Novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins K and L

Article abstract of DOI:10.1021/jm0003440

Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L with IC₅₀ values of 0.37 and 0.45 μM, respectively. Modification of compound 1 by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a K_i value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chemical reactivity of the compound toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with human cathepsin K. The kinetic data obtained showed that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due to an increase of k_on values and that the association of the compound with the enzyme fits an apparent one-step mechanism. ¹³C NMR experiments performed with the enzyme papain showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by ¹³C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC₅₀ value of 0.7 μM. 1-Cyanopyrrolidine represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and proteolysis of bone collagen.

Full text of DOI:10.1021/jm0003440

94
J . Med. Chem. 2001, 44, 94-104
Novel, Non p ep tid ic Cya n a m id es a s P oten t a n d Rever sible In h ibitor s of Hu m a n
Ca th ep sin s K a n d L
J ean-Pierre Falgueyret,† Renata M. Oballa,*,‡ Osamu Okamoto,‡ Gregg Wesolowski,§ Yves Aubin,‡
Robert M. Rydzewski,| Peppi Prasit,‡ Denis Riendeau,† Sevgi B. Rodan,§ and M. David Percival†
Department of Biochemistry and Molecular Biology and Department of Chemistry, Merck Frosst Centre for Therapeutic
Research, 16711 TransCanada Highway, Kirkland, Quebec H9H 3L1, Canada, Department of Bone Biology & Osteoporosis,
Merck Research Laboratories, West Point, Pennsylvania, and Department of Medicinal Chemistry, Axys Pharmaceuticals,
South San Francisco, California
Received August 10, 2000
Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible
inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L
with IC50 values of 0.37 and 0.45 µM, respectively. Modification of compound 1 by replacement
of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide
(2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a Ki value of
50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoaze-
tidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably
due to an enhanced chemical reactivity of the compound toward the thiolate of the active site
of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione
at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions,
there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive
complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl
inhibitors exhibited time-dependent inhibition which allowed us to determine the association
and dissociation rate constants with human cathepsin K. The kinetic data obtained showed
that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due
to an increase of kon values and that the association of the compound with the enzyme fits an
apparent one-step mechanism. 13C NMR experiments performed with the enzyme papain
showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As
predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-
cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by
13C NMR, thereby demonstrating that the formation of the covalent bond with the active site
cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay
involving rabbit osteoclasts and bovine bone with an IC50 value of 0.7 µM. 1-Cyanopyrrolidine
represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and
proteolysis of bone collagen.
In tr od u ction
The physiological role of cathepsins is in intracellular
protein degradation. In general, cathepsins have a broad
tissue distribution, cathepsins L and B being highly
expressed in lysosomes of many cells and secreted
outside lysosomes in diseases such as cancer, muscular
dystrophy, Alzheimer’s disease, and multiple sclerosis.3
Recently, two cathepsins, cathepsins K and S which are
members of the cathepsin L subfamily, have been
described as being more tissue-specific and were found
to be involved in specialized cellular processes. Cathe-
psin S has a specific function in antigen processing4 and
represents a potential target for the treatment of
autoimune diseases. Cathepsin K is almost exclusively
expressed in bone-resorbing osteoclasts.5 Thus, develop-
ment of specific inhibitors for this enzyme could poten-
tially be used as treatment for diseases involving
excessive bone resorption. Moreover, recent work has
demonstrated that nonselective inhibitors of cysteine
proteases which are active against cathepsin K signifi-
cantly reduce bone resorption in in vitro and in vivo
models.6
Cysteine proteases comprise a group of proteolytic
enzymes involved in many physiological processes and
which could potentially play important roles in a
number of pathological situations. Cathepsins are de-
fined as lysosomal proteases, and the majority are
members of the papain-like cysteine protease family.
Eleven different sequences of human cysteinyl cathep-
sins (B, H, L, S, C, K, O, F, V, X, and W) have been
identified to date.1 Cysteine proteases contain a cysteine
and histidine pair at the active site which forms a stable
thiolate-imidazolium ion pair and is required for
enzyme activity.2
* To whom correspondence should be addressed. Tel: (514) 428-
3617. Fax: (514) 428-4900. E-mail: renata_oballa@merck.com.
† Department of Biochemistry and Molecular Biology, Merck Frosst
Centre for Therapeutic Research.
‡ Department of Chemistry, Merck Frosst Centre for Therapeutic
Research.
§ Merck Research Laboratories.
| Axys Pharmaceuticals.
10.1021/jm0003440 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/07/2000

Nonpeptidic Cyanamides as Inhibitors of Cathepsins K/ L
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 95
Sch em e 1a
a
Conditions: (a) NaBH4, MeOH, THF; (b) NaH, DMF, PhCH2Cl, 0 °C; (c) TFA, CH2Cl2; (d) BrCN, Et3N, CH2Cl2; (e) MsCl, Et3N, THF,
0 °C; NaN3, DMSO, 4; (f) H2, Pd/C, MeOH; (g) PhSO2Cl, Et3N, CH2Cl2, 0 °C; (h) NaOH, MeOH; (i) DPPA, Et3N, PhCH3, 4, PhCH2OH;
(j) RC(O)Cl, Et3N, CH2Cl2.
Many chemotypes have been described as inhibitors
of cysteine proteases.7 The majority, if not all, are
dependent for their activity on the presence of an
electrophilic moiety which can form either a reversible
or an irreversible covalent bond with the active site
cysteine of the enzyme. Additionally, most of the com-
pounds described so far for the inhibition of cathepsins
are peptide-based molecules that suffer the liabilities
of high molecular weight and the presence of a peptidic
backbone. While these compounds are potent and selec-
tive inhibitors of the cathepsins, they generally possess
poor pharmacokinetic properties.
The cyanamides 2 and 7-9 were synthesized from the
carbamate intermediate 10 via (i) hydrogenolysis of the
carbamate, (ii) amide or sulfonamide bond formation,
and (iii) conversion of the N-Boc to the desired N-CN
moiety. The carbamate intermediate 10 was, in turn,
synthesized from the ester 3 via Curtius rearrangement
using diphenyl phosphorazidate (DPPA) and trapping
the resultant isocyanate with benzyl alcohol. Finally,
the 2-substituted 1-cyanopyrrolidines 11 and 12 were
obtained from 4 via amide or sulfonamide bond forma-
tion followed by conversion of the N-Boc to the cyana-
mide moiety.
In the present report, we describe a new class of
nonpeptidic and low-molecular-weight compounds with
some degree of selectivity for the inhibition of cathepsins
K and L over cathepsin B. This new class of the
cathepsin L subfamily inhibitors is based on a 1-cyan-
opyrrolidinyl ring, and they form a reversible isothio-
urea ester bond with the enzyme. Furthermore, these
compounds were shown to be potent inhibitors of the
degradation of denaturated collagen by cathepsin K and
of bone resorption in an in vitro model.
The synthesis of the 1-cyanoazetidines is described
in Scheme 2. Starting with tert-butyl 3-hydroxy-1-
azetidinecarboxylate (15), the cyanamides 16 and 17
were synthesized by first forming the benzyl or cyclo-
hexylmethyl ether linkage followed by deprotection of
the Boc group and subsequent cyanamide formation
with BrCN. In the case of the amide and sulfonamide
analogues 18-20, their synthesis originated from the
primary amine 22 which, in turn, was derived by
displacing the mesylate of 15 with NaCN followed by
Raney nickel reduction. The amide or sulfonamide bond
was then formed by reacting the amine 22 with RC(O)-
Cl or PhSO2Cl in the presence of Et3N. Finally, depro-
tection of the Boc-amine and subsequent reaction with
BrCN generated the desired azetidine cyanamides 18-
20.
Ch em istr y
As described in Scheme 1, the synthesis of the
1-cyanopyrrolidines began with either 1-tert-butyl 3-
methyl 1,3-pyrrolidinedicarboxylate (3)11 or the com-
mercially available N-Boc-2-aminomethylpyrrolidine (4).
NaBH4 reduction of 3 and benzylation of the resultant
primary alcohol moiety followed by Boc-deprotection and
treatment with BrCN led to the synthesis of cyanamide
5. Cyanamide 6 was obtained by conversion of the
primary alcohol to an amino group through mesylate
displacement with NaN3 and subsequent hydrogenoly-
sis. The amino group was then converted to the benze-
nesulfonamide, and treatment with TFA followed by
BrCN afforded the 3-substituted 1-cyanopyrrolidine 6.
Bioch em istr y
All compounds were tested against cathepsins K, L,
and B. Purified recombinant cathepsins K and L were
obtained from Axys Pharmaceuticals Inc., and purified
human cathepsin B was obtained from Sigma. To
determine inhibitory potency, compounds were prein-
cubated 30 min with the enzyme, prior to the addition
of the substrate Z-Phe-Arg-pNA (25 µM). All assays

96 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Falgueyret et al.
Sch em e 2a
a
Conditions: (a) NaH, DMF, RCH2Br, 0 °C; (b) TFA, CH2Cl2; (c) BrCN, Et3N, CH2Cl2; (d) MsCl, Et3N, EtOAc, 0 °C; (e) NaCN, DMSO,
130 °C; (f) Raney Ni, NH3, H2, MeOH; (g) RC(O)Cl, Et3N, CH2Cl2; (h) PhSO2Cl, Et3N, CH2Cl2, 0 °C.
Ta ble 1. Inhibition of Human Cathepsins K, L, and B by 1-Cyanopyrrolidinyl Analogues
were performed in 96-wells plates at compound concen-
trations ranging from 100 µM to 1 nM. IC50 values were
determined by fitting experimental values to a four-
parameter logistic model. To determine pre-steady-state
kinetic parameters, enzymatic activity was measured
at room temperature in stirred cells using a fluorometer
(PTI fluorescence system) and Z-Phe-Arg-AMC as sub-
strate.
of potency. However, substitution at the 3-position (i.e.
compounds 2 and 5-9) afforded potent inhibitors of
cathepsins K and L. The amides 7 and 8, in which the
amide substituent is directly attached to the 3-position
of the cyanopyrrolidine, afforded moderate inhibition of
cathepsin K (0.37 and 0.29 µM, respectively). By con-
verting the amide functionality to a benzenesulfonamide
(compound 2) or a benzyl carbamate (compound 9), the
inhibition of cathepsin K increased to 0.05 and 0.04 µM,
respectively. The benzensulfonamide 2 proved to be the
most potent and selective (cathepsins K and L versus
B) compound in the 1-cyanopyrrolidine series. Moreover,
compound 2 inhibits both rat and human cathepsin K
with similar IC50 values.
The SAR in the 1-cyanoazetidine series is depicted
in Table 2. The unsubstituted 1-cyanoazetidine 21
inhibits human cathepsins K and L with IC50 values of
0.1 and 0.43 µM, respectively. Compound 21 is 10-fold
more active than the corresponding unsubstituted ana-
logue 13 in the five-membered ring pyrrolidine series.
This trend of an increase in potency in the four-
Resu lts a n d Discu ssion
As shown in Table 1, the original lead compound 1,
identified through screening of the Merck sample col-
lection, inhibits human cathepsins K and L with IC50
values of 0.37 and 0.45 µM, respectively. If the quinoline
moiety is removed, the unsubstituted 1-cyanopyrrolidine
13 partially retains potency against cathepsin K. How-
ever, the acyclic cyanamide 14 loses all potency against
cathepsins K, L, and B. Thus, the structure-activity
relationship (SAR) of substituted 1-cyanopyrrolidines
was examined. The addition of substituents at the
2-position (i.e. compounds 11 and 12) resulted in loss

Nonpeptidic Cyanamides as Inhibitors of Cathepsins K/ L
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 97
Ta ble 2. Inhibition of Human Cathepsins K, L, and B by 1-Cyanoazetidinyl Analogues
membered ring azetidine series when compared to the
five-membered ring pyrrolidine series is seen across the
board (i.e. compare compound 19 in Table 2 to compound
5 in Table 1). The most potent compound in the
1-cyanoazetidine series, compound 20, possesses a cy-
clohexylamide substituent in the 3-position, and it
inhibits cathepsins K and L with IC50 values of 0.005
and 0.006 µM, respectively.
The 1-cyanopyrrolidines and 1-cyanoazetidines were
also shown to be active in the gelatinase and bone-
resorption assays (see Tables 1 and 2). For example, the
lead compound 1 was equipotent in the gelatinase and
human cathepsin K assays (0.36 and 0.37 µM, respec-
tively). The more potent 3-substituted 1-cyanopyrro-
lidines 2 and 9 (0.05 and 0.04 µM versus cathepsin K,
respectively) were also very potent in the gelatinase
assay with IC50 values of 0.018 and 0.02 µM, respec-
tively. Likewise, in the 1-cyanoazetidine series, the most
potent cathepsin K and L inhibitor 20 had an IC50 value
of 0.01 µM in the gelatinase assay. In the functional in
vitro bone-resorption assay, compounds 2 and 9 were
shown to inhibit bone resorption with IC50 values of 0.75
and 1.41 µM, respectively. In the same assay, E64, a
potent irreversible epoxide inhibitor of cysteine pro-
teases, was found to inhibit bone resorption with an IC50
value of 0.02 µM.
Kin etic Exp er im en ts. Inhibition of cathepsins K
and L by 1-cyanopyrrolidinyl analogues was found to
be time-dependent and fully reversible. Figure 1 shows
the reaction progress curves for the onset of inhibition
of human cathepsin K by compound 19 (Figure 1A) and
compound 1 (Figure 1B). The curves show that for an
equal final concentration of inhibitor, the same steady-
state velocity is obtained either when the reaction is
initiated by the addition of enzyme or when the enzyme
and the inhibitor are preincubated and then diluted into
the assay mixture containing substrate. These kinetics
are consistent with a reversible time-dependent mech-
anism of inhibition. Generally, two possible mechanisms
can describe reversible time-dependent inhibitors. One
mechanism characterizes compounds where the initial
velocity of the enzymatic reaction is independent of the
inhibitor concentration and a linear correlation between
the inhibitor concentration and the apparent first-order
rate constant for the onset of inhibition (kobs) is observed.
This mechanism is often referred to as an apparent
single step binding process8 and is described by eq 1:
E + I ykonz EI*
(1)
koff
The second mechanism involves a two-step process
characterized by an initial rapidly reversible binding,
followed by a second slow formation of a more tightly
bound complex. In this two step process, binding in the
initial complex is tight enough to result in an inhibition
of the initial reaction velocity. This mechanism is
characterized by curved plots of kobs versus inhibitor
concentration, where the asymptote approached at high
inhibitor concentration represents the value of k2 for the
second equilibrium (eq 2):
E + I yk1z EI yk2z EI*
(2)
k-1
k-2
The progress curves for the onset of inhibition (at a
range of inhibitor concentrations) were fitted to the eq
3 (see Experimental Section) describing slow binding
inhibition. The obtained values of kobs were then plotted
versus inhibitor concentration. The results for both the
1-cyanopyrrolidine compounds 1, 2, and 9 and the
1-cyanoazetidine compound 19 showed that the rate of
association of cathepsin K and the inhibitors is linearly
dependent on inhibitor concentration. This is therefore
consistent with an apparent one-step binding mecha-
nism of the inhibitor to the enzyme. From the progress
curves for the onset of inhibition, it is possible to
determine the pre-steady-state kinetics parameters kon
and koff (eq 3) as well as the dissociation constant Ki
(see Experimental Section). The data presented in Table
3 shows that the increase in the potency of the inhibitors
is mainly driven by an increase in the value of kon, while
the value of the koff remains largely unchanged. Al-
though the kinetic data obtained with the 1-cyanopyr-
rolidines fits a one-step mechanism, it is unlikely that
the time dependency of these small molecules is due to
a slow rate of diffusion into the enzyme active site. As
hypothesized for other low-molecular-weight time-de-
pendent inhibitors,9 it is possible that one or more
transient enzyme-inhibitor complexes occurs before the
formation of the final EI complex. However, if the

98 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Falgueyret et al.
Ta ble 4. Effect of GSH on the Inhibitory Activity of
1-Cyanopyrrolidinyl and 1-Cyanoazetidinyl Analogues at pH 5.5
and 7.0 toward Cathepsin Ka
no GSH
+10 mM GSH
compd
pH 5.5
pH 7.0
pH 5.5
pH 7.0
1
9
13
20
21
0.34
0.06
1.63
0.016
0.45
0.28
0.05
2.3
0.038
0.88
0.36
0.09
1.5
0.027
0.48
11.4
0.15
3.4
>100
>100
a
Compounds were preincubated 30 min with GSH prior to the
addition of the enzyme.
site cysteine thiolate anion, IC50 values were determined
in the presence and absence of glutathione (GSH) at pH
5.5 and 7.0. Increasing the pH to 7.0 will favor the
formation of the GSH thiolate anion which is a better
nucleophile and can react with the inhibitor during the
preincubation step. Results shown in Table 4 show that
when the assay was performed at acidic pH (pH 5.5,
i.e. the optimal pH for cathepsins K and L), IC50 values
were not significantly affected by the presence of GSH.
However, at pH 7.0, compounds containing the 1-cy-
anoazetidinyl ring (i.e. compounds 20 and 21) became
completely inactive in the presence of GSH indicating
that these four-membered ring cyanamides possess a
higher reactivity than the five-membered ring cyana-
mides (i.e. compounds 1, 9, and 13) toward the thiolate
of the GSH. This increase in reactivity could in part
explain the lower IC50 values observed with this class
of compounds. Compounds containing a 1-cyanopyrro-
lidinyl ring were only slightly affected by the presence
of GSH at either pH 5.5 or 7.0. In fact, compound 1
which has an IC50 value of 0.4 µM for cathepsin K is
shifted by a factor of 30 in the presence of GSH at pH
7.0, while the IC50 value of compound 9 which is 10-
fold more potent for cathepsin K than compound 1 is
not significantly affected by GSH at pH 7.0. This
observation suggests that the increase in potency ob-
tained with compounds in the 1-cyanopyrrolidinyl series
(Table 1) is probably not due to an increase in reactivity
toward the thiolate anion of the enzyme but likely due
to an increase in the affinity of the compound for the
enzyme.
NMR Exp er im en t. Peptidic nitriles are known to be
potent and reversible inhibitors of cysteine proteases
of the papain family.10 Unlike aldehydes, which are
inhibitors of both serine and cysteine proteases, nitriles
seem to be more specific for cysteine proteases.10d 13C
NMR studies with papain have demonstrated that
peptidic nitriles form a reversible thioimidate ester
adduct with papain.10a,c,d Because of the obvious simi-
larity of the electrophile of the 1-cyanopyrrolidines and
the peptidic nitriles, the 13C analogue of compound 2
was synthesized and used to probe the formation of an
isothiourea bond with the active site thiol of a cysteine
protease as illustrated in Scheme 3. Although compound
2 is more potent for cathepsin K (Table 1), it is also
relatively potent for the inhibition of papain with an
IC50 value of 0.23 µM at pH 5.5. The results of the NMR
studies are presented in Figure 2. Incubation of 0.5
equiv of glutathione and 1 equiv of compound 2 at pH
8.0 led to the formation of a new resonance peak that
appeared at 161.7 ppm (Figure 2A). As previously
described,10d this resonance peak is consistent with the
F igu r e 1. Time-dependent inhibition and reversibilty of
1-cyanoazetidine and 1-cyanopyrrolidinyl compounds. Progress
curves were obtained when the reaction was initiated either
by the addition of 0.5 nM cathepsin K (b) or when cathepsin
K and the inhibitor were preincubated for 15 min and then
diluted 100-fold prior to the initiation of the enzymatic reaction
(O): (A) 130 nM compound 19; (B) 500 nM compound 1; control
reaction (9) without inhibitor.
Ta ble 3. Inhibition of Human Cathepsin K by
1-Cyanopyrrolidinyl and 1-Cyanoazetidinyl Analogues
IC50
compd koff (s-1) ( SE kon (M-1 s-1) ( SE Ki (µM) ( SE (µM)
1
2
9
0.0025 ( 0.0003
0.0019 ( 0.001
0.0056 ( 0.0003
6594 ( 129
19899 ( 1079
67303 ( 7800
0.38 ( 0.084 0.37
0.09 ( 0.001 0.081
0.14 ( 0.017 0.04
19
0.0028 ( 0.0002 140722 ( 24637 0.02 ( 0.0002 0.017
dissociation constant of this intermediate complex is
much higher than the Ki of the final EI complex, its
contribution will be kinetically insignificant and difficult
to observe experimentally.
Rea ctivity of th e Cya n a m id e w ith Th iols. To
determine if the increase in potency observed between
the 1-cyanopyrrolidine series (Table 1) and the 1-cy-
anoazetidine series (Table 2) is due to an increase in
the reactivity of the cyanamide moiety toward the active

Nonpeptidic Cyanamides as Inhibitors of Cathepsins K/ L
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 99
F igu r e 3. Mean plasma levels after intravenous administra-
tion of compound 2 in PEG-200/water (3:2) and after oral
administration of 2 as a 1% methocel suspension in rats.
Dosing volumes for intravenous and oral administration were
1 and 10 mL/kg, respectively.
administered orally in rats at a dose of 20 mg/kg in a
1% methocel suspension, the compound was well-
absorbed with a bioavailability of 38%. The half-life of
compound 2 was determined after intravenous dosing
in PEG-200/water (3:2) and was found to be 2.0 h in
rats. These results clearly demonstrate that compound
2 possesses good pharmacokinetic properties in rats and
could be eventually used for in vivo studies.
F igu r e 2. 13C NMR spectrum of the papain-compound 2
complex: (A) NMR spectrum obtained when 0.5 equiv of GSH
was incubated with 1 equiv of compound 2 (13CN) at pH 8.0;
(B) 1 equiv of compound 2 (13CN) with 1 equiv of papain (2.0
mM); (C) 1 equiv of E64 (2.0 mM) was added to the mixture
obtained in B; (D) 1 equiv of papain (2.0 mM) with no inhibitor
present.
Sch em e 3
Con clu sion
In the present report we have shown that cathepsins
K and L can be inhibited by compounds containing a
1-cyanopyrrolidine or 1-cyanoazetidine moiety. These
cyanamide-based compounds represent a new class of
nonpeptidic cysteine protease inhibitors. They inhibit
cathepsins K and L in a time-dependent manner and
form a reversible isothiourea ester link with the active
site cysteine of the enzyme. The SARs with 1-cyanopy-
rrolidines showed that substitution at the 3-position is
required to increase potency within this class of inhibi-
tors. Addition of a benzenesulfonamide in the 3-position
resulted in compound 2, a potent inhibitor of cathepsins
K and L which was also active in an in vitro model of
bone resorption. Moreover, this inhibitor proved to have
good pharmacokinetic properties in rats and thus would
be suitable for animal studies where the roles of
cathepsins K and L could be studied.
formation of an isothiourea linkage since it falls between
the range for a thioamide carbonyl (200-210 ppm) and
an amide and peptide carbonyl (160-170 ppm). The
chemical shift value was used as a reference when 13C-2
was incubated with papain. When 1.1 equiv of com-
pound 2 was incubated with 1 equiv of papain, we
observed the formation of two resonance peaks at 159.3
and 159.9 ppm and the disappearance of the nitrile peak
at 115.7 ppm (Figure 2B). The existence of two reso-
nance peaks for the isothiourea ester is most probably
due to rotational isomers (i.e. restricted rotation about
the isothiourea ester bond). Further addition of the
irreversible inhibitor E64 to the EI complex totally
abolished the resonance peaks observed at 159.3 and
159.9 ppm while the nitrile peak at 115.7 ppm reap-
peared (C, Figure 2). Consistent with the kinetic results,
this observation demonstrates the reversibility of the
covalent enzyme-inhibitor isothiourea ester bond.
P h a r m a cok in etic P r ofile of th e Cya n a m id e 2. At
neutral pH, 1-cyanopyrrolidinyl compounds might form
stable isothiourea ester bonds with small thiol-contain-
ing molecules such as GSH or protein thiols. To deter-
mine if the reactivity of the cyanamide toward thiols
could prevent this class of compounds from possessing
a suitable pharmacokinetic profile, in vivo dosing stud-
ies were performed with compound 2. The results of this
study are presented in Figure 3. When compound 2 was
Exp er im en ta l Section
Gen er a l. Proton (1H NMR) magnetic resonance spectra
were recorded on a Bruker AM instrument operating at 300,
400 or 500 MHz. All spectra were recorded using residual
solvent (CHCl3 or acetone) as internal standard. Signal
multiplicity was designated according to the following ab-
breviations: s ) singlet, d ) doublet, dd ) doublet of doublets,
t ) triplet, m ) multiplet, br s ) broad singlet, br t ) broad
triplet. A Sciex API-100 mass spectrometer operating in
positive ion APCI was used to obtain the [M + H+]+ ion of the
target molecules. High-resolution mass spectrometry was
carried out on a VG ZAB 2F mass spectrometer using positive
ion FAB at 10000 resolution. 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. Compound 3 was prepared as previ-
ously described.11 BrCN was prepared as described below. The
1-cyanoazetidine 21 was prepared by reacting the commer-

100 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Falgueyret et al.
cially available azetidine with BrCN. Cyanamides 13 and 14
were purchased from Aldrich.
BrCN). The crude material was purified by flash chromatog-
raphy (50% EtOAc in hexane) to afford the desired cyanamide
5 (23 mg, 12% yield). H NMR (400 MHz, acetone-d6): δ 7.88
(m, 2H), 7.62 (m, 3H), 6.68 (br s, 1H), 3.38 (m, 3H), 3.12 (dd,
1H), 2.97 (t, 2H), 2.46 (m, 1H), 2.02 (m, 1H), 1.70 (m, 1H). m/z
(+APCI): 266.0 [M + H+]+. HRMS (+FAB): calcd for C13H17N2O
[MH+] 217.13411, found 217.13409. Anal. (C13H16N2O) H, N;
C: calcd, 72.19; found, 71.59.
1
Cya n ogen Br om id e (Br CN). To a cold (-5 °C), stirred
suspension of bromine (5.5 mL, 1 equiv) in H2O (15 mL) was
added, dropwise over 30 min, a solution of sodium cyanide (5.0
g, 1 equiv) in H2O (15 mL). The temperature of the reaction
mixture during the addition of sodium cyanide was maintained
at -5 to 5 °C. The resultant suspension was stirred an
additional 10 min and was then extracted with CH2Cl2 (3 ×
33.3 mL). The resultant solution of BrCN (1 M in CH2Cl2) was
stored over CaCl2 at 4 °C.
3-[(Ben zyloxy)m eth yl]-1-cya n op yr r olid in e (6). To a re-
fluxing suspension of 1-tert-butyl 3-methyl 1,3-pyrrolidinedi-
carboxylate (3)11 (2.29 g, 1 equiv) and NaBH4 (757 mg, 2 equiv)
in THF (10 mL) was added, dropwise, MeOH (2 mL). After
the addition of MeOH, the resulting mixture was refluxed for
1 h. The mixture was then poured into 10% citric acid and
extracted with EtOAc (3×). The combined organic extracts
were washed with H2O and brine, dried (MgSO4) and concen-
trated under reduced pressure. The resultant oil was purified
using flash chromatography (gradient elution: 50% EtOAc in
hexane to 70% EtOAc in hexane) to afford the desired primary
alcohol (1.54 g, 77% yield). To a cold (0 °C), stirred solution of
this alcohol (188 mg, 1 equiv) in DMF (9 mL) was added NaH
(60% in oil, 56 mg, 1.5 equiv). The suspension was stirred at
0 °C for 40 min followed by the addition of the benzyl chloride
(215 µL, 2 equiv). The suspension was warmed to room
temperature and stirred for 16 h. MeOH was added followed
by saturated aqueous NH4Cl. The aqueous phase was ex-
tracted with EtOAc (3×) and the combined organic extracts
were washed with brine, dried (MgSO4) and concentrated
under reduced pressure. The residue was purified by flash
chromatography (20% EtOAc in hexane) to afford the desired
benzyl ether (228 mg, 84% yield). The N-Boc group of this
benzyl ether was then converted to the cyanamide (N-CN)
following general procedure 1 (i.e. successive treatment with
TFA and BrCN). The crude material was purified by flash
chromatography (gradient elution: 30% EtOAc in hexane to
50% EtOAc in hexane) to afford the desired cyanamide 6 (165
mg, 98% yield). 1H NMR (400 MHz, acetone-d6): δ 7.30 (m,
5H), 4.53 (s, 2H), 3.43 (m, 5H), 3.18 (dd, 1H), 2.59 (m, 1H),
2.03 (m, 1H), 1.74 (m, 1H). m/z (+APCI): 217.1 [M + H+]+.
HRMS (+FAB): calcd for C12H16N3O2S [MH+] 266.09622,
found 266.09632. Anal. (C12H15N2O2S) C, H, N.
ter t-Bu t yl 3-{[(Ben zyloxy)ca r b on yl]a m in o}-1-p yr r o-
lid in eca r boxyla te (10). To a solution of 1-tert-butyl 3-methyl
1,3-pyrrolidinedicarboxylate (3)11 (2.29 g, 1 equiv) in MeOH
(24 mL) was added 1 N NaOH (12 mL, 1.2 equiv). The mixture
was stirred at room temperature for 3 days and then concen-
trated under reduced pressure. The residue was poured into
a solution of 1 N HCl (10 mL) and 10% citric acid (20 mL).
The mixture was extracted with EtOAc (3×) and the combined
organic extracts were washed with H2O and brine, dried
(MgSO4) and concentrated under reduced pressure to yield the
corresponding acid (1.83 g, 85% yield). To a stirred solution of
the acid (1.505 g, 1 equiv) and Et3N (1.17 mL, 1.2 equiv) in
toluene (20 mL) was added DPPA (1.66 mL, 1.1 equiv). After
stirring at room temperature for 10 min, the mixture was
refluxed for 1 h. To this refluxing mixture was added benzyl
alcohol (1.45 mL, 2 equiv) and the refluxing was continued
for 14 h. The resultant mixture was poured into 1 N aqueous
NaOH and extracted with Et2O (3×). The combined organic
extracts were washed successively with H2O, 10% citric acid,
H2O and brine. The organic extracts were dried (MgSO4),
concentrated under reduced pressure and the residue was
purified by flash chromatography (gradient elution: 20%
EtOAc in hexane to 67% EtOAc in hexane) to afford the benzyl
carbamate 10 (2.06 g, 92% yield). 1H NMR (300 MHz, CDCl3):
δ 7.36 (m, 5H), 5.10 (s, 2H), 4.81 (m, 1H), 4.25 (m, 1H), 3.60
(dd, 1H), 3.42 (m, 2H), 3.19 (m, 1H), 2.15 (m, 1H), 1.83 (m,
1H), 1.45 (s, 9 H).
Gen er a l P r oced u r e 1. To a stirred solution of the Boc-
protected amine (1 equiv) in CH2Cl2 (∼1 mL/mmol of the
amine) was added TFA (16 equiv). The solution was stirred
at room temperature for 0.5 h and the CH2Cl2/TFA solvent
was removed under reduced pressure. The residue was diluted
with CH2Cl2 and the solvent was removed once again under
reduced pressure (repeated 2×). The resultant amine was
dissolved in CH2Cl2 (∼5 mL/mmol of amine) and cooled to 0
°C. To this cold solution was added Et3N (1.5 equiv) followed
by a solution of BrCN in CH2Cl2 (1 M, 1.1-5 equiv). The
resultant reaction mixture was stirred at 0 °C for 1 h. The
solution was warmed to room temperature, diluted with EtOAc
and washed with H2O and brine. The organic extract was dried
(MgSO4), concentrated under reduced pressure and the result-
ant residue was purified by flash chromatography to afford
the desired cyanamide.
N -[(1-Cya n o-3-p yr r olid in yl)m e t h yl]b e n ze n e su lfon -
a m id e (5). To a refluxing suspension of 1-tert-butyl 3-methyl
1,3-pyrrolidinedicarboxylate (3)11 (2.29 g, 1 equiv) and NaBH4
(757 mg, 2 equiv) in THF (10 mL) was added, dropwise, MeOH
(2 mL). After the addition of MeOH, the resulting mixture was
refluxed for 1 h. The mixture was then poured into 10% citric
acid and extracted with EtOAc (3×). The combined organic
extracts were washed with H2O and brine, dried (MgSO4) and
concentrated under reduced pressure. The resultant oil was
purified using flash chromatography (gradient elution: 50%
EtOAc in hexane to 70% EtOAc in hexane) to afford the desired
primary alcohol (1.54 g, 77% yield). To a cold (0 °C), stirred
solution of this alcohol (250 mg, 1 equiv) in THF (25 mL) was
added Et3N (346 µL, 2 equiv) followed by MsCl (144 µL, 1.5
equiv). The resultant suspension was stirred at room temper-
ature for 40 min. The suspension was then diluted with EtOAc
and the mixture was washed with 10% citric acid, H2O,
saturated aqueous NaHCO3 and brine. The combined aqueous
washings were back extracted with EtOAc (3×) and the
combined organic extracts were dried over MgSO4 and con-
centrated under reduced pressure to afford the desired mesy-
late which was used immediately in the next reaction. This
crude oil (mesylate) was dissolved in DMSO (3 mL) and NaN3
(173 mg, 2 equiv) was added. The resultant reaction mixture
was stirred at 100 °C for 5 h. The reaction mixture was then
poured into H2O and extracted with Et2O (4×). The combined
organic extracts were washed with H2O and brine, dried over
MgSO4 and concentrated under reduced pressure to afford the
crude azide (295 mg, 98% yield). To a stirred solution of this
crude azide (295 mg, 1 equiv) in MeOH/CHCl3 (2 mL each)
was added 10% Pd on carbon (30 mg, 10%). The solution was
evacuated, placed under a H2 atmosphere (1 atm) and stirred
at room temperature for 16 h. The suspension was then filtered
through Celite and washed with MeOH. The filtrate was
concentrated under reduced pressure to afford the desired
primary amine (∼300 mg, 115% yield). To a stirred solution
of this amine (109 mg, 1 equiv) and Et3N (130 µL, 1.7 equiv)
in CH2Cl2 (7 mL) was added benzenesulfonyl chloride (97 µL,
1.4 equiv). The resultant mixture was stirred at room tem-
perature for 16 h. The mixture was then diluted with EtOAc
and washed successively with 10% citric acid, H2O and brine.
The organic extract was dried (MgSO4) and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (50% EtOAc in hexane) to afford the desired coupled
product (123 mg, 66% yield). The N-Boc group of the coupled
product was then converted to the cyanamide (N-CN) following
general procedure 1 (i.e. successive treatment with TFA and
N-(1-Cya n o-3-p yr r olid in yl)ben zen esu lfon a m id e (2). To
a stirred solution of the benzyl carbamate 10 (1.95 g, 1 equiv)
in MeOH (20 mL) was added 10% palladium on carbon (200
mg, 10%). The solution was evacuated, placed under a H2
atmosphere (35 atm) and shaken for 5 h. The suspension was
then filtered through Celite and washed with MeOH. The

Nonpeptidic Cyanamides as Inhibitors of Cathepsins K/ L
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 101
filtrate was concentrated under reduced pressure to afford the
desired amine (1.358 g, 120% yield). To a stirred solution of
this amine (100 mg, 1 equiv) and Et3N (120 µL, 1.6 equiv) in
CH2Cl2 (5 mL) was added benzenesulfonyl chloride (82 µL, 1.2
equiv). The resultant mixture was stirred at room temperature
for 16 h. The mixture was then diluted with EtOAc and
washed successively with 10% citric acid, H2O and brine. The
organic extract was dried (MgSO4) and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (gradient elution: 10% EtOAc in hexane to 67%
EtOAc in hexane) to afford the desired coupled product (135
mg, 77% yield). The N-Boc group of the coupled product was
then converted to the cyanamide (N-CN) following general
procedure 1 (i.e. successive treatment with TFA and BrCN).
The crude material was purified by flash chromatography
(gradient elution: 33% EtOAc in hexane to 70% EtOAc in
hexane) to afford the desired cyanamide 2 (94 mg, 90% yield).
1H NMR (500 MHz, CDCl3): δ 7.78 (d, 2H), 7.53 (m, 1H), 7.43
(m, 2H), 4.90 (d, 1H), 3.79 (m, 1H), 3.32 (m, 3H), 3.03 (m, 1H),
1.95 (m, 1H), 1.73 (m, 1H). m/z (+APCI): 251.9 [M + H+]+.
HRMS (+FAB): calcd for C11H14N3O2S [MH+] 252.08068,
found 252.08067. Anal. (C11H13N3O2S) H; C: calcd, 51.72;
found, 52.57; N: calcd, 16.29; found, 16.72.
Ben zyl 1-Cyan o-3-pyr r olidin ylcar bam ate (9). To a stirred
solution of the benzyl carbamate 10 (100 mg, 1 equiv) in CH2-
Cl2 (1 mL) was added TFA (1 mL). The solution was stirred at
room temperature for 0.5 h and the CH2Cl2/TFA solvent was
removed under reduced pressure. The residue was diluted with
CH2Cl2 and the solvent was removed once again under reduced
pressure (repeated 2×). The resultant amine was dissolved in
CH2Cl2 (2 mL) and cooled to 0 °C. To this cold solution was
added Et3N (0.22 mL, 5 equiv) followed by a solution of BrCN
in CH2Cl2 (1M, 0.37 mL, 1.2 equiv). The resultant reaction
mixture was stirred at 0 °C for 1 h. The solution was warmed
to room temperature, diluted with EtOAc and washed with
H2O and brine. The organic extract was dried (MgSO4),
concentrated under reduced pressure and the resultant residue
was purified by flash chromatography (50% EtOAc in hexane)
to afford the desired cyanamide 9 (51 mg, 66% yield). 1H NMR
(300 MHz, CDCl3): δ 7.35 (m, 5H), 5.10 (s, 2H), 4.82 (m, 1H),
4.29 (m, 1H), 3.62 (dd, 1H), 3.50 (m, 2H), 3.28 (dd, 1H), 2.19
(m, 1H), 1.90 (m, 1H). m/z (+APCI): 246.0 [M + H+]+. HRMS
(+FAB): calcd for
C
13H16N3O2 [MH+] 246.12419, found
246.12425. Anal. (C13H15N3O2) C, H, N.
N -[(1-Cya n o-2-p yr r olid in yl)m e t h yl]b e n ze n e su lfon -
a m id e (11). To a stirred solution of tert-butyl 2-(aminomethyl)-
1-pyrrolidinecarboxylate (4, commercially available) (200 mg,
1 equiv) and Et3N (0.21 mL, 1.5 equiv) in CH2Cl2 (5 mL) was
added benzenesulfonyl chloride (0.14 mL, 1.1 equiv). The
resultant mixture was stirred at room temperature for 30 min.
The mixture was then diluted with EtOAc and washed
successively with 10% citric acid, H2O and brine. The organic
extract was dried (MgSO4) and concentrated under reduced
pressure. The residue was purified by flash chromatography
(gradient elution: 10% EtOAc in hexane to 33% EtOAc in
hexane) to afford the desired coupled product (350 mg, 103%
yield). The N-Boc group of the coupled product was then
converted to the cyanamide (N-CN) following general proce-
dure 1 (i.e. successive treatment with TFA and BrCN). The
crude material was purified by flash chromatography (gradient
elution: 50% EtOAc in hexane to 67% EtOAc in hexane) to
afford the desired cyanamide 11 (190 mg, 72% yield). 1H NMR
(500 MHz, CDCl3): δ 7.76 (d, 2H), 7.48 (m, 3H), 4.70 (br t,
1H), 3.58 (m, 1H), 3.30 (m, 2H), 3.02 (m, 2H), 1.82 (m, 4H).
HRMS (+FAB): calcd for C12H16N3O2S [MH+] 266.09622,
found 266.09632. Anal. (C12H15N3O2S) C, H, N.
N-(1-Cya n o-3-p yr r olid in yl)ben za m id e (7). To a stirred
solution of the benzyl carbamate 10 (1.95 g, 1 equiv) in MeOH
(20 mL) was added palladium on carbon (200 mg, 10%). The
solution was evacuated, placed under a H2 atmosphere (35
atm) and shaken for 5 h. The suspension was then filtered
through Celite and washed with MeOH. The filtrate was
concentrated under reduced pressure to afford the desired
amine (1.358 g, 120% yield). To a stirred solution of this amine
(105 mg, 1 equiv) and Et3N (160 µL, 2 equiv) in CH2Cl2 (5 mL)
was added benzoyl bromide (90 µL, 1.3 equiv). The resultant
mixture was stirred at room temperature for 10 min. The
mixture was concentrated under reduced pressure and the
residue was purified by flash chromatography (50% EtOAc in
hexane) to afford the desired coupled product (134 mg, 83%
yield). The N-Boc group of the coupled product was then
converted to the cyanamide (N-CN) following general proce-
dure 1 (i.e. successive treatment with TFA and BrCN). The
crude material was purified by flash chromatography (gradient
elution: 50% EtOAc in hexane to 67% EtOAc in hexane) to
1
afford the desired cyanamide 7 (69 mg, 70% yield). H NMR
(300 MHz, CDCl3): δ 7.76 (d, 2H), 7.47 (m, 3H), 6.23 (m, 1H),
4.70 (m, 1H), 3.75 (dd, 1H), 3.57 (m, 2H), 3.39 (dd, 1H),
2.30 (m, 1H), 2.03 (m, 1H). m/z (+APCI): 216.1 [M + H+]+.
HRMS (+FAB): calcd for C12H14N3O [MH+] 216.11358, found
216.11369. Anal. (C12H13N3O) H; C: calcd, 66.96; found, 66.42;
N: calcd, 19.52; found, 18.87.
N-[(1-Cya n o-2-p yr r olid in yl)m eth yl]ben za m id e (12). To
a stirred solution of tert-butyl 2-(aminomethyl)-1-pyrrolidin-
ecarboxylate (4, commercially available) (200 mg, 1 equiv) and
Et3N (0.21 mL, 1.5 equiv) in CH2Cl2 (5 mL) was added benzoyl
bromide (0.13 mL, 1.1 equiv). The resultant mixture was
stirred at room temperature for 30 min. The mixture was then
diluted with EtOAc and washed successively with 10% citric
acid, H2O and brine. The organic extract was dried (MgSO4)
and concentrated under reduced pressure. The residue was
purified by flash chromatography (gradient elution: 10%
EtOAc in hexane to 33% EtOAc in hexane) to afford the desired
coupled product (301 mg, 99% yield). The N-Boc group of the
coupled product was then converted to the cyanamide (N-CN)
following general procedure 1 (i.e. successive treatment with
TFA and BrCN). The crude material was purified by flash
chromatography (gradient elution: 50% EtOAc in hexane to
67% EtOAc in hexane) to afford the desired cyanamide 12 (173
mg, 76% yield). 1H NMR (500 MHz, CDCl3): δ 7.67 (d, 2H),
7.35 (m, 3H), 6.45 (br s, 1H), 3.72 (m, 2H), 3.37 (m, 3H), 1.96
(m, 1H), 1.70 (m, 2H), 1.60 (m, 1H). HRMS (+FAB): calcd for
C13H16N3O [MH+] 230.12935, found 230.12934.
ter t-Bu tyl 3-Hyd r oxy-1-a zetid in eca r boxyla te (15). To
a stirred solution of 1-benzhydryl-3-azetidinol12 (10.6 g, 1
equiv) in EtOH/H2O (100 and 20 mL, respectively) was added
5% Pd on carbon (1 g, 10%). The solution was evacuated, placed
under a H2 atmosphere (60 atm) and stirred at room temper-
ature for 16 h. The suspension was then filtered through Celite
and washed with MeOH. The filtrate was concentrated under
reduced pressure to afford the desired amine. To a cold (0 °C),
stirred solution of the amine (2 g, 1 equiv) in EtOH (35 mL)
was added Et3N (7.5 mL, 2 equiv) followed by Boc2O (6.47 g,
N-(1-Cya n o-3-p yr r olid in yl)[1,1′-bip h en yl]-4-ca r boxa m -
id e (8). To a stirred solution of the benzyl carbamate 10 (1.95
g, 1 equiv) in MeOH (20 mL) was added palladium on carbon
(200 mg, 10%). The solution was evacuated, placed under a
H2 atmosphere (35 atm) and shaken for 5 h. The suspension
was then filtered through Celite and washed with MeOH. The
filtrate was concentrated under reduced pressure to afford the
desired amine (1.358 g, 120% yield). To a stirred solution of
this amine (100 mg, 1 equiv) and Et3N (110 µL, 1.5 equiv) in
CH2Cl2 (5 mL) was added 4-biphenylcarbonyl chloride (152 mg,
1.3 equiv). The resultant mixture was stirred at room tem-
perature for 10 min. The mixture was concentrated under
reduced pressure and the residue was purified by flash
chromatography (50% EtOAc in hexane) to afford the desired
coupled product (106 mg, 54% yield). The N-Boc group of the
coupled product was then converted to the cyanamide (N-CN)
following general procedure 1 (i.e. successive treatment with
TFA and BrCN). The crude material was purified by flash
chromatography (gradient elution: 50% EtOAc in hexane to
75% EtOAc in hexane) to afford the desired cyanamide 8 (73
mg, 87% yield). 1H NMR (500 MHz, CDCl3): δ 7.75 (d, 2H),
7.55 (m, 4H), 7.34 (m, 3H), 6.44 (d, 1H), 4.61 (m, 1H), 3.68 (m,
1H), 3.50 (m, 2H), 3.33 (m, 1H), 2.19 (m, 1H), 1.96 (m, 1H).
HRMS (+FAB): calcd for C18H18N3O [MH+] 292.14505, found
292.14499. Anal. (C18H17N3O) C, H, N.

102 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Falgueyret et al.
1.1 equiv). The resultant solution was stirred at room tem-
perature for 30 min and then concentrated under reduced
pressure. The residue was diluted with EtOAc, washed with
10% citric acid, H2O and brine. The organic extract was dried
over MgSO4 and concentrated under reduced pressure to afford
an oil which was purified by flash chromatography (gradient
elution: 67% EtOAc in hexane to 100% EtOAc) to yield the
Boc-protected azetidine 15 (2.71 g, 58% yield).
suspension was then filtered through Celite and washed with
MeOH. The filtrate was concentrated under reduced pressure
to afford the desired primary amine 22 (856 mg, 116% yield).
1H NMR (500 MHz, CDCl3): δ 3.90 (m, 2H), 3.50 (m, 2H), 2.80
(m, 2H), 2.43 (m, 1H), 1.32 (s, 9H).
N-[(1-Cya n o-3-a zetid in yl)m eth yl]ben za m id e (18). To a
stirred solution of the amine 22 (100 mg, 1 equiv) and Et3N
(0.11 mL, 1.5 equiv) in CH2Cl2 (5 mL) was added benzoyl
bromide (0.07 mL, 1.1 equiv). The resultant mixture was
stirred at room temperature for 10 min. The mixture was then
diluted with EtOAc and washed successively with 10% citric
acid, H2O and brine. The organic extract was dried (MgSO4)
and concentrated under reduced pressure. The residue was
purified by flash chromatography (gradient elution: 50%
EtOAc in hexane to 70% EtOAc in hexane) to afford the desired
coupled product (92 mg, 59% yield). The N-Boc group of the
coupled product was then converted to the cyanamide (N-CN)
following general procedure 1 (i.e. successive treatment with
TFA and BrCN). The crude material was purified by flash
chromatography (gradient elution: 50% EtOAc in hexane to
67% EtOAc in hexane) to afford the desired cyanamide 18 (21
3-(Ben zyloxy)-1-cya n oa zetid in e (16). To a cold (0 °C),
stirred solution of tert-butyl 3-hydroxy-1-azetidinecarboxylate
(15) (173 mg, 1 equiv) in DMF (2 mL) was added NaH (60%
in oil, 60 mg, 1.5 equiv). The suspension was stirred at 0 °C
for 30 min followed by the addition of the benzyl chloride (230
µL, 2 equiv). The resulting suspension was poured into H2O
and extracted with Et2O (3 ×). The combined organic extracts
were washed with H2O and brine, dried (MgSO4) and concen-
trated under reduced pressure. The residue was purified by
flash chromatography (gradient elution: 10% EtOAc in hexane
to 25% EtOAc in hexane) to afford the desired benzyl ether
(273 mg, 104% yield). The N-Boc group of the benzyl ether
was then converted to the cyanamide (N-CN) following general
procedure 1 (i.e. successive treatment with TFA and BrCN).
The crude material was purified by flash chromatography
(gradient elution: 10% EtOAc in hexane to 33% EtOAc in
hexane) to afford the desired cyanamide 16 (148 mg, 79%
yield). 1H NMR (500 MHz, CDCl3): δ 7.22 (m, 5H), 4.34 (s,
2H), 4.25 (m, 1H), 4.10 (m, 2H), 3.97 (m, 2H). HRMS (+FAB):
calcd for C11H13N2O [MH+] 189.10276, found 189.10279. Anal.
(C11H12N2O) C, H, N.
1
mg, 30% yield). H NMR (500 MHz, CDCl3): δ 7.65 (m, 2H),
7.38 (m, 3H), 6.34 (br s, 1H), 4.17 (m, 2H), 3.84 (m, 2H),
3.59 (m, 2H), 2.93 (m, 1H). m/z (+APCI): 216.1 [M + H+]+.
HRMS (+FAB): calcd for C12H14N3O [MH+] 216.11369, found
216.11369. Anal. (C12H13N3O) H; C: calcd, 66.96; found, 66.50;
N: calcd, 19.52; found, 18.74.
N -[(1-C y a n o -3-a ze t id in y l)m e t h y l]b e n ze n e s u lfo n -
a m id e (19). To a cold (0 °C), stirred solution of the amine 22
(100 mg, 1 equiv) and Et3N (0.12 mL, 1.5 equiv) in CH2Cl2 (5
mL) was added benzenesulfonyl chloride (0.08 mL, 1.1 equiv).
The resultant mixture was stirred at room temperature for
16 h. The mixture was then diluted with EtOAc and washed
successively with 10% citric acid, H2O and brine. The organic
extract was dried (MgSO4) and concentrated under reduced
pressure. The residue was purified by flash chromatography
(gradient elution: 30% EtOAc in hexane to 50% EtOAc in
hexane) to afford the desired coupled product (121 mg, 69%
yield). The N-Boc group of the coupled product was then
converted to the cyanamide (N-CN) following general proce-
dure 1 (i.e. successive treatment with TFA and BrCN). The
crude material was purified by flash chromatography (gradient
elution: 30% EtOAc in hexane to 67% EtOAc in hexane) to
1-Cya n o-3-a zetid in yl Cycloh exylm eth yl Eth er (17). To
a
cold (0 °C), stirred solution of tert-butyl 3-hydroxy-1-
azetidinecarboxylate (15) (173 mg, 1 equiv) in DMF (2 mL)
was added NaH (60% in oil, 60 mg, 1.5 equiv). The suspension
was stirred at 0 °C for 30 min followed by the addition of the
cyclohexylmethyl bromide (280 µL, 2 equiv). The resulting
suspension was poured into H2O and extracted with Et2O (3×).
The combined organic extracts were washed with H2O and
brine, dried (MgSO4) and concentrated under reduced pres-
sure. The residue was purified by flash chromatography
(gradient elution: 5% EtOAc in hexane to 50% EtOAc in
hexane) to afford the desired cyclohexylmethyl ether (215 mg,
80% yield). The N-Boc group of the cyclohexymethyl ether was
then converted to the cyanamide (N-CN) following general
procedure 1 (i.e. successive treatment with TFA and BrCN).
The crude material was purified by flash chromatography
(gradient elution: 10% EtOAc in hexane to 20% EtOAc in
hexane) to afford the desired cyanamide 17 (82 mg, 53%
yield). 1H NMR (500 MHz, CDCl3): δ 4.16 (m, 3H), 3.95 (m,
2H), 3.00 (m, 2H), 1.38-1.65 (m, 6H), 0.72-1.19 (m, 5H). m/ z
(+APCI): 195.2 [M + H+]+. HRMS (+FAB): calcd for C11H19N2O
[MH+] 195.14968, found 195.14974. Anal. (C11H18N2O) N; C:
calcd, 68.01; found, 67.25; H: calcd, 9.34; found, 8.60.
1
afford the desired cyanamide 19 (53 mg, 57% yield). H NMR
(400 MHz, CDCl3): δ 7.85 (m, 2H), 7.58 (m, 3H), 5.20 (br t,
1H), 4.16 (t, 2H), 3.81 (dd, 2H), 3.13 (t, 2H), 2.83 (m, 1H). m/z
(+APCI): 251.9 [M + H+]+. HRMS (+FAB): calcd for
C
11H14N3O2S [MH+] 252.08068, found 252.080671.
N -[(1-Cya n o-3-a ze t id in yl)m e t h yl]cycloh e xa n e ca r -
boxa m id e (20). To a cold (0 °C), stirred solution of the amine
22 (100 mg, 1 equiv) and Et3N (0.15 mL, 2 equiv) in CH2Cl2 (5
mL) was added cyclohexanecarbonyl chloride (0.11 mL, 1.5
equiv). The resultant mixture was stirred at room temperature
for 16 h. The mixture was then diluted with EtOAc and
washed successively with 10% citric acid, H2O and brine. The
organic extract was dried (MgSO4) and concentrated under
reduced pressure. The residue was purified by flash chroma-
tography (gradient elution: 50% EtOAc in hexane to 75%
EtOAc in hexane) to afford the desired coupled product (96
mg, 60% yield). The N-Boc group of the coupled product was
then converted to the cyanamide (N-CN) following general
procedure 1 (i.e. successive treatment with TFA and BrCN).
The crude material was purified by flash chromatography
(gradient elution: 50% EtOAc in hexane to 85% EtOAc in
hexane) to afford the desired cyanamide 20 (25 mg, 21%
ter t-Bu tyl 3-(Am in om eth yl)-1-azetidin ecar boxylate (22).
To a cold (0 °C), stirred solution of tert-butyl 3-hydroxy-1-
azetidinecarboxylate (15) (1.23 g, 1 equiv) in EtOAc (10 mL)
was added Et3N (1.28 mL, 1.3 equiv) followed by MsCl (0.66
mL, 1.2 equiv). The resultant suspension was stirred at 0 °C
for 1 h. The undissolved material (Et3N‚HCl) was filtered off
and washed with EtOAc. The organic filtrate was concentrated
under reduced pressure to afford an oil. This crude oil
(mesylate) was dissolved in DMSO (5 mL) and NaCN (0.696
g, 2 equiv) was added. The resultant reaction mixture was
stirred at 130 °C for 2 days. The reaction mixture was then
poured into H2O and extracted with Et2O (2×). The combined
organic extracts were washed with H2O and brine, dried over
MgSO4 and concentrated under reduced pressure to afford a
residue which was purified by flash chromatography (gradient
elution: 25% EtOAc in hexane to 33% EtOAc in hexane) to
yield the intermediate nitrile (735 mg, 57% yield). To a stirred
solution of this nitrile (728 mg, 1 equiv) in MeOH (5 mL) was
added NH3 (2M in MeOH, 10 mL) and Raney nickel (1 mL).
The solution was evacuated, placed under a H2 atmosphere
(40 atm) and stirred at room temperature for 3.5 h. The
1
yield). H NMR (500 MHz, CDCl3): δ 5.5 (br s, 1H), 4.10 (m,
2H), 3.74 (m, 2H), 3.31 (m, 2H), 2.78 (m, 1H), 1.96 (m, 1H),
1.06-1.75 (m, 10 H). m/z (+APCI): 222.2 [M + H+]+. HRMS
(+FAB): calcd for
222.16064.
C
12H20N3O [MH+] 222.16064, found
En zym e In h ibition . To measure enzyme activity, enzy-
matic assays were carried out in 50 mM MES pH 5.5
containing 2.5 mM DTT, 2.5 mM EDTA and 10% DMSO. IC50
values of compounds were determined for cathepsins K, L and

Nonpeptidic Cyanamides as Inhibitors of Cathepsins K/ L
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 103
B using 25 µM of Z-Phe-Arg-pNA as substrate. Prior to the
addition of substrate, different concentrations of the inhibitor
ranging from 100 µM down to 2 nM were preincubated for 30
min with the enzyme (2-4 nM) to allow the establishment of
the enzyme-inhibitor complex. Substrate was then added and
the enzyme activity measured from the increase of OD at 405
nm. The final volume of the reaction was 300 µL. Assays were
performed in 96-well plate format and the plates read using a
Vmax (Molecular Devices) plate reader. The percent inhibition
of the reaction was calculated from a control reaction contain-
ing only the compound vehicle. IC50 curves were generated by
fitting percentage inhibition values to a four-parameter logistic
model using a data analysis computer program (SOFTmax
PRO, Molecular Devices). The inhibition of human cathepsin
K gelatinase activity was measured using a fluorescent assay
based on the release of quenched fluorescein from DQ gelatin
(Molecular Probes). Assays were performed under the same
conditions as for the colorimetric assay. The final concentration
of DQ gelatin was 0.25 mg/mL and the reaction was measured
using excitation and emission wavelengths of 495 and 515 nm,
respectively.
for 1 min and the supernatant withdrawn with a pipet and
retained. 25 mL of medium was added back to the tissue and
rocked again. The second supernatant was combined with the
first. Cells were diluted 1:10 in 2% acetic acid in PBS, counted
by hemacytometer and diluted to 5 × 106 cells/mL in R-MEM
containing 2% FBS. 200-µL aliquots were plated onto 6-mm
diameter × 200-µm bovine bone slices. After 2 h, test com-
pounds were diluted in R-MEM containing 2% FBS, 10 nM
1,25(OH)2D3, the plating medium removed and 200 µL of test
media was added to triplicate wells. The cultures were
incubated for 3 days at 37 °C in a 5% CO2 atmosphere.
The medium was then removed from the cultures and col-
lagen fragments released into the medium measured by the
CROSSLAPS elisa assay (Osteometer Biotech, Herlev, Den-
mark).
Refer en ces
(1) Turk, B.; Turk, D.; Turk, V. Lysosomal cysteine proteases: more
than scavengers. Biochim. Biophys. Acta 2000, 1477, 98-111.
(2) Storer, A. C.; Menard, R. Catalytic mechanism in papain family
of cysteine peptidases. Methods Enzymol. 1994, 244, 486-500.
(3) (a) Cataldo, A. M.; Nixon, R. A. Enzymatically active lysosomal
proteases are associated with amyloid deposits in Alzheimer
brain. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3861-5. (b) Bever,
C. T., J r.; Garver, D. W. Increased cathepsin B activity in
multiple sclerosis brain. J . Neurol. Sci. 1995, 131, 71-3. (c)
Takeda, A.; J imi, T.; Wakayama, Y.; Misugi, N.; Miyake, S.;
Kin etic An a lysis. The measurement of enzyme activity to
determine kinetic parameters was performed in 50 mM MES
pH 5.5 containing 2.5 mM DTT, 2.5 mM EDTA and 10%
DMSO. Z-Phe-Arg-AMC was used as substrate and enzymatic
activity was measured at room temperature in 1 mL stirred
cells using a Quantamaster spectrofluorometer (Photon Tech-
nology International) with excitation and emission wave-
lengths of 355 nm of 460 nm, respectively. Product formation
was measured at different inhibitor concentrations following
initiation of the reaction by the addition of the enzyme (0.1
nM final concentration). The Km value for Z-Phe-Arg-AMC was
determined (30 µM) for cathepsin K, and inhibition assays
were performed at a substrate concentration 10-fold below the
Km value. The formation of product (P) with time for an enzyme
inhibited by a slow-binding inhibitor is described by the
following equation:13
Kumagai, T. Demonstration of cathepsins B,
H and L in
xenografts of normal and Duchenne-muscular-dystrophy muscles
transplanted into nude mice. Biochem. J . 1992, 288, 643-8. (d)
Littlewood-Evans, A. J .; Bilbe, G.; Bowler, W. B.; Farley, D.;
Wlodarski, B.; Kokubo, T.; Inaoka, T.; Sloane, J .; Evans, D. B.;
Gallagher, J . A. The osteoclast-associated protease cathepsin K
is expressed in human breast carcinoma. Cancer Res. 1997, 57,
5386-90.
(4) Riese, R. J .; Mitchell, R. N.; Villadangos, J . A.; Shi, G. P.; Palmer,
J . T.; Karp, E. R.; De Sanctis, G. T.; Ploegh, H. L.; Chapman, H.
A. Cathepsin S activity regulates antigen presentation and
immunity. J . Clin. Invest. 1998, 101, 2351-63.
(5) (a) Shi, G. P.; Chapman, H. A.; Bhairi, S. M.; DeLeeuw, C.;
Reddy, V. Y.; Weiss, S. J . Molecular cloning of human cathepsin
O, a novel endoproteinase and homologue of rabbit OC2. FEBS
Lett. 1995, 357, 129-34. (b) Li, Y. P.; Alexander, M.; Wucherpfen-
nig, A. L.; Yelick, P.; Chen, W.; Stashenko, P. Cloning and
complete coding sequence of a novel human cathepsin expressed
in giant cells of osteoclastomas. J . Bone Miner. Res. 1995, 10,
1197-202. (c) Bromme, D.; Okamoto, K. Human cathepsin O2,
a novel cysteine protease highly expressed in osteoclastomas and
ovary molecular cloning, sequencing and tissue distribution. Biol.
Chem. Hoppe-Seyler 1995, 376, 379-84.
(6) (a) Delaisse, J . M.; Eeckhout, Y.; Vaes, G. In vivo and in vitro
evidence for the involvement of cysteine proteinases in bone
resorption. Biochem. Biophys. Res. Commun. 1984, 125, 441-
7. (b) Everts, V.; Delaisse, J . M.; Korper, W.; Niehof, A.; Vaes,
G.; Beertsen, W. Degradation of collagen in the bone-resorbing
compartment underlying the osteoclast involves both cysteine-
proteinases and matrix metalloproteinases. J . Cell. Physiol.
1992, 150, 221-31. (c) Everts, V.; Delaisse, J . M.; Korper, W.;
Beertsen, W. Cysteine proteinases and matrix metalloprotein-
ases play distinct roles in the subosteoclastic resorption zone.
J . Bone Miner. Res. 1998, 13, 1420-30.
P ) vst - (vs - vo) (1 - e-k )/kobs
(3)
obs
where vs is the rate of the reaction at steady-state, vo is the
initial velocity of the reaction, and kobs is the apparent first-
order rate constant characterizing the establishment of the
steady-state velocity. Experimental values were fitted by
nonlinear regression to eq 3 and fitted parameters vs, vo, and
k
obs were used to estimate pre-steady-state kinetic parameters
kon and koff (eq 1) and the dissociation constant Ki using the
following relationships:13
Ki ) [I]/((vo/vs) - 1)
kon ) kobs/([I] + Ki)
koff ) konKi
(4)
(5)
(6)
(7) Yamashita, D. S.; Dodds, R. A. Cathepsin K and the design of
inhibitors of cathepsin K. Curr. Pharm. Des. 2000, 6, 1-24.
(8) Morrison, J . F.; Walsh, C. T. The behavior and significance of
slow-binding enzyme inhibitors. Adv. Enzymol. Relat. Areas Mol.
Biol. 1988, 61, 201-301.
(9) Duggleby, R. G.; Attwood, P. V.; Wallace, J . C.; Keech, D. B.
Avidin is a slow-binding inhibitor of pyruvate carboxylase.
Biochemistry 1982, 21, 3364-70.
(10) (a) Hanzlik, R. P.; Zygmunt, J .; Moon, J . B. Reversible covalent
binding of peptide nitriles to papain. Biochim. Biophys. Acta
1990, 1035, 62-70. (b) Dufour, E.; Storer, A. C.; Menard, R.
Engineering nitrile hydratase activity into a cysteine protease
by a single mutation. Biochemistry 1995, 34, 16382-8. (c)
Brisson, J . R.; Carey, P. R.; Storer, A. C. Benzoylamidoacetoni-
trile is bound as a thioimidate in the active site of papain. J .
Biol. Chem. 1986, 261, 9087-9. (d) Moon, J . B.; Coleman, R. S.;
Hanzlik, R. P. Reversible covalent inhibition of papain by a
The value of Ki was calculated using eq 4 for each inhibitor
concentration which gave a final steady-state velocity (vs) of
between 20% and 80% that of the initial velocity (vo). The pre-
steady-state constants kon and koff were calculated from the
above values of kobs and Ki using eqs 5 and 6. The values of Ki,
k
on, and koff, determined for at least three inhibitor concentra-
tions, were averaged to give the values shown in Table 3.
Bon e-Resor p tion Assa y. The long bones were aseptically
isolated from a 10-day-old New Zealand White hare (Covance
Research Products, Denver, PA) and the soft tissue removed.
The bones were minced into ∼1-mm pieces with scissors in 10
mL of R-MEM (Gibco BRL, Gaithersburg, MD) containing
penicillin/streptomycin, pH 7.1. The volume was brought to
25 mL and the tissue transferred to a 50-mL tube. The tube
was rocked gently for 60 cycles, the tissue allowed to settle
peptide nitrile. 13C NMR evidence for
a thioimidate ester
adduct. J . Am. Chem. Soc. 1986, 108, 1350-1.

104 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Falgueyret et al.
(11) MacLeod, A. M.; Baker, R.; Freedman, S. B.; Patel, S.; Merchant,
K. J .; Roe, M.; Saunders, J . Synthesis and muscarinic activities
of 1,2,4-thiadiazoles. J . Med. Chem. 1990, 33, 2052-9.
(12) Kozikowski, A. P.; Fauq, A. H. Synthesis of novel four-membered
ring amino acids as modulatorrs of the n-methyl-d-aspartate-
(NMDA) receptor complex. Synlett 1991, 783-4.
(13) Izquierdo-Martin, M.; Stein, R. L. Transition-state structural
features for the association of metalloproteases with phosphorus-
containing inhibitors. J . Am. Chem. Soc. 1992, 114, 1527-8.
J M0003440