Acyclic cyanamide-based inhibitors of cathepsin K

Article abstract of DOI:10.1016/j.bmcl.2005.04.032

Conversion of the proline-derived cyanamide lead to an acyclic cyanamide capable of forming an additional hydrogen bond with cathepsin K resulted in a large increase in inhibitory activity. An X-ray structure of a co-crystal of a cyanamide with cathepsin K confirmed the enzyme interaction. Furthermore, a representative acyclic cyanamide inhibitor 6r was able to attenuate bone resorption in the rat calvarial model.

Full text of DOI:10.1016/j.bmcl.2005.04.032

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3039–3043
Acyclic cyanamide-based inhibitors of cathepsin K
David G. Barrett,a, David N. Deaton,a,* Anne M. Hassell,b Robert B. McFadyen,a
Aaron B. Miller,b Larry R. Miller,c J. Alan Payne,c Lisa M. Shewchuk,b
Derril H. Willard, Jr.d,z and Lois L. Wrighte
aDepartment of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
bDiscovery Research Computational, Analytical, and Structural Sciences, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
cDepartment of Molecular Pharmacology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
dDepartment of Gene Expression and Protein Purification, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
eDiscovery Research Biology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
Received 24 February 2005; revised 12 April 2005; accepted 18 April 2005
Available online 17 May 2005
Abstract—Conversion of the proline-derived cyanamide lead to an acyclic cyanamide capable of forming an additional hydrogen
bond with cathepsin K resulted in a large increase in inhibitory activity. An X-ray structure of a co-crystal of a cyanamide with
cathepsin K confirmed the enzyme interaction. Furthermore, a representative acyclic cyanamide inhibitor 6r was able to attenuate
bone resorption in the rat calvarial model.
Ó 2005 Elsevier Ltd. All rights reserved.
Osteoporosis results from an imbalance in the normally
tightly coupled processes of bone formation and bone
resorption that maintain the adult skeleton.1 This skele-
tal fragility, resulting from the loss of bone mineral den-
sity and integrity, is the leading cause of non-traumatic
fracture. In light of the risks associated with hormone
replacement therapy, there is continued interest in the
development of new agents to treat osteoporosis.2 One
target receiving considerable attention from the pharma-
ceutical industry is the cysteine protease cathepsin K.3
eral have entered clinical trials for the treatment of post-
menopausal osteoporosis.5–7
As part of a sustained effort to develop novel cathepsin
K inhibitors, researchers from these laboratories re-
cently reported the discovery of reversible, competitive,
cyanamide-based cathepsin K inhibitors.8 Starting from
a high throughput screening hit, a key pharmacophore 1
(Ki = 2100 nM) was identified. Addition of P2–P3 sub-
stituents to this pyrrolidine cyanamide led to the devel-
opment of nanomolar cathepsin K inhibitors such as 2
(Ki = 60 nM) and 3 (Ki = 1.8 nM). An X-ray crystal
structure of a cyanamide-based inhibitor bound to
cathepsin K showed that the active site thiol of cathep-
sin K adds to the carbon nitrogen triple bond of the
cyanamide to form an isothiourea, confirming the con-
clusions of researchers at Merck, who applied 13C
NMR studies to determine the mode of inhibition of pa-
pain by cyanamides.9 The crystal structure also revealed
that the proposed P2 substituent did indeed occupy the
S2 pocket, although this interaction was not optimal.
Peptide aldehyde-based inhibitors from these laborato-
ries form a key hydrogen bond between their carbamate
NH and 161Asn of cathepsin K,10,11 whereas cyanamides
such as 1–3 cannot undergo such interactions. Surmising
that additional potency could be achieved by introduc-
ing a hydrogen bond donor into the cyanamide-based
Cells of hematopoietic origin called osteoclasts resorb
bone by adhering to the bone surface and secreting acid
and proteases that degrade the mineral and protein com-
ponents of bone. The cysteine protease cathepsin K is
highly expressed in osteoclasts, and, in complex with
glycosaminoglycans, rapidly hydrolyzes type I collagen,
the major component of bone matrix.4 Cathepsin K
inhibitors have proven effective in protecting skeletal
integrity in animal models of bone resorption, and sev-
Keywords: Cyanamide; Cathepsin K; Cysteine protease inhibitor.
*
Corresponding author. Tel.: +1 919 483 6270; fax: +1 919 315
0430; e-mail: david.n.deaton@gsk.com
Present address: Lilly Forschung GmbH, Essener Str. 93,
22419Hamburg, Germany.
zDeceased.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.032

3040
D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3039–3043
inhibitors, it was decided to break open the pyrrolidine
ring and replace the resulting sp3 carbon atom adjacent
to the nitrogen with a second nitrogen atom.
As shown in Scheme 1, the acyclic cyanamides could be
synthesized from commercially available hydrazines
4a,e, and j–q. Treatment of the hydrazines with cyano-
gen bromide in the presence of a base produced the
azacyanamides, which were then coupled to the chloro-
formates derived from known alcohols 5a11 and 5b11 to
afford the desired cyanamides 6a,e, and j–r.
The cyanamides 6f–i were synthesized as depicted in
Scheme 2. The commercially available carboxylic acids
7a–c were esterified with alcohol 5a. Then, the benzyl
carbamates were cleaved by hydrogenolysis to give the
amines 8a–c. Subsequent treatment with cyanogen bro-
mide in the presence of a base provided the cyanamides
6f–h. Cyanamide 6i was derived from acid 7a and amine
9, which was synthesized from commercially available
alcohol 5c. Mitsunobu reaction of phthalimide with
alcohol 5c, followed by hydrazine cleavage of the imide
protecting group provided the phenethylamine. Then,
exhaustive catalytic hydrogenation of the aryl ring
yielded the amine 9. Amine 9 was coupled to the acid
7a employing the carbodiimide method. This amide
was unmasked, as before, to give the amine 8d. Once
again, reaction of the amine 8d with cyanogen bromide
provided the cyanamide 6i.
Scheme 2. Reagents and conditions: (a) 7a–c, 5a, TsOH, PhH, "#, 81–
94%; or 9, 7a, EDC, HOBt, i-Pr2NEt, THF, 60%; (b) H2/Pd–C,
MeOH, 77–97%; (c) BrCN, K2CO3, MeCN, 94–99%; (d) phthalimide,
DEAD, Ph3P, THF, 93%; (e) NH2NH2, MeOH, D, 84%; (f) H2/PtO2,
AcOH, 89%.
The nitriles 6b–d were prepared as illustrated in
Scheme 3. Reaction of the commercially available amine
10 with the chloroformate derived from alcohol 5a pro-
duced the nitrile 6b. Alternatively, treatment of this
chloroformate with commercially available primary
amides 11a or b, followed by dehydration utilizing triflu-
oroacetic anhydride gave the nitriles 6c and d.
Scheme 3. Reagents and conditions: (a) 5a, 1.93 M COCl2 in PhMe,
pyridine, CH2Cl2, ꢀ20 °C to rt; 10, 11a or b, i-Pr2NEt, THF, 73–97%;
(b) (CF3CO)2O, CH2Cl2, 33–42%.
As shown in Table 1, the acyclic azacyanamide 6a
(Ki = 0.009 nM) was over two orders of magnitude more
potent than the proline-derived cyanamide
3
(Ki = 1.8 nM), in agreement with the original hypothe-
sis. This is in sharp contrast to results from Merck
researchers, wherein the acyclic cyanamide diethyl-
amino-N-carbonitrile was >100-fold less active than
N-pyrrolidine carbonitrile.9
Attempting to confirm the design hypothesis for the syn-
thesis of acyclic cyanamide 6a, the N-methyl derivative
6e (Ki = 62 nM) was prepared. The large decrease in
activity relative to analog 6a supported the hypothesis
that the carbamate NH forms a hydrogen bond to
161Asn of the enzyme. However, the loss in activity
could also be the result of unfavorable steric interactions
between the methyl substituent and the protein. To dis-
tinguish between these two possibilities and to obtain a
more direct comparison of an acyclic azacyanamide and
an acyclic cyanamide, cyanamides 6f–h were prepared.
The potency of the methylene analog 6f (Ki = 26 nM)
Scheme 1. Reagents and conditions: (a) BrCN, Na2CO3, H2O,
CH2Cl2, 67–96%; (b) 5a or b, 1.93 M COCl2 in PhMe, pyridine,
CH2Cl2, ꢀ20 °C to rt; cyanamide, i-Pr2NEt, THF, 52–92%.

D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3039–3043
Table 1. Inhibition of human cathepsin K
3041
potency was surprising, and may be due to more than
the increased electrophilicity of the cyanamide warhead.
As stated above, the conformation afforded by the sp2
character of the cyanamide nitrogen might allow a bet-
ter enzyme fit than the sp3 carbon of the nitrile
inhibitors.
#
Inhibitor
Ki (nM)a
6a
0.009
6b
6c
6d
6e
6f
7900
Replacing the ester oxygen of analog 6f with nitrogen
might still allow a hydrogen bond interaction with
161Asn, but from a different angle, perhaps leading to
an increase in potency relative to 6f. However, cyan-
amide 6i (Ki = 830 nM) proved significantly less active
than 6f or 6a, suggesting that the amide NH is unable
to form a similar hydrogen bond as the carbamate NH
of azacyanamide 6a. The loss in potency of 6i versus
6f is presumably the result of the higher entropic cost
of desolvating an amide versus an ester.
>13,000
>13,000
62
The inhibitory activity of P1 cyanamide analogs is
shown in Table 2. These results parallel the P1 SAR of
aldehyde-based cathepsin K inhibitors recently reported
from this laboratory.10 A comparison of the iso-propyl
analog 6k (Ki = 0.019 nM), cyclohexyl analog 6m
26
6g
6h
6i
2300
(Ki = 0.016 nM),
and
tert-butyl
analog
6l
(Ki = 1500 nM) with the ethyl analog 6j (Ki = 0.003 nM)
reveals that increased steric bulk adjacent to the cyan-
amide nitrogen decreases enzyme inhibition. In contrast,
a comparison of the iso-butyl analog 6o (Ki = 0.004 nM)
and benzyl analogs 6q (Ki = 0.006 nM) with the n-propyl
analog 6n (Ki = 0.004 nM) shows that branching at the
b-position from the cyanamide nitrogen has little effect
on potency.
>13,000
830
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 400 pM cathepsin K with 10 lM Cbz-Phe-Arg-
AMC as substrate in 100 mM NaOAc, 10 mM DTT, 120 mM NaCl,
pH = 5.5. The Ki values are the mean of two or three inhibition
assays, individual data points in each experiment were within a 3-fold
range of each other.
As shown in Table 3, the azacyanamide analogs 6a,k,
and r exhibit moderate selectivity versus cathepsins B
(B/K = 8–70), H (H/K = 20–160), and L (L/K = 1–40).
In contrast, although cyanamide 6f is a less potent
cathepsin K inhibitor, it is more selective versus cathep-
sins B (B/K = 100), H (H/K = >500), and L (L/K = 60).
The ability of these cyanamides to inhibit cathepsin S,
another closely related endopeptidase, was not studied.
is similar to that of the N-methyl derivative 6e, whereas
the (S)-methyl analog 6g (Ki = 2300 nM) is substantially
less potent and the (R)-methyl analog 6h is inactive at
concentrations up to 13,000 nM. Thus, the enzyme
may not be able to fully accommodate a methyl group
projecting in these directions at this site. The enhanced
potency of 6a relative to 6e–h probably arises from the
formation of a key hydrogen bond interaction with
161Asn as well as better enzyme fit. This better fit may
be the result of not only decreased steric repulsion of
hydrogen versus methyl substituents (compare 6a to
6e,g, and h), but also of the sp2 character of the carba-
mate nitrogen, which may allow an energetically more
favorable interaction between inhibitor and enzyme than
does the sp3 a-carbon of the ester (compare 6a to 6f).
Table 2. Inhibition of human cathepsin K by P1 analogs
#
R1
Ki (nM)a
6a
6j
Me
Et
0.009
0.003
0.019
1500
6k
6l
i-Pr
t-Bu
Cy
6m
6n
6o
6p
6q
0.016
0.004
0.004
0.007
0.006
Due to the presence of the electronegative nitrogen at-
tached to the carbon nitrogen triple bond, cyanamides
are stronger electrophiles than are nitriles. To explore
how important this enhanced electrophilicity was to
the inhibitory activity of the azacyanamides, nitrile ana-
logs of 6a were studied. Nitrile 6b (Ki = 7900 nM) is a
micromolar inhibitor of cathepsin K, whereas the con-
formationally biased alanine-derived nitriles 6c (Ki >
13,000 nM) and 6d (Ki > 13,000 nM) were even poorer
inhibitors. This million-fold difference in inhibitory
n-Pr
i-Bu
n-Bu
Bn
a Inhibition of recombinant human cathepsin K activity in a fluores-
cence assay using 400 pM cathepsin K with 10 lM Cbz-Phe-Arg-
AMC as substrate in 100 mM NaOAc, 10 mM DTT, 120 mM NaCl,
pH = 5.5. The Ki values are the mean of two or three inhibition
assays, individual data points in each experiment were within a 3-fold
range of each other.

3042
D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3039–3043
Table 3. Cathepsin B, H, and L inhibition and selectivity
An X-ray crystal structure of cathepsin
K and
t-BuO(C@O)NHN(Me)CN is shown in Figure 1.15
The tert-butyl P2 group occupies the S2 pocket formed
by residues 67Tyr, 68Met, 134Ala, 163Ala, and 209Leu.
This is the deepest, most hydrophobic binding region
of the cathepsin K active site. Also, the carbonyl oxygen
of the carbamate forms a hydrogen bond to the back-
bone NH of 66Gly. Furthermore, as hypothesized, the
carbamate NH also donates its hydrogen to the back-
bone carbonyl oxygen of 161Asn. This interaction
accounts for some of the increased potency of the acyclic
cyanamide inhibitor class compared with the cyclic
cyanamide analogs. The P1 methyl group has limited
interaction with the S1 groove formed from 23Gly,
24Ser, 64Gly, and 65Gly. However, it is clear that there
is limited space available for larger groups at the S1
subsite, helping explain the steric effects of the P1
substituents shown in Table 2.
#
Cat K
Ki (nM)
Cat B
Ki (nM)a
Cat H
Ki (nM)b
Cat L
Ki (nM)c
6a
6f
0.009
26
0.077
2600
1.3
1.4
0.025
1500
0.79
>13,000
0.46
—
6k
6r
0.019
0.032
0.25
0.036
a Inhibition of recombinant human cathepsin B activity in a fluores-
cence assay using 220 pM cathepsin B with 10 lM Cbz-Phe-Arg-
AMC as substrate in 100 mM NaOAc, 10 mM DTT, 120 mM NaCl,
pH = 5.5. The Ki values are the mean of two or three inhibition
assays, individual data points in each experiment were within a 2-fold
range of each other.
b Inhibition of recombinant human cathepsin H activity in a fluores-
cence assay using 107 nM cathepsin H with 50 lM L-Arg-b-naph-
thalamide as substrate in 100 mM NaOAc, 10 mM DTT, 120 mM
NaCl, pH = 5.5.
c Inhibition of recombinant human cathepsin L activity in a fluores-
cence assay using 300 pM cathepsin L with 5 lM Cbz-Phe-Arg-AMC
as substrate in 100 mM NaOAc, 10 mM DTT, 120 mM NaCl,
pH = 5.5.
A covalent isothiourea intermediate is formed by the
cyanamide moiety of the inhibitor and the active site
25Cys of the enzyme. Moreover, the NH of the isothio-
urea is stabilized by two hydrogen bonds to the side
chain carbonyl oxygen of 19Gln and the backbone NH
of 25Cys. This mode of binding is similar to the reaction
of cysteine proteases with aldehydes to form hemithio-
acetals.10 In fact, this cyanamide structure is virtually
superimposable on the t-BuO(C@O)NHCH(Cy)CHO
structure with cathepsin K previously disclosed by these
researchers.10 Direct comparison of cyanamide 6p with
its corresponding aldehyde (Ki = 2.7 nM) reveals that
the cyanamide is >100-fold more potent inhibitor of
cathepsin K. In addition to differences in the electrophi-
licity of the warheads, this discrepancy could be partially
explained by hybridization differences between the
warheads. The sp2 hybridized aldehyde requires a limi-
ted Dunitz–Burgi attack angle by the active site thiol
In contrast to proline-based cyanamides, the acyclic
azacyanamides were rapidly degraded by S9 liver slices
(<10% remaining after 1 h of incubation),12 and, with
the exception of the tert-butyl analog 6l (t1/2 > 12 h),
they were also short-lived in plasma (e.g., 6a t1/2
<
1 h). In addition to potential lability to plasma enzymes
such as amidases, the azacyanamides are probably more
reactive than the proline-based cyanamides to free thi-
ols. At pH = 5.5 in the presence of glutathione, the Kis
of the azacyanamides were not significantly different
from their Kis determined in its absence. In contrast,
in the presence of glutathione at pH = 7.0, the azacyan-
amides were substantially poorer inhibitors of cathepsin
K. This increased reactivity is similar to that seen by
Merck researchers with azetidine-based cyanamides.9
These liabilities will need to be overcome or they may
limit the in vivo utility of these azacyanamides. Despite
these shortcomings, cyanamide 6r (human Ki =
0.032 nM, rat Ki = 0.072 nM) was profiled in an ex vivo
rat calvarial resorption assay.13,14 As measured by
deoxypyridinoline crosslink release from type I collagen,
treatment of rat calvaria with parathyroid hormone
(PTH) enhanced bone resorption (see Table 4). Analog
6r dose-dependently attenuated the release of this mar-
ker of bone resorption by PTH. This efficacy reached
statistical significance at the 3000 nM and 10,000 nM
doses. There is a large discrepancy between the rat ex
vivo assay and the in vitro human and rat inhibition
values. The poor stability of 6r (plasma t1/2 < 30 min)
may partially account for this difference.
Table 4. Rat calvarial assay
Media
PTH
6r
1000 nM
6r
3000 nM
6r
10,000 nM
Figure 1. Active site of the X-ray co-crystal structure of t-
BuO(C@O)NHN(Me)CN complexed with cathepsin K. The cathepsin
K carbons are colored magenta with the inhibitor carbons colored
green. The semi-transparent white surface represents the molecular
surface, while hydrogen bonds are depicted as yellow dashed lines. The
coordinates have been deposited in the Brookhaven Protein Data
Bank, accession number 1YK8. This figure was generated using
PYMOL version 0.97 (Delano Scientific, www.pymol.org).
Av
SD
10.00a
0.21
14.41a
1.35
12.53a,b
1.23
9.45a,c
0.91
8.41a,d
1.23
a Deoxypyridinoline crosslink release from type I collagen.
b DunnettÕs multiple comparison test, p = 0.293.
c DunnettÕs multiple comparison test, p = 0.003.
d DunnettÕs multiple comparison test, p = 0.0008.

D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3039–3043
3043
6. Anonymous (2004). AAE-581, Pharmaprojects, Novem-
ber 12, 2004.
7. Anonymous (2004) 462795, Pharmaprojects, November 5,
2004.
8. Deaton, D. N.; Hassell, A. M.; McFadyen, R. B.; Miller,
A. B.; Miller, L. R.; Shewchuk, L. M.; Tavares, F. X.;
Willard, D. H., Jr.; Wright, L. L. Bioorg. Med. Chem.
Lett. 2005, 15, 1815.
on the carbonyl carbon electrophile; whereas the sp
hybridized cyanamide carbon accommodates less opti-
mal trajectories of assault. This azacyanamide contains
no P3 group, thus no interaction is realized with the S3
subsite. Additional binding energy is obviously obtained
via incorporation of a P3 substituent as in cyanamides
6a and r.
9. Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.; Wesolow-
ski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.;
Riendeau, D.; Rodan, S. B.; Percival, M. D. J. Med.
Chem. 2001, 44, 94.
10. Catalano, J. G.; Deaton, D. N.; Furfine, E. S.; Hassell, A.
M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.;
Shewchuk, L. M.; Willard, D. H., Jr.; Wright, L. L.
Bioorg. Med. Chem. Lett. 2004, 14, 275.
11. Boros, E. E.; Deaton, D. N.; Hassell, A. M.; McFadyen,
R. B.; Miller, A. B.; Miller, L. R.; Paulick, M. G.;
Shewchuk, L. M.; Thompson, J. B.; Willard, D. H., Jr.;
Wright, L. L. Bioorg. Med. Chem. Lett. 2004, 14,
3425.
In summary, very potent acyclic cyanamides such as 6a
were designed and synthesized, starting from the proline
derived cyanamide 3. Verifying the design hypothesis, an
X-ray crystal structure of a cathepsin K/azacyanamide
complex confirmed that these inhibitors form a key
hydrogen bond with 161Asn of the protein. Furthermore,
a representative cyanamide inhibitor 6r is capable of
attenuating bone resorption in a rat calvarial assay.
These promising results warrant additional efforts to im-
prove the metabolic stability and other drug properties
of these novel inhibitors.
12. Iwatsubo, T.; Hirota, N.; Ooie, T.; Suzuki, H.; Shimada,
N.; Chiba, K.; Ishizaki, T.; Green, C. E.; Tyson, C. A.;
Sugiyama, Y. Pharmacol. Ther. 1997, 73, 147.
13. Hahn, T. J.; Westbrook, S. L.; Halstead, L. R. Endocri-
nology 1984, 114, 1864.
Acknowledgments
The authors would like to thank James G. Conway for
statistical analyses.
14. Conaway, H. H.; Grigorie, D.; Lerner, U. H. J. Endocri-
nol. 1997, 155, 513.
15. (t-BuO(C@O))2NN(Me)CN was synthesized in a similar
manner to Scheme 1. Methyl hydrazine was treated with
cyanogen bromide and the resulting product was then
reacted with di-tert-butyl dicarbonate and triethylamine
to provide (t-BuO(C@O))2NN(Me)CN. Co-crystalliza-
tion of (t-BuO(C@O))2NN(Me)CN with cathepsin K at
acidic pH caused the loss of one tert-butyloxycarbonyl
References and notes
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Beresford, S. A. A.; Howard, B. V.; Johnson, K. C.;
Kotchen, J. M.; Ockene, J. JAMA 2002, 288, 321.
3. Deaton, D. N.; Kumar, S. Prog. Med. Chem. 2004, 42, 245.
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5. Stroup, G. B.; Lark, M. W.; Veber, D. F.; Bhattacharyya,
A.; Blake, S.; Dare, L. C.; Erhard, K. F.; Hoffman, S. J.;
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protecting group, resulting in
a
inhibitor/enzyme
co-crystal containing t-BuO(C@O)NHN(Me)CN with
cathepsin K. The parent (t-BuO(C@O))2NN(Me)CN
inhibited cathepsin K with an Ki = 20 nM. In hindsight,
it is probably hydrolyzed under the acidic conditions of
the enzyme assay (pH = 5.5) to produce small quantities
of t-BuO(C@O)NHN(Me)CN. Therefore, the inhibitory
activity of t-BuO(C@O)NHN(Me)CN is likely below
20 nM. Since this X-ray crystal structure was solved
several months after resources were shifted to higher
priority targets, t-BuO(C@O)NHN(Me)CN was never
prepared and tested.