Discovery and optimization of p38 inhibitors via computer-assisted drug design

Article abstract of DOI:10.1021/jm070415w

Integration of computational methods, X-ray crystallography, and structure-activity relationships will be disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.

Full text of DOI:10.1021/jm070415w

4016
J. Med. Chem. 2007, 50, 4016-4026
Discovery and Optimization of p38 Inhibitors via Computer-Assisted Drug Design
Daniel R. Goldberg,*,† Ming-Hong Hao,*,† Kevin C. Qian,† Alan D. Swinamer,† Donghong A. Gao,† Zhaoming Xiong,†
Chris Sarko,† Angela Berry,† John Lord,† Ronald L. Magolda,†,‡ Tazmeen Fadra,† Rachel R. Kroe,§ Alison Kukulka,†
Jeffrey B. Madwed,| Leslie Martin,† Christopher Pargellis, Donna Skow,† Jinhua J. Song,# Zhulin Tan,# Carol A. Torcellini,O
Clare S. Zimmitti,| Nathan K. Yee,# and Neil Moss†
Departments of Medicinal Chemistry, Immunology and Inflammation, Biological and Biomolecular Sciences, CardioVascular, Drug DiscoVery
Support, and Chemical DeVelopment, Boehringer Ingelheim Pharmaceuticals, Inc., Research and DeVelopment Center, 900 Ridgebury Road,
Ridgefield, Connecticut 06877
ReceiVed April 6, 2007
Integration of computational methods, X-ray crystallography, and structure-activity relationships will be
disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.
Introduction
Mitogen-activated protein kinases (MAPKs) are important
components of signal transduction pathways that mediate cell
proliferation, differentiation, and death.1 p38 is a critical member
of this family and is important in enabling the production of
pro-inflammatory cytokines such as TNFR and IL-1â, both of
which are implicated in chronic inflammatory diseases.2 Inject-
able biologic therapies that bind and inactivate TNFR are
effective treatments for rheumatoid arthritis and psoriasis.3 In
animal models of arthritis, treatment with p38 inhibitors leads
to a reduction in pro-inflammatory cytokine levels and a
significant improvement in the severity of inflammation.4
Consequently, inhibition of p38 with a small molecule could
provide an effective oral therapy for the chronic treatment of
these autoimmune diseases.5
Figure 1. Interactions of 1 in p38.
A number of companies have advanced small molecule p38
evac), an Abl kinase inhibitor that also binds in a Phe out
inhibitors into the clinic, including ourselves with BIRB 796,
binding mode,9 helped to reinforce our commitment to the
design of new p38 inhibitors that bind in the Phe out binding
mode.
1.6 As part of our efforts to advance the therapeutic potential
of p38 inhibition and position ourselves with backup compounds
to 1, we have been seeking alternative classes of inhibitors
structurally distinct to 1. One of the approaches we undertook
to accomplish this was to design a new class of p38 inhibitors
by capitalizing on the structural perspective we had gained from
the X-ray cocrystal structure of 1 bound to p38 and the distinct
binding mode revealed by this structure (Figure 1). The binding
mode for 1 involves interactions beyond those typically found
with many ATP site kinase inhibitors.7,8 In addition to the
interactions within the adenine binding site and the adjacent
kinase specificity pocket, 1 also takes advantage of binding sites
made available by the movement of a phenylalanine residue
(Phe169) found in the conserved DFG segment of the kinase
activation loop. The increased opportunity offered by multiple
binding sites for achieving potency encouraged us to focus our
design efforts on this Phe out binding mode. Furthermore, the
clinical and subsequent commercial success of Imatinib (Gle-
Design Process
In considering how best to translate our previously obtained
structural information into the design of a new Phe out p38
inhibitor, we felt that computer assisted methods would facilitate
the generation of new ideas of how to fill and link between the
available binding sites. However, it was not immediately obvious
how to design a new group that would ensure binding to p38
in a Phe out manner. We therefore started with a functionality
that was already precedented to bind to the region of p38 made
available upon the movement of Phe169. For 1, this functionality
includes most notably the tert-butyl pyrazole, with the tert-butyl
group situated tightly in the Phe pocket (the area previously
occupied by Phe169 in the Phe in conformation). In addition to
this hydrophobic interaction, we felt that two hydrogen bonding
interactions were also critical for a Phe out binder, the urea
carbonyl with the backbone N-H of Asp168 plus the urea N-H
with the carboxyl of Glu71. Previous SAR had demonstrated
the essentiality of the urea carbonyl and N-Hs for inhibitor
potency in naphthyl urea-based p38 compounds.10 Furthermore,
the observation that other published Phe out inhibitors9,11 all
contained a related H-bond donor/acceptor arrangement rein-
forced our belief in the necessity of this type of functionality.
We knew that Phe out fragments structurally distinct from
the tert-butyl pyrazole functionality in 1 were possible because
our own SAR investigations with p38 and that of Bayer with
* To whom correspondence should be addressed. For Daniel R.
Goldberg: phone, (203) 778-7828; fax, (203) 791-6072; e-mail, dgoldber@
rdg.boehringer-ingelheim.com. For Ming-Hong Hao: phone, (203) 791-
6033; fax, (203) 791-6072; e-mail, mhao@rdg.boehringer-ingelheim.com.
† Department of Medicinal Chemistry.
‡ Current Address: Wyeth Research, 865 Ridge Road, Room 1007,
Monmouth Junction, NJ 08852.
§ Department of Biological and Biomolecular Sciences.
| Department of Cardiovascular Research.
Department of Immunology and Inflammation.
# Department of Chemical Development.
O Department of Drug Discovery Support.
10.1021/jm070415w CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/20/2007

p38 Inhibitors Via Computer-Assisted Drug Design
Table 1. Initially Designed Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4017
Figure 2. LigBuilder design process.
as a key starting point for applying computer-assisted design
to fill the kinase specificity pocket and ATP site in a novel
way.
Most potent kinase inhibitors partake in a H-bond with the
kinase amino acid that forms an interaction with the N-7 of the
adenine ring of ATP;13 in the case of p38, this residue is Met109.
Given the typical essentiality of this interaction for potent kinase
inhibition, we decided to increase our chances of success in
applying computer-assisted design by positioning a small
fragment to interact with Met109. A common motif in kinase
inhibitors associated with this interaction is a pyridine. Thus,
we positioned a pyridine group into the ATP site in a typically
observed binding orientation. We used the computer program
LigBuilder to act as an initial idea generator, utilizing the linking
algorithm14 to generate structures that would link the pyridine
to the phenylsulfonamide Phe out fragment with the expectation
that the program would use fragments that also take advantage
of the kinase specificity pocket (Figure 2).
LigBuilder adds and links fragments from a library of small
chemical moieties to generate new structures and then ranks
these new structures based on their binding score.14 LigBuilder
generated over 800 different structures bridging the two frag-
ments. However, none of the generated structures were con-
sidered ideal, having a high degree of conformational flexibility
and not being readily amendable to rapid chemical synthesis.
Nonetheless, a common fragment, a 5,6-fused heterocyclic ring,
turned up in a significant number of the computationally derived
molecules. This observation generated the idea of attaching this
fragment directly to the amide of the phenyl sulfonamide group.
Computationally, this amide-linked 5,6-fused heterocyclic ring
reasonably occupied the kinase specificity pocket. Subsequent
modeling identified that a two-atom linker could connect the
5,6-fused heterocyclic ring to the pyridine fragment in the
adenine binding site. Based on synthetic flexibility, compound
rigidity, and optimal positioning of the pyridine ring to interact
with Met109, we chose a carboxamide as a link between the
pyridine and the 5,6-fused heterocyclic ring (Figure 3a). Thus,
compound 8 became the initial key target to test our design.
a Values are the mean of gtwo experiments, standard deviation 0.1-
0.5 °C unless otherwise noted. b Value n ) 1. c Values are the geometric
means from 8 to 12 donor experiments, standard deviation typically (50%
of reported value.
Chemistry
Compounds 3-7 were synthesized from commercially avail-
able acid or acid chlorides through standard coupling reactions
with tert-butyl phenyl sulfonamide 25. To prepare elaborated
compounds with the appropriately substituted 4-amino group,
the 4-nitrobenzothiophene-2-carboxylic acid 26 was prepared
as described in the literature.15 Coupling of 26 with amine 25
produced amide 27. Hydrogenation over 10% Pd/C gave 28,
which was then coupled with nicotinic acid to provide 8
(Scheme 1).
Sorafenib (Raf inhibitor) revealed that a phenyl template was
also a viable option. Table 1 shows one of the more potent
phenyl-based alternatives we had found for p38. The tert-butyl
phenyl sulfonamide derivative 2 (SAR details for this moiety
to be published elsewhere) proved essentially equipotent to 1.12
As the tert-butyl phenyl sulfonamide functionality was structur-
ally distinct from the tert-butyl pyrazole functionality in 1, we
chose this group together with the adjacent N-H and carbonyl

4018 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Goldberg et al.
Figure 3. (a) Designed inhibitor 8. (b) Bound inhibitor and X-ray structure of 5.
Scheme 1a
a Reagents and conditions: (a) TBTU, DIPEA, DMF; (b) H2, 10% Pd/C, EtOAc; (c) EDC, HOBt, nictonic acid, DMF.
Compound 9 was prepared from 2-chloro-3-nitrobenzalde-
hyde 29 (Scheme 2). Addition of the anion of methyl thiogly-
colate to 29 afforded benzothiophene 30. Nitro reduction over
10% Pd/C provided 31. Amide formation with either nicotinic
acid or 6-chloronicotinic acid afforded 32 and 33, respectively,
after acid hydrolysis. The amide coupling of carboxylic acids
32 or 33 with aniline 25 gave 9 and 38a. The N-methyl indole
core was prepared by methylation of 34 with methyl iodide.
Saponification followed by coupling with 25 provided 36.
Subsequent hydrogenation of the nitro group over 10% Pd/C
provided 37, which was coupled with nicotinic acid to provide
10. Amide formation of 37 with 6-chloronicotinic acid produced
38b. Nucleophilic substitution of chlorides 38a or 38b with both
primary and secondary amines provided the compounds listed
in Table 2.
to determine if this new way of occupying and linking to the
kinase specificity pocket would provide useful binding affinity
(Table 1). Somewhat disappointingly, the heterocyclic deriva-
tives 4-7 proved to be over 2 orders of magnitude less potent
than the corresponding naphthyl urea 3 based on data from our
thermal denaturation assay (Table 1).16 We nevertheless sought
some validation of our original design through preparing the
complete compound 8. Surprisingly, 8 proved to be even less
a
potent (Tm ) 48.5 °C) than the truncated analogue 5 (Tm
)
51.4 °C). Puzzled by the apparent complete failure of our initial
design, we eventually recognized an important but subtle flaw
in our design through an X-ray cocrystal structure of the
truncated derivative 5 (Figure 3b). We had assumed that the
benzothiophene in 5 would bind to p38 close to its ground-
state s-trans conformer, with the sulfur on the same side as the
N-H of the adjacent amide, as opposed to the energetically
less-favored s-cis conformation that could create lone pair/lone
Results and Discussion
Before we embarked on the synthesis of designed inhibitor
8, we prepared truncated analogues containing only the 5,6-
heterocycle attached to the phenyl sulfonamide Phe out fragment
a Abbreviations: Tm, thermal denaturation assay; HuWB, human whole
blood assay.

p38 Inhibitors Via Computer-Assisted Drug Design
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4019
Scheme 2a
a Reagents and conditions: (a) K2CO3, methyl thioglycolate, DMF; (b) H2, 10% Pd/C, EtOAc; (c) nicotinic acid or 6-chloronicotinic acid, Et3N, BopCl,
CH2Cl2; (d) LiOH, H2O, THF; (e) 25, HATU, HOBt, DIPEA, DMF; (f) NaH, MeI, THF; (g) NaOH, EtOH; (h) 25, EDC, HOBt, DMF; (i) H2, 10% Pd/C,
EtOAc; (j) nicotinic acid, HOBt, EDC, DMF; (k) 6-chloronicotinic acid, HOBt, EDC, DMF; (l) HNR1R2, ∆.
pair repulsive interactions between the sulfur atom and the
carbonyl oxygen. However, the X-ray cocrystal structure of
compound 5 showed the benzothiophene in the s-cis conforma-
tion. Presumably, preference for the s-cis conformation in the
bound state is due to repulsive interactions in the s-trans
conformation between the carboxyl group of Glu71 and the lone
pairs of the benzothiophene sulfur. Importantly, we now realized
that our initial design did not optimally position the pyridyl
carboxamide attachment. Based on the observed binding
conformation of the benzothiophene of compound 5, the
7-position of the benzothiophene (compound 9) would be the
better site for attaching the pyridyl carboxamide than the
4-postion in our originally designed compound 8.
denaturation temperature. Thus, while the benzothiophene amide
template appeared to be inferior to the naphthyl urea template,
the pyridyl carboxamide attachment appears to compensate for
this deficit so that compound 9 ends up with nearly identical
potency to inhibitor 1.
A subsequent X-ray structure of 9 provides a possible
explanation for this unexpected gain in molecular potency
(Figure 4). In addition to the hydrogen bonds with Met109,
Glu71, and Asp168, a number of new hydrogen bonding
interactions were also observed. The 7-nicotinamide N-H forms
a hydrogen bond with Thr106 (the gatekeeper residue7a), while
the carbonyl oxygen of the 7-nicotinamide interacts with both
Tyr35 and Lys53 through a water-mediated bridge.17 In addition,
Tyr35 also forms a π-stacking interaction with the pyridine
group of the nicotinamide.
We were delighted and somewhat surprised that repositioning
the pyridyl carboxamide attachment from the 4-position to the
7-position of the benzothiophene (compound 9) resulted in a
thermal denaturation temperature with p38 of 59.5 °C (IC50
≈
Given the information gained through the crystal structure
of 9, we were curious why compound 8 was less potent than
even the truncated benzothiophene given it still has the ability
to interact with Met109 albeit in an energetically less-favored
conformation. A cocrystal structure of 8 demonstrated that unlike
9, where the benzothiophene is nearly coplanar to the adjacent
1 nM). This corresponds to a greater than 2 order of magnitude
(8.3 °C) increase in potency compared to the unsubstituted
derivative 5. The magnitude of this increase was initially
surprising considering that addition of the ethoxymorpholino
moiety (compound 1) to the unsubstituted naphthyl urea
derivative 2 provided only a 3.7 °C increase in thermal

4020 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Table 2. 6-Amino Substituted Nicotinamides
Goldberg et al.
14 lost potency. In addition, inhibition of LPS-induced TNFR
in human whole blood also improves as compared to 1 (Table
2; 11-13 and 15-24). A cocrystal structure of 19 confirmed
the new interaction of the N-H and the carbonyl of Met109
(Figure 6). In general, we found that small alkyl or alkoxy
substituted amines (19-24) provided us with the best combina-
tion of in vitro potency (vide infra), likely due to increased
hydrophobic interactions in this region of p38 in addition to
the extra H-bonding interaction.
Given that we know that 1 can inhibit other kinases,19
compound 19 as a representative example from this new class
of molecules is reasonably selective. While potent against both
Lyn (230 nM) and Jnk2 (16 nM), compound 19 shows negligible
inhibition of the following kinases when tested at 3 µM: MKK1,
JNK1, MAPKAP-K1a, MAPKAP-K2, MSK1, PRAK, PKA,
PKCa, PDK1, PKBδPH, SGK, S6K1, GSK3b, ROCK-II,
AMPK, CHK1, CK2, PHOS. KINASE, CDK2/cyclin A, CK1,
DYRK1A, PP2A, and NEK6 and the following tested at 10
µM: Btk, Eck, EGFR, FGFR3, Hek, HGFR, IGF1R, IR, Itk,
JAK3, PDGFRa, Syk, TrkA, TXK, VEGFR1.
Mouse In Vivo and Rat Pharmacokinetic Studies. To
assess the potency of these new p38 inhibitors in vivo, we
utilized a mouse (oral) model of LPS-stimulated TNFR produc-
tion. In this model, compounds were dosed 30 min prior to LPS
challenge, and plasma TNFR levels were determined 1 h later.
While compounds 9 and 10 demonstrated identical in vivo
efficacy to 1 in this model at a dose of 10 mg/kg, the more
potent derivatives 19 and 20 also proved significantly more
potent in vivo, inhibiting TNFR at a dose at or below 3 mg/kg
(Table 3).
Subsequent pharmacokinetic analysis of compounds 9, 10,
19, and 20 demonstrate good oral exposure and bioavailability
at a dose of 10 mg/kg. Compound 20 has a comparable
pharmacokinetic profile to 1, while having an improved in vitro/
in vivo potency profile (Table 4).
Conclusion
A new series of potent Phe out p38 inhibitors have been
discovered through a combination of computer-assisted drug
design, X-ray crystallography, and chemical intuition. Critical
to the discovery of this series of inhibitors was the iterative
nature of the design process based on obtaining timely structural
information throughout the discovery and optimization process.
This process successfully culminated in the identification of
compounds with improved cellular and in vivo potency profiles
to our previous clinical candidate 1.
a Values are the mean of gtwo experiments, standard deviation 0.1-
0.5 °C unless otherwise noted. b Value n ) 1. c Values are the geometric
means from 8 to 12 donor experiments, standard deviation typically (50%
of reported value.
amide carbonyl, the benzothiophene in compound 8 is twisted
by 30°, presumably to reduce lone pair-lone pair interactions
between Glu71 and the sulfur atom. The twist causes a loss in
all of the productive hydrogen bonding interactions within the
adenine site (Figure 5).
Experimental Section
General Methods. 1H NMR spectra were recorded on a Bruker
UltraShield-400 MHz spectrometer operating at 400 MHz in
solvents, as noted. Proton coupling constants (J values) are rounded
to the nearest Hz. All solvents were HPLC grade or better. The
reactions were followed by TLC on precoated Uniplate silica gel
plates purchased from Analtech. The developed plates were
visualized using 254 nm UV illumination or by PMA stain. Flash
column chromatography on silica gel was performed on Redi Sep
prepacked disposable silica gel columns using an Isco Combiflash.
Reactions were carried out under an atmosphere of N2 at room
temperature, unless otherwise noted. Purity was evaluated by
analytical HPLC using a Varian Dynamax SD-200 pump coupled
to a Varian Dynamax UV-1 detector (system A), or a HP 110
Agilant LCMS using a Quaternary G1311A pump coupled to a
Micromass Platform LCZ detector (system B). In system A, the
solvents were the following: (A) water + 0.05% TFA and (B)
acetonitrile + 0.05% TFA, flow 1.2 mL/min. Column Vydac RP-
18, 5 m, 250 × 4.6 mm, Photodiode Array Detector at 220 nm;
In addition to the benzothiophene, the corresponding N-
methyl indole analogue 10 also demonstrated a similar binding
potency to 9 (Table 2). While compounds 9 and 10 represented
a new class of potent p38 inhibitors compared to 1, we felt that
these two inhibitors did not possess the level of in vitro potency
desired in a backup compound. We felt the adenine binding
site was a logical area to further explore, given that a number
of p38 inhibitors gain additional binding interactions in this
region.18
From the X-ray structure of 9, it appeared possible that the
inhibitor could interact not only with the N-H of Met109, but
also with the adjacent carbonyl by incorporating a hydrogen
bond donating group at the 6-position of the pyridine ring. To
test this hypothesis, we appended a variety of amine groups to
this position of the pyridine. In support of our hypothesis, we
found that 11 led to an improvement in molecular potency, while

p38 Inhibitors Via Computer-Assisted Drug Design
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4021
Figure 4. Two views of the cocrystal structure of 9.
N-(3-Amino-5-tert-butyl-2-methoxy-phenyl)-methanesulfona-
mide (25): A total of 210 g (0.82 mole) of 5-tert-butyl-2-methoxy-
1,3-dinitro-benzene was dissolved in ethyl acetate (1 L) in a stainless
steel autoclave. The autoclave was then purged with H2 and
pressured to 40 psi with vigorous stirring. The pressure was
maintained at 40 psi for 4 h, the autoclave was then vented, and
the reaction mixture was filtered through Celite, dried (MgSO4),
and concentrated in vacuo (151 g, 96%). The corresponding diamine
(147 g, 0.76 mole) was dissolved in MTBE (1.5 L), and Hu¨nig’s
base (198 mL, 1.14 mole) was added, followed by the dropwise
addition of methanesulfonyl chloride (58.6 mL, 0.76 mole) over
20 min. The resulting mixture was stirred at room temperature for
2 h, quenched with 1 M NaOH (1.5 L), and stirring was continued
for 10 min until the final pH ) 12. The reaction mixture was
extracted with 1 M NaOH, and the combined aqueous fractions
were then extracted with CH2Cl2. Concentrated HCl (175 mL) was
slowly added with vigorous stirring until the pH ) 1. The organic
fraction was then washed with water and concentrated in vacuo.
The resulting oil was dissolved in ethyl acetate and cooled to 0
°C, after which time HCl (4 M in 1,4-dioxane) was added. The
solution was stirred vigorously for 2 h, over which time the product
precipitated and was collected by filtration to provide a white solid
(127 g, 54%). 1H NMR (DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 3.0
(s, 3H), 3.6 (s, 3H), 4.9 (br s, 2H), 6.6 (s, 2H), 8.7 (s, 1H). ESMS
m/z ) 272 [M + H]+. HPLC purity: system B, tR 3.09 min (99%).
Figure 5. Cocrystal structure of 9 (green) overlaid with 8 (yellow).
N-(5-tert-Butyl-2-methoxy-3-{3-[4-(2-morpholin-4-yl-ethoxy)-
naphthalen-1-yl]-ureido}-phenyl)-methanesulfonamide (2): 4-(2-
Morpholin-4-yl-ethoxy)-naphthalen-1-ylamine (180 mg, 0.7 mmol)
was dissolved in 1:1 CH2Cl2/saturated aqueous NaHCO3 (40 mL).
Phosgene (0.9 mL, 20% solution in toluene, 1.6 mmol) was then
added, and the reaction was stirred vigorously for 20 min. The
reaction was extracted with CH2Cl2, and the organic fraction was
dried (MgSO4), filtered, and concentrated in vacuo. The crude
isocyanate was then dissolved in THF (10 mL), added to a solution
of 25 in THF (5 mL), and the reaction mixture was then stirred for
6 h. The reaction was concentrated in vacuo and the residue was
triturated with ether to afford the title compound as a white solid
Figure 6. Cocrystal structure of 19.
Table 3. In Vivo Studies in LPS-Stimulated Mouse Proof of Principle
Model
inhibition of TNFR
a
Tm
inhibition of TNFR
from LPS-treated mice
at dose (mg/kg)
cmpd
(°C)
from HuWBb (nM)
1
9
10
19
20
63.0
59.5
57.5
63.9
63.6
780
1100
3600
180
60% at 10
60% at 10
60% at 10
98% at 10; 80% at 3
75% at 10; 60% at 3
1
(150 mg, 40%). H NMR (DMSO-d6, 400 MHz): δ 1.2 (s, 9H),
140
2.4 (br s, 2H), 2.8 (br s, 2H), 3.1 (s, 3H), 3.3 (s, 2H), 3.6 (br s,
4H), 3.8 (s, 3H), 4.2 (br s, 2H), 7.0 (br s, 2H), 7.5-7.6 (m, 2H),
7.7 (d, 1H, J ) 2 Hz), 8.1 (d, 1H, J ) 2 Hz), 8.2 (br s, 1H), 8.3 (d,
1H, J ) 2 Hz), 8.6 (s, 1H), 9.0 (s, 1H), 9.1 (s, 1H). ESMS m/z )
571 [M + H]+. HPLC purity: system B, tR 2.66 min (99%).
N-[5-tert-Butyl-2-methoxy-3-(3-naphthalen-1-yl-ureido)-phen-
yl]-methanesulfonamide (3): To a solution of 25 (100 mg, 0.36
mmol) in THF (1 mL) was added 1-isocyanato-naphthalene (62
mg, 0.36 mmol). The reaction mixture was stirred for 12 h and
then concentrated in vacuo. Triturating with ethyl acetate afforded
the title compound as an off-white solid (150 mg, 95%). 1H NMR
(DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 3.1 (s, 3H), 3.6 (s, 3H), 6.9
(d, 1H, J ) 2 Hz), 7.4 (t, 1H, J ) 2 Hz), 7.5-7.7 (m, 4H), 7.9 (d,
1H, J ) 2 Hz), 8.1 (d, 1H, J ) 2 Hz), 8.2-8.3 (m, 1H), 8.9 (s,
1H), 9.1 (s, 1H), 9.3 (s, 1H). HPLC purity: system B, tR 5.31 min
(98%).
a Values are the mean of gtwo experiments, standard deviation 0.1-
0.5 °C unless otherwise noted. b Values are the geometric means from 8 to
12 donor experiments, standard deviation typically (50% of reported value.
Table 4. Sprague-Dawley Rat Pharmacokineticsa
cmpd
1
9
10
19
20
iv Cl (mL/min/kg)
PO Cmax (ng/mL)
F (%)
1.7
10 900
76
19
5200
57
3
4
5
9000
90
2400
51
14 000
51
a 1 mg/kg i.v.; 10 mg/kg p.o.
from 95% to 20% solvent (A) over 25 min. In system B, the solvents
were the following: (A) water + 0.1% formic acid and (B)
acetonitrile + 0.1% formic, flow 1.5 mL/min. Column Waters
Sunfire C-18 Column, 4.6 × 30 mm, 3.5 µm, Photodiode Array
Detector at 200 or 600 nm; from 95% to 5% solvent (A) over 10
min. Elemental analyses were performed by Quantitative Technolo-
gies, Whitehouse, NJ.
Benzofuran-2-carboxylic Acid (5-tert-Butyl-3-methanesulfo-
nylamino-2-methoxy-phenyl)-amide (4): Benzofuran-2-carboxylic
acid (60 mg, 0.37 mmol) was suspended in DMF (2 mL).
Diisopropylethylamine (40 mg, 70 µL, 0.40 mmol) followed by

4022 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Goldberg et al.
TBTU (128 mg, 0.40 mmol) and 25 (100 mg, 0.37 mmol) were
then added. The resulting suspension was stirred for 15 h and then
extracted with ethyl acetate. The organic fraction was washed with
water and brine, dried (MgSO4), filtered, and concentrated in vacuo.
Flash column chromatography on silica gel (2:1 hexanes/ethyl
acetate) afforded the title compound as a pale yellow solid (59 mg,
39%). 1H NMR (DMSO-d6, 400 MHz): δ 1.3 (s, 9H), 3.0 (s, 3H),
3.8 (s, 3H), 7.3 (d, 1H, J ) 2 Hz), 7.4 (t, 1H, J ) 8 Hz), 7.5 (t,
1H, J ) 8 Hz), 7.6 (d, 1H, J ) 2 Hz), 7.7 (d, 1H, J ) 8 Hz), 7.8
(s, 1H), 7.9 (d, 1H, J ) 8 Hz), 9.2 (s, 1H), 9.9 (s, 1H). ESMS m/z
) 417 [M + H]+. HPLC purity: system B, tR 5.31 min (99%).
Anal. Calcd for C21H24N2O5S‚0.25 H2O: C, 59.91; H, 5.87; N, 6.65.
Found: C, 60.85; H, 5.95; N, 6.30.
stirred for 72 h. The reaction was extracted with ethyl acetate, and
the organic fraction was washed with water and brine, dried
(MgSO4), filtered, and concentrated in vacuo. Flash column
chromatography on silica gel (100% ethyl acetate) afforded the title
compound as a pale yellow solid (22 mg, 36%). 1H NMR (DMSO-
d6, 400 MHz): δ 1.1 (s, 9H), 2.9 (s, 3H), 3.5 (s, 3H), 7.1 (d, 1H,
J ) 2 Hz), 7.2 (d, 1H, J ) 2 Hz), 7.4 (d, 1H, J ) 8 Hz), 7.5-7.6
(m, 2H), 7.8 (d, 1H, J ) 8 Hz), 8.2 (d, 1H, J ) 8 Hz), 8.3 (s, 1H),
8.6 (d, 1H, J ) 8 Hz), 9.0 (s, 1H), 9.1 (s, 1H), 10.0 (s, 1H), 10.6
(s, 1H). ESMS m/z ) 553 [M + H]+.
7-Nitro-benzo[b]thiophene-2-carboxylic Acid Methyl Ester
(30): Compound 29 (11 g, 60 mmol) was dissolved in DMF (100
mL) and cooled to 0 °C. To this solution was added K2CO3 (9.1 g,
66 mmol) followed by methyl thioglycolate (6.4 g, 61 mmol), and
the reaction was warmed to room temperature. The reaction mixture
was stirred for 12 h, after which time a solid precipitate had formed.
Water was added to provide a homogeneous solution, and the
reaction mixture was cooled to 0 °C to precipitate the desired
product. The solid was filtered and washed with water until the
filtrate was clear and colorless, providing the title compound as a
white solid (13.3 g, 94%). 1H NMR (DMSO-d6, 400 MHz): δ 3.9
(s, 3H), 7.8 (t, 1H, J ) 8 Hz), 8.4 (s, 1H), 8.5 (d, 1H, J ) 8 Hz),
8.6 (d, 1H, J ) 8 Hz), ESMS m/z ) 238 [M + H]+.
7-Amino-benzo[b]thiophene-2-carboxylic Acid Methyl Ester
(31): A mixture of 30 (1.0 g, 4.2 mmol) and 10% Pd/C (400 mg)
was suspended in ethyl acetate (40 mL). The reaction was placed
under 1 atm of H2 (balloon). The black suspension was stirred for
12 h, then filtered through Celite, and washed with ethyl acetate
until the filtrate was clear and colorless. The filtrate was dried
(MgSO4), filtered, and then concentrated in vacuo to afford the
title compound as a yellow solid (860 mg, 98%). 1H NMR (DMSO-
d6, 400 MHz): δ 3.9 (s, 3H), 5.6 (s, 2H), 6.7 (d, 1H, J ) 8 Hz),
7.1 (t, 1H, J ) 8 Hz), 7.2 (d, 1H, J ) 8 Hz), 8.0 (s, 1H). ESMS
m/z ) 207 [M + H]+.
Benzo[b]thiophene-2-carboxylic Acid (5-tert-Butyl-3-meth-
anesulfonylamino-2-methoxy-phenyl)-amide (5): Compound 5
was prepared as described above from benzo[b]thiophene-2-
carboxylic acid to afford the title compound as a pale yellow solid
(50 mg, 16%). 1H NMR (DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 3.0
(s, 3H), 3.7 (s, 3H), 7.2 (d, 1H, J ) 2 Hz), 7.5-7.6 (m, 3H), 8.0
(d, 1H, J ) 7 Hz), 8.1 (d, 1H, J ) 7 Hz), 8.3 (s, 1H), 9.2 (s, 1H),
10.1 (s, 1H). ESMS m/z ) 433 [M + H]+. HPLC purity: system
B, tR 5.46 min (99%). Anal. Calcd for C21H24N2O4S2‚0.5H2O: C,
57.12; H, 5.71; N, 6.34. Found: C, 56.96; H, 5.47; N, 6.19.
1H-Indole-2-carboxylic Acid (5-tert-Butyl-3-methanesulfony-
lamino-2-methoxy-phenyl)-amide (6): Compound 6 was prepared
as described above from indole-2-carboxylic acid to afford the title
1
compound as a beige solid (9 mg, 6%). H NMR (DMSO-d6, 400
MHz): δ 1.2 (s, 9H), 3.0 (s, 3H), 3.7 (s, 3H), 7.0 (t, 1H, J ) 8
Hz), 7.1-7.2 (m, 2H), 7.3 (s, 1H), 7.4 (d, 1H, J ) 8 Hz), 7.5 (s,
1H), 7.6 (d, 1H, J ) 8 Hz), 9.1 (s, 1H), 9.7 (s, 1H), 11.7 (s, 1H).
ESMS m/z ) 414 [M - H]-.
1-Methyl-1H-indole-2-carboxylic Acid (5-tert-Butyl-3-meth-
anesulfonylamino-2-methoxy-phenyl)-amide (7): Compound 7
was prepared as described above to afford the title compound as a
white solid (50 mg, 20%). 1H NMR (DMSO-d6, 400 MHz): δ 1.3
(s, 9H), 3.1 (s, 3H), 3.8 (s, 3H), 4.0 (s, 3H), 7.1 (t, 1H, J ) 8 Hz),
7.2 (d, 1H, J ) 2 Hz), 7.3-7.4 (m, 2H), 7.5 (d, 1H, J ) 2 Hz), 7.6
(d, 1H, J ) 8 Hz), 7.7 (d, 1H, J ) 8 Hz), 9.2 (s, 1H), 9.8 (s, 1H),
ESMS m/z ) 430 [M + H]+.
4-Nitro-benzo[b]thiophene-2-carboxylic Acid (5-tert-Butyl-3-
methanesulfonylamino-2-methoxy-phenyl)-amide (27): 4-Nitro-
benzo[b]thiophene-2-carboxylic acid (60 mg, 0.27 mmol) was
suspended in DMF (2 mL). Diisopropylethylamine (40 mg, 70 µL,
0.40 mmol) followed by TBTU (128 mg, 0.40 mmol) and 25 (100
mg, 0.37 mmol) was then added. The resulting suspension was
stirred for 15 h and then extracted with ethyl acetate. The organic
fraction was washed with water and brine, dried (MgSO4), filtered,
and concentrated in vacuo. Flash column chromatography on silica
gel (2:1 hexanes/ethyl acetate) afforded the title compound as a
pale yellow solid (59 mg, 39%). 1H NMR (DMSO-d6, 400 MHz):
δ 1.2 (s, 9H), 3.0 (s, 3H), 3.7 (s, 3H), 7.3 (d, 1H, J ) 2 Hz), 7.4
(d, 1H, J ) 2 Hz), 7.7 (t, 1H, J ) 8 Hz), 8.4 (d, 1H, J ) 8 Hz),
8.5 (d, 1H, J ) 8 Hz), 9.1 (s, 1H), 9.2 (s, 1H), 10.7 (s, 1H) ESMS
m/z ) 476 [M - H]-.
7-[(Pyridine-3-carbonyl)-amino]-benzo[b]thiophene-2-car-
boxylic Acid (32): Nicotinic acid (400 mg, 3.3 mmol) was
suspended in CH2Cl2 (20 mL). Triethylamine (330 mg, 3.3 mmol,
445 µL) followed by BopCl (830 mg, 3.3 mmol) was then added,
and the suspension was stirred for 15 min. A solution of 31 (450
mg, 2.2 mmol) in CH2Cl2 (2 mL) was added, and the reaction
mixture was stirred for 12 h. The reaction was concentrated in vacuo
and extracted with ethyl acetate. The organic fraction was washed
with water and brine, dried (MgSO4), filtered, and concentrated in
vacuo to afford the corresponding amide as a yellow solid (460
1
mg, 68%). H NMR (DMSO-d6, 400 MHz): δ 3.9 (s, 3H), 7.6-
7.7 (m, 3H), 7.9 (d, 1H, J ) 8 Hz), 8.2 (s, 1H), 8.4 (d, 1H, J ) 8
Hz), 8.8 (d, 1H, J ) 8 Hz), 9.2 (s, 1H), 10.9 (s, 1H). ESMS m/z )
313 [M + H]+.
The above amide was suspended in THF (25 mL) and water (8
mL) and LiOH‚H2O (200 mg, 4.9 mmol) was added. The reaction
mixture was stirred for 24 h. The solvent was removed in vacuo
and extracted with ethyl acetate. The aqueous fraction was acidified
with 1 M HCl to precipitate the product, which was then collected
1
by filtration to provide a white solid (390 mg, 54%). H NMR
4-Amino-benzo[b]thiophene-2-carboxylic Acid (5-tert-Butyl-
3-methanesulfonylamino-2-methoxy-phenyl)-amide (28): A mix-
ture of 27 (200 mg, 0.42 mmol) and 10% Pd/C (50 mg) was
suspended in ethyl acetate (2 mL). The reaction was placed under
1 atm of H2 (balloon). The black suspension was stirred for 12 h
then filtered through Celite and washed with ethyl acetate until the
filtrate was clear and colorless. The filtrate was dried (MgSO4) and
concentrated in vacuo to afford the title compound as a pale yellow
(DMSO-d6, 400 MHz): δ 7.6-7.7 (m, 3H), 7.9 (d, 1H, J ) 8 Hz),
8.1 (s, 1H), 8.4 (d, 1H, J ) 8 Hz), 8.8 (d, 1H, J ) 8 Hz), 9.2 (s,
1H), 10.8 (s, 1H), 13.5 (s, 1H). ESMS m/z ) 299 [M + H]+.
7-[(6-Chloro-pyridine-3-carbonyl)-amino]-benzo[b]thiophene-
2-carboxylic Acid (33): To a solution of 6-chloronicotinic acid
(490 mg, 3.1 mmol) and triethylamine (800 uL, 5.9 mmol) in CH2-
Cl2 (20 mL) was added BopCl (1.5 g, 5.9 mmol). After 15 min, a
solution of 31 (540 mg, 2.6 mmol) in CH2Cl2 (10 mL) was added.
The reaction was stirred for 12 h and then extracted with ethyl
acetate. The organic fraction was washed with saturated aqueous
NaHCO3, water, and brine, dried (MgSO4), filtered, and concen-
trated in vacuo. Flash column chromatography on silica gel (4:1
hexanes/ethyl acetate) provided the corresponding amide as a light
1
solid (170 mg, 90%). H NMR (DMSO-d6, 400 MHz): δ 1.2 (s,
9H), 3.0 (s, 3H), 3.7 (s, 3H), 5.8 (br s, 2H), 6.5 (d, 1H, J ) 8 Hz),
7.1-7.2 (m, 2H), 7.3 (d, 1H, J ) 2 Hz), 7.7 (d, 1H, J ) 2 Hz), 8.5
(s, 1H), 9.1 (br s, 1H), 9.5 (s, 1H). ESMS m/z ) 448 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phe-
nylcarbamoyl)-benzo[b]thiophen-4-yl]-nicotinamide (8): Nico-
tinic acid (19 mg, 0.11 mmol) and 28 (50 mg, 0.1 mmol) were
dissolved in DMF (2 mL). EDC (21 mg, 0.11 mmol) and HOBt
(15 mg, 0.1 mmol) were then added, and the brown solution was
1
yellow solid (613 mg, 68%). H NMR (DMSO-d6, 400 MHz): δ
3.9 (s, 3H), 7.6-7.5 (m, 2H), 7.8 (d, 1H, J ) 8 Hz), 7.9 (dd, 1H,
J ) 8.0, 2 Hz), 8.3 (s, 1H), 8.4 (dd, 1H, J ) 8, 2 Hz), 9.0 (d, 1H,
J ) 2 Hz), 10.9 (s, 1H).

p38 Inhibitors Via Computer-Assisted Drug Design
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4023
The above amide (91 mg, 0.26 mmol) was dissolved in 1:1 THF/
MeOH (6 mL) and 1 N NaOH (660 µL, 0.66 mmol) was then added.
The solution was stirred for 12 h and then extracted with CH2Cl2.
The aqueous fraction was acidified with 1 N HCl until the pH )
4 and then extracted with CH2Cl2. The organic fraction was washed
with water and brine, dried (MgSO4), filtered, and concentrated in
vacuo to provide the title compound as a light yellow solid (90
mg, 100%). 1H NMR (DMSO-d6, 400 MHz): δ 7.5-7.6 (m, 2H),
7.8 (d, 1H, J ) 8 Hz), 7.9 (d, 1H, J ) 8 Hz), 8.2 (s, 1H), 8.4 (d,
1H, J ) 2 Hz), 9.0 (d, 1H, J ) 2 Hz), 10.9 (s, 1H).
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phenyl-
carbamoyl)-benzo[b]thiophen-7-yl]-nicotinamide (9): Compound
32 (20 g, 67 mmol) and 25 (18.6 g, 67 mmol) were suspended in
DMF (450 mL), and diisopropylethylamine (17 g, 23 mL, 134
mmol) was then added. The reaction mixture was stirred for 20
min, then HATU (30.6 g, 80.5 mmol) and HOBt (10.9 g, 80.5
mmol) were added. The solution was stirred for an additional 2
days. After 2 d, the reaction was poured into water and the aqueous
portion was decanted, and the resultant brown residue was
suspended in CH2Cl2 to afford a yellow suspension that was then
filtered through Celite. The filtrate was dried (MgSO4), filtered,
and concentrated in vacuo to afford the title compound as a yellow
solid (27 g, 73%). 1H NMR (DMSO-d6, 400 MHz): δ 1.3 (s, 9H),
3.1 (s, 3H), 3.7 (s, 3H), 7.3 (d, 1H, J ) 2 Hz), 7.4 (d, 1H, J ) 2
Hz), 7.5-7.6 (m, 2H), 7.7 (dd, 1H, J) 7, 2 Hz), 7.9 (dd, 1H, J)
7, 2 Hz), 8.4 (dt, 1H, J ) 6, 2 Hz), 8.4 (s, 1H), 8.8 (dd, 1H, J) 6,
2 Hz), 9.2 (d, 1H, J ) 2 Hz), 9.2 (s, 1H), 10.2 (s, 1H), 10.9 (s,
1H). ESMS m/z ) 553 [M + H]+.
9H), 3.0 (s, 3H), 3.7 (s, 3H), 3.9 (s, 3H), 5.4 (s, 2H), 6.4 (d, 1H,
J ) 8 Hz), 6.9-7.0 (m, 2H), 7.3 (d, 1H, J ) 2 Hz), 7.4 (d, 1H, J
) 2 Hz), 7.6 (d, 1H, J ) 2 Hz), 9.8 (s, 1H), 11.4 (s, 1H). ESMS
m/z ) 431 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phenyl-
carbamoyl)-1-methylindol-7-yl]-nicotinamide (10): Compound 37
(218 mg, 0.5 mmol), nicotinic acid (74 mg, 0.6 mmol), HOBt (80
mg, 0.6 mmol), and EDC (115 mg, 0.6 mmol) were suspended in
DMF (5 mL). After 12 h, additional nicotinic acid (37 mg, 0.3
mmol), HOBt (41 mg, 0.3 mmol), and EDC (58 mg, 0.3 mmol)
were added, and the reaction mixture was stirred for an additional
5 h. The reaction was then extracted with ethyl acetate, and the
organic fraction was washed with water and brine, dried (Na2SO4),
filtered, and concentrated in vacuo. Preparative HPLC afforded the
title compound as a white solid (160 mg, 59%). 1H NMR (DMSO-
d6, 400 MHz): δ 1.2 (s, 9H), 3.0 (s, 3H), 3.7 (s, 3H), 4.0 (s, 3H),
7.0-7.1 (m, 2H), 7.2 (d, 1H, J ) 2 Hz), 7.3 (s, 1H), 7.5 (d, 1H, J
) 2 Hz), 7.6 (dd, 1H, J ) 8, 5 Hz), 7.7 (dd, 1H, J ) 7, 2 Hz), 8.4
(dd, 1H, J ) 8, 2 Hz), 8.8 (dd, 1H, J ) 5, 2 Hz), 9.2 (m, 2H), 9.8
(s, 1H), 10.6 (s, 1H). ESMS m/z ) 550 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-
phenylcarbamoyl)-benzo[b]thiophen-7-yl]-6-chloro-
nicotinamide (38a): To a solution of 33 (50 mg, 0.15 mmol) in
DMF (1.5 mL) was added EDC (43 mg, 0.22 mmol) and HOBt
(30 mg, 0.22 mmol). The reaction was stirred for 15 min, then 25
(49 mg, 0.18 mmol) was added. The reaction was stirred for 12 h,
and then DMF was removed and the residue was taken up in ethyl
acetate. The organic fraction was washed with water and brine,
dried (MgSO4), filtered, and concentrated in vacuo. Flash column
chromatography on silica gel (5:1 hexanes/ethyl acetate) afforded
the title compound as a white solid (36 mg, 41%). 1H NMR
(DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 3.0 (s, 3H), 3.7 (s, 3H), 7.2
(d, 1H, J ) 2 Hz), 7.4 (d, 1H, J ) 2 Hz), 7.5-7.6 (m, 2H), 7.7 (d,
1H, J ) 8 Hz), 7.9 (dd, 1H, J ) 7, 2 Hz), 8.4-8.5 (m, 2H), 9.0 (d,
1H, J ) 2 Hz), 9.2 (s, 1H), 10.2 (s, 1H), 10.9 (s, 1H).
7-Nitroindole-2-carboxylic Acid (5-tert-Butyl-3-methane-
sulfonylamino-2-methoxy-phenyl)-amide (36): To a suspension
of NaH (60% w/w in mineral oil, 0.3 g, 6.41 mmol) in DMF (20
mL) was added ethyl 7-nitroindole-2-carboxylate (1.5 g, 6.4 mmol).
The reaction mixture was stirred for 1 h and then MeI (0.8 mL,
13.0 mmol) in DMF (4 mL) was added dropwise. After 5.5 h, the
reaction mixture was poured onto ice and extracted with ethyl
acetate. The organic fraction was washed with water and brine,
dried (MgSO4), filtered, and concentrated in vacuo. Flash column
chromatography on silica gel provides 35 as an off-white solid (800
7-[(6-Chloro-pyridine-3-carbonyl)-amino]-1-methyl-indole-2-
carboxylic Acid (5-tert-Butyl-3-methanesulfonylamino-2-meth-
oxy-phenyl)-amide (38b): A mixture of 36 (310 mg, 0.7 mmol),
6-chloro nicotinic acid (170 mg, 1.1 mmol), HOBt (149 mg, 1.1
mmol), and EDC (210 mg, 1.1 mmol) were dissolved in DMF (5
mL) and stirred for 16 h. The reaction mixture was extracted with
ethyl acetate, and the organic fraction was washed with water and
brine, dried (Na2SO4), filtered, and concentrated in vacuo. Flash
column chromatography on silica gel (3:1 hexanes/ethyl acetate)
1
mg, 50%). H NMR (DMSO-d6, 400 MHz): δ 1.5 (t, 3H, J ) 2
Hz), 4.0 (s, 3H), 4.4 (q, 2H, J ) 2 Hz), 7.1 (t, 1H, J ) 2 Hz), 7.4
(s, 1H), 7.8 (d, 1H, J ) 2 Hz), 7.9 (dd, 2H, J ) 8, 2 Hz).
The above ester (0.54 g) was dissolved in EtOH (15 mL) and 2
N aqueous NaOH (5 mL) and heated to reflux for 2.5 h. The
reaction was then cooled to room temperature and stirred for an
additional 12 h. The ethanol was removed in vacuo, and the residue
was acidified with 2 N HCl. A white precipitate formed, was
collected by filtration, washed with water, and dried in vacuo.
Recrystallization from 2-propanol provided the corresponding
carboxylic acid as a white solid (250 mg, 52%). 1H NMR (DMSO-
d6, 400 MHz): δ 3.8 (s, 3H), 7.3 (t, 1H, J ) 2 Hz), 7.4 (s, 1H), 7.8
(d, 1H, J ) 2 Hz), 8.1 (d, 1H, J ) 2 Hz), 13.1 (br s, 1H).
The above acid (1.0 g, 4.9 mmol), 25 (1.3 g, 4.9 mmol), HOBt
(655 mg, 4.9 mmol), and EDC (930 mg, 4.9 mmol) were dissolved
in DMF (25 mL) and stirred for 12 h. The reaction mixture was
then extracted with ethyl acetate. The organic fraction was washed
with saturated aqueous NH4Cl, water, and brine, dried (MgSO4),
filtered, and concentrated in vacuo. Flash column chromatography
on silica gel (4:1 hexanes/ethyl acetate) afforded the title compound
1
afforded the title compound as a yellow foam (350 mg, 86%). H
NMR (DMSO-d6, 400 MHz): δ 1.3 (s, 9H), 3.0 (s, 3H), 3.7 (s,
3H), 4.0 (s, 3H), 7.0-7.1 (m, 2H), 7.2 (d, 1H, J ) 2 Hz), 7.3 (s,
1H), 7.5 (d, 1H, J ) 2 Hz), 7.7 (dd, 1H, J ) 7, 2 Hz), 7.8 (d, 1H,
J ) 8 Hz), 8.4 (dd, 1H, J ) 8, 2 Hz), 9.0 (d, 1H, J ) 2 Hz), 9.2
(s, 1H), 9.9 (s, 1H), 10.6 (s, 1H). ESMS m/z ) 584 [M + H]+.
6-Amino-N-[2-(5-tert-butyl-3-methanesulfonylamino-2-meth-
oxy-phenylcarbamoyl)-benzo[b]thiophen-7-yl]-nicotinamide (11):
Compound 38a (34 mg, 0.06 mmol) was placed in a sealed tube
and 4-methoxybenzylamine (200 µL) was added. The vessel was
purged with argon and then heated to 100 °C. After 4 h, the reaction
mixture was cooled to room temperature and extracted with ethyl
acetate. The organic fraction was washed with water and brine,
dried (MgSO4), filtered, and concentrated in vacuo. Flash column
chromatography on silica gel (1:9 MeOH/CH2Cl2) provided the
corresponding benzyl amine as an off-white solid (26 mg, 65%).
1H NMR (DMSO-d6, 400 MHz): δ 1.3 (s, 9H), 3.0 (s, 3H), 3.7 (s,
6H), 4.5 (d, 2H, J ) 6 Hz), 6.4 (d, 1H, J ) 8 Hz), 6.8 (d, 1H, J )
8 Hz), 7.2-7.3 (m, 3H), 7.4 (d, 1H, J ) 2 Hz), 7.5-7.6 (m, 2H),
7.7-7.8 (m, 3H), 7.9 (dd, 1H, J ) 6, 2 Hz), 8.0-8.1 (m, 1H), 8.3
(s, 1H), 8.7 (d, 1H, J ) 2 Hz), 9.2 (s, 1H), 10.1 (s, 1H), 10.2 (s,
1H). ESMS m/z ) 688 [M + H]+.
1
as a white solid (700 mg, 31%). H NMR (DMSO-d6, 400 MHz):
δ 1.3 (s, 9H), 3.1 (s, 3H), 3.7 (s, 3H), 3.9 (s, 3H), 7.3 (d, 1H, J )
2 Hz), 7.4 (t, 1H, J ) 8 Hz), 7.6-7.7 (m, 2H), 8.3 (m, 2H), 9.3 (s,
1H), 11.8 (s, 1H). ESMS m/z ) 461 [M + H]+.
7-Aminoindole-2-carboxylic Acid (5-tert-Butyl-3-methane-
sulfonylamino-2-methoxy-phenyl)-amide (37): A mixture of 36
(60 mg, 0.13 mmol) and 10% Pd/C (20 mg) were suspended in
ethyl acetate (2 mL). The reaction was placed under 1 atm of H2
(balloon). The black suspension was stirred for 12 h then filtered
through Celite and washed with ethyl acetate until the filtrate was
clear and colorless. Concentration in vacuo followed by triturating
with 1:1 CH2Cl2/ether afforded the title compound was an off-white
The above amine (94 mg, 0.1 mmol) was placed in a sealed
tube and trifluoroacetic acid (800 µL) was added. The solution was
purged with argon and then heated to 75 °C for 12 h. The reaction
was cooled to room temperature, diluted with toluene, and
concentrated in vacuo. The residue was taken up in ethyl acetate
1
solid (55 mg, 98%). H NMR (DMSO-d6, 400 MHz): δ 1.3 (s,

4024 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Goldberg et al.
and washed with saturated aqueous NaHCO3, water and brine, dried
(MgSO4), filtered, and concentrated in vacuo. Flash column
chromatography on silica gel (1:13 MeOH/CH2Cl2) afforded the
title compound as a white solid (54 mg, 70%). 1H NMR (DMSO-
d6, 400 MHz): δ 1.3 (s, 9H), 3.0 (s, 3H), 3.7 (s, 3H), 6.5 (d, 1H,
J ) 9 Hz), 6.7 (s, 2H), 7.3 (d, 1H, J ) 2 Hz), 7.4 (d, 1H, J ) 2
Hz), 7.5-7.6 (m, 2H), 7.9 (d, 1H, J ) 7 Hz), 8.0 (d, 1H, J ) 9
Hz), 8.4 (s, 1H), 8.6 (s, 1H), 9.2 (s, 1H), 10.1 (s, 1H), 10.3 (s, 1H).
HPLC purity: system A, tR 8.94 min (99%). ESMS m/z ) 568 [M
+ H]+.
anesulfonylamino-2-methoxy-phenyl)-amide (18): Compound 18
was prepared as described above (50%, off-white solid). 1H NMR
(DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 2.3-2.4 (m, 6H), 3.0 (s,
3H), 3.4-3.5 (m, 2H), 3.6 (t, 4H, J ) 4 Hz), 3.7 (s, 3H), 4.0 (s,
3H), 6.5 (d, 1H, J ) 9 Hz), 7.0-7.1 (m, 3H), 7.2 (d, 1H, J ) 2
Hz), 7.4 (s, 1H), 7.5 (d, 1H, J ) 2 Hz), 7.6 (dd, 1H, J ) 8, 2 Hz),
7.9 (d, 1H, J ) 8 Hz), 8.7 (d, 1H, J ) 2 Hz), 9.2 (s, 1H), 9.8 (s,
1H), 10.0 (s, 1H). HPLC purity: system A, tR 9.86 min (97%).
ESMS m/z ) 678 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phenyl-
carbamoyl)-benzo[b]thiophen-7-yl]-6-cyclopropylamino-nicotinamide
(19): Compound 19 was prepared as described above to provide
the title compound as an off-white solid (71%). 1H NMR (DMSO-
d6, 400 MHz): δ 0.4-0.5 (m, 2H), 0.7-0.8 (m, 2H), 1.3 (s, 9H),
2.5-2.6 (m, 1H), 3.0 (s, 3H), 3.7 (s, 3H), 6.7 (d, 1H, J ) 9 Hz),
7.2 (d, 1H, J ) 2 Hz), 7.4 (d, 1H, J ) 2 Hz), 7.5-7.6 (m, 3H), 7.9
(dd, 1H, J ) 6, 2 Hz), 8.0 (d, 1H, J ) 9 Hz), 8.3 (s, 1H), 8.7 (d,
1H, J ) 2 Hz), 9.2 (s, 1H), 10.1 (s, 1H), 10.3 (s, 1H). HPLC
purity: system A, tR 9.42 min (99%). ESMS m/z ) 608 [M +
H]+.
7-[(6-Cyclopropylamino-pyridine-3-carbonyl)-amino]-1-
methyl-indole-2-carboxylic Acid (5-tert-Butyl-3-methanesulfo-
nylamino-2-methoxy-phenyl)-amide (20): Compound 20 was
prepared as described above (45%, off-white solid). 1H NMR
(DMSO-d6, 400 MHz): δ 0.4-0.5 (m, 2H), 0.7-0.8 (m, 2H), 1.2
(s, 9H), 2.5-2.6 (m, 1H), 3.0 (s, 3H), 3.7 (s, 3H), 4.0 (s, 3H), 6.6
(d, 1H, J ) 9 Hz), 7.1-7.2 (m, 2H), 7.3 (d, 1H, J ) 2 Hz), 7.4 (s,
1H), 7.5 (d, 1H, J ) 2 Hz), 7.6 (d, 1H, J ) 2 Hz), 7.7 (dd, 1H, J
) 7, 2 Hz), 8.0 (d, 1H, J ) 7 Hz), 8.7 (d, 1H, J ) 2 Hz), 9.1 (s,
1H), 9.8 (s, 1H), 10.0 (s, 1H). HPLC purity: system A, tR 9.33
min (99%), system B, tR 3.18 min (99%). ESMS m/z ) 605 [M +
H]+. Anal. Calcd for C31H36N3O5S: C, 61.57; H, 6.00; N, 13.90.
Found: C, 61.11; H, 5.94; N, 13.46.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phenyl-
carbamoyl)-benzo[b]thiophen-7-yl]-6-(cyclopropylmethyl-amino)-nicotinamide
(21): Compound 21 was prepared as described above (68%, off-
white solid). 1H NMR (DMSO-d6, 400 MHz): δ 0.2-0.3 (m, 2H),
0.4-0.5 (m, 2H), 1.0-1.1 (m, 1H), 1.2 (s, 9H), 3.0 (s, 3H), 3.1-
3.2 (m, 2H), 3.7 (s, 3H), 4.0 (s, 3H), 6.5 (d, 1H, J ) 9 Hz), 7.2 (d,
1H, J ) 2 Hz), 7.3 (s, 1H), 7.4 (d, 1H, J ) 2 Hz), 7.5-7.6 (m,
2H), 7.8 (dd, 1H, J ) 2, 7 Hz), 7.9 (d, 1H, J ) 9 Hz), 8.3 (s, 1H),
8.7 (d, 1H, J ) 2 Hz), 9.2 (s, 1H), 10.1 (s, 1H), 10.2 (s, 1H). HPLC
purity: system A, tR 9.63 min (99%). ESMS m/z ) 622 [M +
H]+. Anal. Calcd for C31H35N5O5S2: C, 59.88; H, 5.67; N, 11.26.
Found: C, 59.47; H, 5.65; N, 11.14.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phe-
nylcarbamoyl)-benzo[b]thiophen-7-yl]-6-methylamino-nicotina-
mide (12): Prepared as described above (30%, off-white solid).
1H NMR (DMSO-d6, 400 MHz): δ 1.3 (s, 9H), 2.8 (d, 3H, J ) 4
Hz), 3.3 (s, 3H), 3.7 (s, 3H), 6.5 (d, 1H, J ) 8 Hz), 7.2 (s, 1H), 7.4
(s, 1H), 7.5 (d, 2H, J ) 4 Hz), 7.8 (d, 1H, J ) 6 Hz), 7.9 (dd, 2H,
J ) 8, 6 Hz), 8.3 (d, 1H, J ) 6 Hz), 8.7 (s, 1H), 9.2 (s, 1H), 10.2
(s, 1H), 10.3 (s, 1H). HPLC purity: system A, tR 9.03 min (99%),
system B, tR 2.98 min (99%). ESMS m/z ) 582 [M + H]+.
1-Methyl-7-[(6-methylamino-pyridine-3-carbonyl)-amino]-in-
dole-2-carboxylic Acid (5-tert-Butyl-3-methanesulfonylamino-
2-methoxy-phenyl)-amide (13): Compound 13 was prepared as
described above (30%, off-white solid). 1H NMR (DMSO-d6, 400
MHz): δ 1.2 (s, 9H), 2.8 (d, 3H, J ) 5 Hz), 3.0 (s, 3H), 3.7 (s,
3H), 4.0 (s, 3H), 6.5 (d, 1H, J ) 9 Hz), 7.0-7.1 (m, 2H), 7.2-7.3
(m, 1H), 7.4 (d, 1H, J ) 2 Hz), 7.5 (s, 1H), 7.6 (d, 1H, J ) 2 Hz),
7.7 (dd, 1H, J ) 8, 2 Hz), 7.9 (d, 1H, J ) 8 Hz), 8.7 (s, 1H), 9.1
(s, 1H), 9.8 (s, 1H), 10.0 (s, 1H). ESMS m/z ) 579 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxyphenyl-
carbamoyl)-benzo[b]thiophen-7-yl]-6-dimethylamino-nicotina-
mide (14): Compound 14 was prepared as described above (50%,
1
off-white solid). H NMR (DMSO-d6, 400 MHz): δ 1.2 (s, 9H),
3.0 (s, 3H), 3.1 (s, 6H), 3.7 (s, 3H), 6.7 (d, 1H, J ) 9 Hz), 7.2 (d,
1H, J ) 2 Hz), 7.4 (s, 1H), 7.5-7.6 (m, 2H), 7.8 (dd, 1H, J ) 9,
3 Hz), 8.1 (dd, 1H, J ) 9, 3 Hz), 8.3 (s, 1H), 8.7 (d, 1H, J ) 2
Hz), 9.2 (s, 1H), 10.1 (s, 1H), 10.3 (s, 1H). HPLC purity: system
A, tR 8.94 min (97%). ESMS m/z ) 596 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phe-
nylcarbamoyl)-benzo[b]thiophen-7-yl]-6-(2-dimethylamino-ethy-
lamino)-nicotinamide (15): Compound 15 was prepared as de-
1
scribed above (34%, off-white solid). H NMR (DMSO-d6, 400
MHz): δ 1.2 (s, 9H), 2.2 (s, 6H), 2.4-2.5 (m, 2H), 3.0 (s, 3H),
3.4-3.5 (m, 2H), 3.7 (s, 3H), 6.9 (d, 1H, J ) 9 Hz), 7.2 (d, 1H, J
) 2 Hz), 7.3 (d, 1H, J ) 2 Hz), 7.4-7.5 (m, 2H), 7.8 (dd, 1H, J
) 6.0, 2 Hz), 7.9 (dd, 1H, J ) 9, 2 Hz), 8.3 (s, 1H), 8.7 (d, 1H, J
) 2 Hz), 9.2 (br s, 1H), 10.1 (s, 1H), 10.2 (s, 1H). HPLC purity:
system A, tR 8.54 min (99%), system B, tR 2.61 min (99%). ESMS
m/z ) 639 [M + H]+.
7-{[6-(2-Dimethylamino-ethylamino)-pyridine-3-carbonyl]-
amino}-1-methyl-indole-2-carboxylic Acid (5-tert-Butyl-3-meth-
anesulfonylamino-2-methoxy-phenyl)-amide (16): Compound 16
was prepared as described above (42%, off-white solid). 1H NMR
(DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 2.1 (s, 6H), 2.3-2.4 (m,
2H), 3.0 (s, 3H), 3.4-3.5 (m, 2H), 3.7 (s, 3H), 4.0 (s, 3H), 6.5 (d,
1H, J ) 9 Hz), 7.0-7.1 (m, 3H), 7.2-7.3 (m, 1H), 7.4 (d, 1H, J
) 2 Hz), 7.5 (d, 1H, J ) 2 Hz), 7.6 (d, 1H, J ) 7, 2 Hz), 7.9 (d,
1H, J ) 7 Hz), 8.7 (d, 1H, J ) 2 Hz), 9.1 (s, 1H), 9.8 (s, 1H), 10.0
(s, 1H). HPLC purity: system A, tR 8.38 min (99%). ESMS m/z )
636 [M + H]+.
7-{[6-(Cyclopropylmethyl-amino)-pyridine-3-carbonyl]-amino}-
1-methyl-indole-2-carboxylic Acid (5-tert-Butyl-3-methanesulfo-
nylamino-2-methoxy-phenyl)-amide (22): Compound 22 was
prepared as described above (63%, off-white solid). 1H NMR
(DMSO-d6, 400 MHz): δ 0.2-0.3 (m, 2H), 0.4-0.5 (m, 2H), 0.9-
1.0 (m, 1H), 1.2 (s, 9H), 3.0 (s, 3H), 3.2 (t, 2H, J ) 6 Hz), 3.7 (s,
3H), 4.0 (s, 3H), 6.5 (d, 1H, J ) 9 Hz), 7.0-7.1 (m, 2H), 7.2 (d,
1H, J ) 2 Hz), 7.3-7.4 (m, 2H), 7.5 (d, 1H, J ) 2 Hz), 7.6 (dd,
1H, J ) 7, 2 Hz), 7.9 (dd, 1H, J ) 7, 2 Hz), 8.6 (d, 1H, J ) 2 Hz),
9.1 (s, 1H), 9.8 (s, 1H), 10.0 (s, 1H). HPLC purity: system A, tR
9.67 min (98%). ESMS m/z ) 619 [M + H]+.
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phenyl-
carbamoyl)-benzo[b]thiophen-7-yl]-6-(2-methoxy-ethylamino)-nicotinamide
(23): Compound 23 was prepared as described above (55%, off-
1
N-[2-(5-tert-Butyl-3-methanesulfonylamino-2-methoxy-phe-
nylcarbamoyl)-benzo[b]thiophen-7-yl]-6-(2-morpholin-4-yl-ethy-
lamino)-nicotinamide (17): Compound 17 was prepared as de-
white solid). H NMR (DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 3.1
(s, 3H), 3.3 (s, 3H), 3.4-3.5 (m, 4H), 3.7 (s, 3H), 6.6 (d, 1H, J )
9 Hz), 7.2 (d, 1H, J ) 2 Hz), 7.3 (s, 1H), 7.4 (d, 1H, J ) 2 Hz),
7.5-7.6 (m, 2H), 7.8 (dd, 1H, J ) 6, 2 Hz), 7.9 (dd, 1H, J ) 9, 2
Hz), 8.3 (s, 1H), 8.7 (d, 1H, J ) 2 Hz), 9.2 (s, 1H), 10.1 (s, 1H),
10.3 (s, 1H). HPLC purity: system A, tR 9.13 min (98%). ESMS
m/z ) 626 [M + H]+. Anal. Calcd for C30H35N5O6S2: C, 57.58;
H, 5.64; N, 11.19. Found: C, 57.60; H, 5.61; N, 10.92.
1
scribed above (40%, off-white solid). H NMR (DMSO-d6, 400
MHz): δ 1.2 (s, 9H), 2.3-2.4 (m, 6H), 3.0 (s, 3H), 3.4-3.5 (m,
2H), 3.6-3.7 (m, 4H), 3.8 (s, 3H), 6.5 (d, 1H, J ) 9 Hz), 7.1-7.2
(m, 1H), 7.3 (d, 1H, J ) 2 Hz), 7.4 (d, 1H, J ) 2 Hz), 7.5-7.6 (m,
2H), 7.8 (dd, 1H, J ) 9, 2 Hz), 7.9 (dd, 1H, J ) 9, 2 Hz), 8.3 (s
1H), 8.7 (d, 1H, J ) 2 Hz), 9.3 (br s, 1H), 10.1 (s, 1H), 10.3 (s,
1H). ESMS m/z ) 681 [M + H]+.
7-{[6-(2-Methoxy-ethylamino)-pyridine-3-carbonyl]-amino}-
1-methyl-indole-2-carboxylic Acid (5-tert-Butyl-3-methanesulfo-
nylamino-2-methoxy-phenyl)-amide (24): Compound 24 was
prepared as described above (50%, off-white solid). 1H NMR
1-Methyl-7-{[6-(2-morpholin-4-yl-ethylamino)-pyridine-3-car-
bonyl]-amino}-indole-2-carboxylic Acid (5-tert-Butyl-3-meth-

p38 Inhibitors Via Computer-Assisted Drug Design
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4025
(DMSO-d6, 400 MHz): δ 1.2 (s, 9H), 3.0 (s, 3H), 3.2 (s, 3H),
3.4-3.5 (m, 4H), 3.7 (s, 3H), 4.0 (s, 3H), 6.4 (d, 1H, J ) 9 Hz),
7.0-7.1 (m, 2H), 7.2 (d, 1H, J ) 2 Hz), 7.3 (s, 1H), 7.5 (d, 1H, J
) 2 Hz), 7.6 (d, 1H, J ) 7, 2 Hz), 7.9 (d, 1H, J ) 9 Hz), 8.3 (s,
1H), 8.7 (d, 1H, J ) 2 Hz), 9.1 (s, 1H), 9.8 (s, 1H), 10.0 (s, 1H).
HPLC purity: system A, tR 9.18 min (96%), system B, tR 3.25
min (96%). ESMS m/z ) 623 [M + H]+.
rats were placed into two groups of three animals per group. The
animals were fasted overnight prior to dosing and food was returned
approximately 4 h post-dose. The animals in group 1 were dosed
via an intravenous injection into the femoral vein catheter (∼15 s
push) at a target dose level of 1 mg/kg for each compound and at
a dose volume of 1 mL/kg. Immediately following dosing, each
catheter was flushed with 0.5 mL of saline and tied off to prevent
re-access. The animals in group 2 were dosed via oral gavage at a
target dose of 10 mg/kg and a dose volume of 10 mL/kg. The
plasma samples, dose formulation samples, and residual dose
formulations were analyzed in a noncompartmental pharmacokinetic
manner using WinNonlin Anal. Calcd for software.
In Vitro Methods. 1. Thermal Denaturation Assay: The details
of the thermal denaturation experiments are found in ref 16. Briefly,
the UV thermal melt experiments were carried out using a Perkin-
Elmer Lambda 40 spectrophotometer. For each measurement, a
quartz cuvette was loaded with 2.5 µM murine p38 MAP kinase
and 25 µM inhibitor in a pH 7.0 buffer containing 10 mM sodium
phosphate, 100 mM NaCl, and 1 mM TCEP. Absorbance data at
230 nM was collected as the temperature was scanned from 25 to
80 °C at a ramp rate of 0.2 °C/min. The melting temperature for
each sample was calculated as the maximum deflection point of
the first derivative of the melting transition using the Perkin-Elmer
Templab software (version 1.62). Generally, the reported Tm values
have an average standard error of (0.5 °C and are the average of
at least two experiments unless otherwise noted. The Tm values
can be converted into Kd values using the equation Tm ) 3.08(log
Ka) + 31.2, where Ka ) 1/Kd. The error associated with Kd
calculations using this equation is (16%. It must be noted that
this equation was developed for pyrazole naphthyl urea-based
inhibitors (e.g., 1) and if the thermodynamics of binding for this
class of inhibitors is significantly different this relationship may
be more of an approximation.
2. Human Whole Blood TNF-(HWB) Assay: This assay,
performed in 96-well polypropylene plates, measures the ability
of test compounds to inhibit the elaboration of TNF-R by human
whole blood following ex vivo stimulation with LPS (Escherichia
coli lipopolysaccharide 0111:B4). Each test well contained the
following components: 15 µL of compound serially diluted in
human type AB serum, 135 µL of freshly drawn heparinized human
whole blood, and 15 µL of LPS diluted in PBR S containing 10%
human serum for a final concentration of 50 ng LPS/mL. The plates
were incubated for 30 min at 37 °C prior to addition of LPS. Control
wells on every plate contained no test compound, both with and
without LPS. Following addition of LPS to test and control wells,
the plates were placed on a rotary shaker for 30 s and then incubated
for 5 h at 37 °C. To harvest plasma, 100 µL of PBR S was added
to all wells, the plates centrifuged to pellet cells, and the diluted
plasma removed for TNFR quantitation by standard ELISA,
DELFIA, or electrochemiluminescence assay methods. TNFR in
each test well was determined by signal interpolation from a TNFR
standard curve run on the same plate. Test compound EC50 values
were determined by nonlinear curve fitting of the data from cross-
plate duplicate 11-point concentration-response curves to a
4-parameter logistic equation. Composite EC50 values were the
geometric mean of EC50 values determined in blood from at least
three donors. The EC50 of a standard inhibitor was determined on
239 independent test days across 113 different blood donors and
returned lower and upper one-standard deviation values that were
29 and 37% of the geometric mean EC50 value.
Acknowledgment. The authors are indebted to Dr. Mathew
Netherton for critical reading of the manuscript.
References
(1) Imajo, M.; Tsuchiya, Y.; Nishida, E. Regulatory mechanisms and
functions of MAP kinase signaling pathways. IUBMB Life 2006, 58
(5-6), 312-317.
(2) Wagner, G.; Laufer, S. Small molecular anti-cytokine agents. Med.
Res. ReV. 2005, 26 (11), 1-62. (b) Calcagni, F.; Elenkov, I. Stress
system activity, innate and T helper cytokines, and susceptibility to
immune-related diseases. Ann. N.Y. Acad. Sci. 2006, 62-76.
(3) Klinkhoff, A. Biological agents for rheumatoid arthritis: Targeting
both physical function and structural damage. Drugs 2004, 64 (12),
1267-1283. (b) Barry, J.; Kirby, B. Novel biologic therapies for
psoriasis. Expert Opin. Biol. Ther. 2004, 6, 975-987. (c) Elliott, M.
J.; Maini, R. N.; Feldmann, M.; Kalden, J. R.; Antoni, C.; Smolen,
J. S.; Leeb, B.; Breedveld, F. C.; Macfarlane, J. D.; Woody, J. N.
Randomized double-blind comparison of chimeric monoclonal
antibody to tumor necrosis factor R (cA2) versus placebo in
rheumatoid arthritis. Lancet 1994, 344, 1105-1110. (d) Cohen, S.
B.; Woolley, J. M. Interleukin 1 receptor antagonist anakinra
improves functional status in patients with RA. J. Rheumatol. 2003,
30, 225-231. (e) Boehncke W. H.; Prinz, J.; Gottlieb, A. B. Biologic
therapies for psoriasis. A systematic review. J. Rheumatol. 2006, 33
(7), 1447-1451.
(4) Saklatvala, J. The p38 MAP kinase pathway as a therapeutic target
in inflammatory disease. Curr. Opin. Pharmacol. 2004, 4 (4), 372-
377.
(5) Ashwell, J. D. The many paths to p38 mitogen-activated protein
kinase activation in the immune system. Nat. ReV. Immunol. 2006,
6 (7), 532-540.
(6) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.;
Graham, A. G.; Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan,
J. Inhibition of p38 MAP kinase by utilizing a novel allosteric binding
site. Nat. Struct. Biol. 2002, 9 (4), 268-272.
(7) (a) Dominguez, C.; Powers, D.; Tamayo, N. p38 MAP kinase
inhibitors: Many are made, but few are chosen. Curr. Opin. Drug
DiscoVery DeV. 2005, 8, 421-430. (b) Cirillo P. F.; Pargellis, C.;
Regan, J. The non-diaryl heterocycle classes of p38 MAP kinase
inhibitors. Curr. Top. Med. Chem. 2002, 2 (9), 1021-1035.
(8) Sullivan, J.; Holdgate, G. A.; Campbell, D.; Timms, D.; Gerhardt,
S.; Breed, J.; Breeze, A. L.; Bermingham, A.; Pauptit, R. A.; Norman,
R. A.; Embrey, K.; Read, J. Prevention of MKK6-dependent
activation by binding to p38a MAP kinase. Biochemistry 2005, 44,
16475-16490.
(9) (a) Schindler, T.; Bornmann, W.; Pellicena, P.; Miller, W. T.;
Clarkson, B.; Kuriyan, J. Structural mechanism for STI-571 inhibition
of Abelson tyrosine kinase. Science 2000, 289 (5486), 1938-1942.
(b) Nagar, B.; Bornmann, W. G.; Pellicena, P.; Schindler, T.; Veach,
D. R.; Miller, W. T.; Clarkson, B.; Kuriyan, J. Crystal structures of
the kinase domain of c-Abl in complex with the small molecule
inhibitors PD173955 and imatinib (STI-571). Cancer Res. 2002, 62,
4236-4243. (c) Tokarski, J. S.; Newitt, J. A.; Chang, C. Y. J.; Cheng,
J. D.; Wittekind, M.; Kiefer, S. E.; Kish, K.; Lee, F. Y. F.; Borzillerri,
R.; Lombardo, L. J.; Xie, D.; Zhang, Y.; Klei, H. E. The structure of
Dasatinib (BMS-354825) bound to activated ABL kinase domain
elucidates its inhibitory activity against Imatinib-resistant ABL
mutants. Cancer Res. 2006, 66, 5790-5797.
(10) Regan, J.; Capolino, A.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.;
Hickey, E.; Kroe, R. R.; Madwed, J.; Moriak, M.; Nelson, R.;
Pargellis, C. A.; Swinamer, A.; Torcellini, C.; Tsang, M.; Moss, N.
Structure-activity relationships of the p38alpha MAP kinase inhibitor
1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-
ethoxy)naphthalen-1-yl]urea (BIRB 796). J. Med. Chem. 2003, 46
(22), 4676-4686.
In Vivo Methods. 1. LPS Challenge Assay: Female Balb/c
mice, weighing approximately 20 g, were used. Mice were
administered test compound in cremophor by oral gavage (volume
of 0.15 mL) approximately 30 min prior to LPS/D-gal administra-
tion. Then, mice were administered LPS (E. coli LPS 0111:B4,
1.0 µg/mouse) plus D-gal (50 mg/kg) intravenously in 0.2 mL of
pyrogen-free saline. After 1 h, each mouse was anesthetized, and
blood was collected by cardiac puncture for serum TNFR and
compound level determinations. Blood samples were centrifuged
at 2500 rpm for 10-15 min, the serum was decanted, and plasma
concentration was determined by HPLC. The concentration of
TNF-R in the serum was measured by a commercially available
ELISA kit (R&D Systems, Minneapolis, MN). ELISA was per-
formed according to the manufacturers assay procedure. All samples
were assayed in duplicate.
2. Rat Pharmacokinetic Assessment: Male Sprague-Dawley

4026 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Goldberg et al.
(11) Niculescu-Duvaz, I.; Roman, E.; Whittaker, S. R.; Friedlos, F.; Kirk,
R.; Scanlon, I. J.; Davies, L. C.; Niculescu-Duvaz, D.; Marais, R.;
Springer, C. J. Novel inhibitors of bRAF based on a disubstituted
pyrazine scaffold. Generation of a nanomolar lead. J. Med. Chem.
2006, 49 (1), 407-416.
Henderson, I.; Barrish, J. C.; Dodd, J. H.; Schieven, G. L.; Leftheris,
K. 5-Cyanopyrimidine derivatives as a novel class of potent, selective,
and orally active inhibitors of p38R MAP kinase. J. Med. Chem.
2005, 48 (20), 6261-6270. (b) Johnson, L. N.; De Moliner, E.;
Brown, N. R.; Song, H.; Barford, D.; Endicott, J. A.; Noble, M. E.
Structural studies with inhibitors of the cell cycle regulatory kinase
cyclin-dependent protein kinase 2. Pharmacol. Ther. 2002, 93 (2-
3), 113-124.
(12) Cirillo, P. F.; Hickey, E. Discovery of the N-phenyl-N′-naphthyl-
urea class of p38 MAP kinase inhibitors. Results to be published at
a later date.
(13) Dominguez, C.; Tamayo, N.; Zhang, D. p38 Inhibitors: Beyond
pyridinylimidazoles. Expert Opin. Ther. Pat. 2005, 15 (7), 801-
816.
(18) Liverton, N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon, D. A.;
Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Selnick, H. G.;
Smith, G. R.; Tebben, A.; Vacca, J. P.; Varga, S. L.; Agarwal, L.;
Dancheck, K.; Forsyth, A. J.; Fletcher, D. S.; Frantz, B.; Hanlon,
W. H.; Harper, C. F.; Hofsess, S. J.; Kostura, M.; Lin, J.; Luell, S.;
O’Neill, E. A.; Orevillo, C. J.; Pang, M.; Parsons, J.; Rolando, A.;
Sahly, Y.; Visco, D. M.; O’Keefe, S. J. Design and synthesis of
potent, selective, and orally bioavailable tetrasubstituted imidazole
inhibitors of p38 mitogen-activated protein kinase. J. Med. Chem.
1999, 42 (12), 2180-2190.
(19) Carter, T.A.; Wodicka, L. M.; Shah, N. P.; Velasco, A. M.; Fabian,
M. A.; Treiber, D. K.; Milanov, Z. V.; Atteridge, C. E.; Biggs, W.
H., III; Edeen, P. T.; Floyd, M.; Ford, J. M.; Grotzfeld, R. M.;
Herrgard, S.; Insko, D. E.; Mehta, S. A.; Patel, H. K.; Pao, W.;
Sawyers, C. L.; Varmus, H.; Zarrinkar, P. P.; Lockhart, D. J.
Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor
kinases. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (31), 11011-11016.
(14) Wang, R.; Gao, Y.; Lai, L. LigBuilder: A multi-purpose program
for structure-based drug design. J. Mol. Model. 2000, 6, 498-516.
(15) Rahaman, L. K. A.; Scrowston, R. A. 7-Substituted benzo[b]-
thiophenes and 1,2-benzisothiazoles. Part 2. Chloro and nitro
derivatives. J. Chem. Soc., Perkin Trans. 1 1984, 19, 385-387.
(16) For this series, a 2.8 °C change in thermal denaturation temperature
corresponds to approximately a 10-fold change in binding affinity.
However, it must be noted that this equation was developed for
pyrazole naphthyl urea-based inhibitors (e.g., 1), and if the thermo-
dynamics of binding for this class of inhibitors is significantly
different, this relationship may be more of an approximation. Kroe,
R. R.; Regan, J.; Proto, A.; Peet, G. W.; Roy, T.; Dickert, L.;
Fuschetto, N.; Pargellis, C. A.; Ingraham, R. H. Thermal denatur-
ation: A method to rank slow binding, high-affinity p38a MAP kinase
inhibitors. J. Med. Chem. 2003, 46 (22), 4669-4675.
(17) (a) Liu, C.; Wrobleski, S. T.; Lin, J.; Ahmed, G.; Metzger, A.; Wityak,
J.; Gillooly, K. M.; Shuster, D. J.; McIntyre, K. W.; Pitt, S.; Shen,
D. R.; Zhang, R. F.; Zhang, H.; Doweyko, A. M.; Diller, D.;
JM070415W