Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.05.006

A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

Full text of DOI:10.1016/j.bmcl.2011.05.006

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3856–3860
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BMCL Digest
Substituted N-aryl-6-pyrimidinones: A new class of potent, selective,
and orally active p38 MAP kinase inhibitors
Balekudru Devadas a, , , Shaun R. Selness a, Li Xing b, Heather M. Madsen a, Laura D. Marrufo a, Huey Shieh b,
⇑
Dean M. Messing c, Jerry Z. Yang d, Heidi M. Morgan e, Gary D. Anderson e, Elizabeth G. Webb e, Jian Zhang e,
Rajesh V. Devraj a, Joseph B. Monahan e,
a Department of Medicinal Chemistry, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
b Structural and Computational Chemistry, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
c Department of Pharmacokinetics and Drug Metabolism, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
d Department of Pharmaceutical Sciences, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
e Inflammation Biology, Pfizer Corporation, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a
substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis
model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a sig-
nificantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat.
Received 5 February 2011
Revised 1 May 2011
Accepted 2 May 2011
Available online 8 May 2011
In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-a in a dose-dependent
manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-
induced arthritis model.
Keywords:
p38 MAP kinase
Pyrimidinone
Ó 2011 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-alpha
Oral bioavailability
Dual Hbond motif
Structure activity relationship
Dissolution rate
Anti-inflammatory
p38 MAP kinase is a widely prosecuted disease target that has
resulted in the discovery of a variety of inhibitor classes with
diverse molecular architecture.1 This enzyme plays a pivotal role
in the MAP kinase signal transduction pathway resulting in the
production of inflammatory cytokines including TNF-
a and
IL-1b.2 Modulation of these cytokines by ATP-competitive small
molecules has led to the generation of a variety of novel p38 inhib-
itors as potential therapeutics for the treatment of inflammatory
conditions including Crohn’s disease, psoriasis, and rheumatoid
arthritis (RA).3 Several of the p38 kinase inhibitors have advanced
into human clinical trials, and our own efforts have led to the
identification of a novel pharmacophore 1, (Fig. 1) as a clinical can-
didate for RA.4,5 This compound exhibits exceptional selectivity for
Figure 1. Structures of pyridinone and pyrimidinone classes of p38 kinase
inhibitors.
p38
a (MAPK14) versus other kinases due to a unique binding
mode involving a dual H-bond motif, engaging Met109 and
Gly110 residues, with a flipped backbone conformation of Gly110
from its apo state.6 The backbone flip occurs in p38
a due to the
presence of the glycine in the hinge region, which has no side chain
and consequently has sufficient conformational flexibility to in-
duce a peptide backbone flip. In the human kinome, only p38b
and Myt-1 contain the corresponding glycine and a threonine at
⇑
Corresponding author.
E-mail address: balekudru.devadas@yahoo.com (B. Devadas).
Present address: Confluence Life Sciences, 4320 Forest Park Avenue, Suite 303,
St. Louis, MO 63108, USA.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.006

B. Devadas et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3856–3860
3857
the gatekeeper position, which provides a structural rationale for
the observed high level of selectivity.6
The 2-pyridinone lead 1(aS atropomer), despite its excellent
in vitro and in vivo activity profiles, suffers from poor solubility
which could lead to dissolution-limited absorption and potential
variability in oral bioavailability at projected clinical doses. Upon
examination of the X-ray structure of the 1-p38 complex,6 it was
evident that the N-methyl group is exposed to the solvent accessi-
ble region, which provided an opportunity to improve solubility
and pharmacokinetic properties. Furthermore, 1 is optically active
by virtue of its asymmetry introduced by restricted rotation
around the C–N bond between the 2-pyridinone ring and the
benzamide moiety. A chiral separation step is necessary for the
isolation of the aS atropomer. In order to overcome these issues
we initiated the synthesis of the corresponding pyrimidinone
analogs with reduced lipophilicity bearing polar amide substitu-
ents, represented by 2. This report discloses the synthesis,
structure–activity relationship studies leading to the discovery of
10 as a clinical candidate, and the X-ray crystallographic structure
of 10 bound to the ATP-binding site of the p38 enzyme.
Scheme 2. Reagents and conditions: (k) NBS,dichloromethane, rt, 5 h; (i) isobutyl-
chloroformate, N-methylmorpholine, DMA 0 °C to rt; (l) 2-aminoethanol.
As shown in Scheme 1, the synthesis of N-arylpyrimidinones
commenced with the formation of the 3-thioureidobenzoate
precursor 4, from methyl-3-amino-4-methylbenzoate 3 and potas-
sium thiocyanate in the presence of 4 N HCl. The pyrimidinone ring
was then assembled by the condensation of 4 with dimethylmalo-
nate in the presence of sodium methoxide, followed by methyla-
tion, to afford the 2-thio-methyl-4-hydroxy intermediate 5.
Reaction of 5 with 2,4-difluorobenzyl chloride in the presence of
potassium carbonate in N-methylpyrrolidine provided 6 in good
yield.
Scheme 3. Synthesis of C-5-methyl/ethyl pyrimidinones. Reagents and conditions:
(m) diethyl-2-alkyl-malonate, 25% NaOMe, 18-crown-6, dioxane, 70 °C; (n) MeI, rt;
(o) 2,4-difluorobenzylbromide, K2CO3, DMF, rt, 12 h; (p) Raney Ni, 70 °C, DMA, 6 h;
(q) 2 N NaOH, rt, citric acid; (r) 2 M Oxalyl chloride, DMF, DCM; (s) RNH2.
Desulfurization of 5 using Raney Ni presented a synthetic chal-
lenge due to over-reduction of the pyrimidinone core and poor
conversion to the desired product 7. However, optimal conditions
were established by carrying out the reaction in 2-propanol or
dimethylacetamide at 60 °C to give the pyrimidinone derivative 7
in 80% yield. In the next step, ester 7 was converted to the corre-
sponding acid 8 and subjected to chlorination using N-chlorosuc-
cinimide in dichloroethane in the presence of catalytic amounts
of dichloroacetic acid to provide 9. The final compounds 10–14
were prepared by activation of 9 via the mixed anhydride forma-
tion, followed by coupling with amines.
Molecular modeling suggested a small hydrophobic pocket
around the 5-position of the pyrimidinone ring. In order to assess
its steric compatibility, the 5-bromo analog 16 was prepared in a
Scheme 1. Synthesis of 5-chloropyrimidinones. Reagents and conditions: (a) KSCN; (b) 4 N HCl, dioxane, 80 °C 20 h; (c) dimethylmalonate, NaOMe; (d) MeI;
(e) 2,4-difluorobenzylchloride, K2CO3 NMP rt; (f) Raney Ni, DMA, 80 °C 12 h; (g) 2 N NaOH, dioxane rt, 1 h, 5% citric acid; (h) NCS, dichloroacetic acid, dichloroethane,
65 °C; (i) isobutylchloroformate, N-methylmorpholine, DMA; (j) RNH2

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B. Devadas et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3856–3860
similar manner as depicted in Scheme 2 using N-bromosuccini-
mide in place of N-chlorosuccinimide.
12 and the amide 14 exhibited improved solubility10 compared
to the hydroxyl-substituted compounds 10 and 13, they showed
significantly higher efflux ratios as measured by Caco-2 cell perme-
ability. While the amide analog 11 maintained excellent potency
To further evaluate the role of steric and electronic effects at the
5-position, the synthesis of 5-methyl and 5-ethyl analogs 23 and
24 were undertaken as outlined in Scheme 3. Condensation of 4
with diethyl-2-methylmalonate in the presence of sodium ethox-
ide resulted in the formation of 17 in 87% yield. The corresponding
ethyl derivative 18 was obtained in 94% yield under similar
conditions.
against p38
chloro analog 10, the corresponding bromo counterpart 16 was
found to be less soluble and less potent against p38
(IC50 = 60 nM). Like 1, these substituted 6-pyrimidinones were
found to be highly selective versus JNK2 (IC50 >200 M).
a it suffered from poor aqueous solubility. Unlike the
a
l
In the next step, the 2,4-difluorobenzyl group was installed
using 2,4-difluorobenzyl bromide in the presence of K2CO3 to pro-
vide 19 and 20. Raney Ni assisted desulfurization provided 21 and
22 in good yields. These were converted to the target compounds
23 and 24 following hydrolysis, acid chloride formation using oxa-
lylchloride, and coupling with 2-aminoethanol.
In an effort to expand the structural diversity of this class of
inhibitors, we synthesized the 5-methyl and 5-ethyl analogs 23
and 24, respectively. While these maintained good potency against
p38a and good inhibition of LPS-induced TNF-a production
(Table 2, we de-prioritized this series due to low aqueous
solubility.
To understand the importance of the oxygen linker at the C-4
The prominent role played by the oxygen linker is revealed by
the sulfur analog 29, which exhibited significantly decreased
position to the p38a binding affinity, preparation of the 4-thio
analog 29 was also carried out as depicted in Scheme 4. The aniline
ester 3 was converted to the cyanoacetamide precursor 25. Treat-
ment of 25 with diethyldithiophosphate furnished 26 in 90% yield.
Subsequent cyclization of 26 with methylformate followed by
installation of the 2,4-difluorobenzyl group provided 27.
potency (IC50 = 193 nM) to p38a.
Due to the superior potency (Ki = 6.4 nM) and low efflux ratio
displayed by 10, it was further evaluated in cellular assays and
in vivo disease models of inflammation.11,12 The cellular potency
of 10 correlated well with its inhibition of recombinant enzyme
Following the conversion of 27 to its acid derivative, chlorina-
tion was carried out to furnish 28. Activation of the carboxyl group
in 28 by conversion to the acid chloride and exposure to 2-amino-
ethanol afforded the final compound 29.
activity, consistent with
(Table 1).
a p38 kinase mechanism of action
The U937 cellular activity of 10 (Table 3)8 was confirmed and
extended using LPS-stimulated human monocytes and human
whole blood. In human monocytes, LPS-stimulated production of
These new analogs 10–147 in general showed good potency
against p38a8 and in vitro metabolic stability.9 Although the diol
TNF-a and IL-1b was inhibited by 10 with an IC50 of 25.6 nM and
Scheme 4. Synthesis of 4-thio analog. (t) methylcyano acetate, DBU, xylene, reflux, 42%; (u) diethyldithiophosphate, dioxane/water, 80 °C 24 h, 74%; (v) methylformate,
sodium methoxide; (w) 2,4-difluorobenzylbromide, K2CO3, rt; (x) N-chlorosuccinimide, i-propanol, reflux, 20 min; (y) 2 N NaOH, dioxane, rt; (z) Oxalyl chloride, DMF,
2-aminoethanol. Following the conversion of 27 to its acid derivative, chlorination was carried out to furnish 28. Activation of the carboxyl group in 28 by conversion to the
acid chloride and exposure to 2-aminoethanol afforded the final compound 29.
Table 1
In vitro potency, metabolic stability, and in vivo activity of 5-chloro-substituted-6-pyrimidinones
Compound #
p38
IC50
a
(
Met. stab.
(% remaining)
Rat LPS (%) Inh.of
TNF @ 5 mpk
Aq solubility (amorphous,
@pH 6.5, lg/mL)
Caco-2 Cell
efflux ratio
Rat SCW (% Inh. of disease
score @30mpk/day)
l
M)
a
10
11
12
13
14
0.029
0.03
0.027
0.012
0.006
99 (Human) 97 (Rat)
100 (Human) 100 (Rat)
97 (Human) 100 (Rat)
96 (Human) 85 (Rat)
77 (Human)72 (Rat)
95.2
84.3
91.5
91.3
92.9
51
14
163
94
147
4.2
ND
32.8
7.5
23.6
94.8
ND
ND
82.4
ND
Table 2
In vitro potency, metabolic stability, and in vivo activity of 5-methyl/ethyl-substituted-6-pyrimidinones
Compound #
R
p38
IC50
a
p38
a
M)
JNK2 IC50
M)
Met. Stab.
(% Remaining)
Aq. solubility (Amorphous,
Rat LPS (%) Inh.
of TNFa @ 5 mpk
(l
M)
Ki(
l
(
l
@pH 6.5,
lg/mL)
23
24
Methyl
Ethyl
0.038
0.011
0.017
0.006
>200
>200
84 (Human) 99 (Rat)
76 (Human) 92 (Rat)
28
20
94.7
91.8

B. Devadas et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3856–3860
3859
Table 3
(96.3%) characteristics. While 10 was shown to inhibit cellular
p38 kinase activity as assessed by p38 kinase dependent phosphor-
ylation of HSP-27, no inhibition of the JNK or ERK pathways was
Cellular activity of 10 and 1 on cytokine production and p38 activity
Assay
Compound 10
Compound 1
observed in U937 cells at a concentration of 1
The crystallographic data (Fig. 2) revealed that 10 binds in
the ATP binding site of p38 , with a characteristic dual H-bond
lM.
U937 TNF
U937 p38 kinase activity IC50 (nM)
Human Monocyte TNF IC50 (nM)
Human Monocyte IL-1b IC50 (nM)
Human Monocyte p38 kinase
activity IC50 (nM)
a
IC50 (nM)
35.5 18.8
12.0 1.2
25.6 4.3
37.2 6.5
16.9 3.4
5.9 1.3
1.05 0.64
3.4 2.0
3.4 0.5
17.4 6.9
a
a
motif, engaging both Met109 and Gly110 residues.13 In analogy
to the pyridinone inhibitor 1, the backbone flip of Gly110 was
observed with 10. The 2,4-difluorobenzyloxyl group occupied
the lipophilic pocket defined by Thr106 as the gatekeeping resi-
due. The N-phenyl group is directed toward the solvent front.
Similar to the binding mode of 1, the binding conformation of
the benzamide moiety in 10 is driven by the polar interaction
between its amide NH and the backbone carbonyl of Gly110.
Although 10 is devoid of the 2-methyl substitution in the pyri-
midinone core, it exhibits orthogonal geometry between N-phe-
nyl and the pyrimidinone ring, a binding conformation that is
driven by the stacking of the N-phenyl onto the peptide back-
bone of Gly110-Ala111.5
Human Whole Blood TNF
a
IC50 (nM)
177 30
85 21
In vivo pharmacological evaluations of 10 were carried out both
in acute and chronic disease models of inflammation. In a rat LPS
model, with a 4 h oral pre-dosing interval regimen, all compounds
showed a dose-dependent inhibition of LPS stimulated TNF-
a
production.11 Compound 10 was found to be the most efficacious
candidate in this series, with an ED50 value of 0.115 mg/kg and
ED80 of 0.259 mg/kg. Based on the superior in vitro and in vivo pro-
files, 10 was further evaluated in the chronic SCW-arthritis model.
Analogue 10 was found to be highly effective in mitigating
SCW-induced inflammation (Table 4).12 It showed
a dose-
dependent inhibition of paw swelling when administered from
10 to 21 days with ED50 and ED80 values of 0.105 mg/kg and
0.649 mg/kg b.i.d. respectively. Anti-inflammatory effects observed
with 10 were comparable to that achieved with dexamethasone.
Furthermore, it was observed that 10 protected ankle joint as
Figure 2. Crystal structure of 10 bound to active site of p38a. Crystals of p38a-10
complex were obtained by soaking experiment. The structure has been refined to a
Rfree of 27.6% at 1.7Å (Rcrystal = 23.9%). The inhibitor is represented with carbon,
nitrogen, oxygen and chlorine in cyan, blue, red, and green respectively. The
hydrogen bonds are shown by dotted red lines.
evidenced by retention of bone integrity measured by lCT analysis.
Cellular studies suggested that the mechanism by which 10
displayed bone protective effect may be through the inhibition of
osteoclast differentiation, a cell type primarily responsible for bone
resorption.14
Table 4
In vivo efficacy of 10 and 1 in acute & chronic inflammation models
Assay
Compound 10
Compound 1
10 was evaluated for its pharmacokinetic (PK) properties in rat
and dog. The oral bioavailability (BA) was 99% and 67% in rat and
dog respectively (Table 5). 10 exhibited low clearance and moder-
ate volume of distribution in both species. The primary contributor
for the observed superior bioavailability for 10 across species ap-
pears to be due to a 20-fold increase in dissolution rate compared
to 1 (Table 6).15 A combination of low clearance with moderate
volume of distribution and excellent oral bioavailability for 10 pre-
dicts human PK properties suitable for q.d. or b.i.d dosing regimen.
ED50/ED80 (mg/kg)
ED50/ED80 (mg/kg)
Rat LPS induced TNF inhib.
Rat SCW Arthritis
0.115/0.259
0.105/0.649
0.07/0/142
0.186/1.61
a
37.2 nM respectively, while in the whole blood assay, TNF-
synthesis was inhibited with an IC50 value of 177 nM. This lower
potency of 10 is consistent with its plasma protein binding
a
Table 5
Summary of PK parameters for 10
Species
Dose (mg/kg)
CL (ml/min/kg)
3.19
Vdss (L/kg)
1.32
t1/2 (h)
6.5
Oral BA (%)
Rat (IV)
Rat (PO)
Dog(IV)
Dog (PO)
0.2
0.2
1
99%
67%
1.59
0.986
8.7
1
Table 6
Effect of dissolution rate on BA
Compound
Solid State
Solubility
Intrinsic Dissolution
logP
PermeabilityPapp
Rat Oral
Dose (mpk)
Rat Oral
BA(suspension)
(pH 6.5
lg/mL)
Rate (
l
g/min/cm2)
10À6cm/s,(Efflux Ratio)
1
10
Crystalline
Crystalline (Form A)
ꢀ10
0.3
6.1
3.35
1.65
15.9 (4.4)
7.99 (4.4)
5
5
9.3 3.2%
99%
23

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B. Devadas et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3856–3860
measuring percent compound remaining by a precipitation procedure followed
In summary we have identified a novel class of potent and
selective p38 kinase inhibitors, originating from an N-aryl-6-pyri-
midinone scaffold. Compound 10 displayed exquisite kinase selec-
tivity for p38
blocked LPS-induced TNF-
by LC–MS analysis.
10. Aqueous solubility is expressed in lg/mL at pH 6.5.
11. Adult male Lewis rats (Harlan Sprague Dawley, Indianapolis, IN) (225–250 g)
were used in these studies. Rats were fasted 18 h prior to oral dosing, and
allowed free access to water throughout the experiment. Each treatment group
a
versus a panel of over 100 related kinases.16 It
a
production in human monocyte and
consisted of five animals. 10 was prepared as
a suspension in a vehicle
showed robust efficacy and target modulation in rat LPS and
SCW arthritis models. The efficacy seen in these models appears
to be driven by down regulation of p38 kinase activity because
10 did not cross over to parallel signaling pathways as measured
by cellular assays. Furthermore, 10 demonstrated remarkable
anti-inflammatory and joint protective activity in rodent chronic
disease models. A dose-dependent inhibition of mechanism bio-
markers was achieved upon oral dosing of 10. On the basis of supe-
rior pharmacodynamic and pharmacokinetic profiles, 10 was
selected for evaluation in humans.
consisting of 0.5% methylcellulose, (Sigma, St. Louis, MO), 0.025% Tween 20
(Sigma). The compound or vehicle was administered by oral gavage in a
volume of 1 mL. Two vehicle groups were used per experiment to control for
intra-experiment variability, and three experiments were performed. LPS
(E. coli serotype 0111:B4, Sigma) was administered four hours later by
intravenous injection at a dose of 1 mg/kg in 0.5 mL sterile saline (Baxter
Healthcare, Deerfield, IL). Blood was collected in serum separator tubes via
cardiac puncture ninety minutes after LPS injection,
corresponding to maximal TNF production (data not shown). After clotting,
serum was withdrawn and stored at –20 °C until it was assayed for TNF . TNF
levels in serum were quantified from a recombinant rat TNF (Biosource
a time point
a
a
a
a
International) standard curve using a four parameter fit generated by an Excel
(Microsoft, Redmond, WA) macro. The limit of detection for the ELISA was
approximately 41 pg TNFa/mL.
12. 10 was assayed in Streptococcal Cell Wall(SCW) induced Arthritis in Rats as
follows: Arthritis was induced in female Lewis rats by a single intraperitoneal
administration of peptidoglycan-polysaccaride complexes isolated from group
Acknowledgement
We thank Sean Juo for technical assistance in obtaining the PDB
deposition number.
a SCW (15 lg/g bodyweight). The SCW preparation was purchased from Lee
Labs. (Grayson, GA). The disease course is biphasic in which an acute
inflammatory arthritis develops within days 1–3 (non-T-cell-dependent
phase) followed by
a chronic erosive arthritis (T-cell- dependent phase)
References and notes
developing on days 14–28. Only animals developing the acute phase were
treated with 10 from days 10 to 21 after SCW injection. Paw volume was
measured on day 21 by using a water displacement plethysmometer. 10 was
prepared as an aqueous suspension in 0.5% methylcellulose and 0.025% Tween
20(Sigma–Aldrich). It was administered by oral gavage in a volume of 0.5 mL
beginning on day 10 post-SCW injection and continuing daily until day 21.
Animals were dosed between 0.015 and 5 mg/kg b.i.d. with 10.
Methylcellulose/Tween 20 vehicle was used for comparison. Group size was
four to eight animals per group. Two paw volumes were taken for each animal.
1. (a) Saklatvala, J. Curr. Opin. Pharm. 2004, 4(4), 372; (b) David, J. D.; Tsung, H. L.
Curr. Topics in Med. Chem. 2005, 5, 953.
2. (a) Adams, J. L.; Badger, A. M.; Kumar, S.; Lee, J. C. Prog. Med. Chem. 2001, 38, 1;
(b) Dinarello, C. A. Curr. Opin. Immulol. 1991, 3, 941.
3. Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. Drug. Discovery 2003, 717–726.
4. Selness, S. R.; Devraj, R. V.; Monahan, J. B.; Boehm, T. L.; Walker, J. K.; Devadas,
B.; Durley, R. C.; Kurumbail, R.; Shieh, H.; Xing, L.; Hepperle, M.; Jerome, K. D.;
Benson, A. G.; Marrufo, L. D.; Madsen, H. M.; Hitchcock, J.; Owen, T. J.; Christie,
L.; Promo, M. A.; Hickory, B. S.; Alvira, E.; Naing, W.; Blevis-Bal, R. Bioorg. Med.
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5. Hope, H. R.; Anderson, G. D.; Burnette, B. L.; Compton, R. P.; Devraj, R. V.;
Hirsch, J. L.; Keith, R. H.; Li, X.; Mbalaviele, G.; Messing, D. M.; Saabye, M. J.;
Schindler, J. F.; Selness, S. R.; Stillwell, L. I.; Webb, E. G.; Zhang, J.; Monahan, J. B.
J. Pharm. Exp. Therap. 2009, 331(3), 882.
Paw volume was measured on day 21 by using
a water displacement
plethysmometer. Three to four paws from each treatment group were
scanned for bone density evaluation. Plasma samples were collected on day
21 for determination of compound levels.
13. The coordinates of compound 10 bound to p38 have been deposited with the
Protein Data Bank and assigned RCSB ID code rcsb065177 and PDB ID code
3ROC.
6. Xing, L.; Shieh, H. S.; Selness, S. R.; Devraj, R. V.; Walker, J. K.; Devadas, B.; Hope,
H. R.; Compton, R. P.; Schindler, J. F.; Hirsch, J. L.; Benson, A. G.; Kurumbail, R.
G.; Stegeman, R. A.; Williams, J. M.; Broadus, R. M.; Walden, Z.; Monahan, J. B.
Biochemistry 2009, 48, 6402.
14. Treatment of rat bone marrow cells with 10 resulted in a dose-dependent
inhibition of osteoclast formation induced by RANKL with an IC50 of 10.1 nM.
15. (a) Horter, D.; Dressman, J. B. Adv. Drug Deliv. Rev. 2001, 46, 75; (b) Intrinsic
Dissolution Rate = Initial Influx/ Total Surface Area of Rotating Disk.
7. All compounds showed 1H NMR and mass spectra consistent with their
structure and were > 95% purity by HPLC.
16. Compound 10 exhibited an IC50 of >10 lM for the following kinases: JNK1,
JNK2, JNK3, ERK2, PRK, MK2, MK3, MKK6, CDK2, IKK1, IKK2, Aurora-A,
Myt1,ALK, TBK, BTK, CK1, CHK2, EGFR, Flt3, Lyn, MAPK1, MEK1, MKK4, MST2,
ROCK II, Syk, Tie-2, MKK7, ZAP70, and inhibited p38b with an IC50 of
8. Durley, R. C.; Devadas, B.; Madsen, H. M.; Hickory, B. S.; Palmquist, K.; Selness,
S. R. WO 2004087677.
9. Metabolic stability was determined in vitro by incubating 2
lM test compound
0.249 lM.
with human or rat liver microsomes, NADPH and buffer at 37 °C for 45 min and