β-substituted cyclohexanecarboxamide: A nonpeptidic framework for the design of potent inhibitors of cathepsin K

Article abstract of DOI:10.1021/jm051059p

A new series of nonpeptidic cathepsin K inhibitors that are based on a β-substituted cyclohexanecarboxamide motif has been developed. Lead optimization yielded compounds with sub-nanomolar potency and exceptional selectivity profiles against cathepsins B, L, and S. Use of fluorine atoms to block metabolism on the cyclohexyl ring led to compounds with excellent pharmacokinetic properties. Considering the well-established role of cathepsin K in osteoclast-mediated bone turnover, compounds such as (-)-34a (hrab Cat K IC₅₀ 0.28 nM; >800-fold selectivity vs Cat B, L, and S; PK data in dogs: F 55%, t_1/2 = 15 h) exhibit great potential for development as an orally bioavailable therapeutic for treatment of diseases that involve bone loss.

Full text of DOI:10.1021/jm051059p

1066
J. Med. Chem. 2006, 49, 1066-1079
â-Substituted Cyclohexanecarboxamide: A Nonpeptidic Framework for the Design of Potent
Inhibitors of Cathepsin K
Sheldon N. Crane,*,† W. Cameron Black,† James T. Palmer,‡ Dana E. Davis,‡ Eduardo Setti,‡ Joel Robichaud,† Julie Paquet,†
Renata M. Oballa,† Christopher I. Bayly,† Daniel J. McKay,† John R. Somoza,‡ Natalie Chauret,† Carmai Seto,† John Scheigetz,†
Greg Wesolowski,§ Frederic Masse´,† Sylvie Desmarais,† and Marc Ouellet†
Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway, Kirkland, Quebec, Canada, H9H 3L1, Celera Genomics,
180 Kimball Way, South San Francisco, California 94080, and Merck Research Laboratories, West Point, PennsylVania 19486
ReceiVed October 20, 2005
A new series of nonpeptidic cathepsin K inhibitors that are based on a â-substituted cyclohexanecarboxamide
motif has been developed. Lead optimization yielded compounds with sub-nanomolar potency and exceptional
selectivity profiles against cathepsins B, L, and S. Use of fluorine atoms to block metabolism on the cyclohexyl
ring led to compounds with excellent pharmacokinetic properties. Considering the well-established role of
cathepsin K in osteoclast-mediated bone turnover, compounds such as (-)-34a (hrab Cat K IC50 0.28 nM;
>800-fold selectivity vs Cat B, L, and S; PK data in dogs: F 55%, t1/2 ) 15 h) exhibit great potential for
development as an orally bioavailable therapeutic for treatment of diseases that involve bone loss.
Introduction
Cathepsin K is a lysosomal cysteine protease that plays a
fundamental role in the osteoclast-mediated degradation of the
collagen framework of bone.1 Consequently, it has become an
attractive target for therapeutic intervention in diseases involving
pathophysiological bone resorption.2 Our efforts in this area have
Figure 1. Replacement of the P2-P3 peptide bond in 1 with an aryl
ring in 2.
been focused on the identification of a once-daily, orally
bioavailable cathepsin K inhibitor for the prevention and
treatment of osteoporosis. We have recently shown that replace-
ment of the P2-P3 amide bond in dipeptide-nitrile inhibitor
13a,b with an aryl ring in 2 (Figure 1) was an effective strategy
for the development of potent and selective cathepsin K
inhibitors that exhibit superior pharmacokinetic profiles as
compared to their peptidic counterparts.4a We now wish to report
that further research in this area has identified a â-substituted
cyclohexanecarboxamide scaffold as a promising new entity for
the construction of cathepsin K inhibitors.
Figure 2. Replacement of the P2-P3 peptide bond with a methyl
thioether.
in the active site of the enzyme.5a The syn-isomer 8a was 5-fold
less potent (hCat K IC50 ) 280 nM) than 8b. Excision of the
P3 benzyl thioether and introduction of a conjugated double
bond in 8c resulted in a dramatic loss of activity against
cathepsin K (hCat K IC50 ) 3400 nM). The difficulties
associated with the preparation of the more potent isomer 8b,
because of the tendency of 7 to expel benzyl mercaptan under
strongly basic conditions, prompted us to transpose the carbon
and sulfur atoms in the P2-P3 linker. This was accomplished
as illustrated in Scheme 2. Starting from trans-cyclohexanecar-
boxylic acid anhydride (9), the warhead portion of the inhibitor
was introduced by reaction with aminoacetonitrile in the
presence of DMAP to yield 10. Reduction of the carboxylic
acid moiety in 10, via treatment of an in situ generated mixed
anhydride with sodium borohydride, was followed by reaction
of the resulting alcohol with tosyl chloride to yield 11.
Nucleophilic displacement of the tosylate with thiophenolate
anion produced 12a (ArdPh). Fortunately, only a modest loss
in potency was associated with reorganization of the methylene-
thioether linker (hCat K IC50 ) 140 nM for 12a vs 52 nM for
8b).
Discovery and Initial SAR Studies
The cyclohexanecarboxamide series of cathepsin K inhibitors
were borne out of an attempt to replace the P2-P3 amide bond
in 33b with a methylene-thioether moiety in 4 (Figure 2).
As shown in Scheme 1, treatment of a mixture of bromoester
5a and benzyl mercaptan with K2CO3 in methanol did not
generate 6 as hoped, but rather produced 7 (syn:anti 13:1) via
a 1,4-addition of the thiolate anion onto the in situ generated
R,â-unsaturated ester 5b. Saponification of the ester functionality
in 7 with NaOH followed by HATU-mediated coupling of the
resulting acid with aminoacetonitrile afforded syn-isomer 8a,
the corresponding anti-isomer 8b, and elimination product 8c
in a ratio of 26:1:14.
Anti-isomer 8b was found to have a potency similar to that
of 3 against human cathepsin K (hCat K IC50 ) 52 and 78 nM,
respectively) despite the loss of a hydrogen-bonding interaction
of the P2-P3 amide N-H with the carbonyl oxygen of Gly66
* To whom correspondence should be addressed. Telephone: (514) 428-
3934. Fax: (514) 428-4939. E-mail: sheldon_crane@merck.com.
† Merck Frosst Centre for Therapeutic Research.
‡ Celera Genomics.
From intermediate 11, we prepared a wide variety of thioether
analogues and studied the effect of varying P3 on human and
humanized-rabbit (hrab) cathepsin K6 inhibition and selectivity
§ Merck Research Laboratories.
10.1021/jm051059p CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/19/2006

â-Substituted Cyclohexanecarboxamide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1067
Scheme 1a
a Conditions: (a) benzyl mercaptan, K2CO3, CH3OH, room temperature; (b) NaOH, H2O, CH3OH, reflux; (c) H2NCH2CN‚HCl, i-Pr2NEt, HATU, DMF,
room temperature.
Scheme 2a
Table 2. Cat K Potency of meta-, Di-, and Trisubstituted Phenyl
Analogues of 12
a Each value represents an average of at least three independent
determinations.
a Conditions: (a) H2NCH2CN‚HCl, Et3N, 4-DMAP, THF, room tem-
perature, 82%; (b) i-BuOCOCl, N-methyl-morpholine, THF, -78 °C, then
Table 3. Cat K Potency of Heteroaromatic Analogues of 12
NaBH4, MeOH, -78 °C to room temperature, 73%; (c) TsCl, pyr, CH3CN,
room temperature, 72%; (d) ArSH, K2CO3, DMF, room temperature.
Table 1. Potency and Selectivity Profiles of para-Substituted Phenyl
Analogues of 12
a Each value represents an average of at least three independent
determinations.
Scheme 3a
a Each value represents an average of at least three independent
determinations.
in our counterscreen against cathepsins B, L, and S. We found
that placing a para-substituted phenyl moiety in P3 yielded the
most potent inhibitors of cathepsin K (Table 1). Additional
tuning of the P3 SAR identified a methylthioether as the optimal
group in the para-position (12f, Table 1). This feature further
distinguishes this series of compounds from their dipeptide
counterparts, which require a basic amine moiety in P3 to attain
similar levels of potency and selectivity.5a,b Introduction of meta-
or di- and trisubstituted aromatics (Table 2), as well as mono-
and bicyclic-heteroaromatic rings (Table 3), in P3 generally
produced less potent inhibitors.
a Conditions: (a) Oxone, NaHCO3, THF, MeOH, H2O, room temperature;
(b) NaSCH3, DMF, room temperature, 93% from 12b.
Hydrocarbon-based P2-P3 linkers were introduced as shown
in Scheme 4. Reaction of known aldehyde 147 with Wittig
reagent 15 afforded alkene 16 (Z/E 5.7:1). Subsequent ester
saponification, double bond hydrogenation, and coupling of the
resulting acid 17 with aminoacetonitrile afforded the fully
saturated hydrocarbon linker in 18. Omission of the double bond
reduction step from this sequence produced the corresponding
alkene analogue 19.
Replacement of the styrene moiety in 19 with a chemically
and more metabolically robust biphenyl in 24a was accom-
plished as shown in Scheme 5. Diels-Alder addition of ortho-
bromocinnamic acid 20 to 1,4-butadiene under forcing condi-
tions in a steel bomb gave 21 in 82% yield. Exposure of 21 to
an atmosphere of hydrogen in the presence of palladium on
charcoal preferentially reduced the double bond in the presence
We next turned our attention to studying the effect of further
modification of the P2-P3 linker on cathepsin enzyme activity.
Oxidation of the thioether in 12b to the corresponding sulfone
with Oxone followed by a SNAr displacement of the fluorine
atom by reaction with 10 equiv of NaSCH3 in DMF afforded
13b in 93% yield for the two steps (Scheme 3). It should be
noted that the electrophilic nitrile functionality was stable to
these reaction conditions.

1068 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Crane et al.
Scheme 4a
Table 4. P2-P3 Linker SAR
a Each value represents an average of at least three independent
determinations.
a Conditions: (a) t-BuOK, THF, PhMe, 50 °C, 62%; (b) LiOH, DME,
MeOH, H2O, 60 °C; (c) 40 PSI H2, 10% Pd/C, EtOAc; (d) H2NCH2CN‚HCl,
PyBOP, Et3N, DMF, room temperature, 51% for 18, 61% for 19.
Scheme 5a
Figure 3. Cathepsin K inhibitors possessing metabolically robust
groups in P3.
mL/min/kg for 24a). Incubation of 13b and 24a with rat or
human hepatocytes produced two major metabolites in each
case. These were identified as the corresponding P3 sulfoxides
and sulfones (25a-d, Figure 3) through LC-MS analysis of
the incubation media, and comparison with authentic standards
prepared by the chemical oxidation of 13b and 24a with
m-CPBA.
a Conditions: (a) 1,4-butadiene, PhMe, 200 °C, steel bomb, 82%; (b) 1
atm H2, 10% Pd/C, EtOAc, room temperature; (c) H2NCH2CN‚HCl, PyBOP,
Et3N, DMF, room temperature, 97%; (d) 4-methylthiophenylboronic acid
or 4-fluorophenylboronic acid, 5 mol % PdCl2(dppf), Na2CO3, H2O, DMF,
85 °C, 66% for 24a, 85% for 24b.
Poor pharmacokinetics was also exhibited by analogues 13c
(t1/2 ) 20 min, Cl ) 42 mL/min/kg) and 24b (t1/2 ) 45 min, Cl
) 50 mL/min/kg) despite the absence of a group susceptible to
metabolic oxidation in P3 (Figure 3). This prompted us to
consider that other portions of these inhibitors were susceptible
to cytochrome P450-mediated metabolism. LCMS analysis of
the rat hepatocyte incubations of 13c and 24b revealed that
several M+16 metabolites were indeed produced. MS-frag-
mentation analysis for each metabolite identified the cyclohexyl
portion as the main site of oxidation. The radiolabeled compound
13d, prepared by palladium-catalyzed tritiation of aryl bromide
28 (Scheme 6), was also incubated with rat hepatocytes. The
additional sensitivity offered by radio-HPLC analysis enabled
us to determine that the metabolic oxidation of 13b was even
more extensive than that revealed by LCMS analysis alone
(Figure 4). Obtaining acceptable pharmacokinetic profiles for
this class of compounds would obviously require a dramatic
improvement in metabolic stability. Thus, a study of the P2-
SAR aimed at achieving this was initiated.
of the hindered ortho-substituted aryl bromide. Subsequent
coupling of the carboxylic acid functionality to aminoacetonitrile
yielded intermediate 23 from which a variety of biaryl analogues
were prepared, including 24a and 24b, via a Suzuki-Miyaura
cross coupling reaction.
A summary of the effect of varying the P2-P3 linker on
cathepsin K potency and anti-target selectivities can be found
in Table 4. Compound 13b, possessing a methylene-sulfone P2-
P3 linker, was equipotent to the methylene-thioether linked
analogue (12f) against cathepsin K, but proved to be slightly
less selective in the counterscreen assays. Replacing the sulfur
atom in 12f with a methylene unit in 18 yielded a 2-fold increase
in potency; however, cathepsin L selectivity was eroded as a
result of this change. Introduction of a unit of unsaturation in
19 produced a sub-nanomolar inhibitor of cathepsin K and
restored cathepsin L selectivity to the level observed with 12f.
Phenyl-linked analogue 24a retained the impressive selectivity
profile exhibited by 12f and 19 while maintaining low nano-
molar potency against cathepsin K.
X-ray Structure and Molecular Modeling
Pharmacokinetics and Metabolism I
An X-ray crystal structure of P3-methoxy analogue 13e bound
to the active site of cathepsin K revealed that this series of
inhibitors adopt a binding orientation similar to their peptidic
counterparts.5a The salient features of this enzyme-inhibitor
complex are illustrated in Figure 5a. These include the thio-
The pharmacokinetic profiles of 13b and 24a were evaluated
by intravenous dosing in male Sprague-Dawley rats. Both of
these compounds were rapidly cleared from circulation (t1/2
)
5 min, Cl ) 38 mL/min/kg for 13b; t1/2 ) 44 min, Cl ) 35

â-Substituted Cyclohexanecarboxamide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1069
Scheme 6a
activated zinc dust gave 31 in 31% overall yield from 21.
Conversion of the ketone functionality in 31 to a gem-difluoro
moiety in 32a was effected with diethylaminosulfur trifluoride
(DAST).9b Under a variety of reaction conditions, 32a was
invariably contaminated with two isomeric halogenated alkenes
(32b,c) that were not separable from the desired product by flash
chromatography. Fortunately, treatment of the crude product
mixture with excess ozone at -78 °C followed by oxidative
workup with 30% H2O2 was successful in converting the
fluoroalkene byproducts to the corresponding diacids, which
were easily removed by washing with an aqueous solution of
sodium carbonate. Compound 32a was converted to 33 by a
two-step sequence involving ester saponification with LiOH
followed by coupling of the resulting acid with aminoacetoni-
trile. Conversion of aryl bromide 33 to a variety of biaryls10
including 34a and 34b was accomplished in good yield using
a Suzuki-Miyaura reaction under standard conditions with
PdCl2(dppf) as catalyst.
a Conditions: (a) K2CO3, MeOH, room temperature, 90%; (b) Oxone,
NaHCO3, THF, MeOH, H2O, 40 °C, >99%; (c) 3H2, 10% Pd/C, Et3N, THF.
imidate moiety formed by covalent union of the nitrile carbon
and the sulfur of Cys25, the hydrogen bond between the
thioimidate N-H and terminal oxygen of Gln19, hydrogen-
bonding contacts between the P1-P2 amide of the inhibitor
with the N-H of Gly66 and the carbonyl oxygen of Asn158. Note
also the deep penetration of the cyclohexyl ring into S2, and
the manner in which the aromatic plate of the inhibitor neatly
fits into the region of S3 bordered by the aromatic ring of Tyr67
and the shelf-like structure formed by Gly65 and Gly66.
Starting from the X-ray structure of 13e bound to cathepsin
K, molecular dynamics calculations were carried out in AM-
BER8 to identify the positions and nature of substituents that
would maintain or improve binding to the S2 pocket. Particular
attention was paid to electron-withdrawing substituents such as
fluorine or chlorine atoms, as a means of reducing oxidative
metabolism of the cyclohexyl ring. Figure 5b shows the modeled
structure of 13f with two fluorine atoms added to the cyclohexyl
ring of 13e, which the calculations indicated would be well
tolerated by the protein.
Both enantiomers of 34a were also accessed using the
chemistry shown in Scheme 7 following resolution of 21 with
(S)- and (R)-R-methylbenzylamine to afford (+)- and (-)-21,
respectively, in 93% ee. The more potent enantiomer of 34a,
prepared from (-)-21, was determined to be the (-)-antipode.
The absolute configurations of (+)-34a and (-)-34a were
established as (1S,2S)- and (1R,2R)-, respectively, by conversion
of (+)-21 to the known compound (+)-35 (Scheme 8).11,12
Results and Discussion
Installation of a gem-difluoro in rac-34a yielded a 5-fold
increase in potency against Cat K (Table 5) as compared to the
nonhalogenated inhibitor 24a. The more potent enantiomer (-)-
34a retained an acceptable selectivity profile against cathepsins
L, B, and S and exhibited good potency (IC50 ) 16 nM, n ) 3)
in our functional bone resorption assay that evaluates the
degradation of the type I collagen matrix of bovine bone by
isolated rabbit osteoclasts.4b Introduction of a gem-chloro in rac-
34c13 resulted in a 5-fold potency boost against cathepsin K;
however, cathepsin S selectivity was diminished as a conse-
quence of this modification. The presence of a single axial or
equatorial fluorine on the cyclohexyl ring in rac-38a and rac-
38b, respectively14 (Scheme 9), had little effect on Cat K IC50.
Thus, it appears that both fluorine substituents act cooperatively
in 34a to produce a synergistic enhancement of inhibitor binding
to S2 of the Cat K active site.
Synthesis of Halogenated P2 Analogues
The synthetic route that was used to prepare the gem-
dihalogenated-cyclohexyl analogues of 24a and 24b is shown
in Scheme 7. Diels-Alder adduct 21 from Scheme 5 was
converted to bromolactone 29 using Iwata’s conditions.9a
Treatment of 29 with sodium methoxide in methanol opened
the lactone to afford bromohydroxyester 30. Oxidation of the
alcohol functionality in 30 with Jones reagent followed by
reductive debromination of the resulting R-bromoketone with
Figure 4. Radio-HPLC and LC-MS chromatograms of rat hepatocyte incubations of 13d.

1070 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Crane et al.
Figure 5. (a) X-ray structure of 13e bound to cathepsin K. (b) Modeled structure of 13f bound to cathepsin K.
Scheme 7a
a Conditions: (a) (CH3)3SiBr, Me2SO, i-Pr2NEt, CHCl3, reflux; (b) NaOMe, MeOH, room temperature; (c) Jones oxidation; (d) Zn, KH2PO4, THF, H2O,
room temperature, 31% from 21; (e) DAST, CH2Cl2, room temperature; (f) LiOH, H2O, MeOH, THF, room temperature; (g) H2NCH2CN‚HCl, PyBOP,
Et3N.
Scheme 8a
55%, t1/2 ) 15 h). gem-Difluorination of the cyclohexyl ring
likewise resulted in an increase in t1/2 in rats from 45 min for
24b to 4.5 h for rac-34b. A comparable enhancement of half-
life in rats was also achieved with chlorine substituents in rac-
34c (t1/2 = 3.1 h). Introduction of a single fluorine at the
5-position in rac-38a improved the half-lives of this class of
Cat K inhibitors from a few minutes to over an hour in rats.
However, the terminal half-life observed in squirrel monkeys
for rac-38a (t1/2 ) 0.8 h) was far shorter than that seen for
(-)-34a (t1/2 ) 8.8 h).
a Conditions: (a) CH2N2, Et2O; (b) 1 atm H2, 10% Pd/C, EtOAc, Et3N;
(c) LiOH, H2O, THF, MeOH, 75% overall.
Pharmacokinetics and Metabolism II. Introduction of the
gem-difluoro group onto the cyclohexyl ring also achieved the
desired improvement in pharmacokinetics (Table 6). Acceptable
terminal half-lives (t1/2) and oral bioavailabilities (%F) were
observed for (-)-34a in the three animal species shown with a
particularly impressive PK profile being observed in dog (F )
In contrast with the metabolic profile presented in Figure 4,
incubation of rac-34b with rat hepatocytes led to the production
of only one major M+16 metabolite. It was not possible to
ascertain by MS-fragmentation analysis if oxygenation was
occurring on the cyclohexyl or aromatic portion of the inhibitor.

â-Substituted Cyclohexanecarboxamide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1071
Table 5. Potency and Selectivity of P2-Halogenated Cathepsin K Inhibitors
a Each value represents an average of at least three independent determinations.
Scheme 9a
Table 6. Phamacokinetic Profiles of P2-Halogenated Cathepsin K
Inhibitors
mixture (125 nM) was preincubated for 15 min, followed by a
500-fold dilution into substrate-containing buffer (0.2 µM
citrate). After dilution, the cathepsin K enzyme activity was
initially >95% inhibited, and slowly recovered with a first-order
rate constant of 0.0081 s-1 (t1/2 ) 86 s). The final steady-state
enzyme activity was ∼80% that of control, demonstrating the
reversibility of the formation of the (-)-34a-cathepsin K
complex.
a Conditions: (a) NaBH4, MeOH, room temperature; (b) DAST, CH2Cl2,
-78 to -10 °C; (c) Scheme 7, steps f, g, and h.
Similar incubation of (-)-34a with rat and human hepatocytes
revealed that oxidative metabolism was predominantly confined
to the P3-thioether. This produced both the corresponding
sulfoxide (39a) and the sulfone (39b) metabolites, which were
determined to be 100-fold less potent than the parent compound.
The structures of 39a and 39b were confirmed by LCMS
analysis through comparison with synthetic standards.
Kinetic Characterization of (-)-34a. The reversibility of
inhibition of humanized-rabbit cathepsin K by (-)-34a was
demonstrated in experiments in which a 1:1 enzyme-inhibitor
Conclusion
A new class of nonpeptidic cathepsin K inhibitors that employ
a nitrile-warhead on a â-substituted cyclohexanecarboxamide
scaffold has been developed. SAR optimization of our initial
lead (8b) culminated in the identification of (-)-34a, a
compound that exhibited sub-nanomolar inhibition of cathepsin
K, excellent selectivity against the closely related cathepsins

1072 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Crane et al.
and observed structure factors have been deposited in the Protein
Data Bank under accession code 2F7D.
B, L, and S, and an acceptable pharmacokinetic profile in a
number of animal species. Thus, the â-substituted cyclohexan-
ecarboxamide moiety has been validated as a promising new
scaffold for the development of orally bioavailable cathepsin
K inhibitors as bone anti-resorptive agents.
Methyl 2-(Benzylthio)cyclohexanecarboxylate (7). A mixture
of 5a (520 mg, 2.35 mmol) and milled K2CO3 (990 mg, 7.20 mmol)
was suspended in MeOH (10 mL). Benzyl mercaptan (0.50 mL,
4.2 mmol) was added, and the mixture was stirred for 6 days at
room temperature before partitioning between ethyl acetate and 1
M NaOH. The organic phase was washed with 1 M NaOH and
brine, then dried over Na2SO4 and concentrated. Purification by
flash chromatography (5/95 to 12/88 ethyl acetate/hexanes) provided
the title compound (syn:anti 13:1) as a faint-yellow oil 157 mg
Experimental Section
General. Zinc dust was activated by successive washing in a
sintered glass filter funnel with dilute HCl solution, water, ethanol,
and ether followed by drying under a stream of nitrogen and finally
under high vacuum. m-Chloroperoxybenzoic acid (m-CPBA) was
purified by washing a benzene solution of commercial material with
two portions of pH 7.4 phosphate buffer followed by recrystalli-
zation from ether/hexanes. All other reagents were obtained
commercially and used without further purification. Proton (1H
NMR) magnetic resonance spectra were recorded on a Bruker 500
MHz instrument. All spectra were recorded in d6-acetone (unless
otherwise indicated) using residual solvent as internal standard.
Signal multiplicity was designated according to the following
abbreviations: s ) singlet, d ) doublet, dd ) doublet of doublets,
ddd ) doublet of doublet of doublets, t ) triplet, q ) quartet, dt
) doublet of triplets, td ) triplet of doublets, dq ) doublet of
quartets, m ) multiplet, br ) broad. Elemental analyses were
provided by Prevalere Life Sciences Inc., Whitesboro, NY. Rota-
tions were measured on a Perkin-Elmer model 241 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.
1
(25%). H NMR: major isomer (syn) δ 7.35 (4H, m), 7.25 (1H,
m), 3.77 (1H, d, J ) 13 Hz), 3.73 (1H, d, J ) 13 Hz), 3.60 (3H,
s), 3.28 (1H, m), 2.73 (1H, td, J ) 3.8, 11 Hz), 1.88 (1H, m), 1.70
(5H, m), 1.45 (1H, m), 1.30 (1H, m); discernible signals for the
minor isomer (anti) δ 3.65 (3H, s), 2.41 (1H, dt, J ) 3.8, 11 Hz).
Alternatively, the title compound can be prepared by refluxing a
mixture of 5b (1.00 g, 7.14 mmol), benzyl mercaptan (1.07 g, 8.63
mmol), and K2CO3 (1.52 g, 11.0 mmol) in acetonitrile (4 mL) for
24 h. Substitution of water for 1 M NaOH in the workup procedure
above and flash chromatography on silica eluting with 12/88 EtOAc/
hexanes provides 7 as a colorless oil (1.62 g, 86%, syn:anti 13:1).
(1R/S,2R/S)-2-(Benzylthio)-N-(cyanomethyl)cyclohexanecar-
boxamide (8a), (1R/S,2S/R)-2-(Benzylthio)-N-(cyanomethyl)-
cyclohexanecarboxamide (8b), and N-(Cyanomethyl)cyclohex-
1-ene-1-carboxamide (8c). A solution of 7 (650 mg, 2.50 mmol)
in a 1:1 mixture of MeOH and THF (4 mL) was treated with 2 M
NaOH (4 mL, 8 mmol) followed by heating to reflux for 1 h (strong
smell of benzyl mercaptan). The mixture was cooled, diluted with
water, and extracted with ether. The aqueous layer was acidified
with 1 M HCl and extracted with ethyl acetate. The organic layer
was washed with brine, dried over MgSO4, and concentrated to
give 370 mg of a colorless oil that was combined with HATU (1.20
g, 3.20 mmol), aminoacetonitrile hydrochloride (0.70 g, 7.6 mmol),
and Hunig’s base (2.0 mL, 12 mmol) in DMF (3 mL) with stirring
at room temperature overnight. The mixture was then added to 1
M HCl and extracted with ethyl acetate (2×). The organic phase
was washed with saturated brine and NaHCO3 aqueous solutions,
dried over Na2SO4, and concentrated to afford a brown syrup that
1H NMR analysis revealed to be composed of the title compounds
in a ratio of 26:14:1 (8a:8b:8c). Purification by flash chromatog-
raphy on silica gel eluting with 2/3 to 3/2 EtOAc/hexanes provided
134 mg of the syn-isomer (8a), 6 mg of the anti-isomer (8b), and
55 mg of the elimination product 8c as colorless solids. Rf values
Enzyme Expression and Purification. See ref 4a.
Enzyme Activity Assays. See ref 15.
Pharmacokinetic and Bioavailability Experiments. Single
compounds were dosed orally as suspensions in 1% methocel and
intravenously via the jugular vein as solutions in PEG-200/H2O
(3:2). Dosing volumes used for oral administration were 10 mL/kg
for rats and rabbits, 1 mL/kg for monkeys, and 5 mL/kg for dogs.
Intravenous dosing volumes of 1 mL/kg were used for all species.
Animals receiving the oral suspensions were fasted overnight prior
to a.m. dosing. Plasma samples were collected at regular intervals
and analyzed by LC-MS following a protein precipitation with
1.5 volumes of acetonitrile and centrifugation. All animal procedures
were conducted in accordance with Institutional Animal Care and
Use Committee guidelines.
Hepatocyte Incubations. For hepatocyte incubations, 1 × 106
cells diluted in 0.5 mL of Krebs-Henseleit buffer were first prepared
at 37 °C for 20 min under 95%:5% O2:CO2 (BOC gases; Montreal,
Canada) in a 48-well plate, and then 5 µL of 10 mM solution of
compound dissolved in acetonitrile was added to each well to a
final concentration of 50 µM. After 2 h of incubation at 37 °C
under 95%:5% O2:CO2 atmosphere, one volume of acetonitrile was
added in each well. A quenched incubation spiked with the parent
compound and a blank were also prepared as controls. Once
transferred, samples were centrifuged for 10 min at 14 000 rpm
using an Eppendorf 5415C centrifuge (Hamburg, Germany), and
the supernatant was used for LC/UV/MS analysis.
X-ray Structure Determination of 13e Bound to Cathepsin
K. A mutant of rabbit cathepsin K was expressed and purified as
described previously.16 Compound 13e was cocrystallized with the
mutant cathepsin K at room temperature using the hanging drop
vapor diffusion method. The reservoir consisted of 0.18 M
magnesium formate, and the drop was formed by 2 µL of a solution
of 10 mg/mL cathepsin K in 25 mM sodium acetate (pH 3.9) and
by 2 µL of the reservoir solution. Crystals formed within 4 days,
and diffraction data were collected on a Rigaku R-AXIS IV area
detector, using X-rays produced by a Rigaku RU200 rotating anode
generator. The data were reduced using the HKL package, and
refinement was carried out by iterating between manual rebuilding
using Quanta (MSI, Inc.) and automated refinement using CNX
(MSI, Inc.). Data and refinement statistics for the final model are
shown in Table S1 of the Supporting Information. The coordinates
1
in 1/1 EtOAc/hexanes: 8a (0.42), 8b (0.50), 8c (0.33). H NMR
for 8a: δ 7.58 (1H, br s), 7.39 (2H, m), 7.31 (2H, m), 7.25 (1H,
m), 4.20 (2H, m), 3.77 (2H, m), 3.26 (1H, m), 2.67 (1H, td, J )
3.9, 9.7 Hz), 1.94 (1H, m), 2.70 (5H, m), 1.45 (1H, m), 1.30 (1H,
m). For 8b: δ 7.87 (1H, br s), 7.38 (2H, m), 7.29 (2H, m), 7.22
(1H, m), 4.26 (2H, d, J ) 5.8 Hz), 3.84 (1H, d, J ) 13 Hz), 3.79
(1H, d, J ) 13 Hz), 2.82 (1H, dt, J ) 3.9, 11 Hz), 2.32 (1H, dt, J
) 3.9, 11 Hz), 2.00 (1H, m), 1.85 (1H, m), 1.72 (2H, m), 1.53
(1H, m), 1.30 (3H, m). For 8c: δ 7.73 (1H, br s), 6.70 (1H, m),
4.25 (2H, d, J ) 5.8 Hz), 2.27-2.24 (2H, m), 2.19-2.14 (2H, m),
1.70-1.64 (2H, m), 1.63-1.57 (2H, m).
(1R/S,2R/S)-2-{[(Cyanomethyl)amino]carbonyl}-
cyclohexanecarboxylic Acid (10). A 0 °C suspension of anhydride
9 (1.00 g, 6.49 mmol), aminoacetonitrile hydrochloride (1.30 g,
14.0 mmol), and 4-(dimethylamino)pyridine (70 mg, 0.57 mmol)
in THF (20 mL) was treated with triethylamine (2.0 mL, 14 mmol)
and stirred at room temperature overnight (16 h). The mixture was
then poured into 1 M HCl and extracted with EtOAc. The organic
phase was washed with brine, dried (Na2SO4), and concentrated to
1
afford the title compound as a colorless solid (1.05 g, 78%). H
NMR: δ 10.60 (1H, br s), 7.71 (1H, br s), 4.32-4.00 (2H, m),
2.62 (1H, m), 2.47 (1H, m), 2.07 (1H, m), 1.88 (1H, m), 1.80-
1.68 (2H, m), 1.43-1.20 (4H, m).
(1R/S,2R/S)-(2-{[(Cyanomethyl)amino]carbonyl}-cyclohexyl)-
methyl-4-methylbenzenesulfonate (11). A solution composed of
10 (970 mg, 4.60 mmol) and N-methylmorpholine (0.51 mL, 4.6
mmol) in THF (10 mL) was cooled to -78 °C and treated with

â-Substituted Cyclohexanecarboxamide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1073
isobutylchloroformate (0.60 mL, 4.6 mmol). The resulting colorless
slurry was stirred at this temperature for 5 min and then at 0 °C
for 30 min. The mixture was recooled to -78 °C, and NaBH4 (450
mg, 12.0 mmol) was added in one portion followed by the dropwise
addition of methanol (10 mL) with slow warming to room
temperature over 1.5 h. The reaction was quenched by the addition
of saturated aqueous NH4Cl, and the mixture was poured into brine
and extracted with ethyl acetate (3×). The combined organics were
dried (Na2SO4) and concentrated, and the residue was suspended
in toluene (15 mL) with stirring at room temperature overnight (16
h). Filtration afforded (1R/S,2R/S)-N-(cyanomethyl)-2-(hydroxy-
methyl)cyclohexanecarboxamide as a colorless solid (660 mg, 73%
yield). 1H NMR (d4-methanol): δ 3.48 (1H, dd, J ) 3.0, 9.4 Hz),
3.33 (2H, s), 3.32 (1H, overlapped dd), 2.09 (1H, br t), 1.93 (1H,
m), 1.81 (1H, m), 1.78-1.72 (3H, overlapped m), 1.52 (1H, br q),
1.40-1.22 (2H, m), 1.15 (1H, br q). A solution of (1R/S,2R/S)-N-
(cyanomethyl)-2-(hydroxymethyl)cyclohexanecarboxamide (372 mg,
1.90 mmol) and toluene-4-sulfonyl chloride (430 mg, 2.30 mmol)
in acetonitrile (6 mL) was treated with pyridine (0.23 mL, 2.8 mmol)
and stirred at room temperature until the solution cleared (3 h).
The reaction vessel contents were then partitioned between water
and ethyl acetate, and the layers were separated. The organic phase
was dried (Na2SO4) and concentrated, and the residue was purified
by flash chromatography on silica gel eluting with 4/96 MeOH/
CHCl3 to afford the title compound as a pale-yellow foam (591
mg, 89%). 1H NMR: δ 7.86 (1H, br s), 7.76 (2H, d, J ) 8.3 Hz),
7.47 (2H, d, J ) 8.3 Hz), 4.15 (1H, dd, J ) 6.0, 17 Hz), 4.05 (1H,
dd, J ) 5.5, 17 Hz), 3.91 (1H, dd, J ) 3.2, 9.6 Hz), 3.79 (1H, dd,
J ) 5.5, 9.6 Hz), 2.45 (3H, s), 2.16 (1H, dt, J ) 3.8, 11 Hz), 1.92
(1H, m), 1.82 (1H, m), 1.75-1.67 (3H, m), 1.39 (1H, dq, J ) 3.6,
13 Hz), 1.39-0.90 (3H, m).
Hz), 2.64 (1H, dd, J ) 9.5, 13 Hz), 2.23-2.13 (2H, m), 1.94 (1H,
m), 1.86 (1H, m), 1.78-1.71 (2H, m), 1.46 (1H, m), 1.28-1.22
(2H, m), 1.05 (1H, m). Anal. (C16H19BrN2OS) C, calcd 52.32; found
51.65; H, calcd 5.21; found 5.00; N, calcd 7.63; found 7.28.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-iodophenylthio)methyl]-
cyclohexanecarboxamide (12e). 2/3 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.87 (1H, br s), 7.64 (2H, d, J ) 8.5 Hz), 7.18
(2H, d, J ) 8.5 Hz), 4.31-4.21 (2H, m), 3.17 (1H, dd, J ) 2.9, 13
Hz), 2.64 (1H, dd, J ) 9.6, 13 Hz), 2.20 (1H, m), 2.14 (1H, m),
1.95 (1H, m), 1.86 (1H, m), 1.79-1.71 (2H, m), 1.47 (1H, m),
1.32-1.21 (2H, m), 1.05 (1H, m). Anal. (C16H19IN2OS) C, calcd
46.38; found 46.50; H, calcd 4.62; found 4.42; N, calcd 6.76; found
6.60.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-(methylthio)phenylthio)-
methyl]cyclohexanecarboxamide (12f). 2/3 EtOAc/hexanes, color-
1
less solid. H NMR: δ 7.82 (1H, br s), 7.32 (2H, d, J ) 8.6 Hz),
7.22 (2H, d, J ) 8.6 Hz), 4.30-4.20 (2H, m), 3.15 (1H, dd, J )
3.1, 13 Hz), 2.62 (1H, dd, J ) 9.5, 13 Hz), 2.49 (3H, s), 2.17 (1H,
m), 1.98-1.90 (2H, m), 1.85 (1H, m), 1.80-1.72 (2H, m), 1.48
(1H, m), 1.33-1.22 (2H, m), 1.06 (1H, m). Anal. (C17H22N2OS2)
C, calcd 61.04; found 60.25; H, calcd 6.63; found 6.23; N, calcd
8.37; found 7.90.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-methylphenylthio)methyl]-
cyclohexanecarboxamide (12g). 2/3 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.80 (1H, br s), 7.26 (2H, d, J ) 8.4 Hz), 7.12
(2H, d, J ) 8.4 Hz), 4.32-4.19 (2H, m), 3.13 (1H, dd, J ) 3.2, 13
Hz), 2.61 (1H, dd, J ) 9.4, 13 Hz), 2.30 (3H, s), 2.23-2.15 (2H,
m), 1.94 (1H, m), 1.84 (1H, m), 1.80-1.71 (2H, m), 1.47 (1H, m),
1.32-1.20 (2H, m), 1.06 (1H, m). Anal. (C17H22N2OS) C, calcd
67.51; found 66.67; H, calcd 7.33; found 6.69; N, calcd 9.26; found
8.88.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-hydroxyphenylthio)meth-
yl]cyclohexanecarboxamide (12h). 55/45 EtOAc/hexanes, color-
less solid. 1H NMR: δ 8.41 (1H, br s), 7.75 (1H, m), 7.26 (2H, d,
J ) 8.6 Hz), 6.81 (2H, d, J ) 8.6 Hz), 4.28-4.17 (2H, m), 3.03
(1H, dd, J ) 3.1, 13 Hz), 2.56 (1H, dd, J ) 9.1, 13 Hz), 2.20-
2.12 (2H, m), 1.90-1.79 (2H, m), 1.77-1.71 (2H, m), 1.46 (1H,
m), 1.29-1.21 (2H, m), 1.04 (1H, m). Anal. (C16H20N2O2S) C, calcd
63.13; found 62.18; H, calcd 6.62; found 6.33; N, calcd 9.20; found
8.83.
General Procedure for the Preparation of 12a-z. A 0.35 M
DMF solution of 11 was treated with the appropriate benzenethiol
(1.1 equiv) and K2CO3 (4 equiv) with stirring at room temperature
overnight (16 h). The mixture was then partitioned between ethyl
acetate and water, and the layers were separated. The organic phase
was washed with brine, dried (Na2SO4), and concentrated. Flash
chromatography on silica gel using the indicated solvent mixture
afforded the desired thioether in high purity and generally in >90%
yield.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(phenylthio)methyl]cyclo-
hexanecarboxamide (12a). 2/3 EtOAc/hexanes, colorless solid. 1H
NMR: δ 7.83 (1H, br s), 7.36 (2H, d, J ) 7.5 Hz), 7.30 (2H, t, J
) 7.5 Hz), 7.17 (1H, t, J ) 7.5 Hz), 4.30-4.22 (2H, m), 3.18 (1H,
dd, J ) 3.0, 13 Hz), 2.64 (1H, dd, J ) 9.3, 13 Hz), 2.23-2.13
(2H, m), 1.95 (1H, m), 1.86 (1H, m), 1.79-1.72 (2H, m), 1.48
(1H, m), 1.30-1.22 (2H, m), 1.06 (1H, m). Anal. (C16H20N2OS)
C, calcd 66.63; found 65.85; H, calcd 6.99; found 6.93; N, calcd
9.71; found 9.30.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-methoxyphenylthio)meth-
yl]cyclohexanecarboxamide (12i). 2/3 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.76 (1H, br s), 7.34 (2H, d, J ) 8.8 Hz), 6.89
(2H, d, J ) 8.8 Hz), 4.27-4.18 (2H, m), 3.80 (3H, s), 3.07 (1H,
dd, J ) 3.1, 13 Hz), 2.58 (1H, dd, J ) 9.2, 13 Hz), 2.19-2.12
(2H, m), 1.92-1.80 (2H, m), 1.78-1.71 (2H, m), 1.46 (1H, m),
1.32-1.19 (2H, m), 1.04 (1H, m). Anal. (C17H22N2O2S) C, calcd
64.12; found 63.83; H, calcd 6.96; found 6.70; N, calcd 8.80; found
8.63.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-fluorophenylthio)methyl]-
cyclohexanecarboxamide (12b). 2/3 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.81 (1H, br s), 7.39 (2H, dd, J ) 5.2, 8.9 Hz),
7.07 (2H, t, J ) 8.9 Hz), 4.25-4.20 (2H, m), 3.10 (1H, dd, J )
3.1, 13 Hz), 2.60 (1H, dd, J ) 9.4, 13 Hz), 2.19-2.08 (2H, m),
1.89 (1H, m), 1.82 (1H, m), 1.76-1.68 (2H, m), 1.44 (1H, m),
1.30-1.17 (2H, m), 1.03 (1H, m). Anal. (C16H19FN2OS) C, calcd
62.72; found 62.01; H, calcd 6.25; found 6.04; N, calcd 9.14; found
8.87.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-aminophenylthio)methyl]-
cyclohexanecarboxamide (12j). 5/95 MeOH/CH2Cl2, thick color-
less syrup. 1H NMR: δ 7.68 (1H, br s), 7.15 (2H, d, J ) 8.5 Hz),
6.63 (2H, d, J ) 8.5 Hz), 4.74 (2H, br s), 4.24-4.16 (2H, m), 2.96
(1H, dd, J ) 3.2, 13 Hz), 2.51 (1H, dd, J ) 8.8, 13 Hz), 2.20-
2.12 (2H, m), 1.89-1.79 (2H, m), 1.74 (2H, m), 1.46 (1H, m),
1.29-1.22 (2H, m), 1.04 (1H, m). Anal. (C16H21N3OS) C, calcd
63.33; found 59.07; H, calcd 6.98; found 6.86; N, calcd 13.85; found
12.89.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-chlorophenylthio)methyl]-
cyclohexanecarboxamide (12c). 2/3 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.85 (1H, br s), 7.37 (2H, d, J ) 8.7 Hz), 7.33
(2H, d, J ) 8.7 Hz), 4.30-4.22 (2H, m), 3.17 (1H, dd, J ) 3.0, 13
Hz), 2.65 (1H, dd, J ) 9.6, 13 Hz), 2.20 (1H, m), 2.15 (1H, m),
1.94 (1H, m), 1.86 (1H, m), 1.80-1.70 (2H, m), 1.47 (1H, m),
1.32-1.19 (2H, m), 1.05 (1H, m). Anal. (C16H19ClN2OS) C, calcd
59.52; found 58.60; H, calcd 5.93; found 5.61; N, calcd 8.68; found
8.26.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(3-fluorophenyl)thio]methyl}-
cyclohexanecarboxamide (12k). 2/3 EtOAc/hexanes, colorless
solid. H NMR: δ 7.87 (1H, br), 7.30 (1H, m), 7.14-7.10 (2H,
m), 6.88 (1H, dt, J ) 2.3, 8.5 Hz), 4.34-4.19 (2H, m), 3.17 (1H,
dd, J ) 3.0, 13 Hz), 2.63 (1H, dd, J ) 9.5, 13 Hz), 2.18 (1H, dt,
J ) 3.6, 12 Hz), 2.13 (1H, m), 1.97 (1H, m), 1.83 (1H, m), 1.78-
1.69 (2H, m), 1.46 (1H, m), 1.26-1.20 (2H, m), 1.03 (1H, m).
Anal. (C16H19FN2OS) C, calcd 62.72; found 62.70; H, calcd 6.25;
found 6.43; N, calcd 9.14; found 9.39.
1
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(4-bromophenylthio)methyl]-
cyclohexanecarboxamide (12d). 2/3 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.86 (1H, br s), 7.47 (2H, d, J ) 8.6 Hz), 7.31
(2H, d, J ) 8.6 Hz), 4.31-4.25 (2H, m), 3.17 (1H, dd, J ) 3.1, 13
(1R/S,2R/S)-2-{[(3-Chlorophenyl)thio]methyl}-N-(cyanometh-
yl)cyclohexanecarboxamide (12l). 1/1 EtOAc/hexanes, colorless
solid. H NMR: δ 7.86 (1H, s), 7.37 (1H, d, J ) 1.0 Hz), 7.33-
7.31 (2H, m), 7.20-7.18 (1H, m), 4.31-4.23 (2H, m), 3.20 (1H,
1

1074 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Crane et al.
1
dd, J ) 3.1, 13 Hz), 2.68 (1H, dd, J ) 9.5, 13 Hz), 2.21 (1H, m),
2.15 (1H, m), 2.08 (1H, overlapped m), 1.97 (1H, m), 1.87 (1H,
m), 1.80-1.72 (2H, m), 1.48 (1H, m), 1.32-1.22 (2H, m), 1.07
(1H, m). Anal. (C16H19ClN2OS) C, calcd 59.52; found 59.52; H,
calcd 5.93; found 5.88; N, calcd 8.68; found 8.53.
(1R/S,2R/S)-2-{[(3-Bromophenyl)thio]methyl}-N-(cyanometh-
yl)cyclohexanecarboxamide (12m). 1/1 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.85 (1H, br m), 7.47 (1H, m), 7.32 (2H,
overlapping t), 7.22 (1H, t, J ) 7.9 Hz), 4.32-4.19 (2H, m), 3.16
(1H, dd, J ) 3.1, 13 Hz), 2.64 (1H, dd, J ) 9.5, 13 Hz), 2.18 (1H,
dt, J ) 3.6, 11 Hz), 2.12 (1H, m), 1.94 (1H, m), 1.83 (1H, m),
1.75-1.70 (2H, m), 1.45 (1H, m), 1.32-1.18 (2H, m), 1.04 (1H,
m). Anal. (C16H19BrN2OS) C, calcd 52.32; found 52.68; H, calcd
5.21; found 5.19; N, calcd 7.63; found 7.68.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(3-hydroxyphenyl)thio]-
methyl}cyclohexanecarboxamide (12n). 1/1 EtOAc/hexanes, col-
orless solid. 1H NMR: δ 8.33 (1H, s), 7.80 (1H, br m), 7.09 (1H,
t, J ) 7.9 Hz), 6.80 (1H, d, J ) 7.8 Hz), 6.78 (1H, m), 6.61 (1H,
dd, J ) 1.9, 7.8 Hz), 4.20-4.17 (2H, m), 3.10 (1H, dd, J ) 3.2,
13 Hz), 2.60 (1H, dd, J ) 9.3, 13 Hz), 2.17 (1H, dt, J ) 3.7, 12
Hz), 2.13 (1H, m), 1.93 (1H, m), 1.82 (1H, m), 1.78-1.68 (2H,
m), 1.46 (1H, m), 1.32-1.20 (2H, m), 1.02 (1H, m). Anal.
(C16H20N2O2S) C, calcd 63.13; found 62.79; H, calcd 6.62; found
6.55; N, calcd 9.20; found 8.95.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(3-methoxyphenyl)thio]-
methyl}cyclohexanecarboxamide (12o). 1/1 EtOAc/hexanes, col-
orless solid. 1H NMR: δ 7.85 (1H, br s), 7.19 (1H, t, J ) 7.9 Hz),
6.91 (2H, m), 6.72 (1H, dd, J ) 2.4, 8.2 Hz), 4.29-4.22 (2H, m),
3.85 (3H, s), 3.20 (1H, dd, J ) 3.0, 13 Hz), 2.60 (1H, dd, J ) 9.7,
13 Hz), 2.22-2.16 (2H, m), 1.96 (1H, m), 1.86 (1H, m), 1.76 (2H,
m), 1.48 (1H, m), 1.26 (2H, m), 1.05 (1H, m). Anal. (C17H22N2O2S)
C, calcd 64.12; found 63.40; H, calcd 6.96; found 6.90; N, calcd
8.80; found 8.50.
orless solid. H NMR: δ 7.93 (1H, br s), 7.70 (1H, s), 7.65 (1H,
s), 4.33-4.25 (2H, m), 3.21 (1H, dd, J ) 2.9, 13 Hz), 2.74 (1H,
dd, J ) 9.8, 13 Hz), 2.25 (1H, m), 2.18 (1H, m), 2.02 (1H,
overlapped m), 1.92 (1H, m), 1.82-1.75 (2H, m), 1.48 (1H, m),
1.34-1.27 (2H, m), 1.12 (1H, m). Anal. (C16H17Cl3N2OS) C, calcd
49.06; found 48.62; H, calcd 4.37; found 4.29; N, calcd 7.15; found
7.03.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(1H-imidazol-2-ylthio)meth-
yl]cyclohexanecarboxamide (12u). 95/5 EtOAc/hexanes, colorless
solid. H NMR: δ 11.4 (1H, br), 8.63 (1H, br s), 7.12 (2H, br s),
4.26 (2H, s), 3.26 (1H, dd, J ) 2.9, 14 Hz), 2.72 (1H, dd, J ) 8.9,
14 Hz), 2.22 (1H, m), 2.10 (1H, overlapped m), 2.01-1.93 (2H,
m), 1.79-1.73 (2H, m), 1.45 (1H, m), 1.33-1.25 (2H, m), 1.13-
1.05 (1H, m). Anal. (C13H18N4OS) C, calcd 56.09; found 55.36;
H, calcd 6.52; found 6.48; N, calcd 20.13; found 19.46.
1
(1R/S,2R/S)-2-[(1,3-Benzothiazol-2-ylthio)methyl]-N-(cyano-
methyl)cyclohexanecarboxamide (12v). 2/3 EtOAc/hexanes, col-
1
orless solid. H NMR: δ 7.96 (1H, d, J ) 8.0 Hz), 7.95 (1H,
overlapped br s), 7.87 (1H, d, J ) 8.0 Hz), 7.48 (1H, t, J ) 7.5
Hz), 7.37 (1H, t, J ) 7.5 Hz), 4.37-4.21 (2H, m), 3.58 (1H, dd, J
) 3.3, 13 Hz), 3.30 (1H, dd, J ) 8.2, 13 Hz), 2.29 (1H, m), 2.16
(1H, m), 2.10 (1H, overlapped m), 1.91 (1H, m), 1.82-1.71 (2H,
m), 1.54 (1H, m), 1.39-1.27 (2H, m), 1.19 (1H, m). Anal.
(C17H19N3OS2) C, calcd 59.10; found 58.22; H, calcd 5.54; found
5.36; N, calcd 12.16; found 11.54.
(1R/S,2R/S)-2-[(1,3-Benzoxazol-2-ylthio)methyl]-N-(cyano-
methyl)cyclohexanecarboxamide (12w). 2/3 EtOAc/hexanes, col-
orless solid. 1H NMR: δ 7.96 (1H, br s), 7.62 (1H, d, J ) 6.9 Hz),
7.56 (1H, d, J ) 7.1 Hz), 7.33 (2H, m), 4.35-4.21 (2H, m), 3.54
(1H, d, J ) 13 Hz), 3.26 (1H, dd, J ) 8.0, 13 Hz), 2.28 (1H, m),
2.16 (1H, m), 2.10 (1H, overlapped m), 1.91 (1H, m), 1.85-1.70
(2H, m), 1.52 (1H, m), 1.35-1.27 (2H, m), 1.19 (1H, m). Anal.
(C17H19N3O2S) C, calcd 61.98; found 59.99; H, calcd 5.81; found
5.74; N, calcd 12.76; found 11.97.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(pyrimidin-2-ylthio)methyl]-
cyclohexanecarboxamide (12x). 95/5 EtOAc/hexanes, colorless
solid. 1H NMR: δ 8.59 (2H, d, J ) 4.8 Hz), 7.84 (1H, br s), 7.15
(1H, t, J ) 4.8 Hz), 4.28-4.22 (2H, m), 3.39 (1H, dd, J ) 3.1, 13
Hz), 3.04 (1H, dd, J ) 8.1, 13 Hz), 2.22 (1H, m), 2.08 (1H,
overlapped m), 1.98 (1H, m), 1.87 (1H, m), 1.80-1.71 (2H, m),
1.51 (1H, m), 1.32-1.25 (2H, m), 1.15 (1H, m). Anal. (C14H18N4-
OS) C, calcd 57.91; found 57.29; H, calcd 6.25; found 5.99; N,
calcd 19.29; found 18.76.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[(1,3-thiazol-2-ylthio)methyl]-
cyclohexanecarboxamide (12y). 3/2 EtOAc/hexanes, colorless
solid. 1H NMR: δ 7.86 (1H, br s), 7.66 (1H, d, J ) 3.4 Hz), 7.47
(1H, d, J ) 3.4 Hz), 4.28-4.16 (2H, m), 3.39 (1H, dd, J ) 3.2, 13
Hz), 3.07 (1H, dd, J ) 8.2, 13 Hz), 2.20 (1H, m), 2.09-1.98 (2H,
overlapped m), 1.86 (1H, m), 1.78-1.69 (2H, m), 1.48 (1H, m),
1.30-1.22 (2H, m), 1.11 (1H, m). Anal. (C13H17N3OS2) C, calcd
52.85; found 52.75; H, calcd 5.80; found 5.59; N, calcd 14.22; found
13.95.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(5-pyridin-4-yl-1,3,4-oxa-
diazol-2-yl)thio]methyl}cyclohexanecarboxamide (12z). 95/5
EtOAc/hexanes, colorless solid. 1H NMR: δ 8.84 (2H, d, J ) 4.5
Hz), 8.00 (2H, dd, J ) 4.4 Hz), 7.92 (1H, br s), 4.31-4.23 (2H,
m), 3.54 (1H, dd, J ) 3.2, 13 Hz), 3.17 (1H, dd, J ) 8.4, 13 Hz),
2.32-2.24 (1H, m), 2.18-2.06 (2H, overlapped m), 1.92 (1H, m),
1.83-1.75 (2H, m), 1.52 (1H, m), 1.34-1.28 (2H, m), 1.19 (1H,
m). Anal. (C17H19N5O2S) C, calcd 57.12; found 56.48; H, calcd
5.36; found 5.00; N, calcd 19.59; found 18.92.
(1R/S,2R/S)-2-{[(3-Aminophenyl)thio]methyl}-N-(cyanometh-
yl)cyclohexanecarboxamide (12p). 5/95 MeOH/CH2Cl2, colorless
1
solid. H NMR: δ 7.79 (1H, br s), 6.97 (1H, t, J ) 7.8 Hz), 6.68
(1H, s), 6.57 (1H, d, J ) 7.6 Hz), 6.47 (1H, dd, J ) 2.1, 8.0 Hz),
4.61 (2H, br s), 4.29-4.21 (2H, m), 3.11 (1H, dd, J ) 3.0, 13 Hz),
2.59 (1H, dd, J ) 9.2, 13 Hz), 2.19 (1H, m), 2.14 (1 H, m), 1.95
(1H, m), 1.84 (1H, m), 1.76-1.74 (2H, m), 1.48 (1H, m), 1.31-
1.24 (2H, m), 1.04 (1H, m). Anal. (C16H21N3OS) C, calcd 63.33;
found 62.87; H, calcd 6.98; found 6.98; N, calcd 13.85; found 13.60.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(3,5-dichlorophenyl)thio]-
methyl}cyclohexanecarboxamide (12q). 2/3 EtOAc/hexanes, col-
orless solid. 1H NMR: δ 7.89 (1H, br s), 7.34 (2H, d, J ) 1.8 Hz),
7.24 (1H, t, J ) 1.8 Hz), 4.31-4.24 (2H, m), 3.21 (1H, dd, J )
3.1, 13 Hz), 2.70 (1H, t, J ) 6.6 Hz), 2.22 (1H, m), 2.15 (1H, m),
1.99 (1H, m), 1.89 (1H, m), 1.80-1.74 (2H, m), 1.48 (1H, m),
1.30-1.26 (2H, m), 1.09 (1H, m). Anal. (C16H18Cl2N2OS) C, calcd
53.78; found 53.38; H, calcd 5.08; found 4.92; N, calcd 7.84; found
7.66.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(2,4-dichlorophenyl)thio]-
methyl}cyclohexanecarboxamide (12r). 2/3 EtOAc/hexanes, col-
orless solid. 1H NMR: δ 7.92 (1H, br s), 7.53 (1H, d, J ) 8.6 Hz),
7.48 (1H, d, J ) 2.2 Hz), 7.36 (1H, dd, J ) 2.2, 8.5 Hz), 4.32-
4.24 (2H, m), 3.19 (1H, dd, J ) 2.9, 13 Hz), 2.69 (1H, dd, J )
9.8, 13 Hz), 2.24 (1H, m), 2.18 (1H, m), 1.98 (1H, m), 1.89 (1H,
m), 1.82-1.72 (2H, m), 1.49 (1H, m), 1.32-1.28 (2H, m), 1.10
(1H, m). Anal. (C16H18Cl2N2OS) C, calcd 53.78; found 53.37; H,
calcd 5.08; found 4.85; N, calcd 7.84; found 7.70.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(2,5-dichlorophenyl)thio]-
methyl}cyclohexanecarboxamide (12s). 2/3 EtOAc/hexanes, col-
orless solid. 1H NMR: δ 7.92 (1H, br s), 7.52 (1H, d, J ) 2.3 Hz),
7.41 (1H, d, J ) 8.5 Hz), 7.19 (1H, dd, J ) 2.4, 8.4 Hz), 4.34-
4.24 (2H, m), 3.20 (1H, dd, J ) 3.0, 13 Hz), 2.73 (1H, dd, J )
9.7, 13 Hz), 2.25 (1H, m), 2.18 (1H, m), 2.05 (1H, overlapped m),
1.92 (1H, m), 1.82-1.75 (2H, m), 1.51 (1H, m), 1.32-1.28 (2H,
m), 1.13 (1H, m). Anal. (C16H18Cl2N2OS) C, calcd 53.78; found
53.45; H, calcd 5.08; found 4.87; N, calcd 7.84; found 7.67.
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(2,4,5-trichlorophenyl)thio]-
methyl}cyclohexanecarboxamide (12t). 2/3 EtOAc/hexanes, col-
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(4-fluorophenyl)sulfonyl]-
methyl}cyclohexanecarboxamide (13a). Compound 12b (105 mg,
0.344 mmol), Oxone (595 mg, 0.970 mmol), and NaHCO3 (595
mg, 7.1 mmol) were stirred together in a mixture of MeOH (2 mL),
THF (2 mL), and H2O (2 mL) at room temperature until completion
of the reaction as judged by TLC (7 h). The mixture was then
poured into water and extracted with ethyl acetate. The organic
phase was dried (Na2SO4) and concentrated to afford the title
1
compound in high purity as a colorless solid (110 mg, 95%). H
NMR: δ 7.95 (2H, m), 7.75 (1H, br s), 7.40 (2H, m), 4.14 (2H, d,

â-Substituted Cyclohexanecarboxamide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1075
J ) 5.8 Hz), 3.14 (1H, dd, J ) 2.4, 14 Hz), 3.05 (1H, dd, J ) 9.4,
14 Hz), 2.31 (1H, m), 2.20 (1H, dt, J ) 3.6, 11 Hz), 2.10 (1H, m),
1.81 (1H, m), 1.76-1.64 (2H, m), 1.42 (1H, m), 1.32-1.15 (3H,
m). Anal. (C16H19FN2O3S) C, calcd 55.71; found 55.50; H, calcd
6.05; found 5.81; N, calcd 7.64; found 7.35.
hexanecarboxylate (14)7 (1.82 g, 10.0 mmol) in THF (10 mL). The
reaction vessel contents were warmed to room temperature and then
slowly to 50 °C for 1 h. Water was added at room temperature,
and the mixture was extracted with ether (2×). The combined
organics were washed with brine, dried (Na2SO4), and concentrated.
Flash chromatography of the residue on silica gel eluting with 1/9
EtOAc/hexanes gave the title compound as a colorless, thick oil
(1R/S,2R/S)-N-(Cyanomethyl)-2-{[(4-(methylthio)-
phenylsulfonyl]methyl}cyclohexanecarboxamide (13b). A mix-
ture of 13a (22 mg, 64 µmol) and sodium thiomethoxide (50 mg,
700 µmol) was stirred together in DMF (1.5 mL) at room
temperature overnight (16 h). The reaction vessel contents were
then partitioned between water and EtOAc, and the layers were
separated. The organic phase was washed with brine and dried (Na2-
SO4). Concentration in vacuo and flash chromatography of the
residue on silica gel eluting with 15/85 acetone/benzene gave the
1
(1.88 g, 62%, Z/E 5.7:1). H NMR: δ 7.29 (2H, d, J ) 8.3 Hz),
7.19 (2H, d, J ) 8.3 Hz), 6.33 (1H, d, J ) 16 Hz), 6.08 (1H, dd,
J ) 8.2, 16 Hz), 4.05-3.95 (2H, m), 2.46 (3H, s), 2.32 (1H, m),
2.20 (1H, m), 1.90-1.68 (4H, m), 1.48 (1H, m), 1.40-1.20 (3H,
m), 1.09 (3H, t, J ) 7.1 Hz); discernible signals for E-isomer, δ
7.25 (2H, d, J ) 8.6 Hz), 7.22 (2H, d, J ) 8.6 Hz), 5.40 (1H, t, J
) 10 Hz), 4.00-3.90 (2H, m).
1
(1R/S,2S/R)-2-{(Z)-2-[4-(Methylthio)phenyl]vinyl}-
cyclohexanecarboxylic Acid (17). A 2 M aqueous solution of
lithium hydroxide (9 mL) was added to a solution of 16 (280 mg,
0.921 mmol) in DME (15 mL) and methanol (2 mL) with rapid
stirring at 60 °C for 5 h. The reaction mixture was then cooled to
room temperature, diluted with water, and extracted with ether prior
to acidification to pH 2 with 2 M HCl and extraction with ethyl
acetate (2×). The combined ethyl acetate extracts were washed with
brine, dried (Na2SO4), and concentrated to afford the title compound
title compound as a colorless solid (21 mg, 93%). H NMR: δ
7.75 (2H, d, J ) 8.6 Hz), 7.74 (1H, br s), 7.45 (2H, d, J ) 8.6 Hz),
4.15 (2H, d, J ) 5.8 Hz), 3.12 (1H, dd, J ) 2.4, 14 Hz), 3.01 (1H,
dd, J ) 9.4, 14 Hz), 2.58 (3H, s), 2.31 (1H, m), 2.20 (1H, dt, J )
3.9, 11 Hz), 2.12 (1H, overlapped m), 1.82 (1H, m), 1.73-1.66
(2H, m), 1.43 (1H, m), 1.29-1.18 (3H, m). Anal. (C17H22N2O3S2)
C, calcd 55.71; found 55.50; H, calcd 6.05; found 5.81; N, calcd
7.64; found 7.35.
Synthesis of Radiolabeled Compound 13d. A 4 M aqueous
solution of sodium nitrite (7.0 mL) was added to a slurry of
2-bromo-4-trifluoromethoxyaniline in 6 M HCl (10 mL) at 0 °C.
This heterogeneous mixture was added dropwise to a 70 °C solution
of potassium xanthate (5.0 g, 31 mmol) in water (70 mL) with
stirring at this temperature for 45 min. The solution was then cooled
to room temperature and extracted with two portions of ether. The
extracts were dried (Na2SO4) and concentrated, the residue was
taken up in a mixture of ethanol (40 mL) and 2 M KOH (40 mL),
and the solution was heated to 85 °C for 2 h, before being cooled
to room temperature and extracted with ether (3×, discard). The
aqueous phase was acidified to pH 2-3 with 6 M HCl and extracted
with ether (3×). The second set of ether extracts were combined,
washed with water and brine, and dried (Na2SO4). Concentration
in vacuo and flash chromatography of the residue on silica gel
eluting with 2/98 ethyl acetate/hexanes yielded 2-bromo-4-trifluo-
romethoxythiophenol as a colorless oil (2.19 g, 31%). A mixture
of this thiophenol (60 mg, 220 µmol), 11 (51 mg, 150 µmol), and
K2CO3 (100 mg, 730 µmol) in DMF (0.40 mL) was stirred together
at room temperature for 22 h. The reaction vessel contents were
then poured into water and extracted with ethyl acetate (3×). The
combined organics were washed with brine, dried (Na2SO4) and
concentrated, and the residue was flashed on silica gel eluting with
1/5 acetone/benzene to give 2-({[2-bromo-4-(trifluoromethoxy)-
phenyl]thio}methyl)-N-(cyanomethyl)cyclohexanecarboxamide (27)
as a colorless solid (59 mg, 90%). Compound 27 (56 mg, 120 µmol)
was dissolved in a mixture of methanol, ethyl acetate, and water
(1 mL each) and treated with NaHCO3 (300 mg, 3.60 mmol) and
Oxone (300 mg, 490 mol) with heating at 40 °C for 1 h. Water
was added to dissolve the salts, and the mixture was extracted with
ethyl acetate (2×). The combined organics were washed with brine,
dried (Na2SO4), and concentrated to afford 2-({[2-bromo-4-
(trifluoromethoxy)phenyl]sulfonyl}methyl)-N-(cyanomethyl)cyclo-
hexanecarboxamide (28) as a colorless solid (60 mg, 99%).
Compound 28 (3.6 mg, 7.5 µmol) was dissolved in THF (0.60 mL)
containing Et3N (50 µL) and stirred with 10% Pd on charcoal (4
mg) under an atmosphere of tritium gas (P ) 598 mmHg) overnight.
The mixture was then filtered (Celite) and the filtrate was
concentrated and the residue was purified by reverse phase HPLC
(C18 radial pack, 65/35 MeOH/10 mM NH4OAc aqueous buffer,
17 mL/min flow rate, λ 220 nM) to afford 13d (1.2 mg, specific
activity 12 Ci/mmol).
1
as a faint-yellow foam (180 mg, 71%, Z/E 5.2:1). H NMR: δ
10.50 (1H, br s), 7.29 (2H, d, J ) 8.3 Hz), 7.18 (2H, d, J ) 8.3
Hz), 6.37 (1H, d, J ) 16 Hz), 6.14 (1H, dd, J ) 8.1, 16 Hz), 2.46
(3H, s), 2.38 (1H, m), 2.25 (1H, m), 1.95 (1H, m), 1.87-1.68 (3H,
m), 1.50 (1H, m), 1.45-1.20 (3H, m); discernible signals for
E-isomer, δ 6.30 (1H, d, J ) 12 Hz), 5.45 (1H, t, J ) 11 Hz), 2.48
(3H, s).
(1R/S,2S/R)-N-(Cyanomethyl)-2-{(Z)-2-[4-(methylthio)phenyl]-
vinyl}cyclohexanecarboxamide (18). A mixture of 17 (180 mg,
0.655 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP, 375 mg, 0.721 mmol), and ami-
noacetonitrile hydrochloride (134 mg, 1.44 mmol) in DMF (5.0
mL) was cooled to 0 °C and treated with triethylamine (0.33 mL,
2.3 mmol). The resulting slurry was stirred at room temperature
for 2.5 h and then poured into saturated NaHCO3 aqueous solution
and extracted with ether (2×). The combined extracts were washed
with brine and dried (Na2SO4). Concentration in vacuo and
chromatography of the residue on silica eluting with 2/3 EtOAc/
hexanes yielded the title compound as a colorless foam (177 mg,
86%, Z/E 3.4:1). 1H NMR: δ 7.31 (2H, d, J ) 8.3 Hz), 7.30 (1H,
overlapped m), 7.19 (2H, d, J ) 8.3 Hz), 6.36 (1H, d, J ) 16 Hz),
6.12 (1H, dd, J ) 7.8, 16 Hz), 4.24-4.05 (2H, m), 2.48 (3H, s),
2.42 (1H, m), 2.18 (1H, m), 1.90-1.65 (4H, m), 1.60 (1H, m),
1.45-1.15 (3H, m); discernible signals for E-isomer, δ 7.34 (2H,
d, J ) 8.2 Hz), 7.23 (2H, d, J ) 8.2 Hz), 5.42 (1H, t, J ) 11 Hz),
4.12-4.02 (2H, m), 2.51 (3H, s). Anal. (C18H22N2OS) C, calcd
68.32; found 68.35; H, calcd 7.64; found 7.57; N, calcd 8.85; found
8.72.
(1R/S,2S/R)-N-(Cyanomethyl)-2-{2-[4-(methylthio)phenyl]-
ethyl}cyclohexanecarboxamide (19). A solution of 17 (410 mg,
0.830 mmol) in a mixture of equal portions of methanol, ethyl
acetate, and acetic acid was stirred under 40 PSI of hydrogen in
the presence of 10% Pd on charcoal (400 mg) until the reaction
was judged complete by TLC (6 h). The reaction flask contents
were filtered through Celite, and the filtrate was concentrated. Flash
chromatography of the residue on silica gel eluting with 1/4 EtOAc/
hexanes containing 0.5% acetic acid gave 220 mg (59%) of material
that was coupled to aminoacetonitrile as for 18 to afford the title
1
compound as a colorless foam (217 mg, 87%). H NMR: δ 7.74
(1H, br s), 7.20-7.07 (4H, m), 4.20 (2H, m), 2.56 (1H, m), 2.44
(3H, s), 2.44 (1H, overlapped m), 2.05-1.92 (3H, m), 1.85-1.59
(4H, m), 1.45 (1H, m), 1.38-1.16 (3H, m), 1.00 (1H, m). Anal.
(C18H24N2OS) C, calcd 68.75; found 67.92; H, calcd 7.05; found
6.83; N, calcd 8.91; found 8.64.
(1R/S,2S/R)-2-{(Z)-2-[4-(Methylthio)phenyl]vinyl}-
cyclohexanecarboxylate (16). A mixture of p-(methylthio)benzyl
bromide (2.24 g, 13.1 mmol) and PPh3 (3.43 g, 13.1 mmol) was
heated at 90 °C for 15 h. The resulting slurry was then cooled to
room temperature and treated with potassium tert-butoxide (11.7
mL, 1.0 M in THF) with stirring for 30 min prior to transfer via
cannula into a 0 °C solution of ethyl (1R/S,2R/S)-2-formylcyclo-
(1R/S,6R/S)-6-(2-Bromophenyl)cyclohex-3-ene-1-carboxylic Acid
(21). A mixture of 2-bromocinnamic acid (20) (90 g, 400 mmol),
toluene (240 mL), 1,4-hydroquinone (0.50 g), and 1,4-butadiene
(150 mL) was heated together in a stainless steel bomb at 200 °C

1076 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Crane et al.
for 24 h. After being cooled to room temperature, the bomb was
cooled in an ice/water bath and the solid that crystallized from the
reaction mixture was collected by suction filtration, washed with
cold toluene (3×), and dried in air, then in vacuo, to afford the
title compound as a colorless solid (92 g, 82% yield). 1H NMR: δ
10.58 (1H, br s), 7.55 (1H, d, J ) 8.6 Hz), 7.41 (1H, d, J ) 7.8
Hz), 7.33 (1H, t, J ) 7.6 Hz), 7.11 (1H, t, J ) 7.8 Hz), 5.78 (2H,
m), 3.59 (1H, m), 3.07 (1H, m), 2.50-2.32 (3H, m), 1.98 (1H,
overlapped m). Resolution procedure: A solution of 21 (10.0 g,
36 mmol) in THF (100 mL) was maintained at 60 °C with stirring
in an oil bath. (R)-Phenethylamine (4.6 mL, 36 mmol) was slowly
added, and the solution was cooled slowly to room temperature
(crystallization started at 45 °C) over 1.5 h while immersed in the
oil bath. The solid was collected by suction filtration and washed
with the minimum amount of THF (3×) required to cover the solid.
The product was dried under a stream of nitrogen and then under
high vacuum to afford the amine salt as a colorless solid (6.66 g).
The free acid was liberated from the salt by treatment with 2 M
HCl and extraction with ethyl acetate to afford (-)-21 as a colorless
solid (2.82 g, [R]D -62 (c ) 1.0, CHCl3)) in 93% ee as determined
by HPLC analysis (Chiralpak AD, 1 mL/min, 2-propanol:hexanes
1:140, λ ) 227 nm) of the corresponding methyl ester. The same
procedure using (S)-phenethylamine gave (+)-21 as a colorless solid
in 93% ee, [R]D +60 (c ) 0.98, CHCl3).
Cl2 (18 mg, 0.022 mmol, 5 mol %), and 2 M Na2CO3 aqueous
solution (0.31 mL, 0.62 mmol) were heated at 85 °C in DMF (1.2
mL). After 16 h at this temperature, the reaction mixture was cooled
to room temperature and partitioned between ethyl acetate and
water, and the layers were separated. The aqueous phase was
extracted with additional ethyl acetate, and the combined organics
were washed with brine and dried (Na2SO4). Concentration in vacuo
and chromatography of the residue on silica gel eluting with 8/92
acetone/benzene gave the title compound as a faint-yellow foam
1
(117 mg, 85%). H NMR: δ 7.53 (1H, br s), 7.44 (2H, m), 7.40
(1H, d, J ) 7.8 Hz), 7.28 (1H, t, J ) 7.4 Hz), 7.17 (3H, m), 7.07
(1H, d, J ) 7.5 Hz), 3.97 (2H, m), 3.04 (1H, m), 2.75 (1H, m),
1.87 (1H, m), 1.76-1.58 (3H, m), 1.42 (1H, m), 1.37-1.23 (2H,
m), 1.08 (1H, m). Anal. (C21H21FN2O) C, calcd 74.98; found 73.99;
H, calcd 6.29; found 6.00; N, calcd 8.33; found 8.18.
(1R/S,2R/S)-N-(Cyanomethyl)-2-({[4-(methylsulfinyl)phenyl]-
sulfonyl}methyl)-cyclohexanecarboxamide (25a) and (1R/S,2R/
S)-N-(Cyanomethyl)-2-({[4-(methylsulfonyl)-phenyl]sulfonyl}-
methyl)cyclohexanecarboxamide (25c). A solution of 13b (35 mg,
96 µmol) in CH2Cl2 (0.5 mL) was treated at 0 °C with m-CPBA
(18 mg, 100 µmol) with stirring at this temperature for 1 h. Calcium
hydroxide (50 mg) was then added, and the mixture was stirred at
room temperature for 15 min before filtration through a plug of
Celite and washing well with CH2Cl2. Concentration of the filtrate
and flash chromatography of the residue on silica gel eluting first
with EtOAc and then 30/70 THF/EtOAc afforded 25a as a colorless
foam (28 mg) and 25c as a colorless solid (5 mg). For 25a (1:1
mixture of diastereomeric sulfoxides): 1H NMR δ 8.09 (2H, d, J
) 8.4 Hz), 7.97 (2H, d, J ) 8.0 Hz), 7.78 (1H, br s), 4.18-4.16
(2H, m), 3.22 (1H, d, J ) 13 Hz), 3.13 (1H, dd, J ) 9.1, 14 Hz),
2.821 (3H, s, methyl of one diastereomer), 2.819 (3H, s, methyl of
the other diastereomer), 2.36 (1H, m), 2.28-2.22 (2H, m), 2.19
(1H, m), 2.09 (1H, overlapped m), 1.85 (1H, m), 1.77-1.70 (2H,
m), 1.44 (1H, m), 1.28 (1H, m). For 25c: 1H NMR δ 8.24 (2H, d,
J ) 8.5 Hz), 8.18 (2H, d, J ) 8.5 Hz), 7.80 (1H, br s), 4.17 (2H,
d, J ) 5.8 Hz), 3.29-3.23 (1H, m), 3.26 (3H,s), 3.19 (1H, m),
2.37 (1H, m), 2.26 (1H, m), 2.19 (1H, m), 1.86 (1H, m), 1.79-
1.71 (2H, m), 1.45 (1H, m), 1.36-1.21 (3H, m).
(1R/S,2R/S)-2-(2-Bromophenyl)cyclohexanecarboxylic Acid
(22). A solution of 21 (5.30 g, 18.9 mmol) in EtOAc (110 mL)
was stirred under an atmosphere of hydrogen in the presence of
10% Pd on charcoal (0.50 g) for 12 h. The mixture was filtered
(Celite) and concentrated to afford the title compound as a pale
1
yellow solid (5.23 g, 98% yield). H NMR: δ 10.45 (1H, br s),
7.55 (1H, d, J ) 8.6 Hz), 7.39 (1H, d, J ) 7.8 Hz), 7.30 (1H, t, J
) 7.6 Hz), 7.09 (1H, t, J ) 7.8 Hz), 3.30 (1H, m), 2.83 (1H, m),
2.12 (1H, m), 1.92-1.68 (3H, m), 1.57 (1H, m), 1.50-1.35 (2H,
m), 1.26 (1H, m).
(1R/S,2R/S)-2-(2-Bromophenyl)-N-(cyanomethyl)cyclohex-
anecarboxamide (23). A mixture of 22 (5.23 g, 18.5 mmol),
PyBOP (10.58 g, 20.3 mmol), and aminoacetonitrile hydrochloride
(3.81 g, 41.2 mmol) in DMF (50 mL) was cooled to 0 °C and treated
with triethylamine (9.0 mL, 65 mmol). The resulting slurry was
stirred at room temperature for 2 h and then poured into saturated
NaHCO3 aqueous solution and extracted with ethyl acetate (2×).
The combined extracts were washed with brine and dried (Na2-
SO4). Concentration in vacuo and chromatography of the residue
on silica eluting with 1/4 acetone/benzene yielded the title
compound as a pale-yellow solid (5.76 g, 97%). 1H NMR: δ 7.67
(1H, br s), 7.52 (1H, d, J ) 8.6 Hz), 7.36 (1H, d, J ) 7.8 Hz), 7.27
(1H, t, J ) 7.6 Hz), 7.06 (1H, t, J ) 7.8 Hz), 4.00 (2H, m), 3.40
(1H, m), 2.80 (1H, m), 2.00 (1H, m), 1.93-1.75 (3H, m), 1.63
(1H, m), 1.54-1.32 (2H, m), 1.21 (1H, m).
(1R/S,2R/S)-N-(Cyanomethyl)-2-[4′-(methylsulfinyl)biphenyl-
2-yl]cyclohexanecarboxamide (25b) and (1R/S,2R/S)-N-(Cya-
nomethyl)-2-[4′-(methylsulfonyl)biphenyl-2-yl]cyclohexanecar-
boxamide (25d). The procedure was the same as for the preparation
of 25a and 25c. Flash chromatography was conducted using 22/78
acetone/benzene to elute 25d and then 1/2 acetone/benzene to elute
25b. For 25b (1:1 mixture of diastereomeric sulfoxides): colorless
1
foam. H NMR: δ 7.77 (1H, d, J ) 8.5 Hz), 7.67-7.64 (2H, m),
7.56 (1H, br m), 7.46 (1H, d, J ) 7.9 Hz), 7.34 (1H, t, J ) 7.5
Hz), 7.23 (1H, t, J ) 7.5 Hz), 7.14 (1H, d, J ) 7.6 Hz), 4.06-3.96
(2H, m), 3.07 (1H, m), 2.80 (1H, overlapped m), 1.93 (1H, m),
1.77-1.71 (2H, m), 1.67 (1H, m), 1.49-1.28 (3H, m), 1.13 (1H,
(1R/S,2R/S)-N-(Cyanomethyl)-2-[4′-(methylthio)biphenyl-2-yl]-
cyclohexanecarboxamide (24a). Compound 23 (498 mg, 1.55
mmol), 4-(methylthio)benzeneboronic acid (286 mg, 1.70 mmol),
PdCl2(dppf)‚CH2Cl2 (62 mg, 0.076 mmol, 5 mol %), and 2 M Na2-
CO3 aqueous solution (1.2 mL, 2.4 mmol) were heated at 85 °C in
DMF (3.0 mL). After 16 h at this temperature, the reaction mixture
was cooled to room temperature and partitioned between ethyl
acetate and water, and the layers were separated. The aqueous phase
was extracted with additional ethyl acetate, and the combined
organics were washed with brine and dried (Na2SO4). Concentration
in vacuo and chromatography of the residue on silica gel eluting
with 1/1 ethyl acetate/hexanes gave the title compound as a faint-
1
m). For 25d: colorless foam. H NMR: δ 7.99 (2H, d, J ) 8.6
Hz), 7.69 (2H, m), 7.56 (1H, m), 7.45 (1H, d, J ) 7.8 Hz), 7.34
(1H, t, J ) 7.4 Hz), 7.22 (1H, t, J ) 7.5 Hz), 7.11 (1H, d, J ) 6.5
Hz), 3.97 (2H, m), 3.19 (3H, s), 3.00 (1H, dt, J ) 2.9, 11 Hz),
2.77 (1H, dt, J ) 3.2, 11 Hz), 1.87 (1H, m), 1.75-1.68 (2H, m),
1.66 (1H, m), 1.45-1.27 (3H, m), 1.12 (1H, m).
(1R,2R,4R,5S)-4-Bromo-2-(2-bromophenyl)-6-oxabicyclo[3.2.1]-
octan-7-one (29). Bromotrimethylsilane (1.96 mL, 15.1 mmol) was
added dropwise to a 0 °C solution of dimethyl sulfoxide (1.10 mL,
15.5 mmol) in chloroform (15 mL) with stirring at this temperature
for 30 min. Compound (-)-21 (4.24 g, 15.1 mmol) was then added
as a solid with stirring at room temperature for 1 h. The mixture
was recooled to 0 °C prior to the addition of diisopropylethylamine
(2.65 mL, 15.2 mmol) followed by heating at reflux for 24 h. The
reaction vessel contents were cooled to room temperature, diluted
with ethyl acetate, and washed in succession with water, 5% HCl,
water, and brine, and the organic phase was dried over sodium
sulfate. Concentration in vacuo afforded an oily solid that was used
directly in the next step. Flash chromatography of a portion on
silica gel eluting with 7.5/92.5 acetone/benzene (Rf ) 0.76) afforded
an analytical sample of the title compound as a colorless foam. 1H
1
yellow foam (370 mg, 66%). H NMR: δ 7.79 (1H, br s), 7.42-
7.33 (3H, m), 7.32-7.28 (2H, m), 7.27 (1H, m), 7.16 (1H, t, J )
7.5 Hz), 7.06 (1H, d, J ) 7.6 Hz), 4.04 (2H, m), 3.11 (1H, m),
2.75 (1H, m), 2.52 (3H, s), 1.87 (1H, m), 1.73-1.59 (3H, m), 1.44
(1H, m), 1.38-1.24 (2H, m), 1.12 (1H, m). Anal. (C22H24N2OS)
C, calcd 72.49; found 71.70; H, calcd 6.64; found 6.59; N, calcd
7.69; found 7.48.
(1R/S,2R/S)-N-(Cyanomethyl)-2-[4′-fluorobiphenyl-2-yl]cyclo-
hexanecarboxamide (24b). Compound 23 (132 mg, 0.410 mmol),
4-fluorobenzeneboronic acid (69 mg, 0.49 mmol), PdCl2(dppf)‚CH2-

â-Substituted Cyclohexanecarboxamide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1077
NMR: δ 7.71 (1H, dd, J ) 1.4, 7.8 Hz), 7.65 (1H, dd, J ) 1.2, 8.0
Hz), 7.45 (1H, dt, J ) 1.2, 7.5 Hz), 7.26 (1H, dt, J ) 1.5, 7.7 Hz),
5.08 (1H, dd, J ) 6.8, 11.5 Hz), 5.00 (1H, dd, J ) 6.8, 11.5 Hz),
3.78 (1H, m), 3.03 (1H, m), 2.74 (1H, dd, J ) 6.7, 16 Hz), 2.57
(1H, ddd, J ) 8.2, 12, 15 Hz), 2.35 (1H, m), 1.98 (1H, d, J ) 13
Hz).
was then diluted with water and extracted with ether prior to
acidification to pH 2 with 2 M HCl and extraction with ethyl acetate
(2×). The combined ethyl acetate extracts were washed with brine,
dried (Na2SO4), and concentrated to afford crude (1R,2R)-2-(2-
bromophenyl)-5,5-difluorocyclohexanecarboxylic acid as a tan foam
(1.63 g). A mixture of the crude acid (820 mg, 2.58 mmol), PyBOP
(1.49 g, 2.86 mmol), and aminoacetonitrile hydrochloride (530 mg,
5.73 mmol) in DMF (5.0 mL) was cooled to 0 °C and treated with
triethylamine (1.26 mL, 9.04 mmol). The resulting slurry was stirred
at room temperature for 2.5 h and then poured into water and
extracted with ethyl acetate (3×). The combined extracts were
washed with brine and dried (Na2SO4). Concentration in vacuo and
chromatography of the residue on silica gel eluting with 2/3 EtOAc/
hexanes yielded the title compound as a faint-yellow foam (579
mg, 63% yield for three steps). 1H NMR: δ 7.83 (1H, br, s), 7.55
(1H, d, J ) 8.0 Hz), 7.39 (1H, d, J ) 7.0 Hz), 7.30 (1H, t, J ) 7.3
Hz), 7.11 (1H, m), 4.02 (2H, m), 3.54 (1H, m), 3.12 (1H, m), 2.30
(1H, m), 2.24-1.93 (4H, m), 1.54 (1H, m).
Methyl (1R,2R,4R,5S)-4-Bromo-2-(2-bromophenyl)-5-hydroxy-
cyclohexanecarboxylate (30). Freshly prepared sodium methoxide
(35 mL, 0.45M in methanol) was added to a methanol (20 mL)
solution of 29 (5.43 g, 15.1 mmol) with stirring at room temperature
for 1.5 h. The mixture was then treated with 0.5 M HCl (50 mL),
and the methanol was removed by rotary evaporation under reduced
pressure. The residue was partitioned between water and ethyl
acetate, and the layers were separated. The aqueous phase was
extracted with additional ethyl acetate, and the combined organics
were washed with water, 5% Na2CO3 (2 × 75 mL) and brine, and
dried (Na2SO4). Concentration in vacuo yielded the title compound
as a faint-yellow solid (2.50 g, 42% yield for two steps) after
trituration with ether/hexanes. 1H NMR: δ 7.57 (1H, dd, J ) 1.2,
8.0 Hz), 7.40 (1H, d, J ) 6.5 Hz), 7.31 (1H, t, J ) 7.6 Hz), 7.12
(1H, dt, J ) 1.7, 7.9 Hz), 4.19 (1H, m), 4.00 (1H, m), 3.82 (1H,
m), 3.43 (3H, s), 3.16 (1H, m), 2.80 (1H, m), 2.18-1.97 (4H, m).
Methyl (1R,2R)-2-(2-Bromophenyl)-5-oxocyclohexanecarboxy-
late (31). Jones reagent (2.7 M, 7.0 mL) was added at 0 °C to an
acetone (25 mL) solution of 30 (2.50 g, 6.38 mmol) with stirring
at room temperature for 40 min. The mixture was then diluted with
water and extracted with ether (3×). The combined extracts were
washed with water, saturated aqueous sodium bicarbonate, and brine
solutions, and dried (Na2SO4). Concentration in vacuo gave the
R-bromoketone product as a colorless, thick syrup (2.49 g) that
was taken up in THF (58 mL) and treated with zinc dust (21 g,
330 mmol) and a 1 M solution of KH2PO4 (32 mL) with rapid
stirring at room temperature for 1 h. The mixture was then filtered
(Celite), and the pad was washed well with ethyl acetate and water.
The filtrate was transferred to a separatory funnel, shaken, and the
layers were separated. The organic phase was washed with brine
and dried (Na2SO4). Concentration in vacuo provided the title
compound as a colorless solid (1.71 g, 86% yield for two steps).
1H NMR: δ 7.59 (1H, dd, J ) 1.1, 8.0 Hz), 7.46 (1H, dd, J ) 1.5,
7.9 Hz), 7.34 (1H, t, J ) 7.6 Hz), 7.15 (1H, dt, J ) 1.6, 7.9 Hz),
3.85 (1H, dt, J ) 3.5, 12 Hz), 3.42 (3H, s), 3.38 (1H, m), 2.78
(1H, t, J ) 14 Hz), 2.70 (1H, dt, J ) 6.3, 14 Hz), 2.52 (1H, m),
2.35 (1H, m), 2.16 (1H, m), 1.85 (1H, dq, J ) 4.3, 14 Hz).
Methyl (1R,2R)-2-(2-Bromophenyl)-5,5-difluorocyclohex-
anecarboxylate (32a). A solution of 31 (1.69 g, 5.46 mmol) in
CH2Cl2 (23 mL) was treated with methanol (22 µL, 10 mol %)
and diethylaminosulfur trifluoride (DAST) (1.73 mL, 13.1 mmol)
with stirring at room temperature for 2 h. The reaction vessel
contents were then diluted with dichloromethane and washed with
water, saturated sodium bicarbonate aqueous solution, and brine,
and dried (Na2SO4). A 20% volume of methanol was introduced
to the dichloromethane solution, and the mixture was cooled to
-78 °C and treated with excess ozone until a blue-green color
persisted (about 30 min). A solution of 30% H2O2 (10 mL) in
methanol (10 mL) was then added, and the reaction vessel contents
were warmed to room temperature and concentrated. The residue
was taken up in ether and washed with 10% Na2CO3 aqueous
solution, water, and brine, and dried (Na2SO4/MgSO4). Concentra-
tion in vacuo afforded a cloudy, faint-yellow syrup (1.81 g) that
was used directly in the next step. Purification of a portion of this
material by flash chromatography on silica gel eluting with 1/9
ethyl acetate/hexanes afforded an analytical sample of the title
(1R/S,2R/S)-N-(Cyanomethyl)-5,5-dichloro-[4′-(methylthio)-
1,1′-biphenyl-2-yl]cyclohexanecarboxamide (rac-34c). A solution
of rac-31 (663 mg, 2.12 mmol) in toluene (7 mL) was added to a
0 °C solution of PCl5 (1.40 g, 6.70 mmol) in toluene (7 mL)
followed by stirring at room temperature for 4 h. The mixture was
then recooled to 0 °C, and a 2 M aqueous solution of NaOH was
added to pH 9 followed by extraction with two portions of ether.
The combined organics were washed with water and saturated NaCl
aqueous solution before drying over MgSO4. Concentration in vacuo
and flash chromatography of the residue on silica gel eluting with
5/95 ethyl acetate/hexanes afforded methyl (1R/S,2R/S)-2-(2-
bromophenyl)-5,5-dichlorocyclohexanecarboxylate as a colorless
solid (377 mg, 43%). 1H NMR: δ 7.57 (1H, d, J ) 7.8 Hz), 7.40-
7.33 (2H, m), 7.18-7.12 (1H, m), 3.50 (1H, m), 3.44 (3H, s), 3.29
(1H, m), 2.84 (1H, m), 2.65-2.50 (3H, m), 1.80 (1H, m), 0.87
(1H, m). A 2 M aqueous solution of lithium hydroxide (5.2 mL)
was added to a solution of the above compound (377 mg, 1.03
mmol) in a mixture of methanol (7 mL) and THF (2.5 mL) with
rapid stirring at room temperature for 15 h. The reaction mixture
was then diluted with water and extracted with ether prior to
acidification to pH 2 with 2 M HCl and extraction with ethyl acetate
(2×). The combined ethyl acetate extracts were washed with brine,
dried (Na2SO4), and concentrated to afford crude (1R/S,2R/S)-2-
(2-bromophenyl)-5,5-dichlorocyclohexanecarboxylic acid as a tan
foam (343 mg). A mixture of this crude material (343 mg, 0.97
mmol), PyBOP (555 mg, 1.07 mmol), and aminoacetonitrile
hydrochloride (198 mg, 2.14 mmol) in DMF (4.8 mL) was cooled
to 0 °C and treated with triethylamine (0.50 mL, 3.6 mmol). The
resulting slurry was stirred at room temperature for 16 h and then
was poured into water and extracted with ethyl acetate (3×). The
combined extracts were washed with brine and dried (Na2SO4).
Concentration in vacuo and chromatography of the residue on silica
gel eluting with 2/3 EtOAc/hexanes yielded the (1R/S,2R/S)-2-(2-
bromophenyl)-N-(cyanomethyl)-5,5-dichlorocyclohexanecarboxa-
1
mide as a colorless solid (343 mg, 91% yield for two steps). H
NMR: δ 7.94 (1H, br m), 7.56 (1H, d, J ) 7.8 Hz), 7.41 (1H, m),
7.32 (1H, m), 7.13 (1H, m), 4.05-4.00 (2H, m), 3.56 (1H, m),
3.30 (1H, m), 2.78 (1H, m), 2.67-2.48 (3H, m), 1.62 (1H, m),
0.87 (1H, m). A mixture of this compound (100 mg, 0.26 mmol),
4-(methylthio)benzeneboronic acid (66 mg, 0.39 mmol), PdCl2-
(dppf)‚CH2Cl2 (11 mg, 0.013 mmol, 5 mol %), and 2 M Na2CO3
aqueous solution (0.46 mL, 0.91 mmol) was heated at 85 °C in
DMF (0.65 mL). After 17 h at this temperature, the reaction mixture
was cooled to room temperature and partitioned between ethyl
acetate and water, and the layers were separated. The aqueous phase
was extracted with additional ethyl acetate, and the combined
organics were washed with brine and dried (Na2SO4). Concentration
in vacuo and chromatography of the residue on silica gel eluting
with 35/65 EtOAc/hexanes gave the title compound as a colorless
1
compound as a colorless syrup. H NMR: δ 7.57 (1H, dd, J )
1.2, 8.0), 7.41 (1H, dd, J ) 1.5, 7.8 Hz), 7.34 (1H, dt, J ) 1.1, 7.5
Hz), 7.14 (1H, m), 3.48 (1H, m), 3.42 (3H, s), 3.13 (1H, m), 2.39
(1H, m), 2.24-2.06 (3H, m), 1.97 (1H, m), 1.64 (1H, m).
(1R,2R)-2-(2-Bromophenyl)-N-(cyanomethyl)-5,5-difluorocy-
clohexanecarboxamide (33). A 2 M aqueous solution of lithium
hydroxide (26 mL) was added to a solution of 32a (1.81 g, 5.46
mmol) in a mixture of methanol (20 mL) and THF (10 mL) with
rapid stirring at room temperature for 15 h. The reaction mixture
1
foam (78 mg, 70%). H NMR: δ 7.77 (1H, br m), 7.43 (1H, d, J
) 7.9 Hz), 7.35-7.28 (5H, m), 7.22 (1H, m), 7.11 (1H, d, J ) 6.9
Hz), 3.99 (2H, m), 3.22 (1H, m), 2.62 (1H, m), 2.54 (3H, s), 2.50
(1H, m), 2.38 (1H, m), 2.18 (1H, m), 1.92-1.79 (2H, m), 0.88

1078 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Crane et al.
(1H, m). Anal. (C22H22Cl2N2OS) C, calcd 60.97; found 62.14; H,
calcd 5.96; found 5.22; N, calcd 6.46; found 5.43.
and rac-37b, respectively14) by treatment of a CH2Cl2 solution (0.25
M in alcohol) with DAST (1.5 equiv) at -78 °C followed by slow
warming to -10 °C over 2 h, aqueous workup, and flash
chromatography on silica gel eluting with 1/9 ethyl acetate/hexanes
(1R,2R)-N-(Cyanomethyl)-5,5-difluoro-[4′-(methylthio)-1,1′-
biphenyl-2-yl]cyclohexanecarboxamide (-)-34a. Compound 33
(579 mg, 1.62 mmol), 4-(methylthio)benzeneboronic acid (342 mg,
2.04 mmol), PdCl2(dppf)‚CH2Cl2 (68 mg, 0.083 mmol, 5 mol %),
and 2 M Na2CO3 aqueous solution (1.2 mL, 2.4 mmol) were heated
at 85 °C in DMF (4.6 mL). After 17 h at this temperature, the
reaction mixture was cooled to room temperature and partitioned
between ethyl acetate and water, and the layers were separated.
The aqueous phase was extracted with additional ethyl acetate, and
the combined organics were washed with brine and dried (Na2-
SO4). Concentration in vacuo and chromatography of the residue
on silica gel eluting with 35/65 EtOAc/hexanes gave the title
compound as a faint-yellow foam (563 mg, 86%), [R]D -10 (c )
1
(Rf ) 0.30). For rac-37a: thick colorless syrup; 25% yield. H
NMR: δ 7.60 (1H, d, J ) 7.7 Hz), 7.43-7.35 (2H, m), 7.15 (1H,
m), 5.05 (1H, dm, doublet JH-F ) 48 Hz, multiplet baseline width
) 15 Hz), 3.46 (1H, overlapped m), 3.45 (3H, s), 3.21 (1H,m),
2.36 (1H, m), 2.11 (1H, overlapped m), 1.98 (1H, m), 1.88 (1H,
m), 1.77 (1H, m), 1.67 (1H, m). For rac-37b: thick colorless syrup;
6% yield. 1H NMR: δ 7.59 (1H, d, J ) 7.9 Hz), 7.40 (1H, d, J )
7.0 Hz), 7.34 (1H, t, J ) 7.5 Hz), 7.14 (1H, t, J ) 6.9 Hz) (1H,
dm, doublet JH-F ) 48 Hz, multiplet baseline width ) 40 Hz),
3.45 (3H, s), 3.40 (1H, m), 3.11 (1H, m), 2.42 (1H, m), 2.22 (1H,
m), 1.98 (1H, m), 1.78 (1H, m), 1.68 (1H, m), 1.52 (1H, m).
(1R/S,2R/S,5S/R)-N-(Cyanomethyl)-5-fluoro-2-[4′-(methylth-
io)biphenyl-2-yl]cyclohexanecarboxamide (rac-38a) and (1R/
S,2R/S,5R/S)-N-(Cyanomethyl)-5-fluoro-2-[4′-(methylthio)bi-
phenyl-2-yl]cyclohexanecarboxamide (rac-38b). Compounds rac-
36a and rac-36b were separately transformed as per steps f, g, and
h in Scheme 7 to yield the title compounds as colorless solids. For
rac-38a: 1H NMR δ 7.65 (1H, br s), 7.44 (1H, d, J ) 7.7 Hz),
7.39-7.31 (5H, m), 7.22 (1H, t, J ) 7.4 Hz), 7.12 (1H, d, J ) 7.5
Hz), 4.90 (1H, dm, doublet JH-F ) 48 Hz, multiplet baseline width
) 16 Hz), 4.04-3.96 (2H, m), 3.18 (1H, m), 3.11 (1H, m), 2.57
(3H, s), 2.14 (1H, m), 1.96 (1H, m), 1.80 (1H, m), 1.74-1.64 (2H,
m), 1.60 (1H, m). Anal. (C22H23FN2OS) C, calcd 69.08; found
67.04; H, calcd 6.06; found 5.93; N, calcd 7.32; found 6.92. For
rac-38b: 1H NMR δ 7.57 (1H, s), 7.43 (1H, d, J ) 7.5 Hz), 7.39-
7.33 (4H, m), 7.30 (1H, d, J ) 8.0 Hz), 7.22 (1H, d, J ) 7.0 Hz),
7.12 (1H, dd, J ) 1.1, 7.5 Hz), 4.67 (1H, dm, doublet JH-F ) 49
Hz, multiplet baseline width ) 40 Hz), 4.01 (2H, m), 3.14 (1H,
m), 2.97 (1H, m), 2.58 (3H, s), 2.25 (1H, m), 2.07 (1H, overlapped
m), 1.84 (1H, m), 1.65 (1H, m), 1.54 (1H, m), 1.34 (1H, m).
(1R,2R)-N-(Cyanomethyl)-5,5-difluoro-2-[4′-(methylsulfinyl)-
biphenyl-2-yl]cyclohexanecarboxamide (39a) and (1R,2R)-N-
(Cyanomethyl)-5,5-difluoro-2-[4′-(methylsulfonyl)biphenyl-2-
yl]cyclohexanecarboxamide (39b). A solution of (-)-34a (147
mg, 0.367 mmol) in CH2Cl2 (0.50 mL) was cooled to -15 °C and
treated with m-CBPA (70 mg, 0.40 mmol). The mixture was stirred
at this temperature for 1 h and then warmed slowly to room
temperature over an additional 2 h. CH2Cl2 (3 mL) containing
dimethyl sulfide (10 µL) was added followed 5 min later by Ca-
(OH)2 (200 mg, 2.7 mmol). After being stirred at room temperature
for 15 min, the mixture was filtered (Celite) and concentrated. Flash
chromatography of the residue on silica gel eluting first with 22/
78, then with 1/2 acetone/benzene, afforded 39a (1:1 mixture of
sulfoxide diastereomers) as a colorless solid (137 mg) after
trituration with ether, and sulfone 39b as a colorless solid (13 mg)
after trituration with a mixture of acetone and hexanes. For 39a:
Rf ) 0.13 (22/78 acetone/benzene). 1H NMR: δ 7.76 (2H, m), 7.75
(1H, br s), 7.63 (2H, m), 7.48 (1H, d, J ) 7.8 Hz), 7.38 (1H, t, J
) 7.6 Hz), 7.29 (1H, d, J ) 7.2 Hz), 7.18 (1H, d, J ) 7.6 Hz),
4.02 (2H, m), 3.20 (1H, m), 3.10 (1H, m), 2.80 (3H, s), 2.43 (1H,
m), 2.05 (1H, overlapped m), 2.00-1.86 (2H, m), 1.83-1.76 (2H,
m). For 39b: Rf ) 0.37 (22/78 acetone/benzene). 1H NMR: δ 8.02
(2H, m), 7.75 (1H, br s), 7.70 (2H, m), 7.50 (1H, d, J ) 7.7 Hz),
7.42 (1H, t, J ) 7.4 Hz), 7.31 (1H, t, J ) 7.7 Hz), 7.19 (1H, d, J
) 8.0 Hz), 4.08-3.98 (2H, m), 2.29 (3H, s), 3.18-3.06 (2H, m),
2.26-2.16 (2H, m), 2.01-1.88 (2H, m), 1.87-1.68 (2H, m).
1
1.2, CHCl3). H NMR: δ 7.67 (1H, br s), 7.41 (1H, d, J ) 7.0
Hz), 7.36-7.28 (5H, m), 7.22 (1H, dt, J ) 1.3, 7.5 Hz), 7.10 (1H,
dd, J ) 1.2, 7.6 Hz), 4.00 (2H, m), 3.20 (1H, m), 3.05 (1H, m),
2.83 (3H, s), 2.18 (1H, m), 2.01 (1H, overlapped m), 1.95 (1H,
m), 1.85 (1H, m), 1.75 (1H, m), 1.68 (1H, m). Anal. (C22H22F2N2-
OS) C, calcd 65.98; found 64.63; H, calcd 5.54; found 5.22; N,
calcd 6.99; found 6.85.
(1R/S,2R/S)-N-(Cyanomethyl)-5,5-difluoro-2-[4′-fluorobiphe-
nyl-2-yl]cyclohexanecarboxamide (rac-34b). 4-Fluorobenzenebo-
ronic acid (27 mg, 0.19 mmol), rac-33 (55 mg, 0.15 mmol),
PdCl2(dppf)‚CH2Cl2 (7 mg, 0.009 mmol, 5 mol %), and 2 M Na2-
CO3 aqueous solution (0.12 mL, 0.24 mmol) were heated at 85 °C
in DMF (0.60 mL). After 16 h at this temperature, the reaction
mixture was cooled to room temperature and partitioned between
ethyl acetate and water, and the layers were separated. The aqueous
phase was extracted with additional ethyl acetate, and the combined
organics were washed with brine and dried (Na2SO4). Concentration
in vacuo and chromatography of the residue on silica gel eluting
with 35/65 EtOAc/hexanes gave the title compound as a faint-
yellow solid (49 mg, 85%). 1H NMR: δ 7.69 (1H, br s), 7.42 (3H,
m), 7.33 (1H, t, J ) 7.6 Hz), 7.22 (1H, t, J ) 7.5 Hz), 7.17 (2H,
m), 7.12 (1H, d, J ) 7.6 Hz), 3.99 (2H, m), 3.14 (1H, m), 3.06
(1H, m), 2.18 (1H, m), 2.01 (1H, overlapped m), 1.91 (1H, m),
1.83 (1H, m), 1.74 (1H, m), 1.66 (1H, m). Anal. (C21H19F3N2O) C,
calcd 67.73; found 66.92; H, calcd 5.14; found 4.88; N, calcd 7.52;
found 7.27.
Methyl (1R/S,2R/S,5R/S)-2-(2-Bromophenyl)-5-hydroxycyclo-
hexanecarboxylate (rac-36a) and Methyl (1R/S,2R/S,5S/R)-2-(2-
Bromophenyl)-5-hydroxycyclohexanecarboxylate (rac-36b). Com-
pound rac-31 (2.05 g, 6.61 mmol) was dissolved in methanol (50
mL) and treated with NaBH4 (0.32 g, 8.4 mmol) portionwise at
room temperature. After 30 min, 2 M HCl (16 mL) was added,
and the methanol was evaporated by rotary evaporation under
reduced pressure. The residue was partitioned between ethyl acetate
and water, and the layers were separated. The organic phase was
1
washed with brine, dried (Na2SO4/MgSO4), and concentrated. H
NMR analysis of the crude mixture revealed it to be composed of
two diastereomeric alcohols rac-36a and rac-36b in a ratio of 15:
1. Flash chromatography of the residue on silica gel eluting with
3/2 ethyl acetate/hexanes afforded analytical samples of major
diastereomer rac-36a (1.48 g, Rf ) 0.35) and minor diastereomer
rac-36b (78 mg, Rf ) 0.40) as thick, colorless syrups. For rac-
36a: 1H NMR δ 7.57 (1H, d, J ) 8.0 Hz), 7.42-7.36 (1H, m),
7.32 (1H, t, J ) 7.5 Hz), 7.13-7.11 (1H, m), 3.92 (1H, m, OH),
3.76 (1H, m, baseline width ) 48 Hz), 3.43 (3H, s), 3.34 (1H, m),
3.02 (1H, m), 2.27 (1H, m), 2.05 (1H, overlapped m), 1.91 (1H,
m), 1.56 (1H, q, J ) 12 Hz), 1.49-1.42 (2H, m). For rac-36b: 1H
NMR δ 7.57 (1H, d, J ) 8.0 Hz), 7.37-7.33 (2H, m), 7.12 (1H,
m), 4.19 (1H, narrow m, baseline width ) 22 Hz), 3.84 (1H, s,
OH), 3.42 (3H, s), 3.40-3.32 (2H, m), 2.15-2.09 (2H, m), 1.88
(1H, m), 1.82-1.69 (2H, m), 1.66 (1H, m).
Supporting Information Available: Data and refinement
statistics for the X-ray structure of 13e bound to the active site of
cathepsin K. This material is available free of charge via the Internet
at http://pubs.acs.org.
Methyl (1R/S,2R/S,5S/R)-2-(2-Bromophenyl)-5-fluorocyclo-
hexanecarboxylate (rac-37a) and Methyl (1R/S,2R/S,5R/S)-2-(2-
Bromophenyl)-5-fluorocyclohexanecarboxylate (rac-37b). Equa-
torial alcohol rac-36a and axial alcohol rac-36b were separately
converted into the axial and equatorial fluoro-compounds (rac-37a
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(6) Humanized rabbit cathepsin K (hrab Cat K) was obtained by mutation
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JM051059P