Novel route to the synthesis of peptides containing 2-amino-1′-hydroxymethyl ketones and their application as cathepsin K inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00611-X

Cathepsin K is highly expressed in human osteoclasts, and is implicated in bone resorption. This makes it an attractive target for the treatment of osteoporosis. Peptides containing 2-amino-1′-hydroxymethyl ketones and 2-amino-1′-alkoxymethyl ketones were

Full text of DOI:10.1016/S0960-894X(02)00611-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2887–2891
Novel Route to the Synthesis of Peptides Containing
2-Amino-10-hydroxymethyl Ketones and Their
Application as Cathepsin K Inhibitors
Rohan V. Mendonca,* Shankar Venkatraman and James T. Palmer
Celera, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 16 April 2002;accepted 15 July 2002
Abstract—Cathepsin K is highly expressed in human osteoclasts, and is implicated in bone resorption. This makes it an attractive
target for the treatment of osteoporosis. Peptides containing 2-amino-10-hydroxymethyl ketones and 2-amino-10-alkoxymethyl
ketones were discovered as potent inhibitors of cathepsin K. A novel synthetic route was devised to facilitate rapid elucidation of
the SAR of these inhibitors. The synthesis and SAR of hydroxymethyl ketones are presented.
# 2002 Elsevier Science Ltd. All rights reserved.
Cysteine proteases have been implicated in various dis-
eases such as cancer metastasis,1 rheumatoid arthritis,2
Chagas disease,3 muscular dystrophy,4 etc. We have
been particularly interested in cathepsin K, as it is
implicated in osteoporosis. Cathepsin K is a member of
papain superfamily of cysteine proteases. Cathepsin K is
highly expressed in human osteoclasts and degrades
Type I collagen under acidic conditions in osteclast
mediated bone resorption.5,6 In addition, pycnodysos-
tosis a rare sclerosing skeletal dysplasia with low bone
turnover or increased bone density, has been shown to
be caused by mutations in the cathepsin K gene.7
Cathepsin K therefore appears to be a novel thera-
peutic target for various osteoclastic-related bone diseases.
inhibit cathepsin K, L, B, and S by the above mentioned
mechanism.
We also devised a new synthetic route (Scheme 2) to
hydoxymethyl (Fig. 1, R¼H) and alkoxymethyl ketones
(Fig. 1, R¼CH3, CH2CH3, CH2Ph) which circumvents
using a potentially explosive reagent used in prior syn-
thetic methodologies.16ꢀ18 This novel synthetic route
(Scheme 2) also eliminates intermediates that are
potential irreversible inhibitors of cathepsins and avoids
racemization of the product caused by the hydrolysis of
the acyloxymethyl ketone to the hydroxymethyl ketone
under basic conditions.
To date a number of reversible and irreversible inhibi-
tors of cathepsins have been reported.8 The known
reversible inhibitors of cysteine proteases are alde-
hydes,9 nitriles,10 a-keto amides,11 cyclopropenones,12
diaminoketones,13 etc. These chemotypes inhibit
cysteine proteases by forming a reversible covalent
bond between the electophilic functionality of the inhi-
bitor and the nucleophilic sulfur atom of the active site
cysteine residue.14,15 Herein we report hydroxymethyl
and alkoxymethyl ketone type inhibitors (1, Fig. 1) that
Chemistry
The syntheses of hydroxymethyl ketones in our labora-
tory were carried out via bromomethyl ketones, which
were synthesized from commercially available amino
acids using literature methods (Scheme 1).16,17 The bro-
momethyl ketones 3 were then converted to the 2,5-
*Corresponding author. Tel.: +1-650-866-6289;fax: +1-650-866-
6654;e-mail: rohan.mendonca@celera.com
Figure 1. R¼H, CH3, CH2CH3, CH2Ph.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00611-X

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N-protected amino acids under typical peptide coupling
procedures to give 6. This was hydrolyzed with catalytic
potassium carbonate in methanol to give the hydroxy-
methyl ketone 7.20
The above mentioned synthetic route (Scheme 1)
involves the use of explosive reagent diazomethane,
making it unsuitable for large scale synthesis. In addi-
tion acyloxymethyl ketones 4 are known irreversible
inhibitors of cysteine proteases.19 This led us to devise
an alternative synthetic route to hydroxymethyl
ketones.
The syntheses of hydroxymethyl ketones and alkox-
ymethyl ketones were carried out via Weinreb amide
(Scheme 2). Commercially available amino acids were
converted to the Weinreb amides 8.21 The Weinreb
amides were then added to alkoxymethyl Grignard,22ꢀ24
generated in situ at ꢀ60 ꢁC and then warmed to room
temperature to obtain the alkoxymethyl ketones 9.25
Catalytic mercuric (II) chloride is crucial for the gen-
eration of the alkoxymethyl Grignard. The alkoxy-
methyl ketones 9 were de-protected to amines 10 and
then coupled further to obtain the final alkoxymethyl
ketones 1. The benzyloxymethyl ketones (1,
R¼CH2Ph) obtained from the above procedure were
reduced to the hydroxymethyl ketones 7 using hydro-
gen transfer conditions.26 The added advantage of this
method is that it yielded alkoxymethyl ketones that can
be used to probe the prime side binding sites of the
enzyme cathepsin K.
Scheme 1. Synthesis of hydroxymethyl ketones via diazomethyl
ketones: (a) 1. IBCF, NMM, CH2N2;(b) HBr/HOAc;(c) 2,5-dichloro-
benzoic acid, KF, DMF;(d) anhyd p-TsOH, CH2Cl2;(e) IBCF,
NMM, THF;(f) cat. K 2CO3, CH3OH.
Results and Discussion
A panel of substituted methyl ketones were screened
against cathepsin K, L, B, and S.27 Table 1 shows var-
ious substituted methyl ketones that incorporate the
same R3 and R2 elements to get a more accurate com-
parison of the effect of the R substituent on cathepsin
binding, potency and selectivity. A similar derivative
incorporating a hydroxymethyl ketone 15 and methyl
ketone 16 is also included in the table for comparison
purposes. It is clear from Table 1 that the hydro-
xymethyl ketone 15 is the most potent of all these
Scheme 2. Synthesis of hydroxymethyl ketone via benzyloxymethyl
ketones: (a) DCC, THF, CH3NHOCH3;(b) ROCH 2Cl, Mg, HgCl2,
THF, ꢀ20–0 ꢁC, 24 h;(c) anhyd p-TsOH, CH2Cl2;(d) IBCF, NMM,
THF;(e) 20% Pd(OH) 2, cyclohexene, EtOH, 80 ꢁC.
dichloro benzoates 4 by potassium fluoride mediated
substitution of the carboxylic acid.19 The de-protection
of ketones 4 were carried out using anhydrous p-TsOH
to yield amines 5. Amines 5 were then coupled to
Table 1. SAR of substituted methyl ketones. Ki app. values are in mM
Compd
R
Cat K mM
Cat L mM
Cat S mM
Cat B mM
12
13
14
15
16
CH2OCH3
CH2OCH2CH3
CH2OCH2Ph
CH2OH
0.607
0.947
1.0
0.025
1.44
22.3
16.7
6.34
0.64
1.96
4.5
0.451
0.188
>150
>150
119
34.7
519
CH3

R. V. Mendonca et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2887–2891
Table 2. SAR of hydroxymethyll ketones. Ki app. values are in mM
2889
R3
R2
R1
Cat K mM
Cat L mM
Cat S mM
Cat B mM
17
18
19
20
21
22
0.067
3
0.811
32.2
0.111
0.11
0.79
0.088
0.11
1.44
1.14
1.25
1.95
0.27
0.76
0.020
0.003
0.004
0.13
0.46
0.017
0.081
0.046
0.18
chemotypes. This is probably due to the stabilization of
the enzyme–inhibitor complex via additional hydrogen
bonds between the a-hydroxyl group and the active site
hydrogen bond acceptors.
will be further explored to yield more potent com-
pounds in future.
Conclusions
Modifications to the side-chain elements were made (R1
and R2) to improve their potency towards cathepsin K
and selectivity over cathepsin B, L, and S. Based on
selectivity issues (15 and 17) the isoleucine-homphenyl-
alanine sequence was chosen for R3 optimization.
Table 2 presents the data from the hydroxymethyl
ketones.
We have shown a novel synthetic route to hydro-
xymethyl and alkoxymethyl ketones, which, in addition
to circumventing various synthetic problems can also be
used to facilitate the identification of potent and selec-
tive inhibitors of cathepsins K, L, B, and S.
Comparison of compounds 18 and 22 in Table 2 also
suggest that basic groups at R3 yielded more potent
compounds for cat K, as they can interact with asp 61
at the S3 subsite of the enzyme.28 The substitution
pattern of aminomethyl groups (21 and 22) on the
benzoic acid is also critical for the interaction with
aspartic acid 61 of the cathepsin K enzyme.28 This fact
Acknowledgements
Celera, 180 Kimball way, South San Francisco, CA 94080,
USA. The authors thank the following individuals for the
in vitro assay data: Kyle Elrod, Cynthia Barrett, Tobee
Chong, Lynne Cregar, Siobhan Loftus, Daun Putnam,
Martin Wong, Eric Springman and Winnie Xu.

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R. V. Mendonca et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2887–2891
the process. The reaction is then quenched by adding sat.
References and Notes
NH4Cl solution slowly with stirring. This was then stirred for
15–30 min or till effervescence ceased. This is then extracted
with ethyl acetate (3ꢂ50 mL), dried with anhydrous Mg2SO4,
filtered and concentrated. This is then purified by flash column
chromatography using silica gel 60 and 15% ethyl acetate in
hexane to yield 317.3 mg of product. 1H NMR (200 MHz,
CDCl3) d 1.43 (s, 9H), 1.74–1.82 (m, 1H), 2.14–2.16 (m, 1H),
2.60–2.67 (m, 2H), 4.13–4.14 (d, 2H), 4.49–4.61 (m, 3H), 5.14–
5.17 (m, 1H), 7.1–7.4 (m, 10H).>99% ee by chiral HPLC
(Chiralpak OJ and OD) using a 50% IPA/50% Hexane gradient.
Note: 6 equiv of Mg turnings were used for protected mono
peptide, 9 equiv for protected dipetide, etc.)
26. General procedure for the reduction of the benzyloxy-
methyl ketone to the hydroxymethyl ketone using hydrogen
transfer conditions: To a solution of the benzyloxymethyl
ketone (214 mg, 0.39 mmol) in 12 mL ethanol was added 5 mL
cyclohexene. To this was added 35 mg 20% palladium
hydroxide on carbon (0.1 equiv by weight or greater). The
reaction was then refluxed for 1–2 h till starting material was
consumed. This was followed by TLC analysis. The reaction
was then cooled to room temperature and filtered through
Celite. The filtrate was then concentrated to give the pure
product 18. 1H NMR (200 MHz, DMSO-d6): d 0.8–1.0 (m,
6H), 1.19–1.3 (m, 1H), 1.5–1.6 (m, 1H), 1.7–1.85 (m, 1H), 1.9–
2.10 (m, 1H), 3.6–3.7 (m, 1H), 4.17–4.19 (d, 2H), 4.29–4.35 (m,
1H), 4.40–4.45 (t, 1H), 5.0–5.15 (m, 1H), 7.14–7.3 (m, 4H),
7.5–7.6 (m, 1H), 8.0–8.10 (m, 2H), 8.37 (s, 1H), 8.5–8.53 (d,
1H). M+H+ (454.20).
27. Materials: Cathepsin B (Calbiochem;San Diego, Cali-
fornia) and cathepsin L (Athens Research and Technology;
Athens, Georgia) were purified from human liver and pur-
chased from the indicated commercial sources. Human cathe-
psins S and K were cloned and expressed as described.29,30 The
enzyme active site concentrations were measured by titration
with the either E-64 or the vinyl sulfone APC-3316, Me-Pip-
Phe-HphVSPh.31
Inhibition Assays: Inhibitor potency measurements were
performed at room temperature using 96-well kinetic plate
readers. Reaction velocities were monitored at varying inhib-
itor concentrations by following the hydrolysis of amino-
methylcoumarin substrates (ex355, em460) as indicated. All
substrates were added at a concentration equal to their Km.
Control reactions in the absence of inhibitor were performed
in parallel. The Ki apparent (Ki0) values were determined by a
nonlinear least squares regression fit of the experimentally
derived data to the Morrison equation for tight-binding inhib-
itors as described32 or by least squared regression fit of the
Henderson equation for tight-binding inhibitors.33 Enzyme
and inhibitor were incubated 30-min prior to initiation of
reaction by the addition of substrate.
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25. General procedure for the syntheses of alkoxymethyl
ketones: In a dry flask, Mg turnings (199 mg, 7.44 mmol), pre-
viously dried in an oven overnight at 100 ꢁC was weighed out,
along with HgCl2 (71 mg, 0.49 mmol). This was then purged
with nitrogen for 10–15 min to ensure an anhydrous atmosphere.
To this was added anhydrous THF under nitrogen. The tem-
perature was lowered to ꢀ40 ꢁC and benzyloxymethyl chloride
(1.1 mL, 7.44 mmol) was added via syringe to the stirring sus-
pension. This was then stirred under nitrogen for 6 h. The tem-
perature was allowed to warm up to 3–5 ꢁC during this time.
The temperature was then lowered to ꢀ60 ꢁC and to this was
added 400 mg (1.24 mmol) of 8 (R+CH2CH2Ph). This was then
stirred overnight and allowed to come to room temperature in
Cathepsin B: Enzyme (5.0 nM) was mixed with inhibitor in
50 mM MES or BES (pH 6.0), 2.5 mM DTT, 2.5 mM EDTA,
0.05% Tween 20 and 10% DMSO. The substrate was Z-Phe-
Arg-AMC (300 mM).
Cathepsin K: Enzyme (3.6 nM) was mixed with inhibitor in
50 mM MES (pH 5.5), 2.5 mM DTT, 2.5 mM EDTA, 0.05%
Tween-20 and 10% DMSO. The substrate was Z-Phe-Arg-
AMC (40 mM).
Cathepsin L: Enzyme (1.3 nM) was mixed with inhibitor in
50 mM MES (pH 5.5), 2.0 mM EDTA, 2 mM DTT, 0.05%
Tween-20 and 10% DMSO. The substrate was Z-Phe-Arg-
AMC (10 mM).
Cathepsin S: Enzyme (1.0 nM) was mixed with inhibitor in
50 mM MES (pH 6.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM
2-mercaptoenthanol, 0.001% bovine serum albumin and 10%
DMSO. The substrate was Z-Val-Val-AMC (10 mM).
28. McGrath, M. E.;Palmer, J. T.;Bromme, D.;Somoza,
J. R. Protein Sci. 1998, 7, 1294.

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