Pharmacokinetic benefits of 3,4-dimethoxy substitution of a phenyl ring and design of isosteres yielding orally available cathepsin K inhibitors

Article abstract of DOI:10.1021/jm301119s

Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH₃O)₂Ph motif, such as 31, were found to have excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and balanced inhibitor (indazole, 38).

Full text of DOI:10.1021/jm301119s

Article
pubs.acs.org/jmc
Pharmacokinetic Benefits of 3,4-Dimethoxy Substitution of a Phenyl
Ring and Design of Isosteres Yielding Orally Available Cathepsin K
Inhibitors
James J. Crawford,*,†,§,∥ Peter W. Kenny,† Jonathan Bowyer,† Calum R. Cook,† Jonathan E. Finlayson,†
Christine Heyes,† Adrian J. Highton,† Julian A. Hudson,† Anja Jestel,‡ Stephan Krapp,‡ Scott Martin,†
Philip A. MacFaul,† Benjamin P. McDermott,† Thomas M. McGuire,† Andrew D. Morley,†
Jeffrey J. Morris,† Ken M. Page,† Lyn Rosenbrier Ribeiro,† Helen Sawney,† Stefan Steinbacher,‡
Caroline Smith,† and Alexander G. Dossetter*,†,∥
†AstraZeneca R&D, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
‡Proteros Biostructures, Am Klopferspitz 19, D-82152 Martinsried, Germany
S
* Supporting Information
ABSTRACT: Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical
properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH3O)2Ph motif, such as 31, were found to have
excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was
identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and
balanced inhibitor (indazole, 38).
AZD4996 (3), a potent and selective inhibitor of Cat K with
good pharmacokinetic properties,12 and the thiazolopiperazine
4 from the phenyl compound 5 (Figure 1), where increasing
the margin to hERG inhibition was improved.13 Herein we
describe the discovery of 5 and the development of the N-
phenylpiperazinyl series of compounds.
INTRODUCTION
■
The lysosomal cysteine protease cathepsin K (Cat K) is highly
expressed in osteoclasts and plays a key role in bone resorption
by the degradation of type I cartilage.1 Cat K knockout mice
exhibit osteopetrosis (abnormally dense bone) and abnormal
joint morphology,2 indicating the potential role of this enzyme
in key bone pathologies such as osteoporosis (OP), osteo-
arthritis (OA), and metastatic bone disease (MBD).3−5
Osteoarthritis is a group of degenerative disorders, charac-
terized by joint pain and loss of function in the absence of
chronic autoimmune or autoinflammatory mechanisms.6 They
have an increasing prevalence with age and are thus a growing
socioeconomic burden.2 Articular cartilage breakdown is a
prominent feature of joint degeneration and, as a result, has
received significant attention from the pharmaceutical industry.
Several companies have trialed compounds in the OP and OA
disease areas. Novartis has completed phase II studies in OP
and OA with balicatib (1; Figure 1).7 The phase II OP trial
reported positive outcomes on bone mineral density measures.
Merck has completed a phase III trial in OP with odanacatib (2;
Figure 1); the trial was stopped early as both safety and primary
efficacy (reduced bone fracture risk) end points were
reached.8−11 Both compounds are electrophilic nitrile-contain-
ing compounds that bind covalently and reversibly to the Cat K
enzyme, preventing type I collagen degradation. We have
previously described efforts that led to the identification of
Drug Hunting Approach. During our initial investigations,
which led to the discovery of 3, it was also found that N-
phenylpiperzinyl-containing compounds were an attractive
alternative series. While not as potent inhibitors as their
carboline counterparts, the (4-fluorophenyl)piperazine com-
pound 5, also made in that first library, was only 20-fold less
active.12 The binding of 5 was confirmed by solving an X-ray
structure complex (PDB 4DMX; Figure 2A). The nitrile bound
covalently to Cys25 in the P1 region, part of the Cys−Asn−His
triad, within the active site of the protein.14 The alkyl nitrile
“warhead” of 1−5, which forms this covalent bond,15,16 has the
advantage of being less electrophilic than nitriles attached to
heteroaromatic rings such as pyrimidine.17,18 The attached
amide was well positioned for protein backbone donor−
acceptor interactions,19 and the trans-cyclohexyldiamide scaf-
fold was a good fit in the P2 pocket (Figure 2B).20 The
remaining amide portion (phenylpiperazinyl) of the molecule
Received: July 31, 2012
Published: September 17, 2012
© 2012 American Chemical Society
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Figure 1. Examples of cathepsin K inhibitors disclosed in the literature, 1−3, plus program start point 5.
efficiency (LLE = pIC50 − log D7.4 = 5.7), but lacked oral
bioavailability in rat (F = 0%, clearance (Cl) 36 9.2 mL/min/
kg, volume of distribution (Vdss) 1.3 1.6 L/kg). Previously we
found that changing the group occupying the P3 groove (in this
case, phenylpiperazine) of our inhibitors improved the physical
and pharmacokinetic properties without resorting to changing
the cyclohexyl bisamide, so we focused our efforts on this
group. Cognizant of the potential for lysosomotropism and
phospholipidosis with basic compounds, we aimed to keep the
inhibitors neutral.21−24 As a result, and reasoning that with
neutral compounds the volume of distribution might be
expected to be around 1 L/kg (1.3 L/kg in 5), we were
acutely aware that low levels of metabolic clearance would be
required to achieve properties consistent with the possibility of
once-daily dosing schedules. Increased inhibition of the Cat K
enzyme would also reduce the dose level, and having achieved
<1.0 nM with carboline 3, we sought to increase potency
(aiming for at least single-digit nanomolar). With this in mind,
we started our efforts with goals of increasing Cat K potency
(<10 nM), improving bioavailability (>40% in rodent), and
driving metabolism down to a minimum (HLM < 2.0 μL/min/
mg), by investigating the structure−activity relationships
(SARs) around the phenyl and piperazine groups.
CHEMISTRY
■
Phenylpiperazines were synthesized in one of two general
methods, either leaving group displacement from a substituted
aryl (6) or employing palladium coupling conditions with
piperazines, either carbamate-protected (7) or unprotected (8)
(Scheme 1). Although diversity was introduced early, the route
was short and convenient, and the use of protecting groups
allowed the location of group R2 as required. We found the
palladium complex [1,3-bis(2,6-diisopropylphenyl)imidazol-2-
ylidene](3-chloropyridyl)palladium(II) dichloride (PEPPSI) to
be an excellent catalyst for the Buchwald−Hartwig couplings.25
Most notably at the time, good yields could be obtained using
Figure 2. Compound 5 bound to cathepsin K (PDB 4DMX): (A)
identification of key H-bonding residues, (B) surface of protein to
illustrate the fit of the cyclohexyl and phenylpiperazine moieties.
occupied the P3 region (known as the glycine shelf, Tyr67−
Gly66−Gly65) of the protein. Compound 5 was a potent
starting point, with good LE (0.37) and ligand-lipophilicity
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a
Scheme 1. Synthesis of Cyclohexyl-1,2-diamides 4−38
a
Reagents and conditions: (a) (i) K2CO3 (5 equiv), DMF, 100 °C, 5 h, 23−49%, or (ii) PEPPSI (10 mol %), KOtBu (2 equiv), DME, 70 °C, 18 h;
then (iii) 5 equiv of 1.0 M HCl in dioxane, rt, 1 h, or TFA, CH2Cl2, overall 15−52% yield; (b) (i) K2CO3 (5 equiv), DMF, 100 °C, 5 h, 45−89%, or
(ii) Pd(OAc)2 (10 mol %), BINAP (10 mol %), NaOtBu (2 equiv), toluene, 90 °C, 18 h, overall 25−68% yield; (c) (i) (R,R)-9, CH2Cl2 or DMF, rt,
1 h; (ii) CH2Cl2 or DMF, DIPEA (5 equiv), HATU (1.1 equiv), 11 or 10 (1.0 equiv), rt, 12 h, 8−84% yield; (d) NH2NH2.H2O, EtOH, rt, 18 h,
92%; (e) Ac2O neat, 10−60 min, 60−83%; (f) CH3I (2 equiv), K2CO3, DMF, rt, 5 h, 61−85%.
Table 1. SAR of Phenylpiperazine Compounds
Cat K
a
Cat S
a
Cat B
a
c
e
f
IC50
IC50
IC50
log
hPPB
aqueous solubility
(μM), pH 7.4
HLM
RLM
c
d
compd
R4
R3
nitrile R1
(μM)
(μM)
(μM)
D7.4
LE
(% free)
(μL/min/mg) (μL/min/mg)
12
13
14
15
16
17
18
19
20
5
H
F
H
H
H
H
CH2CN
CH2CN
CH2CN
CH2CN
CH2CN
CH2CN
CH2CN
CH2CN
c-pr-CN
c-pr-CN
c-pr-CN
c-pr-CN
c-pr-CN
0.007
0.023
1.03
1.68
0.455
0.315
1.8
1.9
0.43
0.39
0.42
0.40
0.39
0.37
0.37
0.36
0.38
0.37
0.36
0.34
0.33
>50
39
640
7.7
115
31
>3700
<2.9
b
b
CH3
0.011
0.572
0.399
OCH3
H
0.006
0.008
0.006
0.025
0.011
0.015
0.015
0.014
0.009
0.018
1.05
1.6
>50
390
7.1
5.6
2.2
77
OCH3
OCH3
H
149
OCH3
CN
1.09
2.6
1.27
6.8
1.8
0.67
2
53
>50
>28
>50
46
140
280
SO2CH3
H
H
1.29
2.26
1.31
2.95
2.29
2.15
1.18
1.44
1.09
1.44
3.06
2.4
<2.0
6.9
<5.3
107
H
2700
F
H
2.1
1.7
1.4
>4300
>2000
>2500
2500
<6.3
<2.0
2.3
35
21
22
23
OCH3
OCH3
SO2CH3
H
48
OCH3
H
45
<7.4
<2.2
>50
<2.0
a
Binding affinity for cathepsin versus FRET substrate, mean of greater than n = 4 tests, unless otherwise stated. All compounds test at >10 μM for
Cat L. Mean of n = 2 tests. Standard methods were used to determine log D7.4 and protein binding.39 Units of kJ mol−1 Da−1. In vitro human
liver microsomal turnover mean of at least n = 2 tests (μL/min/mg), unless otherwise stated. In vitro rat liver microsomal turnover mean of at least
b
c
d
e
f
n = 2 tests (μL/min/mg).
aryl chlorides. Nucleophilic aromatic substitution of a fluoro or
chloro group on phenyl 6, where R3 or R4 was an electron-
withdrawing group, was high yielding but required elevated
temperatures. Following either route, simple deprotection with
acid yielded the arylpiperazines 12a−38a ready for coupling.
Ring-opening of chiral anhydride (R,R)-9 with the piperazines
12a−38a gave an intermediate acid, which taken forward in one
pot gave final compounds 12−38 by amide formation with 1,1-
aminocyclopropylnitrile 10 or 11 and a suitable coupling
reagent, such as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetrame-
thyluronium hexafluorophosphate (HATU) and diisopropyle-
thylamine (DIPEA) in yields of up to 84%.26 Bicyclic
heteroaromatic halides were synthesized by known literature
methods. In short, condensation of diacetylchlorobenzene (40)
with hydrazine formed 41; ring closure of hydroxy imines 42
using acetic anhydride to form a leaving group gave the
required pyrazolo and isoxazolo bicycles 43. Finally, in the case
of 43a, the ring nitrogen could be alkylated with methyl iodide
under basic conditions to yield 43b. Isomeric pyrazoles could
be generated by varying the starting benzyl halide.
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Table 2. SAR for Piperazine Substitution
a
Binding affinity for cathepsin versus FRET substrate, mean of greater than n = 4 tests, unless otherwise stated. All compounds test at >10 μM for
b
c
d
Cat L. Mean of n = 2 tests. Units of kJ mol−1 Da−1. In vitro human liver microsomal turnover mean of at least n = 2 tests (μL/min/mg), unless
otherwise stated. In vitro rat liver microsomal turnover mean of at least n = 2 tests (μL/min/mg).
e
following the introduction of 1,2-(CH3O)2 onto a phenyl group
has also been reported in two large matched pair studies.27,28 A
similar synergistic effect of 1,2-(OCH3)2 substitution with other
rigid secondary amides was also observed in other Cat K
inhibitor series we have explored (see Table S1 in the
Supporting Information). Additionally, the introduction of a
sulfone group showed significant promise. In comparing 19 and
23, the cyclopropyl nitrile-containing example had increased
solubility by nearly 10-fold. In 19, the sulfone reduced
lipophilicity (log D7.4 = 0.67) relative to the unsubstituted
phenyl 12; however, its solubility decreased. We suggest that
the introduction of a sulfone may have increased crystal
packing, as recently reported for a series of GPR 119 agonists.29
In addition, both 19 and 23 had markedly improved metabolic
stability, with the result that when 23 was studied in vivo,
bioavailability improved to 16% (rat in vivo Cl = 22 mL/min/
kg). As a result of these interesting observations, further
analogues to investigate the SAR around the dimethoxy and
sulfone moieties were studied.
In efforts to maximize enzyme affinity, we explored
substitution of the piperazine ring with the aim of making
beneficial hydrophobic contacts with the glycine shelf (Table
2). It was hoped that, in compounds such as 24, where the (R)-
methyl group adopts an axial position adjacent to the amide,
that an increase in binding affinity would be observed due to
contact with Tyr67. 24 proved to be similar to 23 in Cat K
inhibition, with small erosions in selectivity and HLM stability
(9.4 versus <2.0 μL/min/mg for 23). A reduction in affinity
and LE was obtained with the rest of the substituted
RESULTS AND DISCUSSION
■
A small, focused library was prepared to investigate the effect of
substitution on the aryl group and two different electrophilic
nitrile groups (Table 1). These showed that substitution
around the aryl ring was well tolerated with respect to Cat K
potency, as well as both methylene (12−19) and cyclopropyl
(5, 20−23) groups adjacent to the nitrile. Of these, the
cyclopropyl seemed to offer a modest improvement in aqueous
solubility (e.g., matched pair 12 and 20), metabolic stability (15
versus 21), and modest Cat B selectivity over Cat K (e.g., 17,
211-fold, and 22, 340-fold). Unfortunately, despite exhibiting
good metabolic stability in human liver microsomes, poor rat
pharmacokinetic profiles were still observed (e.g., 20, F = 0.2%,
Cl = 19 mL/min/kg; 21, F = 3.5%, Cl > 73 mL/min/kg; both n
= 1 experiments, Table 5). A standout from our initial
investigations was the somewhat unexpected and synergistic
effect of disubstitution at the 3,4-positions of the aryl group.
Specifically, by comparing piperazines 15 with 17, and 21 with
22, we observed that methoxy groups at both the 3- and 4-
positions of the phenyl ring resulted in compounds with greater
resistance toward metabolism in both human and rat liver
microsomes. In the case of 22, enzyme affinity was maintained,
and when profiled, improved oral bioavailability in rat was
observed (41% for 22 vs 3.5% for 21 and 0.2% for 20). This
improvement in metabolic stability was initially surprising,
given that the addition of methoxy groups represents the
introduction of additional potential sites of metabolism.
Consistent with this observation, improved HLM stability
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Table 3. In Vitro Biological and Physical Properties of Substituted Aryl-(R)-2-methylpiperazines
a
a
a
d
e
Cat K IC50
Cat S IC50
(μM)
Cat B IC50
(μM)
log
D7.4
HLM
RLM
c
compd
R4
R3
R2
R1
(μM)
LE
(μL/min/mg)
(μL/min/mg)
12
22
24
29
30
31
H
H
H
c-pr-CN
c-pr-CN
c-pr-CN
c-pr-CN
CH2CN
c-pr-CN
0.007
0.009
0.012
1.03
2.29
0.549
0.455
3.06
1.8
1.4
0.43
0.34
0.33
0.38
0.42
0.35
7.7
115
7.4
OCH3
SO2CH3
H
OCH3
H
H
2.3 0.7
CH3
CH3
CH3
CH3
0.977
9.4
<2.0
>347
>347
5.1
b
b
b
H
0.011
1.30
0.649
2.4
1.9
20
b
b
b
H
H
0.006
1.22
0.462
16
OCH3
OCH3
0.004
1.45
1.85
<2.0 (n = 6)
a
Binding affinity for cathepsin versus FRET substrate, mean of greater than n = 4 tests, unless otherwise stated. All compounds test at >10 μM for
b
c
d
Cat L. Mean of n = 2 tests. Units of kJ mol−1 Da−1. In vitro human liver microsomal turnover mean of at least n = 2 tests (μL/min/mg), unless
otherwise stated. In vitro rat liver microsomal turnover mean of at least n = 2 tests (μL/min/mg).
e
piperazines when combined with the 4-CH3SO2Ph group (25−
28), although the in vitro metabolic stability was retained. In
summary, sulfone 23 appeared the best compound from this
series. When explored in rat PK, 23 also had improved
bioavailability (F = 16%).
In contrast, when the substituted piperazine was combined
with the 3,4-(CH3O)2Ph group, a significant improvement was
observed (Table 3). Compared with 12, the (R)-methyl offered
no improvement in potency or metabolic stability when
combined with either nitrile headgroup (29, 30). However,
dimethoxy compound 31 gave a small increase in potency and
reached the limit of quantification for our standard HLM assay.
To quantify the stability of the compound, 31 was subjected, at
1.0 μM concentration, to a prolonged incubation period (120
min) with HLM in the absence of albumin. This gave a
measured turnover of 0.7 μL/min/kg, demonstrating that 31
has excellent metabolic stability. Repeat testing of Cat K
inhibition for 12 and 31 yielded a mean IC50 of 8.9 3.0 nM
(n = 6) and 4.1 0.9 nM (n = 10), respectively, proving that
the small activity difference was real. We postulate that this was
caused, at least in part, by a conformational lock, which has
occurred as a result of the addition of the (R)-methyl group on
the piperazine. This could result in an optimized entropic
minimum, positioning one methoxy group for improved surface
contact. Equally, with such a small change as this, the result
could be a simple hydrophobic effect. Figure 3 shows a matched
pair analysis across all the piperazine/(R)-2-methylpiperazines
prepared in the program, demonstrating this effect was
consistent.30 The opposite (S)-methyl enantiomer had
considerably reduced binding affinity (0.087 μM at Cat K,
not shown). Overall, the small increase in activity of the
combination of 3,4-(CH3O)2Ph and (R)-2-methylpiperazine, in
addition to metabolic stability improvements, meant that the
3,4-(CH3O)2Ph series showed greater promise than the
sulfones (compare 24 and 31).
When dosed to rats, 31 also had good oral exposure and a
profile similar to that of the the desmethylpiperazine 22 (F =
46% vs 41%, Cl = 21 versus 29 mL/min/kg). Moving to higher
species, both 22 and 31 had high bioavailability (F = 79% and
71%) and low to moderate clearance (12.0 and 8.6 mL/min/kg,
respectively) in dog. The 3,4-(CH3O)2Ph group also caused a
reduction in lipophilicity (mean Δlog D7.4 = −0.45; compare 12
to 22 and 29 to 31), and the free fraction in human plasma was
also good (22 and 31 hu = 45% and 39% free, respectively).
Figure 3. Cat K inhibition for matched pairs of piperazine versus (R)-
2-methylpiperazine. Improvement in mean ΔpIC50(Cat K) = 0.15
0.08, p < 0.05, and n = 27. Colored lines indicate matched pairs.
Both compounds were active in a low-throughput primary
human cell osteoclast “pit” resorption assay at potencies of IC50
= 0.126 and 0.040 μM, respectively (both n = 1). This
technically demanding assay measured the ability of com-
pounds to stop the osteoclast cells dissolving cartilage and
bone, thereby stopping the observed formation of pits in the
bone sample (see the Supporting Information). Results were
only considered qualitatively, as no relationship was found to
pharmacodynamic (PD) end points (data not shown). This
compared well with our recently reported Cat K inhibitor AZD-
4996 (0.032 0.007 μM, n = 3) as well as balicatib (0.0266
0.001 μM n = 2) and odanacatib (0.100 μM, n = 1), tested in
our hands. Overall, in compound 31 we had achieved the
desired high affinity, metabolic stability, and in vivo
pharmacokinetic (PK) properties for confident scaled pre-
diction to man.
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a
Scheme 2. Metabolites Found after in Vitro Incubation of 31 with Rat and Human Hepatocytes
a
See the Supporting Information for mass spectrometry chromatograms.
Unfortunately, in vitro metabolite identification (MetID)
studies showed formation of glutathione (GSH) adducts after
incubation in both rat and human hepatocytes. This indicated
that reactive metabolites were generated, despite the low
metabolic turnover (Scheme 2). In other compounds within
this chemical series, MetID had identified the cyclohexyl ring as
the primary site of metabolism (Scheme 2, 31-met-1 and 2).
Following demethylation at the 4-OCH3 group, a second
oxidation step may occur to produce an electrophile of a type
known to be capable of reacting with GSH (31-met-3 and 4).31
This electrophilic species can react with GSH at four ring
positions to yield glutathione adducts.32,33 Several studies have
been carried out to measure and quantify the risk posed by
reactive metabolites; however, elimination, or at least
minimization, is always desirable.34 The benzo[d][1,3]dioxol-
5-yl compound 32, which would test the effects of the
dimethoxy groups on potency in synergy to the (R)-2-
methylpiperazine, was prepared. When compared to 31, 32
was a less potent inhibitor of Cat K and also confirmed what we
suspected, i.e., that the dimethoxy groups actually imparted in
vitro microsomal stability (both HLM and RLM turnovers were
higher, Table 4). As a consequence, we set out to design
isosteres with a view to mimicking the observed effect of the
3,4-(CH3O)2Ph on PK properties (stability and good
bioavailability) and eliminating the formation of reactive
metabolites.
Our efforts to address the formation of this putatively
reactive metabolite consisted of three main approaches, namely,
(i) replacing the 4-OCH3 with a steric mimic, (ii) making the
aryl ring heteroaromatic in attempts to block ring oxidation and
subsequent GSH reactivity, and (iii) heterocyclic mimics of the
3,4-(CH3O)2Ph motif. The conformation of the 3,4-
(CH3O)2Ph unit was explored using the Cambridge Structural
Database (CSD), and it was found that the bulk of the
structures (>95%) were in fact near planar, with CH3 groups
pointing away from each other such that the oxygen lone pairs
are oriented toward each other (Figure 4). Modeling studies
indicated that a structural change from 4-OCH3 to 4-
cyclopropyl (such as in 33) might yield compounds of similar
shape (Figure 4, structure of 33 overlaid in red), removing the
most likely metabolically vulnerable group. In the second
approach, it was thought that changing the phenyl ring to a
pyridyl should reduce the ability of the methoxy oxygens to
Figure 4. Structure of 3,4-(CH3O)2Ph (as in 31) in green. The CSD
searches were for moieties with methyl groups 180° (τ) apart in the
same plane. Greater than 95% of the structures found (n = 1130) had τ
> 160°. This would place the nonbonding lone pairs of electrons of the
oxygen atoms toward each other. Electrostatic potential minimum
(green spheres, Vmin) calculations using this information predicted a Δ
log P of 0.53, which was consistent with experimental observation in
this study (see the Supporting Information, Tables S2 and S3). The 4-
cyclopropyl-3-methoxyphenyl moiety is overlaid in red (as in 33, Table
4). The structure was calculated at the B3LYP/6-31G* level of
theory,40,41 indicating that this group was a good spatial mimic of the
dimethoxy moiety.
stabilize a radical oxidative mechanism. With this in mind, we
designed 34.35
For the third approach we considered replacing the 3,4-
(CH3O)2Ph group with a bicycle dimethyl heteroaryl group.32
To reduce the synthetic effort required, we studied the
electrostatic potential minima (Vmin) of the heterocycle
isosteres of 3,4-(CH3O)2Ph we envisaged via quantum
mechanical calculations36 (see the Supporting Information).
The two minima for the 3,4-(CH3O)2Ph unit (where the
oxygen lone pairs are directed toward each other, vide supra)
were found to be in close proximity, indicating the possibility of
replacement by a single minimum (such as the aza group of a
heterocycle). Furthermore, these calculations served to explain
the observed reduction in lipophilicity resulting from the pair of
adjacent methoxy groups.37 Of the bicycles available,
phthalazine, benzisoxazole, and indazole looked to be the
most attractive, so 35−38 were synthesized (the magnitude of
the minimum on a nitrogen lone pair from an indazole, 37, was
similar to that of 31).
Table 4 summarizes the results of testing analogues 32−37.
When tested, the 4-cyclopropyl compound 33 maintained high
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Table 4. Data for Dimethoxy Compound 31 and Isosteres Thereof, 32−38
a
Binding affinity for cathepsin versus FRET substrate, mean of greater than n = 4 tests, unless otherwise stated. All compounds test at >10 μM for
b
c
d
Cat L. Mean of n = 2 tests. Units of kJ mol−1 Da−1. In vitro human liver microsomal turnover mean of at least n = 2 tests (μL/min/mg), unless
otherwise stated. In vitro rat liver microsomal turnover mean of at least n = 2 tests (μL/min/mg).
e
Cat K potency but did not have the same in vitro stability to
HLM. Unfortunately, and perhaps as a consequence of its
increased lipophilicity (log D7.4 = 3.5), weak CYP3A4 inhibition
(1.6 μM) was observed for this analogue, and glutathione
adducts were still found following incubation with human and
rat hepatocytes, ruling out 33 as a viable candidate. 34
represented our second approach, i.e., to change the substituted
phenyl into a heterocycle to reduce the formation GSH
adducts. As for 33, good Cat K inhibition was maintained,
along with a small loss of stability to HLM. Cat K potency and
in vitro metabolism were also good for the heterocyclic
replacements 35−38, with the benzopyrazoles having 2-fold
greater affinity than the other isosteres. Some erosion of
selectivity for other cathepsins as well as HLM stability was
observed, which was important for the final compound
selection. As such, 38 was the standout compound with good
HLM stability maintained below the limit of quantification for
the assay.
All the dimethoxy isosteres were progressed to rat PK studies
(Table 5). The 4-cyclopropyl (33) and pyridine (34) analogues
retained the good bioavailability of 31. Despite good HLM
stability, benzopyridazine 35 suffered from high Cl in rat, which
appeared to be due, at least in part, to higher turnover in rat
hepatocytes. Benzisoxazole 36 had moderate levels of
bioavailability and clearance in rat, but the instability in in
vitro human hepatocytes meant this projected into high
clearance for this example when scaled. In contrast, both
indazoles were found to have excellent profiles in rat with high
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Table 5. Serum Protein Binding and Selected Pharmacokinetic Data for Dimethoxy 31 and Isosteres
a
b
compd
species
protein binding (% free)
in vitro hepatocyte Clint (μL/min/106 cells)
Clp (mL/min/kg)
36 9.2
Vdss (L/kg)
bioavailability (%)
0
c
5
20
21
31
rat
>53
28
25
53
39
39
7.2
8.1
41
24
62
49
14
20
30
28
27
25
35 (n = 1)
36 (n = 1)
42 (n = 1)
<2.0 (n = 3)
5.2 (n = 1)
<2.0 (n = 2)
13 8.6
1.3 1.6
c
e
e
e
rat
19
1.1
0.2
c
e
e
e
rat
>73
1.1
3.5
c
rat
21 6.1
8.7 1.0
1.2 0.6
0.8 0.3
46 23
71 22
d
dog
human
c
e
e
33
34
35
36
37
38
rat
39
2.8
57
human
25 4.0
c
e
e
rat
<2.0 (n = 2)
15
0.7
55
human
c
e
e
rat
12 4.0
>73
4.1
1.4
human
<3.0 (n = 2)
9.4 2.5
c
e
e
rat
29
2.1
21
human
30 4.2
c
rat
4.6 (n = 1)
4.7 3.1
19 7.8
9.5 5.3
2.5 0.7
2.5 0.3
87 15
64 17
human
c
rat
<2.0 (n = 3)
<3.0 (n = 2)
human
a
b
All results are a mean of at least n = 2 experiments. Standard methods were used to determine protein binding.39 The standard deviation is shown
where possible. The number of repeat experiments where the result was below the limit of detection is shown in parentheses. All pharmacokinetic
c
experiments are reported as single-dosed compounds in at least two experiments involving two animals in each. Alderley Park Han Wistar male rats,
dosed to fed animals, po 2.0 mg/kg as a suspension in 5% DMSO/95% HPMC/TWEEN, iv dosed as a solution in 40% DMA/water at 2.0 mg/kg.
d
Beagle dog, dosed to fasted animals, po 1.0 mg/kg as a suspension in 5% DMSO/95% HPMC/TWEEN, iv dosed as a solution in 10% DMSO/90%
e
Sorensons at 1.0 mg/kg. Single experiment, mean of two animals.
bioavailability and low clearance, exhibiting profiles reminiscent
of the 3,4-(CH3O)2Ph 31. The low clearance was explained at
least in part by relatively good stability of 37 and 38 in rat
microsomes and hepatocytes. Figure 5 shows a head-to-head
excellent solubility, and cathepsin selectivity (greater than 100-
fold over cathepsins L, S, and B). Perhaps most importantly,
GSH adducts were not observed following incubation with
human and rat hepatocytes in vitro for both 37 and 38.
In summary, we have identified novel cathepsin K inhibitors
that show excellent pharmacokinetic profiles. Compounds with
the 3,4-(CH3O)2Ph motif were identified, most notably 31, and
were found to have unexpectedly advantageous PK properties.
Metabolite identification studies subsequently identified a
reactive metabolite risk with this motif, and structure-based
design of isosteres has yielded compounds maintaining many of
the good properties. Consistent with our design hypothesis,
indazoles 37 and 38 exhibited desirable profiles and appear to
have removed the propensity of these compounds to form
glutathione-reactive metabolites. Of the two indazoles, 38 was
selected as the compound with the most balanced profile to be
progressed into further studies, which will be reported in due
course.
EXPERIMENTAL SECTION
■
All solvents and chemical used were reagent grade. Anhydrous solvents
tetrahydrofuran (THF) and dimethoxyethane (DME) were purchased
from Aldrich. Purity and characterization of the compounds were
established by a combination of low-resolution mass spectrometry
(LC−MS) (Waters liquid chromatography−mass spectrometry
system), where purity was determined by UV absorption (254 nm)
and the mass ion was determined by electrospray ionization
(Micromass instrument), and NMR analytical techniques. All test
Figure 5. Comparative rat oral pharmacokinetic profiles for selected
○
compounds, adjusted for protein binding: , free [31]; +, free [33];
●
△
, free [36]; , free [37]; ×, free [38]. Alderley Park Han Wistar
male rat, dosed to fed animals, po 2.0 mg/kg as a suspension in 5%
DMSO/95% HPMC/TWEEN, single-dosed compounds in at least
two experiments involving two animals in each study.
1
compounds were >95% pure. H NMR spectra were recorded using a
Varian AV400 FT spectrometer or via the flow NMR process using an
Avance 500 FT spectrometer, and using DMSO-d6 or CDCl3 with the
data expressed as chemical shifts (ppm) from internal standard TMS
on the δ scale. Splitting patterns are indicated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; br, broad peak. Compound 38 when
measured by H NMR at ambient temperature showed differential
rotameric peaks around the tertiary amide group. In these cases
assignments for protons, which were measured distinctly, have been
comparison of rat oral pharmacokinetic profiles, adjusted for
protein binding, for 31, 33, and 36−38. Most notably, indazole
38 showed an improvement with a sustained higher blood level
than the 3,4-(CH3O)2Ph lead 31. Overall, while 38 was slightly
less potent (approximately 2-fold) than 31, it maintained the
other excellent druglike properties, such as good rat PK,
1
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Article
expressed as fractions (e.g., 0.5H). The reversed-phase column used
was a 4.6 mm × 50 mm Phenomenex Synergi Max-RP 80 Å, and the
solvent system was water containing 0.1% formic acid and acetonitrile
unless otherwise stated. A typical run was 5.5 min with a 4.0 min
gradient from 0% to 95% acetonitrile. Purification by column
chromatography was typically performed using silica gel (Merck
7734 grade), and solvent mixtures and gradients are recorded herein.
Purification by reversed-phase high-performance chromatography was
typically performed using a Perkin-Elmer instrument using UV
detection at 254 nm and a C18 1500 × 21.2 mm Phenomenex
column, 100 Å. Acidic conditions (0.1−0.5% formic acid) or basic
conditions (ammonia to pH 10) were used with gradient solvent
mixtures of acetonitrile and water. Strong cation exchange (SCX)
columns were supplied from International Sorbent Technology.
General Procedure for the Preparation of 22, 24, 31, and 38.
An appropriate amine (1.0 equiv) was added to (3aR,7aR)-
hexahydroisobenzofuran-1,3-dione (9; 1.0 equiv) and DIPEA (3.0
equiv) in CH2Cl2 to a concentration of 0.1 M. The resulting solution
was stirred at 20 °C for 18 h, and then either aminoacetonitrile
hydrochloride (10) or 11 (3.0 equiv) was added followed by HATU
(1.1 equiv) and further DIPEA (5.0 equiv). The resulting suspension
was stirred at 20 °C for 72 h. The solution was diluted with CH2Cl2
(equal volume), partitioned with 50% brine (equal volume), then
dried (Na2SO4), concentrated in vacuo, and adsorbed onto silica. Flash
chromatography (silica, 0−100% EtOAc in isohexane) yielded the
desired compound usually as a solid or alternative by preparative
HPLC where stated.
(1R,2R)-2-{[4-(3,4-Dimethoxyphenyl)piperazin-1-yl]-
carbonyl}cyclohexanecarboxylic Acid (1-Cyanocyclopropyl)-
amide (22). 9 (1.00 g, 5.81 mmol) and 1-(3,4-dimethoxyphenyl)-
piperazine (1.10 g, 6.10 mmol), combined with 11 (0.82 g, 6.97
mmol), HATU (3.10 g, 8.13 mmol), and DIPEA (3.00 mL, 17.4
mmol) in DMF (20 mL), yielded 22 (193 mg, 58%) as a white foam:
1H NMR (400 MHz, CDCl3) δ 1.08−1.20 (m, 2H), 1.24−1.65 (m,
6H), 1.55−1.90 (m, 4H), 2.58 (td, J = 10.5, 3.5 Hz, 1H), 2.78 (td, J =
12.3, 3.5 Hz, 1H), 3.02−3.07 (m, 3H), 3.14−3.19 (m, 1H), 3.60−3.70
(m, 2H), 3.83 (s, 3H), 3.86 (s, 3H), 3.70−3.92 (m, 2H), 6.46 (dd, J =
8.6, 2.7 Hz, 1H), 6.57 (d, J = 2.7 Hz, 1H), 6.59 (s, 1H), 6.78 (d, J = 8.7
Hz, 1H); HRMS (ES+) m/z for C24H33O4N4 (M+ + H), calcd
441.2496, found 441.2498.
1H), 2.85 (dd, J = 11.7, 3.7 Hz, 1H), 3.24 (ddd, J = 11.7, 9.5, 2.6 Hz,
2H), 3.36 (d, J = 11.5 Hz, 1H), 3.84 (s, 3H), 3.88 (s, 3H), 3.94 (d, J =
13.8 Hz, 1H), 4.34 (s, 1H), 6.42 (dd, J = 8.6, 2.6 Hz, 1H), 6.53 (d, J =
2.6 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H).
(R)-1-(3,4-Dimethoxyphenyl)-3-methylpiperazine Hydro-
chloride (31a). 39 (0.400 g, 1.19 mmol) was added to hydrochloric
acid in methanol (methanol reagent 10) (15 mL, 1.19 mmol), and the
resulting solution was stirred at room temperature for 16 h. The
resulting mixture was evaporated to dryness, and the residue was
azeotroped with CH2Cl2 to afford crude 31a (100%): MS (+ve ESI) tR
= 1.22 min, m/z 237.30 (M + H)+.
(1R,2R)-N-(1-Cyanocyclopropyl)-2-{[(R)-4-(3,4-dimethoxy-
p h e n y l ) - 2 - m e t h y l p i p e r a z i n - 1 - y l ] c a r b o n y l } -
cyclohexanecarboxamide (31). 9 (159 mg, 1.04 mmol) was added
to 31a (338 mg, 1.24 mmol) in DCM (3 mL) at room temperature.
The resulting solution was stirred at room temperature for 3 days. To
the reaction mixture were added HATU (509 mg, 1.20 mmol), 11
(159 g, 1.20 mmol), and DIPEA (0.50 mL, 2.86 mmol). The resulting
solution was stirred at room temperature for 16 h to afford 31 (320
mg, 57%) as a cream solid: 1H NMR (400.132 MHz, DMSO) δ 0.91−
1.47 (m, 12H), 1.64−1.82 (m, 4H), 2.37 (m, 1H), 2.54−2.96 (m, 3H),
3.37−3.54 (m, 2H), 3.68 (s, 3H), 3.75 (s, 3H), 3.91−4.67 (m, 2H),
6.46 (dd, J = 8.6, 2.6 Hz, 1H), 6.61 (d, J = 8.7, 2.6 Hz, 1H), 6.81 (d, J
= 8.7 Hz, 1H), 8.69 (br, 1H); HRMS (ES+) m/z for C25H35O4N4 (M+
+ H), calcd 455.2653, found 455.2654.
(R)-4-(1,3-Dimethyl-1H-indazol-5-yl)-2-methylpiperazine
(38a). 5-Bromo-1,3-dimethyl-1H-indazole38 (0.448 g, 1.99 mmol) was
reacted with (R)-1-N-Boc-2-methylpiperazine (0.399 g, 1.99 mmol) to
afford 40 (0.363 g, 52.9%) as a white solid after isolation (preparative
HPLC) and workup: MS (+ve ESI) tR = 2.58 min, m/z 345.55 (M +
H)+; 1H NMR (400 MHz, CDCl3) δ 1.37 (d, J = 6.7 Hz, 3H), 1.50 (s,
9H), 2.52 (s, 3H), 2.72 (td, J = 11.8, 3.8 Hz, 1H), 2.88 (dd, J = 11.8,
3.8 Hz, 1H), 3.29 (ddd, J = 16.4, 11.7, 5.0 Hz, 2H), 3.41 (d, J = 10.2
Hz, 1H), 3.96−4.01(m, 4H), 4.37 (s, 1H), 7.00 (d, J = 2.0 Hz, 1H),
7.14 (dd, J = 9.0, 2.0 Hz, 1H), 7.25 (d, J = 9.0 Hz, 3H). 40 (0.363 g,
1.05 mmol) was added to hydrochloric acid in methanol (methanol
reagent 10) (15 mL, 1.05 mmol), and the resulting solution was stirred
at room temperature for 16 h to afford 38a (80%) as a yellow gum
after workup and isolation: MS (+ve ESI) tR = 1.27 min, 245.50 (M +
1
H)+; H NMR (400 MHz, CDCl3) δ 1.21 (d, J = 6.4 Hz, 3H), 2.05
(br, 1H), 2.44 (dd, J = 10.3, 10.7 Hz, 1H), 2.53 (s, 3H), 2.78 (td, J =
11.4, 3.4 Hz, 1H), 2.91−3.30 (m, 3H), 3.44 (d, J = 12.1 Hz, 2H), 3.96
(s, 3H), 7.03 (d, J = 1.9 Hz, 1H), 7.18 (dd, J = 9.0, 1.9 Hz, 1H), 7.03
(d, J = 9.0 Hz, 1H).
(1R,2R)-N-(1-Cyanocyclopropyl)-2-{[4-[4-(methylsulfonyl)-
phenyl]piperazin-1-yl]carbonyl}cyclohexanecarboxamide (23).
9 (159 mg, 1.04 mmol) was added to 4-[4-(methylsulfonyl)phenyl]-
piperazine (250 mg, 0.98 mmol) in DCM (3 mL) at room
temperature. The resulting solution was stirred at room temperature
for 3 days. To the reaction mixture were added HATU (509 mg, 1.20
mmol), 11 (159 g, 1.20 mmol), and DIPEA (0.50 mL, 2.86 mmol).
The resulting solution was stirred at room temperature for 16 h to
afford 23 as a colorless gum (165 mg, 35% yield): MS (+ve ESI) m/z
459 (M + H)+; 1H NMR (400.13 MHz, CDCl3) δ 1.12−1.20 (m, 2H),
1.32 (d, 1H), 1.36−1.45 (m, 2H), 1.46−1.52 (m, 2H), 1.60 (m, 1H),
1.83−1.85 (m, 4H), 2.51−2.58 (m, 1H), 2.94−3.02 (m, 1H), 2.97−
3.01 (s, 3H), 3.14−3.19 (m, 1H), 3.39 (m, 2H), 3.47 (m, 2H), 3.65−
3.68 (m, 1H), 3.87−3.98 (m, 2H), 6.76 (s, 1H), 6.91 (m, 2H), 7.76−
7.80 (m, 2H).
(1R,2R)-N-(1-Cyanocyclopropyl)-2-{[(R)-4-(1,3-dimethyl-1H-
indazol-5-yl)-2-methylpiperazin-1-yl]carbonyl}-
cyclohexanecarboxamide (38). 9 (0.065 g, 0.42 mmol) was added
to 38a (0.103 g, 0.42 mmol) in DCM (3 mL) at room temperature.
The resulting solution was stirred at room temperature for 5 h. To the
reaction mixture were added HATU (0.224 g, 0.59 mmol), 1-amino-1-
cyclopropanecarbonitrile hydrochloride (0.065 g, 0.55 mmol), and
DIPEA (0.220 mL, 1.26 mmol). The resulting solution was stirred at
room temperature for 3 d to afford 38 (134 mg, 69%) as a cream solid
after workup and isolation: 1H NMR (400.132 MHz, DMSO) δ 0.91−
1.08 (m, 2H), 1.12−1.48 (m, 9H), 1.65−1.84 (m, 4H), 2.41 (s, 3H),
2.54 (m, 1H), 2.60−3.00 (m, 3H), 3.38 (m, 1H), 3.40−3.55 (m, 2H),
3.90 (s, 3H), 3.97 (d, J = 13.8 Hz, 0.5H), 4.24 (d, J = 11.9 Hz, 1.0H),
4.69 (s, 0.5H), 7.04 (m, 1H), 7.20 (dd, J = 9.1, 7.2 Hz, 1H), 7.45 (d, J
= 9.1 Hz, 1H), 8.69 (s, 0.5H), 8.71 (s, 0.5H); HRMS (ES+) m/z for
C26H35O2N6 (M+ + H), calcd 463.2816, found 463.2817.
(R)-tert-Butyl 4-(3,4-dimethoxyphenyl)-2-methylpiperazine-
1-carboxylate (39). Palladium(II) acetate (0.052 g, 0.230 mmol) and
(R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.115 g, 0.180
mmol) were added to 4-bromoveratrole (0.662 mL, 4.61 mmol), (R)-
1-N-Boc-2-methylpiperazine (0.923 g, 4.61 mmol), and sodium tert-
butoxide (0.664 g, 6.91 mmol) in anhydrous toluene (12 mL) under
argon. The resulting solution was stirred at reflux for 16 h. The
reaction mixture was diluted with Et2O and filtered through Celite.
The resulting mixture was evaporated to dryness to afford crude (R)-
tert-butyl 4-(3,4-dimethoxyphenyl)-2-methylpiperazine-1-carboxylate.
The crude product was purified by flash silica chromatography,
elution gradient 0−25% EtOAc in isohexane. Pure fractions were
evaporated to dryness to afford 39 (0.871 g, 56%) as a beige solid: MS
ASSOCIATED CONTENT
■
S
* Supporting Information
Table S1 detailing the data from a series of tetrahydroisoquino-
line and isoquinolines with and without 1,2-(OCH3)2 groups,
procedures for the preparation of 5, 12−21, 24−30, and 32−
37, human and rat in vitro hepatic incubations and metabolite
identification studies, procedures for the testing of compounds
in cathepsin K, S, L, and B and the osteoclast cell assay, and
1
(+ve ESI) tR = 2.57 min, m/z 337.31 (M + H)+; H NMR (400.132
MHz, CDCl3) δ 1.33 (d, 3H), 1.49 (s, 9H), 2.67 (td, J = 11.7, 3.4 Hz,
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Carolyn; Petrovic, R.; Santora, A. C.; Ince, B. A.; Lombardi, A.
Odanacatib in the treatment of postmenopausal women with low bone
mineral density: three-year continued therapy and resolution of effect.
J. Bone Miner. Res. 2011, 26, 242−251.
CCDB search data for the τ angle between 1,2-(CH3O)2-Ph
and Vmin to Δ log P calculations. This material is available free
of charge via the Internet at http://pubs.acs.org.
(10) Stoch, S. A.; Zajic, S.; Stone, J.; Miller, D. L.; Van, D., K.;
Gutierrez, M. J.; De, D., M.; Liu, L.; Liu, Q.; Scott, B. B.; Panebianco,
D.; Jin, B.; Duong, L. T.; Gottesdiener, K.; Wagner, J. A. Effect of the
cathepsin K inhibitor odanacatib on bone resorption biomarkers in
healthy postmenopausal women: two double-blind, randomized,
placebo-controlled phase I studies. Clin. Pharmacol. Ther. (N. Y., NY,
U. S.) 2009, 86, 175−182.
(11) Update on phase III trial for odanacatib, Merck’s investigational
Cat-K inhibitor for osteoporosis. Press release, 2012. http://www.
merck.com/newsroom/news-release-archive/research-and-
development/2012_0711.html.
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0) 7720685539 (A.J.D.); +1 650 467 8886
(J.J.C.). E-mail: aldossetter@gmail.com (A.J.D.); crawford.
james@gene.com (J.J.C.).
■
Present Address
§Genentech Inc., 1 DNA Way, South San Francisco, CA 94080.
Author Contributions
∥These authors contributed equally to this work.
(12) Dossetter, A. G.; Beeley, H.; Bowyer, J.; Cook, C. R.; Crawford,
J. J.; Finlayson, J. E.; Heron, N. M.; Heyes, C.; Highton, A. J.; Hudson,
J. A.; Jestel, A.; Kenny, P. W.; Krapp, S.; Martin, S.; MacFaul, P. A.;
McGuire, T. M.; Gutierrez, P. M.; Morley, A. D.; Morris, J. J.; Page, K.
M.; Ribeiro, L. R.; Sawney, H.; Steinbacher, S.; Smith, C.; Vickers, M.
(1R,2R)-N-(1-Cyanocyclopropyl)-2-(6-methoxy-1,3,4,5-
tetrahydropyrido[4,3-b]indole-2-carbonyl)cyclohexanecarboxamide
(AZD4996): a potent and highly selective cathepsin K inhibitor for the
treatment of osteoarthritis. J. Med. Chem. 2012, 55, 6363−6374.
(13) Dossetter, A. G.; Bowyer, J.; Cook, C. R.; Crawford, J. J.;
Finlayson, J. E.; Heron, N. M.; Heyes, C.; Highton, A. J.; Hudson, J.
A.; Jestel, A.; Krapp, S.; MacFaul, P. A.; McGuire, T. M.; Morley, A.
D.; Morris, J. J.; Page, K. M.; Ribeiro, L. R.; Sawney, H.; Steinbacher,
S.; Smith, C. Isosteric replacements for benzothiazoles and
optimization to potent cathepsin K inhibitors free from hERG channel
inhibition. Bioorg. Med. Chem. Lett. 2012, 22, 5563−5568.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
PEPPSI, [1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-
chloropyridyl)palladium(II) dichloride; OA, osteoarthritis; OP,
osteoporosis; MBD, metastatic bone disease; Cat K, cathepsin
K; Cat S, cathepsin S; Cat B, cathepsin B; Cat L, cathepsin L;
HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate; PYBOP, (benzotriazolyloxy)-
tripyrrolidinophosphonium hexafluorophosphate; DIPEA, dii-
sopropylethylamine; LLE, ligand lipophilicity efficiency; Hep,
hepatocyte; HLM, human liver microsome; RLM, rat liver
microsome; CTX-1, C-telopeptide fragment of type 1 collagen;
QFRET, quenched fluorescent resonance energy transfer
technology; CCP4, collaborative computational project,
number 4; CSD, Cambridge Structural Database (http://
www.ccdc.cam.ac.uk/products/csd/); Vmin, electrostatic poten-
tial minima; Cl, clearance; Vdss, volume of distribution
(14) Lecaille, F.; Kaleta, J.; Bromme, D. Human and parasitic papain-
̈
like cysteine proteases: their role in physiology and pathology and
recent developments in inhibitor design. Chem. Rev. 2002, 102, 4459−
4488.
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