Optimization of a potent class of arylamide colony-stimulating factor-1 receptor inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N -[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1 H -imidazole-2-carboxamide (JNJ-28312141)

Article abstract of DOI:10.1021/jm200900q

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC₅₀ = 0.69 nM, cell assay IC₅₀ = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

Full text of DOI:10.1021/jm200900q

Article
pubs.acs.org/jmc
Optimization of a Potent Class of Arylamide Colony-Stimulating
Factor-1 Receptor Inhibitors Leading to Anti-inflammatory Clinical
Candidate 4-Cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)-
acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamide
(JNJ-28312141)
Carl R. Illig,* Carl L. Manthey, Mark J. Wall, Sanath K. Meegalla, Jinsheng Chen, Kenneth J. Wilson,
Shelley K. Ballentine, Renee L. DesJarlais, Carsten Schubert, Carl S. Crysler, Yanmin Chen,
Christopher J. Molloy, Margery A. Chaikin, Robert R. Donatelli, Edward Yurkow, Zhao Zhou,
Mark R. Player, and Bruce E. Tomczuk
Johnson & Johnson Pharmaceutical Research & Development, Welsh & McKean Roads, Spring House, Pennsylvania 19477,
United States
S
* Supporting Information
ABSTRACT: A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-
1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and
pharmacodynamic properties and potential toxicological liabilities. Early stage absorption,
distribution, metabolism, and excretion assays were employed to ensure the incorporation
of druglike properties resulting in the selection of several compounds with good activity in
a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II
collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory
development candidate JNJ-28312141 (23, FMS IC50 = 0.69 nM, cell assay IC50
=
2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has
entered phase I clinical trials.
given parenterally. Thus, there is a continuing need for an oral
agent that effectively relieves the symptoms of RA, prevents
structural damage, and is better tolerated than methotrexate.
Of the many cellular components of the inflammatory
process, macrophages are known to play a critical role5 where
they contribute to antigen presentation and immunomodula-
tion via the production of growth factors and activating
cytokines such as TNF, IL-1, and IL-6. Of cell types present in
rheumatoid synovium, macrophage numbers present in
synovial biopsies provide the strongest correlation with clinical
symptoms, including scores for knee pain.6 The change in the
number of these macrophages also correlates significantly with
changes in the 28-joint count Disease Activity Score (DAS28)
associated with disease therapy.7 Synovial inflammation in RA
is chronic and eventually organizes to form granulation tissue
known as pannus, which is a highly invasive tissue that
progressively erodes cartilage, bone, tendons, and ligaments and
is associated with structural damage and irreversible loss of joint
function. Moreover, pannus macrophages can differentiate to
osteoclasts required for bone invasion. Therefore, the
modulation of proliferation and activity of macrophages and
osteoclasts is a potentially attractive approach for the treatment
INTRODUCTION
■
Inflammation comprises a complex series of biochemical events
that can be initiated by a number of stimuli including noxious
chemical agents, pathogens, and autoimmune responses and, in
pathological situations, may lead to the destruction of cells,
tissues, and organs. Among autoimmune diseases, rheumatoid
arthritis (RA) represents an area of significant unmet medical
need and affects about 1% of the population. The annual
economic burden caused by this disease in 2006 was estimated
to be $58 billion in Europe and $54 billion in the United
States,1 while the global market for arthritis drugs was
estimated to be as high as $35 billion for 2008.2
Low-dose methotrexate is the first-line therapy in RA.
Although methotrexate has a very low associated cost, patients
must be monitored several times a year for the development of
hepatitis. Additional monitoring is also necessary to guard
against the development of renal, pulmonary, or bone marrow
disease. Many patients on this regimen experience side effects
such as fatigue and nausea, and approximately half of all
patients do not respond sufficiently to methotrexate mono-
therapy.3 In these patients, methotrexate may then be
combined with a biologic therapy, which includes anti-tumor
necrosis factor (TNF) agents4 such as infliximab or etanercept.
Biologics, despite their effectiveness in reducing symptoms and
structural damage, are of significantly higher cost and must be
Received: July 8, 2011
Published: October 31, 2011
© 2011 American Chemical Society
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a
Scheme 1
a
Reagents and conditions: (a) Pd(PPh3)4, LiCl, 2 M Na2CO3, dioxane, 80 °C, 2 h. (b) H2, 10% Pd/C, MeOH, 20 psi, 1 h. (c) NBS, CH2Cl2, room
temperature, 10 h. (d) Pd(PPh3)4, 2 M Na2CO3, toluene, EtOH, 80 °C, 3 h. (e) EDCI, HOBt, DIEA, CH2Cl2, room temperature, 10 h. (f) TFA,
CH2Cl2, room temperature, 1 h.
of RA, and as such, we became interested in one of the key
signaling pathways for the macrophage lineage.
rodents, researchers have demonstrated decreases in overall
progression of clinical scores, destruction of bone and cartilage,
and macrophage numbers using specific oral FMS inhib-
itors,19−21 as well as other nonspecific kinase inhibitors
possessing known FMS inhibitory activity such as imatinab
mesylate,22,23 and also with CSF-1-specific antibodies.18 FMS
inhibitors may also find therapeutic applications for other
immune-modulated inflammatory or remodeling diseases with
high macrophage or osteoclast involvement, such as metastatic
bone disease.24
The colony-stimulating factor-1 receptor (CSF-1R) is
alternatively known as FMS, that is, the product of the cellular
proto-oncogene homologue of the feline McDonough sarcoma
virus oncogene (v-fms). FMS is a member of the class III
receptor tyrosine kinases that include FMS-like tyrosine kinase-
3 (FLT-3), stem cell factor receptor (KIT), and platelet-derived
growth factor receptors (PDGFR) α and β. FMS is the
exclusive cell-surface protein receptor for colony-stimulating
factor-1 (CSF-1, or macrophage colony-stimulating factor, M-
CSF),8 which, upon binding to the extracellular domain of
FMS, induces dimerization and trans-autophosphorylation of
the intracellular FMS kinase domain on Tyr723 and other
tyrosine residues. These phosphorylated sites subsequently act
as binding sites for several cytoplasmic Src homology-2 (SH2)
domain-containing signaling molecules, which promote de
novo gene expression, proliferation, and differentiation. High
expression of FMS is restricted to monocytes, tissue macro-
phages, oocytes, trophoblasts, and osteoclasts, and CSF-1 is the
predominant growth factor for macrophages and macrophage-
lineage cells including osteoclasts.
There is evidence that CSF-1 may drive the macrophage
population in RA.9 Although CSF-1 is expressed constitutively,
expression is strongly induced in fibroblasts, endothelial cells,
and chondrocytes by TNF and IL-1.10,11 Endothelial cells from
rheumatoid synovium express more CSF-1 than endothelial
cells purified from other anatomical sites,12 and CSF-1 levels
are significantly elevated in rheumatoid plasma, synovium, and
synovial fluid.13−15 Furthermore, it has been shown that
administered CSF-1 will exacerbate arthritis in rodents,16−18
and CSF-1 deficient (op/op) mice were highly resistant to
arthritis in a collagen-induced model.18 Thus, it is expected that
FMS inhibitors could intervene advantageously in diseases
where osteoclasts and macrophages are pathogenic, particularly
RA. In fact, in collagen-induced arthritis (CIA) models in
CHEMISTRY
■
The methodology for constructing the core structure of the
compounds is outlined in Scheme 1. The synthesis commences
with a Suzuki−Miyaura coupling25 between the anilinoboronic
ester 1 and the enol triflate ester derivative of N-Boc-protected
piperidinone 2.26 After hydrogenation of the olefin, the
resulting aniline 3 was brominated with N-bromosuccinimide
(NBS) to give 4. This set up the next Suzuki−Miyaura coupling
with 1-cyclohexeneboronic acid to afford the tricyclic aniline 5.
The potassium salt of the trimethylsilylethoxymethyl (SEM)-
protected imidazole-2-carboxylate 6 previously reported in
these laboratories27 is coupled to 5 using N-(3-dimethylami-
nopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), 1-
hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine
(DIEA) to provide amide 7. Simultaneous removal of both the
Boc and the SEM groups with trifluoroacetic acid (TFA)
afforded a common intermediate 8 that was widely employed
for derivatizations of the piperidine ring.
Scheme 2 outlines the chemistry used to introduce the
various N-alkyl and N-aryl substitutions on the piperidine
nitrogen. Reaction of 8 with 2-fluoropyridine at 120 °C
afforded the aminopyridine 9. Conjugate addition of 8 to
acrylonitrile led to the cyanoethyl derivative 10. Reductive
amination of the appropriate aldehyde or ketone with 8 in the
presence of sodium triacetoxyborohydride was useful for the
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a
Scheme 2
a
Reagents and conditions: (a) 2-Fluoropyridine, DMA, 120 °C. (b) Acrylonitrile, Et3N, MeOH, DCE, 80 °C. (c) Aldehyde or ketone, NaBH(OAc)3,
DIEA, 1,2-dichloroethane, room temperature. (d) DIEA, CH2Cl2, room temperature. (e) TFA, EtOH, CH2Cl2, 0 °C to room temperature. (f) 4-(2-
Chloroethyl)morpholine HCl, NaI, DMA, 80 °C.
addition of a 2-pyridylmethyl group as on 11, a hydroxyethyl
group as on 12, and, after deprotection of 13, a 1,3-dihydroxy-
2-propyl group as on 14. Direct alkylation chemistry was also
employed to produce a number of piperidines appended with a
methylsulfonylethyl group as in 15, a carbamylmethyl group as
in 16, and a carboxymethyl group, along with its corresponding
t-butyl ester, as in compounds 18 and 17, respectively. In some
instances, protection of the imidazole was beneficial during
reactions with the piperidine; thus, selective deprotection of the
Boc group was effected by careful treatment of the doubly
protected 7 with TFA in the presence of ethanol to afford 19.
Alkylation of 19 with 4-(2-chloroethyl)-morpholine in DMA at
80 °C followed by final acidic removal of the SEM group
afforded the morpholinoethyl analogue 20.
The numerous N-acyl analogues of the core structure were
synthesized as shown in Scheme 3. Reaction of 8 with acetic
anhydride afforded N-acetylpiperidine 21. Acylation of 8 with
the acid chlorides of nicotinic acid and N,N-dimethylglycine
produced compounds 22 and 23, respectively. The remainder
of the analogues was synthesized by means of amide coupling
chemistry with the corresponding carboxylic acids. Thus,
various derivatives, including N-acetyl derivatives substituted
α to the carbonyl with amino, methylsulfonyl, various pyridyl
N-oxides, imidazoyls, and pyridyls, were all made by reaction of
the appropriate carboxylic acids and EDCI resulting in
compounds 24, 26, and 30−38. Hydrolysis of the ketal of 24
with aqueous HCl afforded 2,3-dihydroxypropionyl compound
25. Deprotection of N-Boc-protected 26 with TFA followed by
sodium triacetoxyborohydride and glyoxal gave N-hydroxye-
thylglycinyl derivative 28. Further treatment of 28 with sodium
triacetoxyborohydride and formalin then gave the N-methyl
analogue of 28, compound 29. Similarly, bromotripyrrolidino-
phosphonium hexafluorophosphate (PyBrop) was used as the
coupling agent in the production of the morpholinoacetyl
analogue 39, the N-Boc-protected amine 40, as well as
compound 42, where SEM-protected compound 19 was the
precursor. Removal of the Boc group in 40 and simultaneous
removal of both the Boc and the SEM groups in 42 with TFA
yielded the final compounds 41 and 43, respectively. When
intermediate 19 was treated with triphosgene followed by 2-
aminoethanol and then deprotected as before, N-(2-
hydroxyethyl)urea 44 was produced. Finally, the unsubstituted
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a
of arylamides as represented by 46 in Figure 1. It seemed
evident from the initial collection of screening hits that an
ortho-aminophenyl heteroarylcarboxamide comprised the basic
pharmacophore of this series. Subsequent hit-to-lead efforts29
resulted in significant improvements in binding affinity as in, for
example, 47, and eventually resulted in an initial lead
compound 48. This compound, in addition to improved
druglike properties, also stabilized the crystal packing of an
engineered chimeric form of the FMS kinase domain, thereby
providing our first crystallographic information on the binding
mode of this interesting new series30 and supplying us with an
avenue to rapid structure-based optimization.
Scheme 3
Further optimization studies and investigation of the
structure−activity relationship (SAR) produced proof-of-
concept compounds, for example, furans and pyrroles 49−52,
of excellent potency (Figure 1),31 which permitted confirma-
tion of in vivo efficacy for this class of compounds. Compound
52, for example, in addition to possessing subnanomolar
potency, displayed a very favorable pharmacokinetic profile and
demonstrated significant efficacy in a collagen-induced model
of arthritis in mice at doses as low as 5 mg/kg po BID.
Histological evaluation of the tissues was consistent with the
reductions in arthritis clinical scores, revealing that this agent
reduced pannus invasion as well as destruction of both bone
and cartilage, competitive in degree to that seen with biological
anti-TNF agents in similar models. Moreover, the significant
reductions in the number of macrophages in the joints of 52-
treated mice were consistent with both the observed macro-
scopic results and the hypothesized mechanism of action for
these agents, viz., FMS inhibition.
Despite these promising data, it was important to remain
mindful of the latent liability of a 1,2,4-phenylenetriamine core
structure, possessed by these proof-of-concept compounds, to
undergo possible in vivo oxidation to form reactive
quinonediimine metabolites. Such reactive species could
potentially haptenize proteins as well as deplete glutathione
concentrations, culminating in tissue damage via an idiosyn-
cratic drug reaction (IDR).32 In this respect, in vitro glutathione
conjugation assays33 partly corroborated this prospective risk.
Hoping to circumvent this anticipated liability and armed
with the knowledge of the binding mode of this class from
X-ray studies, a focused structure-based design effort was
initiated to replace two of the three nitrogen substituents on
the benzene core to derive the 2- and 4-substituted carbon-
based analogues.34 Although the challenge of instituting such
major structural changes was recognized with regard to
maintaining potency and good pharmacological properties, it
was deemed necessary to ensure the long-term safety
characteristics for a drug candidate.
The SAR at the 4-position proved tolerant to replacement of
the piperazine with a range of lipophilic rings and substituents
(data not shown) where the direct replacement of a piperazine
with a 4-piperidine resulted in derivatives, such as 53, with no
loss in potency. This was consistent with structural evidence
indicating that substituents at the 4-position have close contact
with a lipophilic region of the protein proximal to the
attachment point at the benzene ring. The region more distal
to the attachment point is largely directed out into solvent and
is therefore open for more variation.
a
Reagents and conditions: (a) Et3N, CH2Cl2, room temperature. (b)
DIEA, CH2Cl2, room temperature. (c) EDCI, HOBt, DIEA or Et3N,
CH2Cl2, room temperature. (d) EDCI, HOBt, DIEA, DMF, room
temperature. (e) PyBrop, DIEA, CH2Cl2, room temperature. (f)
Triphosgene, DIEA, H2N(CH2)2OH, CH2Cl2, −78 °C, then TFA,
EtOH, CH2Cl2, room temperature. (g) TMS-NCO, Et3N, CH2Cl2,
room temperature. (h) 2 M HCl, MeOH−H2O, room temperature. (i)
TFA, EtOH, CH2Cl2, room temperature. (j) Na(OAc)3BH, glyoxal,
CH2Cl2, room temperature. (k) Na(OAc)3BH, CH2O, MeOH, room
temperature.
urea 45 was derived by treatment of 8 with trimethylsilyliso-
cyanate followed by hydrolysis during aqueous purification by
reverse HPLC.
RESULTS AND DISCUSSION
■
We embarked on this discovery program with a focused
screening effort within our chemical library collection. This
endeavor resulted in three promising chemical classes: a series
of quinolones28 and pyrimidinopyridones,20 both of which have
been reported previously, and, the subject of this article, a series
Unlike the 4-position, the SAR surrounding the 2-position
revealed that a piperidine group was very specific in its steric
and conformational requirements. Hence, the best carbocyclic
analogue that could be obtained for the piperidine, a
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Figure 1.
1-cyclohexenyl group, resulted in an unavoidable 20−30-fold
erosion of the potency as illustrated by 54. Ultimately, this
significant loss of potency was mitigated by further
optimization of the terminal acyl heterocycle.
the first time and shown in Figure 2C, confirmed the
hypothesis for increased potency where the two complemen-
tary internal and external hydrogen bonds were unmistakably
present.
It was hypothesized through modeling that the equipotent
nature of cyanofurans and cyanopyrroles in this acyl
heterocyclic location was a result of two different hydrogen-
bonding scenarios. An intramolecular hydrogen bond was
thought to form between the amide NH and the furan oxygen,
which assisted in holding the inhibitor in the conformation
required for binding. This hydrogen bond was confirmed in the
now-available X-ray cocrystal structure (PDB ID: 3KRL) of
4935 in FMS as shown in Figure 2A. In the contrasting case of
the pyrrole 5035 modeled into FMS (Figure 2B), the internal
hydrogen bond cannot form. However, the anticipated
reduction in binding affinity from the loss of this interaction
was not observed. This appears to have been the result of an
alternate intermolecular hydrogen bond that can form between
the pyrrole NH and the carbonyl of Glu664, which, from
modeling, would exist where the dashed line in Figure 2B is
placed. Using this information, it was possible to combine the
individually advantageous hydrogen-bonding characteristics of
the two heterocycles. The result was the corresponding class of
cyanoimidazoles, for example, 8 and 21, which show
approximately 10-fold improvement in affinity over either the
furan or the pyrrole analogues compensating for the loss
induced by replacement of the piperidine in the 2-position with
the suboptimally potent 1-cyclohexenyl group. A cocrystal
structure of 8 in FMS (PDB ID: 3KRJ), also reported here for
As shown in Table 1, these molecules also demonstrated low
nanomolar potency in a cellular assay that measured the ability
of compounds to inhibit the proliferation of murine bone
marrow-derived macrophages (BMDM).20 More importantly,
these compounds did not show the potential for reactive
metabolite formation as gauged by the glutathione conjugation
assay previously described.33
Secure in the knowledge that these were potent inhibitors,
which were less likely to present an IDR liability, we
investigated the pharmacokinetic (PK) and pharmacodynamic
(PD) properties of 8 and 21. A PD assay in mice20,24 was
developed based on the ability of CSF-1 to induce the
expression of c-fos mRNA in macrophages.39 Tail-vein injection
of CSF-1 caused an increase in c-fos mRNA concentrations of
more than 10-fold in the spleen within 15 min but returned
toward baseline by 30 min. The CSF-1-induced expression of c-
fos was dose-dependent and specific as it was blocked
completely in mice predosed with a neutralizing antibody to
CSF-1.24 Table 2 shows the PD effect of compounds 8, 21, and
the earlier proof-of-concept compound 51, at a time of 8 h
following an oral dose of 50 mg/kg. This PD assay was used
routinely throughout the program. With initial assessment at
relatively high doses, compounds were often triaged on the
basis of the presence or absence of robust (80−100%)
inhibition of c-fos induction. Thus, the 70% inhibition of
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Table 1. FMS and Cell Potency and Potential for Reactive
Metabolite Formation of Early Leads 8 and 21
mean IC50 (nM)
a
compd
R
FMS
BMDM assay glutathione conjugation
8
H
1.1 (n = 31)
2.4 (n = 2)
7.2 (n = 5)
5.6 (n = 2)
no
no
21
Ac
a
The test compound at 20 or 40 μM was incubated with NADPH
(1 mM), GSH (5 mM) in PBS (0.1 mM), and 1 mg/mL of mouse
liver microsomes at 37 °C for 1 h. The products were then analyzed by
LC/MS for the glutathione conjugate of the parent (P + GSH), the
amide bond cleavage product (ACP + GSH), and their fragments.
the oral exposures of 51 at 4 and 8 h postdose were robust as
compared to 8 and 21 and provided the apparent explanation
of the superior PD activity. The superior exposures were largely
a consequence of the good oral bioavailability of 51 vs the poor
oral bioavailability of 8 and 21. All three compounds exhibited
moderate to high clearance relative to liver blood flow, together
with moderate to high volumes of distribution. It seemed
apparent from these results, in particular the failure of both 8
and 21 to attain adequate bioavailability, that the early
evaluation of the in vivo properties of subsequent candidates
would be essential for the rapid progression of this program.
To investigate the potential for side effects, 8 and 21 at
10 μM were examined in a battery of 50 counterscreen assays40
for activity against a wide variety of receptors, transporters, and
ion channels. Compound 8 hit nine targets with greater than
50% inhibition of specific control ligand binding, whereas 21
gave greater than 50% inhibition at only three targets. It is not
uncommon to see significant levels of off-target binding with
relatively basic compounds such as piperidine 8 (calculated
pKa = 10.2 ± 0.1).41 Many groups have investigated the target
promiscuity of compounds as a function of their basicity and, in
general, it seems clear that increased basicity as well as the
potential for a positive charge at physiological pH are strong
contributors to off-target activity.42
Of particular concern was that higher levels of binding at ion
channels, in particular sodium43 and calcium ion channels,44 for
basic compounds may lead to an increased risk of adverse
cardiovascular effects. Indeed, it was demonstrated45 that the
highly basic compound 8 inhibited Ca2+ channel, L, reference
compound binding by 91% and Na+ channel, site 2 reference
compound binding by 96% at 10 μM, and had somewhat
greater undesirable effects in a guinea pig right atrium assay
than the less basic amide derivative 21, which inhibited Ca2+
channel and Na+ channel binding by 46 and 27%, respectively.
The strategy of reducing the basicity of compounds to mitigate
off-target ion channel effects has been used for calcium and
sodium channels46 and for potassium channels such as human
ether-a-go-go-related gene potassium channel (hERG).47 Thus,
we hypothesized that moderating the basicity of future
analogues would be beneficial and, so, the majority of analogues
in this paper were selected with that concept in mind.
Leads also were optimized against a battery of in vitro tests
for ADMET properties including the in vitro metabolic stability
in human and rodent liver microsomes as a means to separate
out compounds that may not withstand first-pass metabolism.
A compound stability of at least 70% remaining after a 10 min
exposure to the microsomal mixture was preferred. In addition,
in vitro assays for inhibition of recombinant cytochrome P450
(CYP) enzymes provided a means to predict future problems
with drug−drug interactions,48 and we routinely monitored
Figure 2. (A) Crystal structure of FMS, cyan carbons, with furan-
containing compound 49, brown carbons (PDB ID: 3KRL).36 (B)
Model of FMS, cyan carbons, with pyrrole-containing compound 50,
salmon carbons. (C) Crystal structure of FMS, cyan carbons, with
imidazole-containing compound 8, orange carbons (PDB ID: 3KRJ).37
Hydrogen bonds between the inhibitor and FMS and the inhibitor
intramolecular hydrogen bonds are shown as green dashed lines. The
figures were prepared with PyMol.38
compound 8 and the 24% inhibition of compound 21 fell short
of this criterion, while compound 51, by comparison, which had
excellent oral efficacy in a mouse CIA model,31 had strong
inhibition (96%) even at a dose that is significantly lower
(20 mg/kg) than for either of the two candidates 8 and 21. Table 2
also presents some of the PK properties of 8 and 21. In mice,
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Table 2. PK and PD Data for Compounds 8, 21, and 51
b
c
PD (po)
PK (iv)
PK (po)
c-fos assay (%
Cmax
AUClast
concn at 4 h concn at 8 h
a
compd inhib at 8 h ) t1/2 (h) Vz (L/kg) Cl (mL/min/kg)
t1/2 (h)
F (%) Tmax (h) (ng/mL) (min ng/mL)
(ng/mL)
(ng/mL)
d
8
70 (50
mg/kg)
7.2
1.2
3.0
32
52
27
46
2.0
12
15
75
0.25
50
209
285
5715
27218
69711
7
BLQ
21
24 (50
mg/kg)
2.8
1.9
3.1
0.5
54
9
e
51
96 (20
mg/kg)
12.3
0.5−4.0
183
64
a
b
See the text for details of PD assay. Compounds 8 and 51 administered in 20% HPβCD (pH 2 by HCl) at 0.5 mg/kg iv and 5 mg/kg po.
c
Compound 21 administered in 20% DMSO/80% PEG400 at 0.5 mg/kg iv and in 1% Tween 80/0.5% hydroxypropylmethylcellulose at 5 mg/kg po.
d
e
Below the limit of quantitation. PK values for 51 are the mean of multiple studies (n = 4).
four of the CYP enzyme subtypes (CYP1A2, CYP2C9,
CYP2C19, and CYP3A4) responsible for metabolism of a
majority of drugs.49 IC50 values below 3 μM were considered
suspect requiring follow-up in secondary assays, and values
below 1 μM can often prove problematic.
As mentioned above, many of the derivatives for this
optimization process were selected to modulate the basicity of
the piperidine nitrogen. The predictable effects on basicity of
various electron-withdrawing groups, electronegative hetero-
atoms, and heteroaryl rings placed two to three bonds away from
an amine nitrogen are well-documented,50 and selections based
on these principles resulted in a group of N-alkyl and N-aryl
piperidines (compounds 9−20). The data for these analogues
are displayed in Table 3 where the unsubstituted piperidine
lead 8 is included as a reference.
syndrome.51 None of the compounds tested gave results
that merited concern. Thus, the six compounds from the
N-alkylpiperidine analogues with the greatest cell potency
(10−12, 15, 16, and 20) were selected for further evaluation.
Data for the corresponding collection of N-acylpiperidine
derivatives (compounds 22−45) are displayed in Table 4.
Because acylation of the piperidine to give nonbasic amides
could potentially impact the solubility, some acyl groups were
selected that still contained amines to retain a solubilizing
feature. As discussed above, however, the proximity of the
added amine to the acyl carbonyl or other heteroatoms was
maintained to modulate the pKa and limit ion-channel and
other off-target activity. As before, one of the early leads,
N-acetyl piperidine 21, is included as an N-acyl reference for
this group.
As a first screening criterion, we sought compounds with an
IC50 potency better than 15 nM in the cellular BMDM
proliferation assay based on experience that compounds with
higher values often did not exhibit PD activity at reasonable
(<50 mg/kg) doses. In general, the data in the tables are
formatted in bold when they met the criteria as discussed above
for a particular assay. Thus, bolded values for all assay results
within a given row indicate compounds of greater interest for
secondary studies.
It is immediately apparent that this entire group of
compounds displayed good in vitro stability in the presence
of both human and mouse liver microsomal preparations. It is
possible that the additional functionality present on the various
N-alkyl groups prevents metabolism commonly observed with
simpler N-alkyl groups. In vitro potency in the FMS enzyme
assay was also excellent with the only exception being the
2-pyridylpiperidine 9 (IC50 = 22 nM), which was further
compromised by significant inhibition of CYP3A4 (IC50 = 1.7 μM).
Of the remaining candidates, the highest IC50 values in the group
did not exceed 2.4 nM.
In the cellular BMDM assay, only two compounds (14 and
18) failed to give IC50 values below 15 nM. In the case of the
carboxymethyl derivative 18, the low pKa expected for an
aminoacid of this type likely resulted in insufficient
concentration of the free acid at physiological pH to provide
adequate cell membrane penetration.
The values for inhibition of the four cytochrome P450
enzymes assayed also did not strongly rule out any additional
compounds. A number of the compounds were also tested for
inhibition of [3H]-astemizole binding at the hERG potassium
channel as a predictor for compounds that may prolong cardiac
action potential duration, potentially resulting in long QT
All compounds in this series also exhibited excellent in vitro
potency in the FMS enzyme assay with no IC50 values
exceeding 3.8 nM. Five compounds were discarded based on
relatively modest cellular potency, viz., the isonicotinic amide
N-oxide 32, the 4-imidazoyl amides 33 and 34, the amino-
alkylamide 41, and the primary urea 45. Additionally, the
4-imidazoyl amides, 33 and 34, showed greater CYP inhibition
with 33 being a particularly potent inhibitor of CYP2C9 and
CYP2C19. With regard to the relatively weak cellular potency
of 41, one may postulate that the more strongly basic nature of
the α-branched amine results in a more charged species at
physiological pH that does not pass easily through cell
membranes, whereas, by comparison, the closer proximity of
the amino group to the carbonyl in 43 attenuates the pKa such
that 43 does not suffer the same fate and retains good cellular
activity.
When the remaining 14 candidates were subsequently
incubated in the presence of liver microsomes, a significant
number demonstrated less-than-desirable stability. All of the
pyridylmethyl amides, 35−37, were very unstable in both
species. In addition, nicotinic amide 22, the two hydroxyethyl-
aminomethyl amides 28 and 29, both morpholinoalkyl amides
38 and 39, as well as the hydroxyethylurea 44 all suffered
varying degrees of reduced stability in the presence of one or
both species of microsomes, which did meet our threshold for
desired stability. Nicotinic amide 22 also displayed submicro-
molar inhibition of CYP2C9.
Finally, of the remaining five most potent and stable
derivatives within this group, nicotinic amide N-oxide 31 and
aminoalkylamide 43 showed low micromolar CYP inhibition.
More importantly, the N-oxide 31, despite its apparent in vitro
microsomal stability, also carried the liability that any in vivo
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a
Table 3. FMS and Cell Potency and Early ADMET Properties of N-Alkyl/N-Aryl Piperidines
a
For comparative purposes, data in the table are formatted in bold when they met desired subjective criteria (FMS IC50 < 10 nM (bolded but not
b
used as a selection criterion), BMDM IC50 < 15 nM, liver microsomal stability 70−100%, CYP450 IC50 > 3 μM, and hERG IC50 > 3.2 μM). na, not
assayed.
metabolic loss of the N-oxide would result in the formation of
the potent CYP2C9 inhibitor 22, a concern that also removed it
from further consideration. Again, of the compounds tested for
inhibition of binding of astemizole to hERG, none appeared to
have a hERG liability. The remaining compounds appeared
quite promising in their profile. Unfortunately, the attrition of
compounds from this group of N-acyl derivatives shown in
Table 4 was significantly greater than from the N-alkyl and
N-aryl compounds shown in Table 3, resulting in only three
analogues (23, 25, and 30) for advancement to further studies.
At this stage, there remained a collection of nine promising
candidates from Tables 3 and 4, and it was clear from the
shortcomings of 8 and 21 that an early assessment of in vivo
efficacy would be useful for the most efficient selection of
improved candidates. Because the activity in the PD model was
presumably an integrated function of potency and tissue
exposure, the performance in the PD model was deemed the
most efficient way to prioritize compounds for further
advancement. However, the resource-intensive nature of this
assay at multiple doses and time points required an abbreviated
approach be used to screen this collection. Therefore, the top
candidates were investigated in this model initially at a single
dose of 50 mg/kg and at a time of 8 h after oral administration.
The results of these PD screening studies are presented in
Table 5. Of the nine compounds, two (11 and 25) were not
tested. After Cerep counterscreening for other activities,
compound 11 demonstrated greater than 50% inhibition
against 12 targets at 10 μM, and compound 25, as a chiral
compound, was also excluded. Lead compounds 8 and 21 are
included again in this table for reference. To ensure that the in
vitro screens based on cellular activity and microsomal stability
were not excluding potentially useful compounds, a selection of
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a
Table 4. FMS and Cell Potency and Early ADMET Properties of N-Acyl Piperidines
a
For comparative purposes, data in the table are formatted in bold when they met desired subjective criteria (FMS IC50 < 10 nM (bolded but not
b
used as a selection criterion), BMDM IC50 < 15 nM, liver microsomal stability 70−100%, CYP450 IC50 > 3 μM, and hERG IC50 > 3.2 μM). na, not
assayed. Insufficient solubility in PBS to determine. On different occasions, hERG assays were run with a maximum concentration that limited the
c
d
determination of IC50 values to either 3.2 or 10 μM.
compounds that did not meet the cutoff criteria were also
added as controls. These included 35−37 and 44, which had
excellent cellular potency for inhibition of BMDM proliferation
but which had marginal to poor stability in mouse liver
microsomes. Also added as a control was 34, which, by contrast,
had good mouse microsomal stability but poor cellular BMDM
activity.
The negative control compounds 34−37 and 44 gave little or
no PD response, as expected. Also, the seven top primary
screen candidates did not all perform equally well in this screen.
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Table 5. PD, PK, and Solubility Properties of Selected Compounds
b,d
PK parameters
iv
po
Cmax (ng/mL) AUClast (min ng/mL)
PD assay % inhibition at
t1/2
(h)
t1/2
(h)
solubility in
PBS (μM)
a
compd
8 h (50 mpk po)
Vz (L/kg) Cl (mL/min/kg)
% F
8
21
10
12
15
16
20
23
30
34
35
36
37
44
70
7.16
1.21
na
32
51.7
27.0
na
2.00
1.88
na
50
5715
27218
na
12
38.8
26.1
11.8
>40
4.2
c
c
c
c
c
c
24
2.84
na
209
na
15
28
na
76
na
na
na
na
na
na
na
88−93
<51
86
1.44
1.39
0.23
1.36
1.20
0.54
na
3.84
6.43
8.99
7.11
1.58
3.85
na
30.7
53.3
46.6
60.6
23.9
81.8
na
1.55
0.88
2.37
2.33
1.55
0.40
na
866
55
127845
8445
93173
61365
7500
6075
na
88
9.8
101
83
28.6
25.9
37.3
0.91
36.7
1.6
461
296
88−100
33
c
c
c
c
c
c
24
2.3
13
81
na
13
na
na
18
36
0
na
na
na
na
na
na
10.4
0.39
8.2
0
0.41
2.41
7.27
10.3
205
49.2
0.23
1.63
118
24
4080
7740
23
9.3
a
b
PD model in B6C3F1 mice with all compounds administered po in 20% HPβCD (see the details in the Experimental Section). PK studies in 30−
40 g male CD-1 mice. All compounds administered in 20% HPβCD (pH 2 by HCl) at 0.5 mg/kg iv and 5 mg/kg po except where indicated
otherwise. Compounds administered in 20% DMSO/80% PEG400 at 0.5 mg/kg iv and in 1% Tween 80/0.5% hydroxypropyl- methylcellulose at
5 mg/kg po. na, not assayed.
c
d
Table 6. Kinase Inhibition by 23
kinases inhibited more
than 50% by 0.1 μM 23
kinases not inhibited 50% by 0.1 μM
23 but more than 50% by 1 μM 23
kinases not inhibited 50% by 1 μM 23
FMS, FLT3, KIT, AXL,
TRKA, and LCK
ALK, BLK, BMX, FES, FGR, FYN,
PDGFRA, PDGFRB, TRKB, and
YES
Abl1, Akt1, Akt2, Akt3, Arg, Brk, BTKC, aMKII-α, CHK1, CHK2, CK2-α1, MET, Csk, EGFR,
EphA3, EphB4, ERK1, ERK2, FGFR1, FGFR2, FGFR3, FGFR4, GSK3-α, GSK3-β, Hck, Hyl,
IGF1R, IRAK4, Lyn A, Lyn B, MAPKAP-K2, MAPKAP-K3, MAPKAP-K5, NEK2, PHKG2,
PKA, PKC (α, β I, β II, δ, ϵ, η, γ, ι, θ, ζ), RSK2, Src, AMPK, Aurora-A, CaMKIV, CDK/cyclinB,
CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE, CDK5/p35, CDK6/cyclinD3, CDK7/cyclinH,
CK1, CK1d, c-RAF, CSK, EphB2, IKKα, IKKβ, IR, JNK1α1, JNKα2, JNK3, MAPK1, MAPK2,
MEK1, MKK4, MKK6, MKK7b, MSK1, MST2, NEK2, p70S6K, PAK2, PAR-1Ba, PDK1, PKBα,
PKBβ, PKBγ, PRAK, PRK2, ROCK-II, Ros, Rsk1, Rsk3, SAPK2a, SAPK2b, SAPK3, SAPK4,
SGK, Syk, Tie, and ZAP-70
Ultimately, three candidates, 15, 20, and 23, gave good to
excellent results.
80%) and by high volumes of distribution resulting in good oral
half-lives (1.6−2.4 h).
In an effort to understand some of the factors that
contributed to the ineffectiveness of some compounds in this
assay, PK parameters and solubility values in PBS are included
in this table for selected compounds. In general, it seems clear
that nearly all of the compounds within this series suffer from
moderate to high clearance rates, ranging from values of 24 up
to as high as 82 mL/min/kg. Nevertheless, in some cases,
correspondingly high volumes of distribution result in
compounds that still retain iv half-lives of reasonable length.
Control compound 44, which was below the desired threshold
for stability in the mouse microsomal assay, had low
bioavailability and low Cmax values in plasma, consistent with
the poor PD activity seen, and although poor microsomal
stability may often be a predictor of high clearance, compounds
with good microsomal stability may still suffer from high
clearance for other reasons (such as phase II metabolism), as
shown by control compound 34, which possessed one of the
highest clearance values of those tested. Among the remaining
compounds, the poor Cmax and bioavailability of compounds 16
and 30 appear to explain the low PD inhibition effects
observed. The three compounds that demonstrated the most
promising PD activities, 15, 20, and 23, all possessed good PK.
Moderate to high clearance values of each compound were
compensated for by excellent bioavailabilities (all greater than
Solubility may play a role in the PK behavior observed. It can
be seen that the solubility in phosphate buffered saline (PBS)
of the control compounds 35−37 and 44 and for test
compounds 10 and 30 was lower than for the reference
compounds 8 and 21 and may have been an additional
contributing factor in the poor PK observed for compounds 30
and 44. However, good solubility is only one helpful
characteristic and, clearly, somewhat lower solubility did not
impact the good PK seen for test compound 15.
To assess kinase selectivity, compounds 15, 20, and 23 were
screened against a large number of tyrosine, serine-threonine,
or mixed kinases. Differences in the profiles of the three
compounds were not pronounced (data not shown). For
example, as shown in Table 6, compound 23 was assessed at
1 μM for activity vs 39 tyrosine kinases and 79 serine-threonine
or mixed kinases. Seventy-eight of the serine-threonine or
mixed kinases were inhibited less than 50%; one serine-
threonine kinase, ALK, was inhibited by 51%. Sixteen of the 39
tyrosine kinases were inhibited by more than 50% by 1 μM 23.
Of these, six were inhibited by more than 50% at 0.1 μM, and
the IC50 values of 23 vs these six kinases were determined and
are reported in Table 7. IC50 values are also reported in Table 7
for 15 and 20. All three compounds were found to be relatively
potent inhibitors of FLT3, AXL, KIT, and TRKA with modest
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each group, relative to the vehicle-treated disease control group
and, when achieved, is indicated in the charts with an asterisk. A
separate, overall, four-paw, animal score based on the sum of
the four histological scores was also calculated to provide a
more generalized indication of efficacy.
Table 7. Kinase Selectivities of 15, 20 and 23
IC50 (nM)
kinase
15
1.5
20
1.1
23
6.0
23
FMS
KIT
0.69
5.0
12
16
The vehicle-treated disease control animals developed
arthritis symptoms with histological changes in most paws
with scores ranging from minimal (1) to severe (5). As shown
in Figure 3, the vehicle-treated disease control group displayed
a greatly elevated four-paw summary score (mean = 5.45)
relative to the disease-free naive group. Compound 15 at
60 mg/kg produced a significant 61% reduction in the four-paw
score relative to the vehicle-treated disease control. This was
the result of significant inhibition, ranging from 57 to 63%, in
all four of the measured individual histological parameters as
shown in Figure 4. Compound 20 proved the most efficacious
of the three candidates in this model. However, the 60 mg/kg
dose of 20 caused acute weight loss in a subset of mice and
resulted in the deaths of two animals by day 17. As a result of
this toxicity and the subsequent deaths, the dose of compound
20 was reduced to 40 mg/kg beginning day 18 and maintained
at the lower dose through day 26. Even so, compound 20
produced a significant overall reduction of 82% in the summary
score. This is a result of marked reductions in inflammation,
pannus, cartilage damage, and bone damage, all in the range of
77−88%. When the dose of 20 was reduced to 20 mg/kg, the
toxicity seen at the higher dose was not seen, but the overall
efficacy was still impressive with a summary score of 74%
inhibition. The inhibition of the individual histological
parameters for 20 at this lower dose displayed a range of
69−77% reduction relative to the disease controls. Compound
23 at 60 mg/kg produced a significant 73% reduction of the
four-paw score (Figure 3). The inhibition of individual
histological parameters ranged from 65 to 79% (Figure 4)
with the reductions in the scores for both pannus formation
(79%) and bone erosion (79%) of particular note. When
administered at the lower dose of 20 mg/kg, compound 23 was
less effective overall, providing a nonsignificant 20% reduction
in the summary score. This reflected nonsignificant reductions
in inflammation and cartilage damage of 16 and 18%,
respectively, and significant 25% reductions in pannus
formation and bone erosions (Figure 4). With the exception
of the 60 mg/kg dose of 20, the candidates had no deleterious
effects on body weight or any clinical signs of toxicity.
Although compound 20 showed a clear advantage in terms of
efficacy over 15 and 23, the toxicity observed at the high dose
was worrisome. In further studies, 20 and 23 were examined in
a battery of 50 counterscreen assays40 for activity against a wide
variety of receptors, transporters, and ion channels. While 23
hit four targets with greater than 50% inhibition of specific
control ligand binding, 20 gave greater than 50% inhibition at
seven targets. The apparent increased off-target activity of 20
was also reflected in unambiguous conductance effects in
guinea pig acute cardiovascular (CV) safety56 and guinea pig
right atrium studies.57 Consequently, despite the lack of overt
toxicity at the lower dose of 20 mg/kg in the arthritis study, the
potential for safety issues was of significant concern, and the
decision was made to eliminate 20 as a candidate. Thus,
compound 23, which was superior to 15 at a comparable dose
for every histological parameter and in its summary score, was
chosen as the best candidate in the group for further evaluation.
To better characterize the potential of compound 23 as a
AXL
12
TRKA
FLT3
LCK
30
19
23
15
30
88
58
860
200
selectivities for FMS ranging from 7- to 43-fold. The inhibition
of FLT3 and KIT can suppress functions in dendritic cells and
mast cells, suggesting that the presence of these activities may
have beneficial effects toward inflammation. However, there is
also evidence that potent inhibition of KIT can produce
myelosuppression and anemia.52 We also tested these
compounds in cell-based assays designed to exhibit dependence
on these kinases (Table 8). While the selectivity for FMS vs
FLT3 and KIT was modest for compound 23 and the
selectivity for FMS vs FLT3 was minimal for compound 20,
compound 15 exhibited the highest selectivity for FMS among
the three compounds with FMS selectivity in cells ranging from
ca. 32- to 161-fold. Although compound 23 was a potent
inhibitor of AXL and LCK enzyme assays, only high (>2 μM)
concentrations inhibited these targets in cellular assays.
Compounds 15, 20, and 23 had properties from the
abbreviated in vivo screening studies that more than qualified
each to advance to more extensive efficacy studies. Therefore,
to better characterize and compare them, a more detailed
dose−response PD study was performed. Each compound was
dosed at high and medium doses of 50 and 20 mg/kg,
respectively, and at a lower dose of either 10 mg/kg for
compounds 20 and 23 or at 8 mg/kg for compound 15 (Table 9).
Differences in the in vivo activity of the three compounds
became apparent in the dose−response comparison. By the
mid-dose of 20 mg/kg, the performance of 15 had fallen off
sharply, while the responses of 20 and 23 remained robust
(77−79% inhibition). At the lowest dose, 20 and 23 still
showed substantial responses with 20 (51% inhibition)
appearing somewhat superior to 23 (32% inhibition).
The three candidates, compounds 15, 20, and 23, were
subsequently evaluated in a type II CIA model in mice.53,54
This model has been particularly useful in evaluating the
beneficial effects of soluble TNF receptors and other cytokine
inhibitors.55 Although 15 had demonstrated a reduced response
relative to the other two candidates in the previous PD assay, it
was still included in the CIA study at a high dose.
Male B10RII mice were administered Freund's complete
adjuvant containing bovine type II collagen and supplemental
Mycobacterium tuberculosis H37 RA at the base of the tail on day
0 and again on day 15. Groups of 15 mice received twice daily
oral administration of either the vehicle (20% HPβCD) in a
disease control group or the test compound on days 12−26.
Compounds 20 and 23 were tested at doses of 60 and 20 mg/kg
po BID and 15 at a single dose of 60 mg/kg po BID. One
untreated naive group was used as a normal (nondisease)
control. Efficacy was based on four individual histopathological
parameters, that is, inflammation, pannus formation, cartilage
damage, and bone erosion. Histopathology scores were
recorded for each category in a range of 0 (normal) to 5
(severe), and the mean value was calculated for the four paws.
Statistical significance (p < 0.05, Student's t test) was based, in
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a
Table 8. Cell-Based Kinase Selectivities of 15, 20, and 23
a
IC50 (nM)
target
FMS
cells/cell line
stimulus
CSF-1
end point
15
20
23
b
BMDM
proliferation
proliferation
proliferation
proliferation
IL-2
3.1
98
3.2
8.0
2.6
21
c
ITD-FLT 3
KIT
MV-4−11
none
c
f
Mo7e
SCF
210
500
na
na
41
c
TRKA
LCK
TF-1
NGF
na
na
na
150
3900
d
Jurkat
anti-CD3, PMA
GAS6
e
AXL
AXL/HEK
phospho-AXL
na
2600
a
b
c
d
Assays were performed as described in the Experimental Section or as described previously.24 Mouse BMDMs. Leukemia cell lines. T-cell
e
f
lymphoma cell line. HEK cells were transfected to overexpress AXL. na, not assayed.
Table 9. PD Dose−Response Studies of 15, 20, and 23
PD assay
a
% inhibition at 8 h (po)
mg/kg
compd
50
20
10
8
b
15
20
23
93
86
5
77
79
na
51
32
0
na
na
88−100
a
PD model in B6C3F1 mice with all compounds administered in 20%
b
HPβCD (see the details in the Experimental Section). na, not
assayed.
potential treatment for RA, it was subsequently characterized in
additional models of this disease.
Figure 3. Compounds 15, 20, and 23 reduced four-paw histopathol-
ogy summary scores in mouse CIA. CIA was induced in mice as
described in the Experimental Section. Group mean values (SEM) are
plotted, and percent inhibition values relative to the vehicle-treated
disease control are indicated in text within the bars (mpk = mg/kg);
*p < 0.01 vs disease control (vehicle).
Rat adjuvant arthritis is an experimental model of
polyarthritis that has been widely employed for preclinical
testing of antiarthritic agents.58−61 Two characteristics of this
model are the reliable onset and progression of robust, easily
measurable, periarticular inflammation, and marked bone
resorption. Cartilage destruction also occurs but is dispropor-
tionally mild in comparison to the inflammation and bone
destruction that takes place. This is a fairly aggressive model
especially when used, as in this case, in a therapeutic mode for
evaluation of compounds administered after the symptoms of
the disease have emerged.
Arthritis was induced in male Lewis rats by injection of
Freund's complete adjuvant containing lipoidal amine [N,N-
dioctyldecyl-N′,N-bis(2-hydroxyethyl)propanediamine] at the
base of the tail on day 0. Clinical signs of disease began to
emerge on day 8 postinjection. Beginning on day 8, randomized
groups of rats received twice daily oral administration of either
the vehicle (0.5% hydroxypropylmethylcellulose/0.5% Tween
80) or compound 23 at 5, 20, and 60 mg/kg. All adjuvant-
injected control rats developed clinical signs of arthritis as
evidenced by increased ankle joint diameter. Mean body
weights were similar in all groups at the start of the study, and
none of the treatment groups with established arthritis had
significant mean body weight changes as compared to the
established arthritis vehicle controls (data not shown).
After euthanization on day 14, the hind paws were removed,
weighed, processed, and scored for histopathology assessment.
Paw weights were increased nearly 2-fold in rats with adjuvant
arthritis (Figure 5A). The increase in paw weights was reduced
by 23 in a dose-dependent fashion with 60 mg/kg reducing the
gain in paw weight by 49%. Suppression of paw weight increase
by 23 correlated with suppression in the histological scores of
inflammation (Figure 5B). The abundance of periarticular
macrophages was assessed in sections following immunohis-
Figure 4. Compounds 15, 20, and 23 reduced the histopathology
evidence of inflammation, pannus, cartilage, and bone damage. CIA
was induced in mice, and scores for each histopathology parameters
were determined as described in the Experimental Section. Group
mean values (SEM) are plotted. *p < 0.01 vs disease control
(vehicle).
tochemical detection of ED-1 antigen (CD68), a marker of
macrophage lineage cells. ED-1+ macrophages were reduced by
23 in a dose-dependent fashion with significant reductions at all
doses (Figure 5C). Splenomegally, an additional manifestation
of the immune inflammatory response to adjuvant, was reduced
by 23 in a similar dose-dependent fashion (Figure 5D).
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Paws were examined further for histopathological evidence of
bone erosion (Figure 5E). By day 14, all 16 evaluated tibio-
tarsal joints of the vehicle-treated group exhibited signs of bone
erosion ranging from minimal to severe. Loss of medulary
trabecular bone was readily observed at low power in most
paws from vehicle-treated diseased rats. Osteoclast numbers per
400× field were averaged from examining the five fields of
greatest bone resorption. In vehicle-treated rats, areas of bone
erosion were characterized by numerous osteoclasts (average of
12.6 osteoclasts per 400× field). The occurrence of erosions
and erosion-associated osteoclasts was completely prevented in
rats dosed with 60 or 20 mg/kg 23. Even in rats receiving
5 mg/kg 23, 11 of 16 evaluated joints were characterized by an
absence of bone erosion, and the remaining five joints had mild
erosions. At all three doses of 23, osteoclast numbers were
similar to disease-free joints. Adjuvant arthritis was additionally
associated with periosteal new bone formation. The formation
of new bone was proportional to inflammation scores and was
reduced 67% by a dose of 60 mg/kg 23 but was not reduced by
lower doses.
In a third model, polyarthritis was induced in female Lewis
rats following intraperitoneal (ip) administration of strepto-
coccal cell wall (SCW) proteoglycan-polysaccharides (PG-PS).
This model has an acute nonerosive phase (days 3−7) that is
complement- and neutrophil-dependent and that resolves. A
chronic erosive phase then begins at about day 10 and is
dependent on macrophages and on the development of specific
T-cell immunity to SCW.62,63 In an initial study, dosing of 23
was commenced on day 8 when the acute phase was resolving
and transitioning to the chronic phase. Rats treated with vehicle
progressively developed a severe arthritis until study termi-
nation (day 19). Administration of 23 at 20 mg/kg (po, BID)
blocked the emergence of the chronic arthritic phase with a
significant effects on ankle thickness (Figure 6A) and clinical
scores (Figure 6B) observed as early as day 12, 4 days after
dose initiation. Histological assessment of the ankles of vehicle-
treated rats revealed recruitment of numerous ED-1 positive
macrophages into the periarticular tissues together with
fibroplasia (Figure 6C). Also notable were the greatly increased
numbers osteoclasts (also ED-1 positive) eroding many large
areas of trabecular bone in all vehicle-treated rats (Figure 6C).
Compound 23 reduced the recruitment of macrophages into
the periarticular tissues (Figure 6D). Furthermore, osteoclast
counts and distribution were normal, and the large areas of
trabecular bone loss observed in all vehicle-treated rats were
not observed in rats treated with 23. The bone protective
effects of 23 were corroborated when imaged by micro-
radiography (Figure 7). When compared to disease-free
controls (Figure 7A), the ankle bones of vehicle-treated rats
appeared pitted and deformed (Figure 7B). Consistent with the
observed histological effects, joints of rats dosed from day 8 to
day 19 with 20 mg/kg 23 appeared normal (Figure 7C) with
the exception of one rat that exhibited minimal signs of pitting.
On the basis of a four-point severity scale, the mean (SD) bone
deformation score in vehicle-treated rats was 2.83 ± 0.41 vs
0.17 ± 0.41 for rats dosed with 23.
39). On day 39, the ankle thickness measurements for the SCW
vehicle group rats were 13.78 ± 0.57 mm, and the ankle
thickness measurements of 2 mg/kg 23 group rats were
11.56 ± 1.54; the ankle thickness measurements of 6 mg/kg 23
group rats were 9.91 ± 0.41 mm; the ankle thickness
measurements of 20 mg/kg 23 group rats were 9.30 ± 0.39
̈
and 7.58 ± 0.03 mm for naive disease-free rats.
Collectively, 23 was shown to prevent osteoclastogenesis and
to reduce synovial and periarticular macrophages in three
models of RA together with reductions in histopathology scores
or joint swelling and erosion. These data sugget the potential
for substantial disease modification with 23 in RA as a single
agent. Furthermore, because the antirheumatic effects of low-
dose methotrexate (MTX) are currently ascribed to the
capacity of the drug to suppress the development of a fully
activated T-cell phenotype, 23 and MTX might be expected to
have complementary effects in suppressing autoimmune
disease, macrophage-derived inflammation, and bone erosion.
Because of the overall promising preclinical profile of 23 and
the potential to provide benefit to RA patients, 23 was selected
as a candidate for clinical studies in humans pending the results
of safety testing.
CONCLUSION
■
Two previous lead FMS inhibitors, 8 and 21, which possessed
excellent in vitro potency against FMS and cells, were found to
have poor PK characteristics resulting in less than ideal in vivo
PD activity. A sequence of analogues of the piperidine group
was prepared to optimize both the ADME and the PK/PD
properties of this series, resulting in three potent and efficacious
compounds, 15, 20, and 23, with relatively good selectivity
against a number of kinases. In vivo PD dose−response
behavior, CIA efficacy, and toxicity observations were used to
ultimately select 23 as the most promising clinical candidate.
Compound 23 was shown to effectively reduce inflammation
and bone erosion in mouse CIA as well as in rat adjuvant and
SCW models of arthritis, indicating that this agent may be
beneficial in the treatment of RA.
EXPERIMENTAL SECTION
Chemistry. Reagents and solvents were obtained from commercial
suppliers and used without further purification. H NMR spectra were
■
1
measured in the indicated solvent with tetramethylsilane (TMS) or the
residual solvent peak as the internal standard on a Bruker B-ACS-120
(400 MHz) spectrophotometer at room temperature. Chemical shifts
are given in ppm (δ) relative to the internal standard, and coupling
constants (J) are in Hertz (Hz). Abbreviations for the multiplicities of
the signals are as follows: s (singlet), d (doublet), t (triplet), q (quartet),
quin (quintet), sep (septet), m (multiplet), br s (broad singlet), and
combinations thereof. All reactions were monitored by thin-layer
chromatography (TLC) carried out on EMD silica gel plates
(2.5 cm × 7.5 cm, 250 μm thick, 60 F254), visualized by using UV
(254 nm) or stains such as KMnO4, p-anisaldehyde, and ceric
ammonium molybdate (CAM). All organic solutions were dried over
anhydrous MgSO4 or Na2SO4 and concentrated on a rotary
evaporator. Hydrogenation was performed at atmospheric pressures
under a balloon of hydrogen and at higher pressures on a shaker type
hydrogenation apparatus (Parr Instrument Co.). Flash chromatog-
raphy was performed using Fisher Chemical Silica Gel Sorbent (230−
400 mesh, grade 60) or on prepacked silica solid-phase extraction
(SPE) columns (Biotage IST Isolute or Waters Sep-Pak columns).
Preparative TLC was performed on Analtech Silica Gel GF plates
(1000 or 2000 μm, 20 cm × 20 cm). Preparative high-performance
liquid chromatography (HPLC) was carried out on a Varian Prep Star
system equipped with a Varian model 325 UV detector (monitoring
In a second study, dosing was commenced on day 18 when
chronic arthritis was well-established (Figure 8). Treatment
with 23 induced a dose-dependent decrease in arthritis scores
with significant effects observed by day 21 for the 6 and 20 mg/kg
dose groups and for all dose groups by day 26. Both ankle
swelling and arthritic scores continued to decrease in the 6 and
20 mg/kg dose groups 1 day prior to study termination (day
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Figure 5. Compound 23 reduced disease activity in rats with adjuvant arthritis. Adjuvant arthritis was induced in rats as described in the
Experimental Section. Commencing on day 8, adjuvant-induced rats (Adj) received twice daily oral doses of vehicle (veh) or 5, 20, or 60 mg/kg of
23 until study termination on day 14. An additional group of rats was not adjuvant induced and served as the disease-free (normal) control. All end
points were determined as described in the Experimental Section. (A) Hind paws were severed at the level of the medial and lateral malleolus and
weighed. (B) H&E-stained sections of formalin-fixed hind paws were scored for inflammation by histopathology assessment using a five-point scale.
(C) Soft tissue regions of sections stained for ED-1 antigen (CD68) positive macrophages were assessed for the number of areas (hot spots) with
marked numbers of positive cells. (D) Spleens were weighed at necropsy. (E) H&E-stained sections of formalin-fixed hind paws were scored for
bone erosions using a five-point scale. All values represent group means (n = 16 hind limbs or 8 spleens). Error bars represent standard errors of the
means. Percent inhibition values relative to adjuvant-induced, vehicle-treated rats were provided as white text within bars. *p < 0.05.
at 254 nm) and a Varian Pro Star fraction collector and using a
ThermoElectron Betasil C18 (150 mm × 21.2 mm, 10 μm particle
size,) reverse phase column. The solvents, both acetonitrile (MeCN)
and water, contained 0.05% of trifluoroacetic acid (TFA, v/v). LC-MS
was performed on a system consisting of an electrospray ionization
(ESI) source on a Finnigan LCQ Classics ion trap mass spectrometer,
an Agilent 1100 DAD UV detector (214 nm wavelength), an Agilent
1100-LC binary gradient pumping system, a Gilson 215 configured as
an autosampler, and a PrincetonSPHER HTS 60A (Princeton Chro-
matography Inc.) C12 HPLC column (5 μm, 50 mm × 3.0 mm). The
purities of the key target compounds were determined on the Finnigan
LCQ instrument, which gave both the MS and LC trace of the
compound in a 5 min run. The HPLC method used was as follows:
column, PrincetonSPHER HTS 60A C12 HPLC column (5 μm,
50 mm × 3.0 mm); column temperature, ambient; flow rate, 1.5 mL/min;
and gradient, 10% acetonitrile in water to 100% acetonitrile in water
in 5 min, both MeCN and water contained 0.05% of TFA, v/v. All
compounds used for biological assays were at least of 95% purity based
on HPLC analytical results, unless otherwise noted.
dioxaborolan-2-yl)-phenylamine 1 (4.0 g, 18 mmol), 4-trifluorome-
thanesulfonyloxy-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl
ester64 2 (7.4 g, 22 mmol), and 2 M aqueous Na2CO3 (80 mL) in
toluene (160 mL) and EtOH (80 mL) was placed under Ar and heated
to 80 °C for 3 h. The mixture was washed with 1 M aqueous NaOH,
and the organic layer was removed, dried (Na2SO4), and concentrated
in vacuo. The residue was purified by silica gel chromatography,
eluting with 20% EtOAc/hexanes to afford 3.2 g (63%) of the title
1
compound as a yellow foam. H NMR (CDCl3, 400 MHz): δ 7.18−
7.23 (m, 2H, J = 8.4 Hz), 6.64−6.69 (m, 2H, J = 8.6 Hz), 5.90 (br s,
1H), 4.02−4.08 (m, 2H), 3.68 (s, 2H), 3.62 (t, 2H, J = 5.6 Hz), 2.48
(br s, 2H), 1.49 (s, 9H). Mass spectrum (ESI, m/z): calcd for
C16H22N2O2, 275.2 (M + H); found, 275.1.
b. 4-(4-Amino-phenyl)-piperidine-1-carboxylic Acid tert-Butyl
Ester (3). A solution of 4-(4-amino-phenyl)-3,6-dihydro-2H-pyri-
dine-1-carboxylic acid tert-butyl ester (0.350 g, 1.28 mmol) (as
prepared in the previous step) in methanol was hydrogenated over
10% Pd/C at 20 psi for 1 h. The solution was filtered through
diatomaceous earth, and the filtrate was concentrated to give 0.35 g
1
(100%) of the title compound 3 as a yellow solid. H NMR (CDCl3,
4-Cyano-1H-imidazole-2-carboxylic Acid (2-Cyclohex-1-
enyl-4-piperidin-4-yl-phenyl)-amide Trifluoroacetic Acid Salt
(8). a. 4-(4-Amino-phenyl)-3,6-dihydro-2H-pyridine-1-carboxylic
Acid tert-Butyl Ester. A solution of 4-(4,4,5,5-tetramethyl-[1,3,2]-
400 MHz): δ 6.96−7.01 (d, 2H, J = 8.4 Hz), 6.62−6.67 (d, 2H, J =
8.4 Hz), 4.21 (br s, 2H), 3.58 (br s, 2H), 2.77 (t, 2H, J = 12.6 Hz),
2.53 (tt, 1H, J = 12.1, 3.5 Hz), 1.77 (d, 2H, J = 12.3 Hz), 1.52−1.59
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Figure 6. Compound 23 prevented the chronic phase of SCW arthritis and the recruitment of macrophages and osteoclasts and bone erosion.
Arthritis was induced in rats following a single ip injection of SCW as described in the Experimental Section. Following partial resolution of the acute
phase, twice daily oral dosing was commenced on day 8 with vehicle or with 20 mg/kg compound 23. Ankle thickness was determined using calipers
(A), and clinical scores were assigned as described in the Experiment Section (B). Immunohistochemistry was used to assess formalin-fixed sections
of hind-limb ankles for ED-1 antigen positive cells. Representative low power (20×) fields of rats treated with vehicle (C) or with 20 mg/kg
compound 23 (D) are provided. The arrow head indicates periarticular tissue with intense infiltration of macrophages. Arrows indicated erosions in
trabecular bone associated with large numbers of osteoclasts. All values represent group means (n = 6). Error bars represent standard errors of the
means. *p < 0.05.
(m, 2H), 1.48 (s, 9H). Mass spectrum (ESI, m/z): calcd for
C16H24N2O2, 277.2 (M + H); found, 277.1.
1.85 (m, 8H), 1.50 (s, 9H). Mass spectrum (ESI, m/z): calcd for
C22H32N2O2, 357.2 (M + H); found, 357.1.
e. (4-{[4-Cyano-1-(2-trimethylsilanyl-ethoxymethyl)-1H-imida-
zole-2-carbo-nyl]-amino}-3-cyclohex-1-enyl-phenyl)-piperidine-1-
carboxylic Acid tert-Butyl Ester (7). To a solution of 4-cyano-1-(2-
trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carboxylate potassium
salt27 6 (3.34 g, 10.9 mmol) in 20 mL of CH2Cl2 were added DIEA
(3.80 mL, 21.8 mmol) and PyBroP (5.59 g, 12.0 mmol), and the
reaction was stirred at 25 °C for 15 min. A solution of 4-(4-amino-3-
cyclohex-1-enyl-phenyl)-piperidine-1-carboxylic acid tert-butyl ester
(3.92 g, 11.0 mmol) (as prepared in the previous step) in 10 mL of
CH2Cl2 was added, and the reaction was stirred for 8 h at 25 °C. The
reaction was diluted with EtOAc (60 mL) and washed with saturated
aqueous NaHCO3 (2 × 60 mL) and brine (100 mL), and the organic
layer was dried over Na2SO4 and then concentrated. The residue was
purified by flash chromatography (silica gel, 2% EtOAc/CH2Cl2) to
c. 4-(4-Amino-3-bromo-phenyl)-piperidine-1-carboxylic Acid tert-
Butyl Ester (4). To a solution of 4-(4-amino-phenyl)-piperidine-1-
carboxylic acid tert-butyl ester (0.20 g, 0.71 mmol) (as prepared in the
previous step) in CH2Cl2 (3 mL) was added N-bromosuccinimide
(NBS) (0.13 g, 0.71 mmol), and the reaction was stirred at room
temperature for 10 h. The reaction was diluted with EtOAc (10 mL)
and washed with saturated aqueous NaHCO3 (2 × 10 mL) and brine
(10 mL). Concentration of the organic layer gave 0.26 g (100%) of the
1
title compound 4 as a yellow foam. H NMR (CDCl3, 400 MHz): δ
7.27 (d, 1H, J = 2.1 Hz), 6.96 (dd, 1H, J = 8.1, 1.9 Hz), 6.73 (d, 1H,
J = 8.1 Hz), 4.24 (br s, 2H), 4.01 (br s, 2H), 2.78 (t, 2H, J = 12.2 Hz),
2.53 (tt, 1H, J = 12.2, 3.3 Hz), 1.79 (d, 2H, J = 12.6 Hz), 1.52−1.59
(m, 2H), 1.50 (s, 9H). Mass spectrum (ESI, m/z): calcd for
C16H23BrN2O2, 355.1 (M + H); found, 355.1.
1
give 5.5 g (85%) of the title compound 7 as a yellow oil. H NMR
d. 4-(4-Amino-3-cyclohex-1-enyl-phenyl)-piperidine-1-carboxylic
Acid tert-Butyl Ester (5). 4-(4-Amino-3-bromo-phenyl)-piperidine-1-
carboxylic acid tert-butyl ester (0.13 g, 0.36 mmol) (as prepared in the
previous step), cyclohex-1-enyl boronic acid 4 (0.060 g, 0.48 mmol),
Pd(PPh3)4 (0.04 g, 10 mol %), aqueous 2 M Na2CO3 (1.5 mL),
ethanol (1.5 mL), and toluene (3 mL) were heated at 80 °C for 3 h.
The reaction was diluted with EtOAc (10 mL) and washed with
saturated aqueous NaHCO3 (2 × 10 mL) and brine (10 mL), and the
organic layer was dried over Na2SO4 and then concentrated. The
residue was purified on a 20 g SPE cartridge (silica) with 30% EtOAc/
hexane to give 0.10 g (85%) of the title compound 5 as a yellow oil. 1H
NMR (CDCl3, 400 MHz): δ 6.90 (dd, 1H, J = 8.1, 2.1 Hz), 6.85 (d,
1H, J = 1.9 Hz), 6.67 (d, 1H, J = 8.1 Hz), 5.76 (dq, 1H, J = 3.5, 1.8
Hz), 4.23 (br s, 2H), 3.71 (s, 2H), 2.79 (t, 2H, J = 12.7 Hz), 2.54 (tt,
1H, J = 12.3, 3.4 Hz), 2.22−2.29 (m, 2H), 2.16−2.22 (m, 2H), 1.62−
(CDCl3, 400 MHz): δ 9.68 (s, 1H), 8.25 (d, 1H, J = 8.4 Hz), 7.78 (s,
1H), 7.12 (dd, 1H, J = 8.6, 2.1 Hz), 7.02 (d, 1H, J = 2.1 Hz), 5.96 (s,
2H), 5.83 (dt, 1H, J = 3.6, 1.9 Hz), 4.25 (br s, 2H), 3.63−3.69 (m,
2H), 2.80 (t, 2H, J = 11.7 Hz), 2.63 (tt, 1H, J = 12.2, 3.5 Hz), 2.27−
2.33 (m, 2H), 2.20−2.27 (m, 2H), 1.77−1.87 (m, 6H), 1.56−1.68 (m,
2H), 1.49 (s, 9H), 0.95−1.00 (m, 2H), 0.01 (s, 9H). Mass spectrum
(ESI, m/z): calcd for C33H47N5O4Si, 606.2 (M + H); found, 606.2.
f. 4-Cyano-1H-imidazole-2-carboxylic Acid (2-Cyclohex-1-enyl-4-
piperidin-4-yl-phenyl)-amide Trifluoroacetic Acid Salt (8). To a
solution of 4-(4-{[4-cyano-1-(2-trimethylsilanyl-ethoxymethyl)-1H-
imidazole-2-carbonyl]-amino}-3-cyclohex-1-enyl-phenyl)-piperidine-1-
carboxylic acid tert-butyl ester 7 (1.50 g, 2.48 mmol) (as prepared in
the previous step) in 10 mL of CH2Cl2 and 0.3 mL of EtOH was
added 3 mL of TFA, and the solution was stirred for 3 h at 25 °C.
The reaction was diluted with 5 mL of EtOH and then concentrated.
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carboxylic acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide
TFA salt 8 (75.0 mg, 0.153 mmol), K2CO3 (84.7 mg, 0.613 mmol),
2-fluoropyridine (26.4 μL, 0.306 mmol), and 0.3 mL of N,N-
dimethylacetamide were stirred for 8 h at 120 °C. The reaction was
diluted with 3 mL of H2O, and the residue was purified by RP-HPLC
(C18), eluting with 30−50% CH3CN in 0.1% TFA/H2O over 9 min to
1
give 50 mg (75%) of the title compound as a white solid. H NMR
(CD3OD, 400 MHz): δ 8.18 (d, 1H, J = 8.4 Hz), 8.06 (m, 1H), 8.02
(s, 1H), 7.94 (dd, 1H, J = 6.3, 1.6 Hz), 7.48 (d, 1H, 9.5 Hz), 7.22 (dd,
1H, J = 8.5, 2.2 Hz), 7.12 (d, 1H, J = 2.1 Hz), 6.98 (t, 1H, J = 7.1 Hz),
5.82 (m, 1H), 4.32 (m, 2H), 3.46 (m, 2H), 3.00 (m, 1H), 2.30 (m,
4H), 2.18 (m, 2H), 1.96−1.74 (m, 6H). Mass spectrum (ESI, m/z):
calcd for C27H28N6O, 453.2 (M + H); found, 453.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {4-[1-(2-Cyano-
ethyl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl}-amide Tri-
fluoroacetic Acid Salt (10). 4-Cyano-1H-imidazole-2-carboxylic
acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8
(77.0 mg, 0.157 mmol), Et3N (21.9 μL, 0.157 mmol), acrylonitrile
(12.4 μL, 0.188 mmol), 0.1 mL of MeOH, and 1.0 mL of 1,2-
dichloroethane were stirred for 1 h at 80 °C. The reaction was
concentrated, and the residue was purified by RP-HPLC (C18), eluting
with 30−50% CH3CN in 0.1% TFA/H2O over 12 min to give 83 mg
(95%) of the title compound as a white solid. 1H NMR (CD3OD, 400
MHz): δ 8.18 (d, 1H, J = 8.6 Hz), 8.06 (m, 1H), 7.22 (dd, 1H, J = 8.4,
1.9 Hz), 7.12 (d, 1H, J = 1.9 Hz), 5.82 (m, 1H), 3.76 (m, 2H), 3.60
(m, 2H), 3.28 (t, 2H, J = 12.6 Hz), 3.12 (t, 2H, J = 7.2), 2.92 (m, 1H),
2.30 (m, 4H), 2.18−1.98 (m, 4H), 1.92−1.74 (m, 4H). Mass spectrum
(ESI, m/z): calcd for C25H28N6O, 429.2 (M + H); found, 429.2.
4-Cyano-1H-imidazole-2-carboxylic Acid [2-Cyclohex-1-
enyl-4-(1-pyridin-2-ylmethyl-piperidin-4-yl)-phenyl]-amide
Trifluoroacetic Acid Salt (11). 4-Cyano-1H-imidazole-2-carboxylic
acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8
(88.0 mg, 0.180 mmol), pyridine-2-carbaldehyde (20.5 μL, 0.216
mmol), Et3N (30.1 μL, 0.216 mmol), sodium triacetoxyborohydride
(53.4 mg, 0.252 mmol), and 0.8 mL of 1,2-dichloroethane were stirred
for 10 h at 25 °C. The solvent was evaporated, and the residue was
purified by RP-HPLC (C18), eluting with 30−50% CH3CN in 0.1%
TFA/H2O over 20 min to give 81 mg (78%) of the title compound as
a white solid. 1H NMR (CDCl3, 400 MHz): δ 9.62 (s, 1H), 8.62 (ddd,
1H, J = 4.9, 1.7, 0.8 Hz), 8.25 (d, 1H, J = 8.4 Hz), 7.72−7.77 (m, 2H),
7.57 (d, 1H, J = 7.7 Hz), 7.26−7.31 (m, 1H), 7.17 (dd, 1H, J = 8.5, 2.0
Hz), 7.05 (d, 1H, J = 1.9 Hz), 5.84 (dt, 1H, J = 3.5, 1.8 Hz), 3.99 (s,
2H), 3.51 (s, 1H), 3.28 (d, 2H, J = 11.6 Hz), 2.59 (tt, 1H, J = 11.8, 4.0
Hz), 2.44−2.54 (m, 2H), 2.27−2.34 (m, 2H), 2.21−2.27 (m, 2H),
1.75−2.05 (m, 8H). Mass spectrum (ESI, m/z): calcd for C28H30N6O,
467.2 (M + H); found, 467.2.
Figure 7. Compound 23 prevented the development of bone
deformation. Following necropsy on day 19, microradiographic images
were prepared of tibial tarsal joints. Representative images are shown
obtained from a disease-free rat (A), a rat with adjuvant arthritis
treated with vehicle (B), and a rat with adjuvant arthritis treated with
20 mg/kg 23 (C). Bones in vehicle-treated rats with adjuvant arthritis
appeared pitted and lacked fine definition, while bones from rats
treated with 23 appeared normal.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-hydroxy-ethyl)-piperidin-4-yl]-phenyl}-amide Tri-
fluoroacetic Acid Salt (12). The title compound was prepared in
85% yield from 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-
enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8 and hydroxyacetalde-
1
hyde according to the procedure in the preparation of 11. H NMR
(CD3OD, 400 MHz): δ 8.18 (d, 1H, J = 8.6 Hz), 8.02 (s, 1H), 7.22
(dd, 1H, J = 8.4, 2.1 Hz), 7.14 (d, 2H, J = 2.1 Hz), 5.82 (m, 1H), 3.94
(m, 2H), 3.74 (m, 2H), 3.30 (m, 2H), 3.18 (t, 2H), 2.92 (m, 1H), 2.30
(m, 4H), 2.20−1.98 (m, 4H), 1.96−1.74 (m, 4H). Mass spectrum
(ESI, m/z): calcd for C24H29N5O2, 420.2 (M + H); found, 420.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-hydroxy-1-hydroxymethyl-ethyl)-piperidin-4-yl]-
phenyl}-amide Trifluoroacetic Acid Salt (14). To a slurry of 4-
cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperidin-
4-yl-phenyl)-amide trifluoroacetic acid salt 8 (81 mg, 0.16 mmol) in
CH2Cl2 (3 mL) was added Et3N (33 μL, 0.24 mmol). The solution
was then treated with 2,2-dimethyl-[1,3]dioxan-5-one (31 mg, 0.24
mmol), and the reaction was allowed to stir for 3 h. At this time,
NaBH(OAc)3 (51 mg, 0.24 mmol) was added in one portion, and the
reaction was allowed to stir for an additional 4 h. The reaction was
diluted with H2O (10 mL) and extracted with EtOAc (2 × 25 mL).
The organic extracts were dried (Na2SO4) and concentrated in vacuo.
Purification by silica gel preparative TLC (10% MeOH−CHCl3)
afforded 22 mg (28%) of 4-cyano-1H-imidazole-2-carboxylic acid
Figure 8. SCW model of arthritis. Dosing commenced after the
establishment of chronic arthritis.
The residue was crystallized from methanol and ethyl ether to give
0.85 g (70%) of the title compound as a white solid. 1H NMR
(CD3OD, 400 MHz): δ 8.18 (d, 1H, J = 8.4 Hz), 8.04 (s, 1H), 7.22
(dd, 1H, J = 8.6, 2.1 Hz), 7.12 (d, 1H, J = 2.3 Hz), 5.76 (m, 1H), 3.54
(m, 2H), 3.16 (m, 2H), 2.92 (m, 1H), 2.30 (m, 4H), 2.10 (m, 2H),
1.87 (m, 6H). Mass spectrum (ESI, m/z): calcd for C22H25N5O, 376.2
(M + H); found, 376.2.
4-Cyano-1H-imidazole-2-carboxylic Acid [2-Cyclohex-1-
enyl-4-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-4-yl)-phenyl]-
amide Trifluoroacetic Acid Salt (9). 4-Cyano-1H-imidazole-2-
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{2-cyclohex-1-enyl-4-[1-(2,2-dimethyl-[1,3]dioxan-5-yl)-piperidin-4-
yl]-phenyl}-amide (13) as an off-white semisolid. Mass spectrum (ESI,
m/z): calcd for C28H35N5O3, 490.2 (M + H); found, 490.6.
amino]-3-cyclohex-1-enyl-phenyl}-piperidin-1-yl)-acetic acid tert-butyl
ester 17 was eluted with 25% EtOAc/CH2Cl2. This compound was
dissolved in 1 mL of CH2Cl2, 20 μL of EtOH and 1 mL of TFA were
added, and the reaction was stirred for 3 h at 25 °C. The residue was
purified by RP-HPLC (C18), eluting with 30−50% CH3CN in 0.1%
TFA/H2O over 12 min to give 10 mg (40%) of the title compound
To a solution of 4-cyano-1H-imidazole-2-carboxylic acid {2-
cyclohex-1-enyl-4-[1-(2,2-dimethyl-[1,3]dioxan-5-yl)-piperidin-4-yl]-
phenyl}-amide 13 (22 mg, 0.045 mmol, as prepared above) in THF−
H2O (1 mL, 4:1 v/v) was added TFA (0.4 mL), and the reaction was
allowed to stir for 1 h. Removal of the solvent under vacuum afforded
1
as a white solid. H NMR (CD3OD, 400 MHz): δ 8.16 (d, 1H, J =
8.4 Hz), 8.02 (s, 1H), 7.22 (dd, 1H, J = 8.5, 2.0 Hz), 7.10 (d, 1H, J =
1.9 Hz), 5.72 (m, 1H), 4.04. (s, 2H), 3.76 (m, 2H), 3.22 (m, 2H),
2.90 (m, 1H), 2.29 (m, 4H), 2.10 (m, 4H), 1.82 (m, 4H). Mass
spectrum (ESI, m/z): calcd for C24H27N5O3, 434.2 (M + H); found,
434.2.
1
14 mg (60%) of the title compound as an amber foam. H NMR
(CD3OD, 400 MHz): δ 8.17 (d, 1H, J = 8.4 Hz), 8.03 (s, 1H), 7.22
(dd, 1H, J = 8.4, 2.0 Hz), 7.13 (d, 1H, J = 2.0 Hz), 5.83 (s, 1H), 3.98
(d, 4H, J = 4.9 H), 3.74−3.78 (m, 2H), 3.46−3.53 (m, 2H), 3.37−3.40
(m, 1H), 2.88−2.96 (m, 1H), 2.29 (br s, 4H), 2.03−2.16 (m, 4H),
1.78−1.90 (m, 4H). Mass spectrum (ESI, m/z): calcd for C25H31N5O3,
450.2 (M + H); found, 450.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-methanesulfonyl-ethyl)-piperidin-4-yl]-phenyl}-
amide (15). a. Methanesulfonic Acid 2-Methanesulfonyl-ethyl
Ester. To a solution of methanesulfonyl chloride (484 mg, 4.23
mmol) in 15 mL of CH2Cl2 at 0 °C was added 2-methanesulfonyl-
ethanol (500 mg, 4.03 mmol) in 10 mL of CH2Cl2 followed by DIEA
(1.05 mL, 6.05 mmol) under Ar. The mixture was warmed to room
temperature and stirred for 20 h under Ar. The mixture was treated
with 100 mL of EtOAc and washed with H2O (3 × 20 mL) and brine
(20 mL) and was dried (Na2SO4). Removal of the solvent in vacuo
gave 534 mg (66%) of the title compound as a brown oil, which was
used directly in the following step without purification. 1H NMR
(CDCl3, 400 MHz): δ 4.67 (d, 2H, J = 5.5 Hz), 3.46 (d, 2H, J = 5.5
Hz), 3.11 (s, 3H), 3.04 (s, 3H).
4-Cyano-1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-
carboxylic Acid (2-Cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide
Trifluoroacetic Acid Salt (19). To a solution of 4-(4-{[4-cyano-1-
(2-trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carbonyl]-amino}-
3-cyclohex-1-enyl-phenyl)-piperidine-1-carboxylic acid tert-butyl ester
7 (81 mg, 0.12 mmol) in 18 mL of CH2Cl2 was added 1 mL of EtOH
followed by 5 mL of TFA at 0 °C. The resulting solution was stirred at
room temperature for only 0.5 h and treated with 20 mL of EtOH
followed by 20 mL of n-PrOH and 5 mL of H2O. The mixture was
then concentrated under reduced pressure to give a slightly yellow
solid. Flash chromatography of the compound on silica gel (2−4%
MeOH/CH2Cl2) gave 0.87 g (85%) of the title compound as a white
1
solid. H NMR (CDCl3, 400 MHz): δ 9.70 (s, 1H), 9.66 (br s, 1H),
9.15 (m, 1H), 8.29 (d, 1H, J = 8.3 Hz), 7.78 (s, 1H), 7.13 (dd, 1H, J =
8.3, 2.2 Hz), 7.03 (d, 1H, J = 2.2 Hz), 5.95 (s, 2H), 5.83 (m, 1H), 3.66
(t, 2H, J = 8.4 Hz), 3.55 (d, 2H, J = 12.3 Hz), 2.95−3.11 (m, 2H), 2.76
(m, 1H), 2.18−2.33 (m, 4H), 1.99−2.15 (m, 4H), 1.82 (m, 4H), 0.97
(t, 2H, J = 8.3 Hz), 0.00 (s, 9H). Mass spectrum (ESI, m/z): calcd for
C28H39N5O2Si, 506.3 (M + H); found, 506.1.
b. 4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-enyl-
4-[1-(2-methanesulfonyl-ethyl)-piperidin-4-yl]-phenyl}-amide
(15). To a solution of 4-cyano-1H-imidazole-2-carboxylic acid (2-
cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide trifluoroacetic acid salt
8 (85 mg, 0.17 mmol) and DIEA (91 μL, 0.52 mmol) in 3 mL of
CH2Cl2 at room temperature was added 2-methanesulfonic acid 2-
methanesulfonyl-ethyl ester (as prepared in the previous step, 42 mg,
0.21 mmol). The resulting mixture was stirred at room temperature for
3 h. Treated with 50 mL of EtOAc, the mixture was washed with H2O
(2 × 20 mL) and brine (10 mL) and was dried (Na2SO4). Removal of
the solvent in vacuo followed by flash chromatography of the residue
on silica gel (1−3% MeOH/CH2Cl2) gave 54 mg (65%) of the title
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-morpholin-4-yl-ethyl)-piperidin-4-yl]-phenyl}-
amide Bis-trifluoroacetic Acid Salt (20). a. 4-Cyano-1-
(2-trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carboxylic Acid
{2-Cyclohex-1-enyl-4-[1-(2-morpholin-4-yl-ethyl)-piperidin-4-yl]-
phenyl}-amide. 4-Cyano-1-(2-trimethyl-silanyl-ethoxymethyl)-1H-
imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phe-
nyl)-amide TFA salt 19 (830 mg, 1.34 mmol), K2CO3 (600 mg,
4.34 mmol), sodium iodide (40.2 mg, 0.268 mmol), 4-(2-chloro-
ethyl)-morpholine hydrochloride (260 mg, 1.40 mmol), and 5.0 mL of
N,N-dimethylacetamide were stirred for 8 h at 80 °C. The reaction was
diluted with EtOAc (50 mL), washed with saturated aqueous
NaHCO3 (2 × 50 mL) and brine (50 mL), and concentrated. The
residue was purified by flash chromatography (silica gel, 5% MeOH/
CH2Cl2) to give 650 mg (78%) of the title compound as a white solid.
1H NMR (CDCl3, 400 MHz): δ 9.67 (s, 1H), 8.23 (d, 1H, J = 8.6 Hz),
1
compound as a white solid. H NMR (CDCl3, 400 MHz): δ 9.54 (s,
1H), 8.25 (d, 1H, J = 8.4 Hz), 7.72 (s, 1H), 7.15 (dd, 1H, J = 8.4, 2.0
Hz), 7.04 (d, 1H, J = 2.0 Hz), 5.85 (m, 1H), 3.21 (t, 2H, J = 6.5 Hz),
3.09 (s, 3H), 3.02−3.11 (m, 2H), 2.92 (t, 2H, J = 6.5 Hz), 2.52 (dddd,
1H, J = 12.1, 12.1, 3.3, 3.3 Hz), 2.18−2.34 (m, 4H), 2.18 (t, 2H, J =
10.8 Hz), 1.64−1.94 (m, 9H). Mass spectrum (ESI, m/z): calcd for
C25H31N5O3S, 482.2 (M + H); found, 482.2.
4-Cyano-1H-imidazole-2-carboxylic Acid [4-(1-Carbamoyl-
methyl-piperidin-4-yl)-2-cyclohex-1-enyl-phenyl]-amide Tri-
fluoroacetic Acid Salt (16). 4-Cyano-1H-imidazole-2-carboxylic
acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8
(50.0 mg, 0.102 mmol), Et3N (32.7 μL, 0.235 mmol), 2-
bromoacetamide (16.9 mg, 0.123 mmol), and 0.5 mL of CH2Cl2
were stirred for 4 h at 25 °C. The reaction was concentrated, and the
residue was purified by RP-HPLC (C18), eluting with 30−50%
CH3CN in 0.1% TFA/H2O over 12 min to give 42 mg (75%) of the
title compound as a white solid. 1H NMR (CD3OD, 400 MHz): δ 8.14
(d, 1 H, J = 8.6 Hz), 8.00 (s, 1H), 7.19 (d, 1H, J = 8.4 Hz), 7.11 (s,
1H), 5.75−5.83 (m, 1H), 3.99 (s, 2H), 3.73 (d, 2H, J = 11.4 Hz),
3.14−3.29 (m, 2H), 2.78−2.97 (m, 1H), 2.19−2.32 (m, 4H), 2.01−
2.16 (m, 4H), 1.71−1.90 (m, 4H). Mass spectrum (ESI, m/z): calcd
for C24H28N6O2, 433.2 (M + H); found, 433.2.
7.78 (s, 1H), 7.14 (dd, 1H, J = 8.5, 2.0 Hz), 7.04 (d, 1H, J = 1.9 Hz),
5.95 (s, 2H), 5.78−5.85 (m, 1H), 3.70−3.74 (m, 4H), 3.62−3.68 (m,
2H), 2.58−2.67 (m, 4H), 2.46−2.57 (m, 4H), 2.24−2.40 (m, 4H),
2.10−2.20 (m, 4H), 1.73−1.92 (m, 7H), 1.60−1.68 (m, 2H), 0.92−
1.00 (m, 2H), 0.00 (s, 9H). Mass spectrum (ESI, m/z): calcd for
C34H50N6O3 Si, 619.4 (M + H); found, 619.3.
b. 4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-enyl-
4-[1-(2-morpholin-4-yl-ethyl)-piperidin-4-yl]-phenyl}-amide Tri-
fluoroacetic Acid Salt (20). To a solution of 4-cyano-1-(2-
trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carboxylic acid {2-cy-
clohex-1-enyl-4-[1-(2-morpholin-4-yl-ethyl)-piperidin-4-yl]-phenyl}-
amide 19 (650 mg, 1.05 mmol) (as prepared in the previous step) in
10 mL of CH2Cl2 were added 0.3 mL of EtOH and 3.0 mL of TFA,
and the reaction was allowed to proceed for 2 h at 25 °C. The reaction
was diluted with 10 mL of EtOH and concentrated. The residue was
purified by RP-HPLC (C18), eluting with 30−50% CH3CN in 0.1%
TFA/H2O over 9 min to give 600 mg (80%) of the title compound as
(4-{4-[(4-Cyano-1H-imidazole-2-carbonyl)-amino]-3-cyclo-
hex-1-enyl-phenyl}-piperidin-1-yl)-acetic Acid Trifluoroacetic
Acid Salt (18). 4-Cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-
1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8 (33 mg, 0.067
mmol), t-butyl bromoacetate (9.90 μL, 0.0675 mmol), Et3N (18.8
μL, 0.135 mmol), and 0.25 mL of CH2Cl2 were stirred for 10 h at 25
°C. The reaction mixture was loaded on a 5 g SPE cartridge (silica),
and 23 mg (70%) of (4-{4-[(4-cyano-1H-imidazole-2-carbonyl)-
1
a white solid. H NMR (CD3OD, 400 MHz): δ 8.18 (d, 1H, J = 8.4
Hz), 8.04 (s, 1H), 7.24 (dd, 1H, J = 8.4, 2.1 Hz), 7.14 (d, 1H, J = 2.1
Hz), 5.84 (m, 1H), 3.84 (m, 4H), 3.76 (m, 2H), 3.50 (m, 2H), 3.30−
3.10 (m, 4H), 2.92 (m, 5H), 2.30 (m, 4H), 2.20−2.00 (m, 4H), 1.90−
1.74 (m, 4H). Mass spectrum (ESI, m/z): calcd for C28H36N6O2,
489.2; found, 489.2.
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4-Cyano-1H-imidazole-2-carboxylic Acid [4-(1-Acetyl-piper-
idin-4-yl)-2-cyclohex-1-enyl-phenyl]-amide (21). 4-Cyano-1H-
imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phe-
nyl)-amide trifluoroacetic acid salt 8 (22.3 mg, 0.0456 mmol) in
CH2Cl2 (1 mL) was treated with Et3N (22.2 μL, 0.159 mmol) and
acetic anhydride (6.03 μL, 0.0638 mmol) at room temperature for
30 min using a procedure similar to that used in the preparation of 23.
Purification of the mixture on a 10 g silica gel SPE cartridge with 10%
MeOH/CH2Cl2 afforded the title compound (20 mg, 95%) as a white
solid. 1H NMR (CDCl3, 400 MHz): δ 13.12 (br s, 1H), 9.58 (s, 1H),
8.34 (d, 1H, J = 8.4 Hz), 7.76 (s, 1H), 7.21 (dd, 1H, J = 8.6, 2.1 Hz),
7.05 (d, 1H, J = 2.1 Hz), 5.86 (s, 1H), 4.84 (m, 1H), 4.00 (m, 1H),
3.22 (m, 1H), 2.72 (m, 2H), 2.30 (m, 4H), 2.21 (s, 3H), 1.80 (m, 8H).
Mass spectrum (ESI, m/z): calcd for C24H27N5O2, 418.2 (M + H);
found, 418.1.
(as prepared above, 18 mg, 0.090 mmol), EDCI (19 mg, 0.090 mmol),
HOBt (13 mg, 0.090 mmol), and DIEA (42 μL, 0.24 mmol). The
resulting mixture was stirred at room temperature for 6 h. Water
(10 mL) was added, and the organic layer was separated, dried
(Na2SO4), and concentrated. The residue obtained was chromato-
graphed on silica (2% MeOH/CH2Cl2) to obtain the title compound
1
(47 mg, 97%). H NMR (CDCl3, 400 MHz): δ 9.57 (d, 1H, J = 7.9
Hz), 8.31 (dd, 1H, J = 8.4, 6.0 Hz), 7.76 (s, 1H), 7.18 (dd, 1H, J = 8.6,
1.9 Hz), 7.05 (s, 1H), 5.84−5.88 (m, 1H), 4.76−4.82 (m, 2H), 4.45
(dt, 1H, J = 8.4, 5.7 Hz), 4.30 (d, 1H, J = 13.3 Hz), 4.06−4.21 (m,
2H), 3.08−3.29 (m, 1H), 2.67−2.85 (m, 2H), 2.31 (br s, 2H), 2.25 (br
s, 2H), 1.90−2.02 (m, 2H), 1.73−1.90 (m, 6H), 1.43 (d, 6H, J = 4.4
Hz). Mass spectrum (ESI, m/z): calcd for C28H33N5O4, 504.2 (M +
H); found, 503.9.
b. 5-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-enyl-
4-[1-(R)-(+)-(2,3-dihydroxy-propionyl)-piperidin-4-yl]-phenyl}-
amide(25). To a solution of 5-cyano-1H-imidazole-2-carboxylic acid
{2-cyclohex-1-enyl-4-[1-(R)-(+)-(2,2-dimethyl-[1,3]dioxolane-4-car-
bonyl)-piperidin-4-yl]-phenyl}-amide 24 (as prepared in the previous
step, 45 mg, 0.090 mmol) in MeOH (1 mL) was added aqueous 2 N
HCl (2 mL). The resulting mixture was stirred at room temperature
for 12 h. Solvents were removed in vacuo, and the resulting residue
was placed under vacuum for 4 h. Ether (10 mL) was added, and the
mixture was sonicated for 5 min. The ether was removed in vacuo, and
the residue was placed under vacuum for 12 h to obtain the title
compound (21.3 mg, 52%). 1H NMR (DMSO, 400 MHz): δ 14.1 (br
s, 1H), 9.85 (s, 1H), 8.32 (s, 1H), 7.92 (d, 1H, J = 8.4 Hz), 7.18 (dd,
1H, J = 8.4, 2.1 Hz), 7.13 (d, 1H, J = 2.1 Hz), 5.72 (br s, 1H), 4.51 (m,
1H), 4.33 (m, 1H), 4.15 (m, 1H), 3.55 (m, 1H), 3.43 (m, 1H), 3.08
(m, 1H), 2.81 (m, 1H), 2.63 (m, 1H), 2.12−2.24 (m, 4H), 1.31−1.38
(m, 10 H). Mass spectrum (ESI, m/z): calcd for C25H29N5O4, 464.2
(M + H); found, 464.1.
4-Cyano-1H-imidazole-2-carboxylic Acid (2-Cyclohex-1-
enyl-4-{1-[2-(2-hydroxy-ethylamino)-acetyl]-piperidin-4-yl}-
phenyl)-amide Trifluoroacetic Acid Salt (28). a. [2-(4-{4-[(4-
Cyano-1H-imidazole-2-carbonyl)-amino]-3-cyclohex-1-enyl-phe-
nyl}-piperidin-1-yl)-2-oxo-ethyl]-carbamic Acid tert-Butyl Ester
(26). A solution of N-Boc-glycine (286 mg, 1.63 mmol) in CH2Cl2
(10 mL) was treated with DIEA (854 μL, 4.90 mmol), HOBt (265 mg,
1.96 mmol), and EDCI (376 mg, 1.96 mmol). The mixture was stirred
at room temperature for 10 min and added to a suspension of 4-cyano-
1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperidin-4-yl-
phenyl)-amide trifluoroacetic acid salt 8 (800 mg, 1.63 mmol) in
CH2Cl2 (20 mL). The solution was stirred at room temperature for 17
h. Solvents were evaporated in vacuo. Silica gel chromatography (50%
EtOAc in hexanes) afforded the title compound (0.41 g, 47%) as a
white solid. 1H NMR (CDCl3, 400 MHz): δ 9.53 (s, 1H), 8.26 (d, 1H,
J = 8.4 Hz), 7.80−7.78 (m, 1H), 7.71 (s, 1H), 7.45−7.43 (m, 1H),
7.06 (d, 1H, J = 8.4 Hz), 7.00 (s, 1H), 5.83 (br s, 1H), 5.76 (br s, 1H),
4.78−4.68 (m, 1H), 3.96−3.85 (m, 2H), 3.17−3.03 (m, 1H), 2.78−
2.63 (m, 2H), 2.29 (br s, 2H), 2.22 (br s, 2H), 1.95−1.87 (m, 2H),
1.86−1.72 (m, 4H), 1.70−1.55 (m, 2H), 1.44 (s, 9H). Mass spectrum
(ESI, m/z): calcd for C29H36N6O4 533.3 (M + H); found, 532.9.
b. 4-Cyano-1H-imidazole-2-carboxylic Acid (2-Cyclohex-1-enyl-
4-{1-[2-(2-hydroxy-ethylamino)-acetyl]-piperidin-4-yl}-phenyl)-
amide Trifluoroacetic Acid Salt (28). A solution of [2-(4-{4-
[(4-cyano-1H-imidazole-2-carbonyl)-amino]-3-cyclohex-1-enyl-phenyl}-
piperidin-1-yl)-2-oxo-ethyl]-carbamic acid tert-butyl ester 26 (as
prepared in the previous step, 0.410 g, 0.770 mmol) in CH2Cl2
(20 mL) was treated with EtOH (0.2 mL) and TFA (6 mL). The
mixture stirred at room temperature for 45 min, and the solvents were
evaporated in vacuo to afford 4-cyano-1H-imidazole-2-carboxylic acid
{4-[1-(2-amino-acetyl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl]-
amide trifluoroacetic acid salt 27, which was used directly in the next
step. Mass spectrum (ESI, m/z): calcd for C24H28N6O2, 433.2 (M +
H); found, 433.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(pyridine-3-carbonyl)-piperidin-4-yl]-phenyl}-amide
(22). A solution of 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclo-
hex-1-enyl-4-piperidin-4-yl-phenyl)-amide trifluoroacetic acid salt 8
(75.0 mg, 0.152 mmol) in CH2Cl2 (10 mL) was treated with Et3N
(64.1 μL, 0.460 mmol) and cooled to 0 °C. The mixture was treated
with nicotinoyl chloride hydrochloride (30.0 mg, 0.169 mmol) and
stirred at 0 °C for 15 min and then at room temperature for 17 h. The
reaction mixture was adsorbed directly onto silica gel. Silica gel
chromatography (10% MeOH in EtOAc) afforded the title compound
(61.0 mg, 83%) as a white solid. 1H NMR (CDCl3, 400 MHz): δ 9.51
(br s, 1H), 8.77 (s, 1H), 8.70−8.66 (m, 1H), 8.32 (d, 1H, J = 8.4 Hz),
7.86−7.81 (m, 1H), 7.70 (s, 1H), 7.42−7.37 (m, 1H), 7.17 (d, 1H, J =
8.4 Hz), 7.06−7.04 (m, 1H), 5.87−5.82 (m, 1H), 4.98−4.87 (m, 1H),
3.94−3.84 (m, 1H), 3.29−3.18 (m, 1H), 2.98−2.86 (m, 1H), 2.86−
2.76 (m, 1H), 2.34−2.20 (m, 4H), 1.94−1.72 (m, 9H). Mass spectrum
(ESI, m/z): calcd for C28H28N6O2, 481.2 (M + H); found, 481.3.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-dimethylamino-acetyl)-piperidin-4-yl]-phenyl}-
amide (23). A suspension of 4-cyano-1H-imidazole-2-carboxylic acid
(2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide trifluoroacetic acid
salt 8 (655 mg, 1.34 mmol) in CH2Cl2 (15 mL) was cooled to 0 °C,
and DIEA (0.932 mL, 5.35 mmol) was added. Dimethylaminoacetyl
chloride hydrochloride (211 mg, 1.34 mmol) was then added portion
wise over 10 min. The reaction mixture was stirred at 0 °C for 30 min
and allowed to warm to room temperature and stir for 2 h. The solvent
was removed in vacuo, and the resulting residue was partitioned
between brine and CH2Cl2. The organic layer was separated, dried
(Na2SO4), and concentrated. The residue obtained was purified on
silica (5% MeOH:CH2Cl2) to obtain 432 mg (70%) of the title
1
compound as a white solid. H NMR (CDCl3, 400 MHz): δ 9.49 (s,
1H), 8.24 (d, 1H, J = 8.3 Hz), 7.70 (s, 1H), 7.12 (dd, 1H, J = 8.4, 2.1
Hz), 7.01 (d, 1H, J = 2.1 Hz), 5.82 (m, 1H), 4.75 (d, 1H, J = 13.4 Hz),
4.13 (d, 1H, J = 13.4 Hz), 3.57 (d, 1H, J = 14.2 Hz), 3.18 (d, 1H, J =
14.2 Hz), 3.12 (td, 1H, J = 13.3, 2.4 Hz), 2.73 (dddd, 1H, J = 11.9,
11.9, 3.8, 3.8 Hz), 2.65 (ddd, 1H, J = 13.3, 13.3, 2.4 Hz), 2.40 (s, 6H),
2.18−2.32 (m, 4H), 1.60−1.98 (m, 9H). Mass spectrum (ESI, m/z):
calcd for C26H32N6O2, 461.3 (M + H); found, 461.2.
5-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(R)-(+)-(2,3-dihydroxy-propionyl)-piperidin-4-yl]-
phenyl}-amide (25). a. 5-Cyano-1H-imidazole-2-carboxylic Acid
{2-Cyclohex-1-enyl-4-[1-(R)-(+)-(2,2-dimethyl-[1,3]dioxolane-4-car-
bonyl)-piperidin-4-yl]-phenyl}-amide (24). To a solution of methyl
(R)-(+)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (0.16 mL, 1.0 mmol)
in MeOH (2 mL), 2 N KOH (0.50 mL, 1.0 mmol) was added. The
resulting solution was stirred at room temperature for 20 min and
concentrated in vacuo. The residue obtained was suspended in ether
(10 mL) and sonicated for 5 min. The ether was then removed, and the
resulting residue was placed under high vacuum for 4 h to obtain
(R)-(+)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid potassium salt
(173 mg, 94%), which was directly used in the next step without
purification.
A suspension of 4-cyano-1H-imidazole-2-carboxylic acid {4-[1-(2-
amino-acetyl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl]-amide tri-
fluoroacetic acid salt 27 (0.420 g, 0.769 mmol) in CH2Cl2 (20 mL)
was treated with Na(OAc)3BH (0.326 g, 1.54 mmol) and solid glyoxal
(44.6 mg, 0.769 mmol). The mixture was stirred at room temperature
To a solution of 4-cyano-1H-imidazole-2-carboxylic acid (2-
cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide trifluoroacetic acid
salt 8 (40 mg, 0.082 mmol) in CH2Cl2 (1.5 mL) was added a mixture
of (R)-(+)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid potassium salt
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for 1 h, and the solvent was evaporated in vacuo. The residue was
taken up in MeOH, the solids were filtered off, and the filtrate was
concentrated in vacuo. Reverse phase HPLC (C-18 column) (20−60%
acetonitrile in water with 0.1% TFA over 30 min) afforded the title
1.75−1.89 (m, 6H). Mass spectrum (ESI, m/z): calcd for C28H28N6O3,
497.2 (M + H); found, 497.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-1H-imidazol-4-yl-acetyl)-piperidin-4-yl]-phenyl}-
amide Trifluoroacetic Acid Salt (33). The title compound was
prepared from 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-
enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8 in 60% yield according
to the procedure for the preparation of 35 using (1H-imidazol-4-yl)-
1
compound (83 mg, 19% over two steps) as a white solid. H NMR
(CD3OD, 400 MHz): δ 8.06 - 8.16 (m, 1H), 7.99−8.05 (m, 1H), 7.18
(m, 1H), 7.07 (m, 1H), 5.81 (m, 1H), 4.64 (m, 1H), 3.74−4.24 (m,
4H), 3.18−3.29 (m, 2H), 2.77−2.93 (m, 2H), 2.27 (m, 4H), 1.52−
2.00 (m, 10H). Mass spectrum (ESI, m/z): calcd for C26H32N6O3,
477.2 (M + H); found, 477.2.
1
acetic acid. H NMR (CD3OD, 400 MHz): δ 8.88 (s, 1H), 8.12 (d,
1H, J = 1.6 Hz), 8.02 (s, 1H), 7.44 (s, 1H), 7.20 (dd, 1H, J = 8.4, 2.1
Hz), 7.10 (d, 1H, J = 2.1 Hz), 5.82 (m, 1H), 4.70 (m, 1H), 4.18 (m,
1H), 4.06 (m, 2H), 3.36 (m, 1H), 2.84 (m, 2H), 2.30 (m, 4H), 2.00−
1.70 (m, 7H), 1.64 (m, 1H). Mass spectrum (ESI, m/z): calcd for
C27H29N7O2, 484.2 (M + H); found, 484.2.
4-Cyano-1H-imidazole-2-carboxylic Acid (2-Cyclohex-1-
enyl-4-{1-[2-(2-hydroxy-ethyl)-methyl-amino-acetyl]-piperi-
din-4-yl}-phenyl)-amide Trifluoroacetic Acid Salt (29). A
solution of 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-
enyl-4-{1-[2-(2-hydroxy-ethylamino)-acetyl]-piperidin-4-yl}-phenyl)-
amide trifluoroacetic acid salt 28 (50 mg, 0.085 mmol) in MeOH
(3 mL) was treated with Na(OAc)3BH (40 mg, 0.19 mmol) and 37%
aqueous formaldehyde (8.2 μL, 0.10 mmol). The mixture was stirred
at room temperature for 5.5 h, and the solvents were removed in
vacuo. Reverse phase HPLC (C-18 column) (10−50% acetonitrile in
water with 0.1% TFA over 30 min) afforded the title compound (19.5
4-Cyano-1H-imidazole-2-carboxylic Acid (2-Cyclohex-1-
enyl-4-{1-[2-(1-methyl-1H-imidazol-4-yl)-acetyl]-piperidin-4-
yl}-phenyl)-amide Trifluoroacetic Acid Salt (34). The title
compound was prepared from 4-cyano-1H-imidazole-2-carboxylic
acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8 in
66% yield according to the procedure for the preparation of 35 using
(1-methyl-1H-imidazol-4-yl)-acetic acid. 1H NMR (CD3OD, 400
MHz): δ 8.82 (s, 1H), 8.10 (d, 1H, J = 8.4 Hz), 8.00 (s, 1H), 7.42 (s,
1H), 7.16 (dd, 1H, J = 8.6, 2.1 Hz), 7.06 (d, 1H, J = 2.1 Hz), 5.80 (m,
1H), 4.66 (m, 1H), 4.12 (m, 1H), 4.04 (m, 2H), 3.92 (s, 3H), 3.28 (m,
1H), 2.82 (m, 2H), 2.26 (m, 4H), 2.00−1.70 (m, 7H), 1.64 (m, 1H).
Mass spectrum (ESI, m/z): calcd for C28H31N7O2, 498.2 (M + H);
found, 498.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-pyridin-2-yl-acetyl)-piperidin-4-yl]-phenyl}-amide
Trifluoroacetic Acid Salt (35). 4-Cyano-1H-imidazole-2-carboxylic
acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt
(25.0 mg, 0.0511 mmol) 8, pyridin-2-yl-acetic acid hydrochloride (9.75
mg, 0.0562 mmol), EDCI (11.7 mg, 0.0613 mmol), HOBt
(8.28 mg, 0.0613 mmol), DIEA (26.7 μL, 0.153 mmol), and 0.2 mL of
DMF were stirred at 25 °C for 10 h. The reaction was diluted with 2 mL
of H2O, and the residue was purified by RP-HPLC (C18), eluting with 30−
50% CH3CN in 0.1% TFA/H2O over 9 min to give 22 mg (70%) of the
title compound as a white solid. 1H NMR (CD3OD, 400 MHz): δ 8.82 (d,
1H, J = 5.6 Hz), 8.52 (td, 1H, J = 7.8, 1.7 Hz), 8.14 (d, 1H, J = 8.4 Hz),
8.04 (s, 1H), 7.96 (m, 2H), 7.20 (dd, 1H, J = 8.4, 2.1 Hz), 7.10 (d, 1H, J =
2.1), 5.82 (m, 1H), 4.68 (m, 1H), 4.32 (m, 2H), 4.18 (m, 1H), 3.40 (m,
1H), 2.88 (m, 2H), 2.30 (m, 4H), 2.06−1.60 (m, 8H). Mass spectrum
(ESI, m/z): calcd for C29H30N6O2, 495.2 (M + H); found, 495.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-pyridin-3-yl-acetyl)-piperidin-4-yl]-phenyl}-amide
Trifluoroacetic Acid Salt (36). The title compound was prepared
from 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-
piperidin-4-yl-phenyl)-amide TFA salt 8 in 68% yield according to
the procedure for the preparation of 35 using pyridin-3-yl-acetic acid.
1H NMR (CD3OD, 400 MHz): δ 8.80 (s, 1H), 8.77 (d, 1H, J = 5.7
Hz), 8.54 (d, 1H, J = 8.0 Hz), 8.10 (d, 1H, J = 8.4 Hz), 8.06 (dd, 1H,
J = 7.8, 6.1 Hz), 7.98 (s, 1H), 7.18 (dd, 1H, J = 8.4, 2.0 Hz), 7.08 (d,
1H, J = 2.0 Hz), 5.78 (m, 1H), 4.68 (m, 1H), 4.20 (m, 1H), 4.18 (s,
2H), 3.36 (m, 1H), 2.84 (m, 2H), 2.28 (m, 4H), 2.06−1.70 (m, 7H),
1.62 (m, 1H). Mass spectrum (ESI, m/z): calcd for C29H30N6O2, 495.2
(M + H); found, 495.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-pyridin-4-yl-acetyl)-piperidin-4-yl]-phenyl}-amide
Trifluoroacetic Acid Salt (37). The title compound was prepared
from 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-
piperidin-4-yl-phenyl)-amide TFA salt 8 in 58% yield according to
the procedure for the preparation of 35 using pyridin-4-yl-acetic acid.
1H NMR (CD3OD, 400 MHz): δ 8.78 (d, 2H, J = 5.1 Hz), 8.12 (d,
1H, J = 8.4 Hz), 8.03 (s, 1H), 7.99 (d, 2H, J = 6.1 Hz), 7.18 (dd, 1H,
J = 8.4, 1.9 Hz), 7.08 (d, 1H, J = 1.9 Hz), 5.80 (m, 1H), 4.66 (m, 1H),
4.22 (s, 2H), 4.18 (m, 1H), 3.34 (m, 1H), 2.84 (m, 2H), 2.24 (m, 4H),
2.00−1.70 (m, 7H), 1.64 (m, 1H). Mass spectrum (ESI, m/z): calcd
for C29H30N6O2, 495.2 (M + H); found, 495.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(3-morpholin-4-yl-propionyl)-piperidin-4-yl]-phe-
nyl}-amide (38). 3-Morpholin-4-yl-propionic acid potassium salt
1
mg, 47%) as a white solid. H NMR (CD3OD, 400 MHz): δ 8.12 (d,
1H, J = 8.4 Hz), 8.02 (s, 1H), 7.19 (dd, 1H, J = 8.4, 2.0 Hz), 7.09 (d,
1H, J = 2.0 Hz), 5.84−5.79 (m, 1H), 4.72−4.64 (m, 1H), 4.39−4.23
(m, 2H), 3.84−3.79 (m, 1H), 3.31−3.21 (m, 1H), 3.03−2.94 (m, 6H),
2.92−2.80 (m, 2H), 2.32−2.24 (m, 4H), 2.00−1.90 (m, 2H), 1.90−
1.76 (m, 5H), 1.78−1.59 (m, 2H). Mass spectrum (ESI, m/z): calcd
for C27H34N6O3, 491.3 (M + H); found, 491.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-methanesulfonyl-acetyl)-piperidin-4-yl]-phenyl}-
amide (30). Methanesulfonyl-acetic acid (14 mg, 0.10 mmol), EDCI
(30 mg, 0.15 mmol), HOBt (14 mg, 0.10 mmol), DIEA (36 μL,
0.20 mmol), and 0.5 mL of CH2Cl2 were stirred at 25 °C. After 10 min, a
solution containing 4-cyano-1H-imidazole-2-carboxylic acid (2-cyclo-
hex-1-enyl-4-piperidin-4-yl-phenyl)-amide TFA salt 8 (40 mg,
0.082 mmol) and Et3N (14 μL, 0.10 mmol) in 0.5 mL of CH2Cl2 was
added, and the reaction was allowed to proceed for 10 h at 25 °C. The
reaction mixture was loaded on a 5 g SPE cartridge (silica) and eluted
with 10% EtOH/EtOAc to give 10 mg (25%) of the title compound as
1
a white solid. H NMR (CDCl3, 400 MHz): δ 11.60 (br s, 1H), 9.52
(s, 1H), 8.30 (dd, 1H, J = 8.4, 1.6 Hz), 7.74 (d, 1H, J = 2.0 Hz), 7.15
(dd, 1H, J = 8.6, 1.9 Hz), 7.03 (d, 1H, J = 2.0 Hz), 5.86 (m, 1H), 4.84
(m, 1H), 4.18 (s, 2H), 4.12 (m, 1H), 3.32 (m, 1H), 3.20 (s, 3H), 2.82
(m, 2H), 2.30 (m, 4H), 1.98 (m, 2H), 1.84 (m, 5H), 1.72 (m, 1H).
Mass spectrum (ESI, m/z): calcd for C25H29N5O4S, 496.2 (M + H);
found, 496.2.
4-Cyano-1H-imidazole-2-carboxylic Acid [2-Cyclohex-1-
enyl-4-[1-(1-oxy-pyridine-3-carbonyl)-piperidin-4-yl]-phenyl}-
amide (31). The title compound was prepared in 32% yield from
4-cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperi-
din-4-yl-phenyl)-amide trifluoroacetic acid salt 8 and nicotinic acid-
1
N oxide according to the procedure for the preparation of 35. H
NMR (CDCl3, 400 MHz): δ 13.95 (br. s., 1H), 9.44 (s, 1H), 8.52 (s,
1H), 8.32 (d, J = 6.1 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.74 (s, 1H),
7.51−7.56 (m, 1H), 7.41−7.50 (m, 1H), 6.95 (s, 1H), 6.86−6.93 (m,
1H), 5.79 (br. s., 1H), 4.80 (br. s., 1H), 3.88 (d, J = 12.6 Hz, 1H), 3.17
(br. s., 1H), 2.86 (br. s., 1H), 2.66 (t, J = 11.6 Hz, 1H), 2.12−2.33 (m,
4H), 2.00 (br. s., 1H), 1.47−1.88 (m, 7H). Mass spectrum (ESI, m/z):
calcd for C28H28N6O3, 497.2 (M + H); found, 497.2.
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(1-oxy-pyridine-4-carbonyl)-piperidin-4-yl]-phenyl}-
amide (32). The title compound was prepared in 42% yield from 4-
cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperidin-
4-yl-phenyl)-amide trifluoroacetic acid salt 8 and 1-oxy-isonicotinic
acid in 42% yield according to the procedure for the preparation of 35.
1H NMR (CDCl3): δ 13.64 (br s, 1H), 11.17 (br s, 1H), 9.50 (s, 1H),
8.53 (d, 2H, J = 7.0 Hz), 8.30 (d, 1H, J = 8.6 Hz), 7.73 (s, 1H), 7.46
(d, 2H, J = 7.0 Hz), 7.28 (m, 1H), 7.09−7.18 (m, 1H), 7.03 (d, 1H, J =
1.9 Hz), 5.80−5.88 (m, 1H), 3.62−3.76 (m, 2H), 3.11 (qd, 2H, J =
7.3, 4.9 Hz), 2.81 (t, 1H, J = 12.8 Hz), 2.29 (br s, 2H), 2.23 (br s, 2H),
7878
dx.doi.org/10.1021/jm200900q|J. Med. Chem. 2011, 54, 7860−7883

Journal of Medicinal Chemistry
Article
(93.8 mg, 0.475 mmol, prepared from 3-morpholin-4-yl-propionic acid
ethyl ester and 6 N KOH as described in the preparation of 39), 4-
cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-piperidin-4-
yl-phenyl)-amide trifluoroacetic acid salt 8 (179 mg, 0.366 mmol), EDCI
(83.7 mg, 0.439 mmol), and HOBT (69.2 mg, 0.512 mmol) were taken
up in DMF (4 mL). To the stirred slurry was added DIEA
(157 μL, 0.901 mmol), and the reaction was allowed to stir overnight.
The reaction was diluted with H2O (10 mL) and extracted with EtOAc
(2 × 25 mL). The combined organic extracts were dried (Na2SO4)
and concentrated in vacuo, and the crude product was purified by silica
gel preparative TLC (6% of 7 M NH3 in MeOH/CH2Cl2) to afford
10.4 mg (6%) of the title compound as a white solid. 1H NMR
(CDCl3, 400 MHz): δ 9.51 (s, 1H), 8.26 (d, 1H, J = 8.2 Hz), 7.70 (s,
1H), 7.09−7.13 (m, 1H), 6.99−7.00 (m, 1H), 6.81−6.82 (m, 1H),
5.81−5.82 (m, 1H), 4.76−4.81 (m, 2H), 3.97−4.02 (m, 2H), 3.72 (br
s, 4H), 3.10−3.17 (m, 2H), 2.74−2.76 (m, 4H), 2,58−2.63 (m, 3H),
2.52 (br s, 4H), 2.22−2.31 (m, 3H), 1.49−1.93 (m, 5H). Mass
spectrum (ESI, m/z): calcd for C29H36N6O3, 517.3 (M + H); found,
517.3.
of CH2Cl2 and 0.10 mL of EtOH at 0 °C was added 1.0 mL of TFA,
and the mixture was warmed to room temperature and stirred for 3 h.
The reaction was diluted with 3 mL of n-PrOH and then concentrated
in vacuo. The residue was purified by flash chromatography (silica gel,
3−8% MeOH/CH2Cl2) to give 33.5 mg (99%) of the title compound
as a white solid. 1H NMR (CDCl3, 400 MHz): δ 13.3 (s, 1H), 9.52 (s,
1H), 8.57 (br s, 2H), 8.26 (d, 1H, J = 8.6 Hz), 7.69 (s, 1H), 7.02 (dd,
1H, J = 8.6, 1.7 Hz), 6.98 (d, 1H, J = 1.7 Hz), 5.78 (m, 1H), 4.67 (br
d, 1H, J = 13.4 Hz), 3.88 (br d, 1H, J = 13.4 Hz), 3.10 (m, 1H), 2.55−
2.85 (m, 4H), 2.23 (m, 4H), 1.72−2.01 (m, 8H), 1.50 (s, 6H). Mass
spectrum (ESI, m/z): calcd for C27H34N6O2, 475.3 (M + H); found,
475.1.
5-Cyano-1H-imidazole-2-carboxylic Acid {4-[1-(2-Amino-2-
methyl-propionyl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl}-
amide Trifluoroacetic Acid Salt (43). a. {2-[4-(4-{[4-Cyano-1-(2-
trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carbonyl]-amino}-
3-cyclohex-1-enyl-phenyl)-piperidin-1-yl]-1,1-dimethyl-2-oxo-
ethyl}-carbamic Acid tert-Butyl Ester (42). To a solution of 4-(4-{[4-
cyano-1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carbonyl]-
amino}-3-cyclohex-1-enyl-phenyl)-piperidine-1-carboxylic acid tert-
butyl ester 7 (231 mg, 0.380 mmol) in 2.5 mL of CH2Cl2 and
0.4 mL of EtOH was added 700 μL of TFA, and the solution stirred
for 3 h at 25 °C. The reaction was diluted with 4 mL of EtOH and
then concentrated to give ca. a 2:1 mixture of 5-cyano-1-(2-
trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carboxylic acid (2-cy-
clohex-1-enyl-4-piperidin-4-yl-phenyl)-amide trifluoroacetic acid salt
4-Cyano-1H-imidazole-2-carboxylic Acid {2-Cyclohex-1-
enyl-4-[1-(2-morpholin-4-yl-acetyl)-piperidin-4-yl]-phenyl}-
amide (39). To a solution of morpholin-4-yl-acetic acid ethyl ester
(117 mg, 0.675 mmol) in ethanol (4 mL) was added 6 N KOH
(110 μL, 0.660 mmol) via syringe, and stirring was continued for 3 h.
Concentration in vacuo afforded 122 mg (100%) of morpholin-4-yl-
acetic acid potassium salt. To a mixture of morpholin-4-yl-acetic acid
potassium salt (29.2 mg, 0.160 mmol), 4-cyano-1H-imidazole-2-
carboxylic acid 8 (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide
trifluoroacetic acid salt (65.1 mg, 0.133 mmol), and bromotri-
(pyrrolidino)phosphonium hexafluorophosphate (PyBroP) (93.0 mg,
0.199 mmol) in CH2Cl2 (4 mL) was added DIEA (51.0 μL, 0.293
mmol), and the reaction was allowed to stir overnight. The reaction
was diluted with CH2Cl2 (50 mL), washed with H2O (2 × 25 mL),
dried (Na2SO4), and concentrated in vacuo. Purification of the crude
product by silica gel preparative TLC (6% of 7 M NH3 in MeOH/
CH2Cl2) afforded 8.1 mg (12%) of the title compound as a white
1
19 and starting material by H NMR and LC/MS, which was used in
the following step without further purification. The mixture in 3 mL of
CH2Cl2 was added to a solution of 2-tert-butoxycarbonylamino-2-
methyl-propionic acid (53.0 mg, 0.261 mmol), DIEA (122 μL,
0.700 mmol), and PyBroP (144 mg, 0.309 mmol) in 3 mL of CH2Cl2,
and the reaction was stirred at 25 °C overnight. The reaction was diluted
with EtOAc (25 mL) and washed with saturated aqueous NaHCO3
(1 × 25 mL) and brine (25 mL), and the organic layer was dried over
Na2SO4 and then concentrated. Purification of the residue by
preparative TLC (50% EtOAc−hexanes) afforded 40.0 mg (15%) of
1
1
the title compound as a white solid. H NMR (CDCl3, 400 MHz): δ
solid. H NMR (CDCl3, 400 MHz): δ 9.48 (s, 1H), 8.27 (d, 1H, J =
9.67 (s, 1H), 8.24 (d, 1H, J = 8.4 Hz), 7.78 (s, 1H), 7.68−7.76 (m,
1H), 7.48−7.59 (m, 1H), 7.10 (dd, 1H, J = 8.5, 1.9 Hz), 7.00 (d, 1H,
J = 2.1 Hz), 5.96 (s, 2H), 5.78−5.85 (m, 1H), 4.24−4.38 (m, 1H),
3.62−3.76 (m, 3H), 2.66−2.77 (m, 1H), 2.28 (br s, 2H), 2.22 (br s,
2H), 1.42−1.57 (s, 18H), 0.94−1.01 (m, 2H), 0.81−0.92 (m, 6H),
0.01 (s, 9H). Mass Spectrum (ESI, m/z): calcd for C37H54N6O5Si,
691.3 (M + H); found, 691.1.
8.1 Hz), 7.73 (s, 1H), 7.12 (dd, 1H, J = 0.6, 8.0 Hz), 7.00 (s, 1H), 5.82
(s, 1H), 4.73−4.77 (m, 1H), 4.16−4.22 (m, 1H), 3.72 (br s, 4H),
3.35−3.40 (m, 1H), 3.09−3.23 (m, 6H), 2.53−2.78 (m, 5H), 2.20−
2.29 (m, 4H), 1.68−2.04 (m, 5H). Mass spectrum (ESI, m/z): calcd
for C28H34N6O3, 503.3 (M + H); found, 503.1.
4-Cyano-1H-imidazole-2-carboxylic Acid {4-[1-(3-Amino-3-
methyl-butyryl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl}-
amide Trifluoroacetic Acid Salt (41). a. [3-(4-{4-[(4-Cyano-1H-
imidazole-2-carbonyl)-amino]-3-cyclohex-1-enyl-phenyl}-piperidin-
1-yl)-1,1-dimethyl-3-oxo-propyl]-carbamic Acid tert-Butyl Ester
(40). To a mixture of 4-cyano-1H-imidazole-2-carboxylic acid (2-
cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide trifluoroacetic acid salt
8 (40.0 mg, 0.0818 mmol), 3-tert-butoxycarbonylamino-3-methyl-
butyric acid (21.4 mg, 0.0981 mmol), and PyBroP (55.0 mg,
0.0981 mmol) in 1,2-dichloroethane (2 mL) was added DIEA (42.7 μL,
0.245 mmol), and the resulting mixture was stirred at room
temperature for 24 h under Ar. The mixture was diluted with
EtOAc (30 mL), washed with H2O (2 × 10 mL) and brine (10 mL),
dried over Na2SO4, and then concentrated in vacuo. The residue was
purified by flash chromatography (silica gel, 10−40% EtOAc/hexane)
to give 33.0 mg (70%) of the title compound as a colorless oil. 1H NMR
(CDCl3, 400 MHz): δ 12.93 (br s, 1H), 9.55 (s, 1H), 8.28 (d, 1H, J =
8.4 Hz), 7.74 (d, 1H, J = 2.5 Hz), 7.15 (dd, 1H, J = 8.5, 2.1 Hz), 7.02
(d, 1H, J = 2.1 Hz), 5.84 (dt, 1H, J = 3.5, 2.0 Hz), 5.60 (br s, 1H), 4.85
(d, 1H, J = 13.5 Hz), 4.17 (d, 1H, J = 13.9 Hz), 3.16 (t, 1H, J = 12.1
Hz), 2.56−2.84 (m, 4H), 2.29 (d, 2H, J = 3.4 Hz), 2.23 (br s, 2H), 1.67−
1.94 (m, 8H), 1.45 (d, 6H, J = 1.7 Hz), 1.41 (s, 9H). Mass spectrum (ESI,
m/z): calcd for C32H42N6O4, 575.3 (M + H); found, 574.8.
b. 5-Cyano-1H-imidazole-2-carboxylic Acid {4-[1-(2-Amino-2-
methyl-propionyl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl}-amide
Trifluoroacetic Acid Salt (43). To a solution of {2-[4-(4-{[4-cyano-1-
(2-trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-carbonyl]-amino}-
3-cyclohex-1-enyl-phenyl)-piperidin-1-yl]-1,1-dimethyl-2-oxo-ethyl}-
carbamic acid tert-butyl ester 42 (40.0 mg, 0.0579 mmol, as prepared
in the previous step) in 2 mL of CH2Cl2 and 20 μL of EtOH was
added 1.5 mL of TFA. The solution was stirred for 3 h at 25 °C,
diluted with 2 mL of EtOH, and concentrated in vacuo. Trituration of
the residue with ether afforded 8.4 mg (29%) of the title compound as
1
a white solid. H NMR (CD3OD, 400 MHz): δ 8.10 (d, 1H, J = 8.4
Hz), 8.00 (s, 1H), 7.16 (d, 1H, J = 8.4 Hz), 7.07 (s, 1H), 5.79 (s, 1H),
4.55−4.48 (m, 1H), 3.30 (s, 6H), 2.89−2.87 (m, 2H), 2.40−2.25 (m,
4H), 1.96−1.93 (m, 2H), 1.86−1.83 (m, 6H), 1.64−1.61 (m, 2H).
Mass spectrum (ESI, m/z): calcd for C26H32N6O2, 461.3 (M + H);
found, 461.3.
4-{4-[(4-Cyano-1H-imidazole-2-carbonyl)-amino]-3-cyclo-
hex-1-enyl-phenyl}-piperidine-1-carboxylic Acid (2-Hydroxy-
ethyl)-amide (44). a. 4-(4-{[4-Cyano-1-(2-trimethylsilanyl-ethoxy-
methyl)-1H-imidazole-2-carbonyl]-amino}-3-cyclohex-1-enyl-phe-
nyl)-piperidine-1-carboxylic Acid (2-Hydroxy-ethyl)-amide. A sol-
ution of 4-cyano-1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazole-2-
carboxylic acid (2-cyclohex-1-enyl-4-piperidin-4-yl-phenyl)-amide tri-
fluoroacetic acid salt 19 (116 mg, 0.192 mmol) and DIEA (134 μL,
0.770 mmol) in 4 mL of CH2Cl2 was added slowly to solution of
triphosgene (22.8 mg, 0.0770 mmol) in 4 mL of CH2Cl2 at −78 °C
under Ar. The mixture was stirred at −78 °C for 15 min, warmed to
b. 4-Cyano-1H-imidazole-2-carboxylic Acid {4-[1-(3-Amino-3-
methyl-butyryl)-piperidin-4-yl]-2-cyclohex-1-enyl-phenyl}-amide
Trifluoroacetic Acid Salt (41). To a solution of [3-(4-{4-[(4-cyano-
1H-imidazole-2-carbonyl)-amino]-3-cyclohex-1-enyl-phenyl}-piperi-
din-1-yl)-1,1-dimethyl-3-oxo-propyl]-carbamic acid tert-butyl ester
(33.0 mg, 0.0574 mmol) (as prepared in the previous step) in 3 mL
7879
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Journal of Medicinal Chemistry
Article
room temperature, stirred for 15 min, and cooled to −78 °C again. A
suspension of 2-amino-ethanol (350 μL, 5.77 mmol) in 4 mL of THF
was added, and the resulting mixture was warmed to room
temperature and stirred for 20 h under Ar. Treated with 100 mL of
EtOAc, the mixture was washed with H2O (3 × 20 mL) and brine
(20 mL) and was dried (Na2SO4). Removal of the solvent in vacuo
followed by flash chromatography of the residue on silica gel (10%
EtOAc/CH2Cl2 then 5% MeOH/CH2Cl2) gave 95 mg (83%) of the
title compound as a colorless oil. 1H NMR (CDCl3, 400 MHz): δ 9.68
(s, 1H), 8.25 (d, 1H, J = 8.4 Hz), 7.77 (s, 1H), 7.12 (dd, 1H, J = 8.4,
2.2 Hz), 7.01 (d, 1H, J = 2.2 Hz), 5.94 (s, 2H), 5.83 (m, 1H), 4.96 (t,
1H, J = 5.6 Hz), 4.11 (d, 2H, J = 13.3 Hz), 3.75 (ddd, 2H, J = 4.4 Hz),
3.66 (t, 2H, J = 8.3 Hz), 3.44 (ddd, 2H, J = 5.0 Hz), 3.36 (t, 1H, J =
4.6 Hz), 2.91 (ddd, 2H, J = 13.0, 2.2 Hz), 2.66 (dddd, 1H, J = 12.2,
12.2, 3.3, 3.3 Hz), 2.18−2.33 (m, 4H), 1.75−1.91 (m, 6H), 1.67
(dddd, 2H, J = 12.9, 12.9, 12.9, 4.0 Hz), 0.97 (t, 2H, J = 8.3 Hz), 0.00
(s, 9H). Mass spectrum (ESI, m/z): calcd for C31H44N6O4Si, 593.3
(M + H); found, 593.1.
b. 4-{4-[(4-Cyano-1H-imidazole-2-carbonyl)-amino]-3-cyclohex-
1-enyl-phenyl}-piperidine-1-carboxylic Acid (2-Hydroxy-ethyl)-amide
(44). To a solution of 4-(4-{[4-cyano-1-(2-trimethylsilanyl-ethoxy-
methyl)-1H-imidazole-2-carbonyl]-amino}-3-cyclohex-1-enyl-phenyl)-
piperidine-1-carboxylic acid (2-hydroxy-ethyl)-amide (as prepared in
the previous step, 95 mg, 0.16 mmol) in 3 mL of CH2Cl2 was added
0.10 mL of EtOH followed by 1.0 mL of TFA. The resulting solution
was stirred at room temperature for 6 h. Removal of the solvent under
reduced pressure followed by flash chromatography of the residue on
silica gel (2−8% MeOH/CH2Cl2) gave 68 mg (92%) of the title
compound as a white solid. 1H NMR (CD3OD, 400 MHz): δ 8.09 (d,
1H, J = 8.4 Hz), 8.00 (s, 1H), 7.15 (dd, 1H, J = 8.4, 2.2 Hz), 7.06 (d,
1H, J = 2.2 Hz), 5.79 (m, 1H), 4.15 (dd, 2H, J = 13.3, 1.1 Hz), 3.61 (t,
2H, J = 5.9 Hz), 3.27−3.32 (m, 2H), 2.90 (ddd, 2H, J = 13.0, 13.0, 2.5
Hz), 2.73 (dddd, 1H, J = 12.1, 12.1, 2.6, 2.6 Hz), 2.26 (m, 4H), 1.73−
1.88 (m, 6H), 1.62 (dddd, 2H, J = 12.6, 12.6, 12.6, 4.0 Hz). Mass
spectrum (ESI, m/z): calcd for C25H30N6O3, 463.2 (M + H); found,
463.2.
then received 10 μL of a 1:1:3 mixture of 10× antiphosphotyrosine
antibody, 10× PTK green tracer, and fluorescence polarization dilution
buffer (catalog no. P2837, Invitrogen, Carlsbad, CA). The plates were
incubated for 30 min at room temperature, and the fluorescence
polarization was read (485 nm excitation, 530 nm emission) on an
Analyst plate reader (Molecular Devices). Fluorescence polarization
values for positive (EDTA) and negative (DMSO) controls were used
to define 100 and 0% inhibition of the FMS reaction.
Cellular Assays. Functional inhibition of CSF-1R was assessed
using murine bone marrow-derived macrophages as previously
described.24 Macrophages were plated in 96-well culture plates at a
density of 5000 cells/well in 100 μL of α-MEM containing 10% FCS.
After overnight culture, wells were supplemented with the addition of
50 μL of media containing 15 ng/mL CSF-1, 3 μM indomethacin, and
a dilution series of test compound. The cells were cultured for 30 h at
37 °C and 5% CO2. During the final 6 h, cultures were supplemented
with an additional 30 μL of media containing a 1:500 dilution of
bromodeoxyuridine (BrDU) reagent, and incorporation of BrDU into
cellular DNA was quantified using a specific ELISA (catalog no.
X1327K) from Exalpha Corporation (Watertown, MA). Inhibition of
cellular LCK was determined as described.65 Jurkat cells (105/well)
(ATCC) with graded concentrations of 23 were plated into wells
precoated with CD3ε antibody (R&D Systems). PMA was added to a
final concentration of 10 ng/mL, and IL-2 secreted into 24 h culture
supernatants was measured by ELISA (R&D Systems). As described
previously in detail, inhibition of cellular AXL was based on
measurement of GAS6-induced AXL phosphorylation in HEK cells
transfected to express AXL, and cellular ITD-FLT3, KIT, and TRKA
inhibition were based assays of ITD-FLT3, KIT, and TRKA-
dependent cell proliferation.24
MV-4-11 and M-o7e Leukemia Cell Line Proliferation Assays. The
functional impact on cellular FLT3 activity was determined by
measuring compound inhibition of MV-4-11 (ATCC no. CRL-9591)
cell proliferation, and M-07e (DSMZ no. ACC 104) cells were used to
assess compound effects on cellular KIT activity. MV-4-11 cells grew
independent of growth factor due to expression of a constitutive active
FLT3 mutation, and M-07e cells were driven to proliferate in a KIT-
dependent fashion by 25 ng/mL stem cell factor. Following a culture
period of 72 h, relative cell numbers were determined using
CellTiterGlo reagent (Promega).
4-{4-[(4-Cyano-1H-imidazole-2-carbonyl)-amino]-3-cyclo-
hex-1-enyl-phenyl}-piperidine-1-carboxylic Acid Amide
(45). 4-Cyano-1H-imidazole-2-carboxylic acid (2-cyclohex-1-enyl-4-
piperidin-4-yl-phenyl)-amide TFA salt (51.0 mg, 0.104 mmol) 8,
Et3N (21.7 μL, 0.156 mmol), trimethylsilyl isocyanate (15.5 μL, 0.114
mmol), and 1.0 mL of CH2Cl2 were stirred for 10 h at 25 °C. The
solvent was evaporated, and the residue was purified by RP-HPLC
(C18), eluting with 35−60% CH3CN in 0.1% TFA/H2O over 11 min
Biochemical PD Mouse Model. Groups of six B6C3F1 mice
(Taconic Farms) were given oral doses of vehicle [aqueous 20%
hydroxypropyl-β-cyclodextrin (HPβCD)] or test article in vehicle.
Eight hours later, mice were administered saline alone or saline
containing 1 μg of recombinant mouse CSF-1 (Cell Biosciences Inc.,
Norwood, MA) via the tail vein. Fifteen minutes after CSF-1 injection,
mice were sacrificed, and spleens were isolated and snap-frozen on dry
ice. The frozen tissue was homogenized in 1 mL of Trizol (Invitrogen)
per 50 mg of tissue, RNA was purified, and RT-PCR was performed to
assess c-fos mRNA expression as described previously.24
1
to give 30 mg (70%) of the title compound as a white solid. H NMR
(DMSO-d6, 400 MHz): δ 14.28 (br s, 1H), 9.76 (s, 1H), 8.34 (d, 1H,
J = 2.56 Hz), 7.84 (d, 1H. J = 8.1 Hz), 7.18 (dd, 1H, J = 8.5, 2.2 Hz),
7.08 (d, 1H, J = 1.9 Hz), 6.00 (br s, 2H), 5.72 (m, 1H), 4.18 (m, 2H),
2.80−2.60 (m, 3H), 2.24−2.10 (m, 4H), 1.80−1.60 (m, 6H), 1.50 (m,
2H). Mass spectrum (ESI, m/z): calcd for C23H26N6O, 419.2 (M +
H); found, 419.0.
CIA-Induced Arthritis Model. The CIA model was performed by
Bolder BioPath, Inc. (Boulder, CO). Male B10RIII (Jackson
Laboratories) were immunized with type II collagen (Elastin Products),
in Freund's complete adjuvant (with supplemental M. tuberculosis
4 mg/mL, Difco) on days 0 and 15. On day 12, animals were randomized
by body weight into groups (n = 15), and dosing was initiated and
continued every day (BID, 12 h intervals) for a total of 14 days. On day
26, mice were euthanized, paws were fixed in buffered formalin, and
toluiodine blue stained sections were prepared for histopathology.
Inflammation, cartilage damage, pannus invasion, and bone erosion
were each scored using five-point scales corresponding to minimal, mild,
moderate, marked, and severe changes. Inflammation was scored as the
presence of inflammatory cells and edema. Pannus was scored as tissue
invasion of cartilage and subchondral bone and destruction of joint
architecture. Cartilage damage was scored as the loss of toluiodine blue
staining, chondrocyte loss, and collagen disruption. Bone resorption was
scored for areas of resorption of medulary trabecular and cortical bone
and for the abundance of osteoclasts.
Biology. Kinase Assays. Assays for FMS and the other kinases
listed in Table 5 were carried out using a fluorescence polarization
competition immunoassay format and have been described elsewhere
in detail.24 The full cytoplasmic regions of FMS (538−972)
encompassing the tyrosine kinase domain were expressed and purified
from a baculovirus system.30 The FMS kinase assay measured FMS-
mediated phosphorylation of tyrosine residues present on a synthetic
FMS555−568 peptide (SYEGNSYTFIDPTQ). To each well of a black
96-well microplate (catalog no. 42-000-0117, Molecular Devices,
Sunnyvale, CA), 5 μL of compound solution (in 4% DMSO in assay
buffer) was mixed with 2 μL of 3.5 nM FMS, 25 mM MgCl2 in assay
buffer (100 mM HEPES, pH 7.5, 1 mM DTT, and 0.01% Tween-20),
and 2 μL of 1540 μM peptide in assay buffer. The kinase reaction was
initiated by adding 1 μL of 10 mM ATP in assay buffer. The final
concentrations in the 10 μL reaction mixture were 100 mM HEPES,
pH 7.5, 1 mM DTT, 0.01% Tween-20, 2% DMSO, 308 μM
SYEGNSYTFIDPTQ, 1 mM ATP, 5 mM MgCl2, and 0.7 nM FMS.
The plates were incubated at room temperature for 80 min. Reactions
were stopped by the addition of 1.2 μL of 50 mM EDTA. Each well
Adjuvant-Induced Arthritis Model. The adjuvant-induced arthritis
study was performed by Bolder BioPath, Inc. Male Lewis rats (Harlan
7880
dx.doi.org/10.1021/jm200900q|J. Med. Chem. 2011, 54, 7860−7883

Journal of Medicinal Chemistry
Article
catalog no. 1439444), 165−185 g, were anesthetized with isoflurane
and injected with 100 μL of Freund's complete adjuvant (FCA,
Sigma)/lipoidal amine (LA, Sigma) at the base of the tail on day 0.
Twice daily oral dosing was initiated on day 8 (the first day of clinical
disease) with 23 or vehicle (0.5% hydroxypropylmethylcellulose/0.5%
Tween-80). Rats were sacrificed on day 14, and spleens and hind paws
were weighed. Both hind paws were then fixed in formalin, decalcified
in 5% formic acid, and processed for H&E microscopy. H&E sections
of paws were scored for bone resorption using a five-point scale as
described in detail previously.20 Sections were scored additionally for
inflammation using a five-point scale for minimal, mild, moderate,
marked, and severe leukocytic infiltration and edema. Osteoclast
counts (n = 5, 400× fields) were performed on ankles in the areas of
greatest bone resorption. Histological sections prepared for ED1
immunohistochemistry were treated for 3 min with proteinase K to
expose masked epitopes, and the endogenous peroxidase activity was
deactivated with 3% hydrogen peroxidase. Sections were blocked for
1 h with 5% horse serum and for 30 min with Avidin−Biotin solution
(SP-2001, Vector Corporation, Burlingane, CA) prior to a 1 h of
incubation with ED1 primary antibody (mouse antirat CD68,
MCA341R, Serotec, Oxford, United Kingdom). Bound antibody was
detected using the ABC-AP Rat kit (PK-6102, Vector) and a DAB
Substrate kit (SK-4100, Vector). The sections were lightly counter-
stained, and the each section was visually scanned under 40×
magnification. Hot spots were defined as areas with markedly
increased ED1 positive cell density containing >30 ED1 positive
cells. Only hot spots in the soft tissue were counted to avoid inclusion
of ED1 positive osteoclasts.
SCW-Induced Arthritis Model. Female Lewis rats (80−100 g each)
were purchased from Charles River. SCW peptidoglycan-polysaccharide
polymers (PG-PS 10S) were purchased from Becton Dickinson (catalog
no. 210866). On day 0, rats were anesthetized using isoflurane and
injected ip with PG-PS 10S equivalent to 15 μg of rhamnose/gram
(body weight) in the lower left quadrant of the abdomen. Control rats
were treated in a similar manner with sterile saline. On day 5, rats
injected with PG-PS 10S that showed a robust acute phase arthritic
response based on joint swelling were randomized into the treatment
groups. In an initial study, animals were dosed orally BID beginning on
day 8 until sacrifice on day 19. In a second study, animals were dosed
orally BID beginning on day 18 until sacrifice on day 39. Compound 23
was formulated in 20% HPβCD, and the dose volume was 6 mL/kg.
Calipers were used to measure the width (anterior to posterior surface)
of the left and right hind ankles of each rat. Each ankle was measured
three times and averaged. Hind paw inflammation clinical scores were
assigned based on swelling and erythema as follows: 1 = ankle
involvement only; 2 = involvement of ankle and proximal 1/2 of tarsal
joint; 3 = involvement of the ankle and entire tarsal joint down to the
metatarsal joints; and 4 = involvement of the entire paw including the
digits. Scores of both hind paws were summed for a maximal score of 8.
Digital radiographs of the left paws were prepared using a VetTek
Universal AP500 (Del Medical Imaging Corporation, Franklin Park, IL).
Hind paws were assessed for ED1 positive cells using immunohis-
tochemistry as described above for the adjuvant arthritis model.
Statistical Analysis. Ankle thickness, bone erosion scores,
osteoclast counts, and c-fos expression values (means ± SEs) were
analyzed for group differences using the Student's t test. Significance
was set at p ≤ 0.05 unless indicated otherwise.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: cillig@its.jnj.com.
ACKNOWLEDGMENTS
■
We thank Alison Bendele and Mike Bendele at Bolder BioPath
Inc. for performing the mouse CIA and the rat adjuvant-
induced arthritis studies.
ABBREVIATIONS
■
BMDM, bone marrow-derived macrophage (cellular assay);
CIA, collagen-induced arthritis; CSF-1, colony-stimulating
factor-1; CSF-1R, colony-stimulating factor-1 receptor; CV,
cardiovascular; CYP, cytochrome P450 enzyme; EDCI, N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
FMS, the product of the cellular proto-oncogene homologue
of the feline McDonough sarcoma virus oncogene; H&E stain,
hematoxylin and eosin stain; hERG, human ether-a-go-go-
related gene potassium channel; HOBt, 1-hydroxybenzotria-
zole; HPβCD, 2-hydroxypropyl-β-cyclodextrin; IDR, idiosyn-
cratic drug reaction; M-CSF, macrophage colony-stimulating
factor; M-CSFR, macrophage colony-stimulating factor recep-
tor; MTX, methotrexate; PD, pharmacodynamic; PK, pharma-
cokinetics; QT, the interval in a heartbeat measured from the
beginning of the QRS complex to the end of the T wave; SCW,
streptococcal cell wall; RA, rheumatoid arthritis; SAR,
structure−activity relationship; SEM, trimethylsilylethoxymeth-
yl; TNF, tumor necrosis factor
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