic parameters

now recognized in this structural class. The inhibitors

presented in this paper should serve as excellent

compounds for evaluation in both rat and primate

models to determine the potential role of cathepsin K

inhibition for the treatment of diseases associated with

bone loss in humans. These studies will be reported in

detail elsewhere.^26,27

Exp er im en ta l Section

Gen er a l. Except where indicated materials and reagents

were used as supplied. Nuclear magnetic resonance spectra

were recorded at either 250 or 400 MHz using, respectively, a

Bruker AM 250 or Bruker AC 400 spectrometer. Mass spectra

were taken on a PE Syx API III instrument using electrospray

(ES) ionization techniques. Elemental analyses were obtained

using a Perkin-Elmer 240C elemental analyzer. Reactions

were monitored by TLC analysis using Analtech silica gel GF

or E. Merck silica gel 60 F-254 thin layer plates. Flash

chromatography was carried out on E. Merck Kieselgel 60

(230-400 mesh) silica gel.

Allylp en t-4-en ylca r ba m ic Acid Ben zyl Ester (8). To a

suspension of NaH (1.83 g, 76.33 mmol of 90% NaH) in DMF

was added benzyl N-allyl-N-1-pentenylcarbamate (7.3 g, 38.2

mmol) in a dropwise fashion. The mixture was stirred at room

temperature for 10 min whereupon 5-bromo-1-pentene (6.78

mL, 57.24 mmol) was added in a dropwise fashion. The

reaction was heated to 40 °C for approximately 4 h whereupon

it was partitioned between dichloromethane and water. The

organic layer was washed with water (2×), brine, dried

(MgSO₄), filtered and concentrated. Column chromatography

of the residue (10% ethyl acetate:hexanes) provided 10.3 g

(104%) of 8 as an oil: ¹H NMR (400 MHz, CDCl₃) δ 7.3-7.4

(m, 5H), 5.8 (m, 2H), 5.4 (m, 4H), 5.0 (m, 2H), 3.8 (m, 2H), 3.4

(m, 2H), 2.1 (m, 2H), 1.7 (m, 2H); MS (ESI) 260 (M + H)⁺.

2,3,4,7-Tetr a h yd r oa zep in e-1-ca r boxylic Acid Ben zyl

Ester (9). To a degassed solution of diene 8 (50 g, 192 mmol)

in CH₂Cl₂was added bis(tricyclohexylphosphine)benzyli-

dineruthenium(IV)dichloride (5.0 g, 6.1 mmol). The reaction

was heated to reflux until complete consumption of the starting

material was observed by TLC analysis. The mixture was

concentrated and chromatographed (1:1 CH₂Cl₂:hexanes) to

provide 35 g (70%) of azepine 9 as a brown oil: ¹H NMR (400

MHz, CDCl₃) δ 7.3 (m, 5H), 5.7 (m, 2H), 5.1 (m, 2H), 4.0 (dd,

2H), 3.6 (m, 2H), 2.2 (m, 2H), 1.7 (m, 2H); MS (ESI) 232 (M +

H)⁺. Anal. (C₁₆H₂₁N₄O₂) C, H, N.

Con clu sion s

In this paper we have described the synthesis, in vitro

activities, small-molecule and cathepsin K-bound crystal

structures, and pharmacokinetic properties of a series

of configurationally stable azepanone-based inhibitors

of the cysteine protease cathepsin K. Expansion of the

five- or six-membered ring ketones to a seven-membered

ring has led to a dramatic increase in the configura-

tional stability of the C-4 chiral center contained within

these inhibitors. The incorporation of functional groups

previously shown to improve in vitro and cell-based

potencies in the acyclic 1,3-diaminopropanone series

produced potent inhibitors of both human and rat

cathepsin K in this azepanone class of compounds. The

X-ray co-crystal structures of 20 and 24 bound to

cathepsin K confirmed our initial design hypotheses.

Molecular modeling studies provided a valid azepanone-

binding model in advance of the crystal structure and

also provided information crucial to solution of the

crystal structure. The increased configurational stability

of this class has permitted the examination of the

pharmacokinetic, physiochemical, and permeability prop-

8-Oxo-3-azabicyclo[5.1.0]octan e-3-car boxylic Acid Ben -

zyl Ester (10). To a 5 °C solution of 9 (39 g, 168 mmol) in

CH₂Cl₂(1.2 L) was added m-CPBA (55 g) in a portionwise

manner. Upon complete addition the mixture was maintained

at 5 °C for 15 min. The mixture was then stirred at room

temperature overnight. The following day dimethyl sulfide (3

mL) was added and the mixture maintained at room temper-

ature for 1 h. This mixture was filtered and concentrated. The

residue was dissolved in EtOAc and washed with 10% K₂CO₃

(3×), brine (3×), dried (Na₂SO₄), filtered and concentrated to

provide 39 g (94%) of the crude epoxide 10. This material was

of sufficient purity to carry on to the following reaction with

no further purification: ¹H NMR (400 MHz, CDCl₃) δ 7.4 (m

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1391

5H), 5.2 (s, 2H), 4.1 (m, 1H), 3.6 (m, 2H), 3.2 (m, 2H), 2.9 (m,

1H), 2.1 (m, 1H), 1.9 (m, 1H), 1.7 (m, 2H); MS (ESI) 248 (M +

H)⁺.

leucine (0.63 g). The reaction was maintained at room tem-

perature until complete consumption of the starting material

was observed by TLC analysis. The reaction was diluted with

ethyl acetate and washed with 1 N HCl, saturated K₂CO₃,

water, brine, dried (MgSO₄), filtered and concentrated. Column

chromatography of the residue (3% methanol:dichloromethane)

gave 1.0 g (77%) of 4(R,S)-((S)-2-tert-butoxycarbonylamino-4-

methylpentanoylamino)-3(R,S)-hydroxyazepan-1-carboxylic acid

benzyl ester: ¹H NMR (400 MHz, CDCl₃, reported as a mixture

of diastereomers) 7.35 (m, 5H), 6.7 (bs, 1H), 5.15 (m, 3H), 4.0-

3.0 (m, 10H), 2.0-1.7 (m, 5H), 1.4 (s, 9H), 0.9 (m, 6H); MS

(ESI) 478 (M + H)⁺.

To a solution of 4(R,S)-((S)-2-tert-butoxycarbonylamino-4-

methylpentanoylamino)-3(R,S)-hydroxazepan-1-carboxylic acid

benzyl ester (18.0 g, 37.7 mmol) and 10% Pd/C (catalytic) in

ethyl acetate:methanol (300 mL of a 2:1 solution) was affixed

a balloon of hydrogen. The reaction was stirred until complete

consumption of the starting material was observed by TLC

analysis. The reaction was filtered to remove the catalyst and

the filtrate was concentrated to provide 12.6 g (97%) of 17 as

a white powder: ¹H NMR (400 MHz, CDCl₃, reported as a

mixture of diastereomers) δ 5.5 (br s, 1H), 5.2 (br s, 1H), 4.1

(m, 1H), 4.0 (m, 1H), 3.75 (m, 3H), 3.1 (m, 2H), 2.9 (m, 1H),

1.7 (m, 5H), 1.45 (s, 9H), 0.9 (m, 6H); MS (ESI) 344 (M + H⁺).

4(R,S)-Ben zofu r a n -2-ca r boxylic Acid 4-{(S)-3-Meth yl-

1-[3(R ,S )-h yd r ox y-1-(p yr id in -2-ylsu lfon yl)a ze p a n -4-

ylca r ba m oyl]bu tyl}a m id e (18). To a solution of amine 17

(12 g, 34.9 mmol) in CH₂Cl₂was added triethylamine (5.8 mL,

41.9 mmol) followed by the dropwise addition of 2-pyridine-

sulfonyl chloride (7.4 g, 41.9 mmol). The reaction was stirred

until complete as determined by TLC analysis. The mixture

was washed with saturated NaHCO₃, water, brine, dried (Na₂-

SO₄), filtered and concentrated. Column chromatography (75%

ethyl acetate:hexanes to 100% ethyl acetate) of the residue

provided 15 g (88%) of 4(R,S)-{(S)-1-[3(R,S)-hydroxy-1-(pyridin-

2-ylsulfonyl)azepan-4-ylcarbamoyl]-3-methylbutyl}carbamic acid

tert-butyl ester: MS (ESI) 484 (M + H)⁺.

To a solution of 4(R,S)-{(S)-1-[3(R,S)-hydroxy-1-(pyridin-2-

ylsulfonyl)azepan-4-ylcarbamoyl]-3-methylbutyl}carbamic acid

tert-butyl ester (15.7 g, 32.4 mmol) in CH₃OH (50 mL) was

added 4 M HCl in dioxane (50 mL). The reaction was

maintained at room temperature until complete consumption

of the starting material was observed. The mixture was

concentrated to provide 13.7 g (100%) of 4(R,S)-{(S)-2-amino-

4-methylpentanoic acid [3(R,S)-hydroxy-1-(pyridin-2-ylsulfo-

nyl)azepan-4-yl]}amide hydrochloride as a white powder: MS

(ESI) 385 (M + H)⁺.

To a solution of 4(R,S)-{(S)-2-amino-4-methylpentanoic acid

[3(R,S)-hydroxy-1-(pyridin-2-ylsulfonyl)azepan-4-yl]}amide hy-

drochloride (8.7 g, 20.7 mmol) in CH₂Cl₂(50 mL) were added

EDC (4.0 g, 20.9 mmol), HOBt (2.5 g, 19.0 mmol), TEA (5.3

mL, 37.9 mmol) and benzofuran-2-carboxylic acid (3.4 g, 20.9

mmol). The mixture was maintained at room temperature

until complete consumption of the starting material was

observed as determined by TLC analysis. The mixture was

concentrated, diluted with EtOAc and washed with 1 N HCl,

saturated K₂CO₃, H₂O, brine, dried (MgSO₄), filtered and

concentrated. Column chromatography of the residue provided

10 g (91%) of 18 as a white powder: ¹H NMR (400 MHz, CDCl₃,

reported as a mixture of diastereomers) δ 8.7 (m, 1H), 8.0 (m,

2H), 7.6 (m, 1H), 7.4 (m, 4H), 7.2-7.3 (m, 3H), 5.0 (m, 1H),

4.7 (m, 2H), 4.0 (m, 1H), 3.7 (dd, 1H), 2.7 (m, 1H), 2.2 (m, 2H),

1.5-2.1 (m, 5H), 1.0 (m, 6H); MS (ESI) 529 (M + H)⁺.

4(R,S)-Ben zofu r a n -2-ca r boxylic Acid {(S)-3-Meth yl-1-

[3-oxo-1-(p yr id in -2-ylsu lfon yl)a zep a n -4-ylca r b a m oyl]-

bu tyl}a m id e (19). To a solution of alcohols 18 (10 g, 18.8

mmol) in DMSO (20 mL) were added TEA (10.5 mL, 75.7

mmol) and pyridine sulfur trioxide complex (6.02 g, 37.8

mmol). The mixture was maintained at room temperature for

approximately 2 h whereupon it was diluted with EtOAc and

washed with H₂O, brine, dried (MgSO₄), filtered and concen-

trated. Column chromatography (3% CH₃OH/CH₂Cl₂) provided

8.2 g (81%) of the ketones 19 as a 1:1 mixture of diastereomers

as determined by analytical HPLC (Chiralpak AD; 4.6 × 250

4(R,S)-Azido-3(R,S)-h ydr oxyazepan e-1-car boxylic Acid

Ben zyl Ester (11). To a solution of the epoxide 10 (2.0 g, 8.1

mmol) in methanol:water (8:1 solution) were added NH₄Cl (1.3

g, 24.3 mmol) and sodium azide (1.6 g, 24.3 mmol). The

reaction was heated to 70 °C until complete consumption of

the starting epoxide was observed by TLC analysis. The

majority of the solvent was removed in vacuo and the remain-

ing solution was partitioned between ethyl acetate and pH 4

buffer. The organic layer was washed with saturated NaHCO₃,

water, and brine, dried (MgSO₄), filtered, and concentrated.

Column chromatography (20% ethyl acetate:hexanes) of the

residue provided 1.3 g (55%) of 11: ¹H NMR (400 MHz, CDCl₃)

δ 8.0 (s, 1H), 7.4 (m, 5H), 5.2 (s, 2H), 4.0 (m, 1H), 3.6 (m, 2H),

3.2 (m, 1H), 3.0 (m, 2H), 2.2 (m, 1H), 2.0 (m, 1H), 1.7 (m 2H);

MS (ESI) 291 (M + H)⁺.

4(R,S)-Am in o-3(R,S)-h ydr oxyazepan e-1-car boxylic Acid

Ben zyl Ester (13). To a solution of 11 and 12 (12.3 g, 42.5

mmol) in methanol (400 mL) were added triethylamine (17.8

mL, 127.4 mmol) and 1,3-propanedithiol (12.8 mL, 127.4

mmol). The reaction was maintained at room temperature

until complete consumption of the starting material was

observed as determined by TLC analysis. The reaction was

concentrated and chromatographed (20% methanol:ethyl ac-

etate) to provide 6.8 g (61%) of amino alcohol 13 as a clear

viscous oil: ¹H NMR (400 MHz, CDCl₃) δ 7.3 (m, 5H), 5.1 (m,

2H), 3.7 (M, 1H), 3.4 (s, 1H), 3.3 (m, 2H), 3.0 (m, 1H), 2.6 (m,

1H), 2.4 (b, 2H), 1.9 (m, 2H), 1.7 (m, 1H), 1.4 (m, 1H); MS (ESI)

265 (M + H)⁺. Anal. (C₁₆H₂₁N₄O₂) C, H, N.

((S )-1-{1-[4(R ,S )-((S )-2-Be n zyloxyca r b on yla m in o-4-

m e t h ylp e n t a n oyla m in o)-3(R ,S )-h yd r oxya ze p a n -1-yl]-

m eth a n oyl}-3-m eth ylbu tyl)ca r ba m ic Acid Ben zyl Ester

(14). To a solution of the amino alcohol 13 (3.0 g, 11.3 mmol)

in CH₃OH (25 mL) was added 10% Pd/C (0.03 g). The mixture

was shaken under 35 psi of hydrogen for approximately 5 h

whereupon it was filtered through Celite. The filter cake was

washed several times with CH₃OH and CH₂Cl₂. Concentration

of the filtrate provided 0.67 g (46%) of 4(R,S)-aminoazepan-

3(R,S)-ol as a clear oil: ¹H NMR (400 MHz, CD₃OD, reported

as a mixture of diastereomers) δ 3.31-3.30 (m, 3H), 3.01-

2.69 (m, 6H), 1.82-1.56 (m, 5H); MS (ESI) 131.2 (M + H)⁺.

To a solution of 4(R,S)-aminoazepan-3(R,S)-ol (0.67 g, 5.13

mmol) in CH₂Cl₂(30 mL) were added EDC (2.17 g, 11.33 mmol)

and Cbz-leucine (2.74 g, 10.30 mmol). The reaction was

maintained at room temperature until complete consumption

of the starting material was observed by TLC analysis. The

mixture was concentrated, diluted with EtOAc and washed

with saturated K₂CO₃, 1 N HCl, brine, dried (MgSO₄) filtered

and concentrated. Column chromatography of the residue (4:1

EtOAc:hexanes) provided 1.4 g (44%) of alcohols 14 as a white

powder: MS (ESI) 625.3 (M + H)⁺, 644.3 (M + Na)⁺.

((S )-1-{1-[4(R ,S )-((S )-2-Be n zyloxyca r b on yla m in o-4-

m et h ylp en t a n oyla m in o)-3-oxoa zep a n -1-yl]m et h a n oyl}-

3-m eth ylbu tyl)ca r ba m ic Acid Ben zyl Ester (15 a n d 16).

To a solution of the alcohols 14 (0.20 g, 0.32 mmol) in CH₂Cl₂

(5.0 mL) was added Dess-Martin periodinane (0.27 g, 0.64

mmol). The mixture was maintained at room temperature for

30 min whereupon saturated NaHCO₃and 10% Na₂S₂O₃were

added. The aqueous layer was extracted with CH₂Cl₂. The

combined organic layers were dried (MgSO₄), filtered and

concentrated. Column chromatography (2:1 EtOAc:hexanes)

provided 0.16 g (83%) of 6 as a white solid. Separation of this

mixture by preparative HPLC provided the faster eluting

diastereomer 15 (t_R) 16.9 min; >98% purity): MS (ESI) 623.3

(M + H)⁺. Anal. (C₃₄H₄₆N₄O₇‚0.4H₂O) C, H, N, O. And the

slower eluting diastereomer 16 (t_R) 21.8 min; >96% purity):

MS (ESI) 623.1 (M + H)⁺. Anal. (C₃₄H₄₆N₄O₇‚0.3H₂O) C, H,

N, O.

4(R,S)-[(S)-1-(3(R,S)-Hyd r oxya zep a n -4-ylca r ba m oyl)-3-

m et h ylbu t yl]ca r b a m ic Acid ter t-Bu t yl E st er (17). To a

solution of the amino alcohol 13 (0.72 g, 2.72 mmol) in CH₂-

Cl₂were added EDC (0.52 g,), HOBt (0.37 g,) and N-Boc-

1392 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9

Marquis et al.

mm; 10 µm; 100% CH₃CN; 1 mL/min flow rate with UV

detection at 254 nm). Preparative HPLC separation of the

mixture 19 (Chiralpak AD; 50 × 250 mm; 20 µm; 100% CH₃-

CN; 108 mL/min flow rate with UV detection at 320 nm)

provided the faster eluting diastereomer 20 (t_R) 5.6 min,

>95% purity): ¹H NMR (DMSO-d₆, 400 MHz) 8.74 (d, 1H),

8.60 (d, 1H), 8.33 (d, 1H), 8.11 (t, 1H), 7.97 (d, 1H), 7.77 (d,

1H), 7.71-7.66 (m, 2H), 7.62 (s, 1H), 7.46 (t, 1H), 7.33 (t, 1H),

4.80 (m, 1H), 4.59 (m, 1H), 4.36 (d, 1H), 3.86 (m, 2H), 2.86 (m,

1H), 1.88-1.57 (m, 7H), 0.92 (m, 6H); MS (ESI) 527 (M + H⁺,

100%). Anal. (C₂₆H₃₀N₄O₆S) C, H, N, S. And the slower eluting

diastereomer 21 (t_R) 9.9 min, >95% purity): MS (ESI) 527

(M + H⁺, 100%). Anal. (C₂₆H₃₀N₄O₆S) C, H, N, S.

4(R,S)-5-(2-Mor p h olin -4-ylet h oxy)b en zofu r a n -2-ca r -

boxylic Acid ((S)-3-Meth yl-1-{3(R,S)-h yd r oxy-1-[2-(3-p y-

r id in -2-ylp h en yl)a cet yl]a zep a n -4-ylca r b a m oyl}b u t yl)-

a m id e (22). To a solution of amino alcohol 17 (10.0 g, 29.1

mmol) in CH₂Cl₂(200 mL) were added HOBt (4.32 g, 32.0

mmol), EDC (6.14 g, 32.0 mmol) and 2-(3-pyridin-2-ylphenyl)-

acetic acid (4.23 g, 32.0 mmol). The reaction was maintained

at room temperature until complete consumption of the

starting material was observed. The mixture was washed with

H₂O, saturated NaHCO₃and brine. The organic layer was

dried (MgSO₄), filtered and concentrated. Column chromatog-

raphy (5% CH₃OH:CH₂Cl₂) of the residue provided 13.6 g (86%)

of 4(R,S)-((S)-1-{3(R,S)-hydroxy-1-[2-(3-pyridin-2-ylphenyl)e-

thanoyl]azepan-4-ylcarbamoyl}-3-methylbutyl)carbamic acid

tert-butyl ester as a white foam: MS (ESI) 539 (M + H)⁺.

To a solution of 4(R,S)-((S)-1-{3(R,S)-hydroxy-1-[2-(3-pyri-

din-2-ylphenyl)ethanoyl]azepan-4-ylcarbamoyl}-3-methylbutyl)-

carbamic acid tert-butyl ester (13.0 g, 24.0 mmol) in methanol

(100 mL) was added 4 M HCl in dioxane (100 mL). The mixture

was stirred until complete consumption of the starting mate-

rial was observed as determined by TLC analysis. The mixture

was concentrated in vacuo to provide 10.5 g (85%) of 4(R,S)-

(S)-2-amino-4-methylpentanoic acid {3(R,S)-hydroxy-1-[2-(3-

pyridin-2-ylphenyl)ethanoyl]azepan-4-yl}amide hydrochloride

as a white powder: MS (ESI) 439 (M + H)⁺.

To a solution of the 4(R,S)-(S)-2-amino-4-methylpentanoic

acid {3-hydroxy-1-[2-(3-pyridin-2-ylphenyl)ethanoyl]azepan-4-

yl}amide hydrochloride (9.0 g, 17.6 mmol) in CH₂Cl₂(150 mL)

were added HOBt (2.4 g, 17.6 mmol), EDC (3.4 g, 17.6 mmol)

and 5-[2-(4-morpholinyl)ethoxy]-2-benzofuran-2-carboxylic acid

(5.12 g, 17.6 mmol). The mixture was maintained at room

temperature until complete consumption of the starting mate-

rial was observed by TLC analysis. Standard workup and

column chromatography (10% CH₃OH:CH₂Cl₂) provided 13.4

g (91%) of alcohols 22 as a white foam: MS (ESI) 712 (M +

H)⁺.

4(R,S)-5-(2-Mor p h olin -4-ylet h oxy)b en zofu r a n -2-ca r -

b oxylic Acid ((S)-3-Met h yl-1-{3-oxo-1-[2-(3-p yr id in -2-

ylp h en yl)a cetyl]a zep a n -4-ylca r ba m oyl}bu tyl)a m id e (23).

To a solution of alcohols 22 (13.0 g, 18.3 mmol) in DMSO (200

mL) were added triethylamine (7.60 mL, 54.8 mmol) and

pyridine sulfur trioxide complex (8.72 g, 54.8 mmol). The

mixture was maintained at room temperature for approxi-

mately 2 h whereupon it was diluted with EtOAc and washed

with H₂O, brine, dried (MgSO₄), filtered and concentrated.

Column chromatography (5% CH₃OH:CH₂Cl₂) of the residue

provided 8.9 g (68%) of ketones 23 as a 1:1 mixture of

diastereomers as determined by analytical HPLC (Whelk O-1

(R,R); 4.6 × 250 mm; 10 µm; 100% CH₃OH with 50 mM NH₄-

OAc; 1 mL/min flow rate with UV detection at 254 nm).

Preparative HPLC separation of the diastereomers 23 (Whelk

O-1 (R,R); 21.2 × 250 mm; 10 µm; 100% CH₃OH with 50 mM

NH₄OAc; 20 mL/min flow rate with UV detection at 335 nm)

provided the faster eluting diastereomer 24 (t_R) 9.2 min,

>97% purity by HPLC analysis) and the slower eluting

diastereomer 25 (t_R) 10.4 min, >95% purity by HPLC

analysis).

5-(2-Mor p h olin -4-ylet h oxy)b en zofu r a n -2-ca r b oxylic

Acid ((S)-3-Meth yl-1-{(R)-3-oxo-1-[2-(3-p yr id in -2-ylp h en -

yl)a cetyl]a zep a n -4-ylca r ba m oyl}bu tyl)a m id e (25). Anal.

(C₄₀H₄₇N₅O₇‚1.4H₂O) C, H, N.

{(S)-1-[Hyd r oxy-3-(p yr id in -2-ylsu lfon yla m in o)p r op yl-

ca r ba m oyl]-3-m eth ylbu tyl}ca r ba m ic Acid ter t-Bu tyl Es-

ter (27). To a solution of 1,3-diamino-2-hydroxypropane 26

(3.37 g, 37.5 mmol) in DMF (65 mL) were added N-Boc-^L-

leucine (9.34 g, 37.5 mmol), HOBt (5.5 g, 40.7 mmol) and EDC

(7.77 g, 40.7 mmol). The mixture was maintained at room

temperature until complete consumption of the starting mate-

rial was observed as determined by TLC analysis. The mixture

was diluted with EtOAc and washed with water, brine, dried,

filtered and concentrated. To a solution of the residue in CH₂-

Cl₂were added NMM (4.4 mL, 140 mmol) and 2-pyridinesulfo-

nyl chloride (3.7 g, 20.84 mmol) and maintained at room

temperature for 2 h. Workup and column chromatography (1:1

EtOAc:hexanes to 100% EtOAc) provided 4.3 g (25%) of alcohol

27 as a white foam: ¹H NMR (400 MHz, CDCl₃) δ 8.70 (d,

1H), 8.03 (d, 1H), 7.94 (t, 1H), 7.52 (dd, 1H), 7.14 (m, 1H), 6.51

(m, 1H), 5.48-5.33 (m, 1H), 3.84 (m, 1H), 3.54-3.11 (m, 5H),

1.68-1.49 (m, 5H) 1.40 (s, 9H),0.92 (m, 6H).

Ben zofu r a n -2-ca r boxylic Acid {(S)-3-Meth yl-1-[2-oxo-

3-(p yr id in -2-ylsu lfon yla m in o)p r op ylca r b a m oyl]b u t yl}-

a m id e (28). To a solution of 27 (2.1 g, 4.73 mmol) in CH₂Cl₂

(30 mL) was added TFA (30 mL). This mixture was maintained

at room temperature for 2 h whereupon it was concentrated.

The residue was azeotroped with CH₂Cl₂(2×), toluene (2×),

washed with ether (2×) and filtered to provide 2.1 g of the

TFA amine salt as a white powder. This material was of

sufficient purity to carry on to the next step with no further

purification.

To a solution of the amine salt (0.4 g, 0.87 mmol) in DMF

(4 mL) was added benzofuran-2-carboxylic acid (0.11 g, 0.7

mmol), NMM (0.3 mL, 2.8 mmol) and HBTU (0.27 g, 0.7 mmol).

The mixture was maintained at room temperature for 3 h

whereupon it was diluted with EtOAc and washed with water,

brine, dried, filtered and concentrated. Column chromatogra-

phy (5% CH₃OH:CH₂Cl₂) of the residue provided 0.42 g (71%)

of benzofuran-2-carboxylic acid {(S)-3-methyl-1-[2-hydroxy-3-

(pyridin-2-ylsulfonylamino)propylcarbamoyl]butyl}amide as a

white foam: ¹H NMR (400 MHz, CDCl₃) δ 8.58 (m, 1H), 7.90

(m, 1H), 7.77 (m, 1H), 7.61 (m, 1H), 7.48-7.23 (m, 6H), 6.45

(m, 1H), 4.71 (m, 1H), 3.95 (m, 1H), 3.54-3.08 (m, 5H), 1.75

(m, 4H), 0.93 (m, 6H); MS (ESI) 489.2 (M + H)⁺.

To a solution of benzofuran-2-carboxylic acid {(S)-3-methyl-

1-[2-hydroxy-3-(pyridin-2-ylsulfonylamino)propylcarbamoyl]-

butyl}amide (0.3 g, 0.61 mmol) in CH₂Cl₂(5.0 mL) was added

Dess-Martin periodinane (0.5 g, 1.18 mmol). The mixture was

maintained at room temperature for approximately 2 h

whereupon saturated Na₂S₂O₃and saturated NaHCO₃were

added and the mixture stirred for 10 min. Workup and column

chromatography (70% EtOAc:30% hexanes) provided 0.12 g

which was further purified by HPLC to provide 0.048 g (8%)

of ketone 28 was a white foam: ¹H NMR (400 MHz, CDCl₃) δ

8.63 (d, 1H), 7.93 (d, 1H), 7.84 (t, 1H), 7.66 (d, 1H), 7.51-7.18

(m, 6H), 6.06 (br s, 1H), 4.77 (m, 1H), 4.21 (m, 4H), 1.82-1.75

(m, 4H), 0.98 (m, 6H); MS (ESI) 487.2 (M + H)⁺. Anal.

(C₂₃H₂₆N₄O₆S‚1.1H₂O) C, H, N.

Cr ysta lliza tion of th e Com p lex of Ca th ep sin K w ith

In h ibitor 24. Crystals of mature activated cathepsin

K

complexed with the inhibitor were grown by the vapor diffusion

method from a solution of 10% PEG 8000, 0.1 M imidazole

pH 8.0 and 0.2 M calcium acetate. Crystals of the complex are

orthorhombic, space group P2₁2₁2₁, with cell constants a ) 71.8

Å, b ) 75.9 Å and c ) 114.8 Å. The structure was determined

by molecular replacement with a model consisting of all protein

atoms from the previously determined cathepsin K/E-64

complex.⁴⁰The structure was refined at 2.8 Å resolution. The

final R_cwas 0.260.

5-(2-Morpholin-4-ylethoxy)benzofuran-2-carboxylic Acid

((S)-3-Met h yl-1-{(S)-3-oxo-1-[2-(3-p yr id in -2-ylp h en yl)-

a cet yl]a zep a n -4-ylca r b a m oyl}b u t yl)a m id e (24). Anal.

(C₄₀H₄₇N₅O₇‚0.35H₂O) C, H, N.

Cr ysta lliza tion of th e Com p lex of Ca th ep sin K w ith

In h ibitor 20. Crystals of mature activated cathepsin

K

complexed with the inhibitor were grown by the vapor diffusion

method from a solution of 30% MPD, 0.1 M MES pH 7.0, 0.1

Azepanone-Based Inhibitors of Cathepsin K

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1393

M tris buffer at pH 7.0 and 90 mM DTT. Crystals of the

complex are tetragonal, space group P4₃, with cell constants

a ) 100.9 Å and c ) 53.7 Å. The structure was determined as

described above and refined at 2.0 Å. The final R_cwas 0.263.

dosed as single components (28 was dosed as a mixture of 4

compounds). Blood samples were obtained from a lateral tail

vein and plasma isolated by centrifugation. Plasma concentra-

tions of test compounds were quantified by liquid chromatog-

raphy/tandem mass spectroscopy, with lower limits of quan-

titation of 5-10 ng/mL. Standard data analysis techniques

were used to derive pharmacokinetic parameters from the

plasma concentration versus time data.⁴⁴

Molecu la r Mod elin g. Model building and conformational

searches were performed with MacroModel⁴¹in the context of

the MMFF⁴²with the GB/SA solvent model.⁴³Applying energy

and conformational difference (based on visual inspection of

superimposed conformers) as selection criteria, seven conform-

ers were chosen as representative of the low energy structures

observed in the two searches. Both diastereomers were rep-

resented, and based on inspection of crystal structures of

related complexes, conformers with equatorial and axial

peptide were selected. The models were superimposed into the

active sites of cathepsin K complexes of the related compounds,

and then visually inspected for goodness-of-fit to the cathepsin

K active site, which proved especially critical on the unprime

side of the active site. Models that formed good interactions

with the enzyme were used as starting points for elaboration

of the complete inhibitor from the model structure. This

procedure, in conjunction with manual adjustment of the

initial and progressive binding models, led to the unambiguous

selection of a binding model based on an axial conformer of

20. The selected conformer of the model compound allowed for

straightforward elaboration to the full inhibitor in the active

site, and satisfied several key interactions with the enzyme.

Model elaboration from other starting points proved unproduc-

tive, although in some cases extensive manual adjustment

ultimately yielded a binding conformation very similar to that

obtained from simple extension of the selected model.

Ma ss Sp ect r a l An a lysis of Ca t h ep sin K-In h ib it or

Com p lexes. The inhibitor, dissolved in DMSO, was diluted

in water (Milli-Q 18 MΩ) to a molar concentration 5-fold that

of the enzyme, with a 20% final volume of DMSO making sure

that the inhibitor was completely dissolved. Human cathepsin

K was provided in buffer (100 mM sodium acetate, 100 mM

NaCl, 2 mM l-cysteine pH 5.5). An aliquot containing 1 nmol

of the enzyme was mixed 1:1 with inhibitor solution in a 0.65-

mL plastic centrifuge tube, vortexed, and a minimum of 120-

min incubation time was allowed before analysis by LC-

ESIMS. The entire reaction mixture was injected onto a

peptide trap and the trap washed with 1 mL of water (Milli-Q

18 MΩ) manually before being back eluted onto the analytical

column.

Kin etic Stu d y of Ep im er iza tion . The rates of epimer-

ization were determined by HPLC analysis utilizing a

MetaChem Inertsil ODS-3, 5 µm, 250 × 4.6 mm; eluent: 1/1

acetonitrile/20 mM pH 7.0 phosphate buffer; flow rate, 1 mL/

min; detection, UV at 215 and 254 nm. Each compound was

initially prepared in DMSO as a 10 mM stock solution. An

appropriate volume of the stock was diluted into the aqueous

buffer, pH ranging from 7 to 12.5, at the final concentration

of 50 µM. 20-µL aliquots of the resulting solution were

withdrawn at designated elapsed times and immediately

injected into the HPLC for analysis. For all compounds tested,

the epimerization rates were negligible at pH 7, the injection

of the test solution into the HPLC eluent, which was 50/50

acetonitrile/pH 7 buffer, essentially stopped the reaction. With

the assumption of a first-order kinetics and a final 1:1

equilibrium concentration ratio, the epimerization rate con-

stant k can be determined based on the following equation:

A(t)/A(0) ) (e^-2kt+ 1)/2, where A(0) and A(t) are chromato-

graphic peak areas at time ) 0 and t, respectively.

P h a r m a cok in etic An a lysis. Animal procedures reported

in these studies were approved by the Animal Care and Use

Committee at SmithKline Beecham Pharmaceuticals. All

dosages were prepared as solutions in water with up to 20%

Encapsin and up tp 3% DMSO. Previous experience with the

vehicles has shown them to have minimal effect on pharma-

cokinetics in the rat. Studies were conducted in a crossover

fashion on 2 separate days, 2 days apart. On study day 1 rats

received a 30-min intravenous infusion of the test compound,

and on study day 2, the same rats received an oral bolus

gavage of the same compound. All compounds except 28 were

Ack n ow led gm en t. The authors thank Drs. J ohn

Gleason and Brian Metcalf for their support of this

work.

Su p p or tin g In for m a tion Ava ila ble: Synthesis and char-

acterization of inhibitors 2 and 36 and X-ray crystallographic

data for 20. This material is available free of charge via the

Internet at http://pubs.acs.org.

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J M000481X

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Azepanone-based inhibitors of human and rat cathepsin K

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