The discovery of (R)-2-(sec-butylamino)-N-(2-methyl-5-(methylcarbamoyl)phenyl) thiazole-5-carboxamide (BMS-640994)-A potent and efficacious p38α MAP kinase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2008.02.031

A novel structural class of p38α MAP kinase inhibitors has been identified via iterative SAR studies of a focused deck screen hit. Optimization of the lead series generated 6e, BMS-640994, a potent and selective p38α inhibitor that is orally efficacious in rodent models of acute and chronic inflammation.

Full text of DOI:10.1016/j.bmcl.2008.02.031

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1762–1767
The discovery of (R)-2-(sec-butylamino)-N-
(2-methyl-5-(methylcarbamoyl)phenyl) thiazole-5-carboxamide
(BMS-640994)—A potent and efficacious p38a
MAP kinase inhibitor
John Hynes, Jr.,* Hong Wu, Sidney Pitt, Ding Ren Shen, Rosemary Zhang,
Gary L. Schieven, Kathleen M. Gillooly, David J. Shuster, Tracy L. Taylor,
XiaoXia Yang, Kim W. McIntyre, Murray McKinnon, Hongjian Zhang,
Punit H. Marathe, Arthur M. Doweyko, Kevin Kish, Susan E. Kiefer,
John S. Sack, John A. Newitt, Joel C. Barrish, John Dodd and Katerina Leftheris
Bristol-Myers Squibb, Princeton, NJ 08543-4000, USA
Received 17 January 2008; revised 11 February 2008; accepted 13 February 2008
Available online 16 February 2008
Abstract—A novel structural class of p38a MAP kinase inhibitors has been identified via iterative SAR studies of a focused deck
screen hit. Optimization of the lead series generated 6e, BMS-640994, a potent and selective p38a inhibitor that is orally efficacious
in rodent models of acute and chronic inflammation.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The protein-based pharmacological blockade of pro-
inflammatory signaling molecules, specifically TNF-a
is emerging as an important therapeutic option for the
treatment of rheumatoid arthritis (RA) and other
inflammatory diseases.1,2 Upon activation, p38 mito-
gen-activated protein kinase (MAPK), a serine-threo-
nine kinase consisting of 4 isoforms, up-regulates the
biosynthesis of two pro-inflammatory cytokines, TNFa
and IL-1b.3 Accordingly, modulation of the p38 signal-
ing cascade has emerged as an attractive target for the
design of novel chemotherapeutic agents for the treat-
ment of inflammatory diseases.4–6 Expression of the
p38 MAPK isoforms varies across cell types of the im-
mune system and it is believed that the predominant iso-
form involved in inflammation is p38a.7
ery, in vitro SAR, and in vivo efficacy of 2-alkylamino-
5-carboxamidoaniline thiazole-derived p38a inhibitors.
A focused screen of internal kinase inhibitors identified
C2-alkylaminothiazole 2 (Fig. 1) as a moderately potent
p38a kinase inhibitor (IC50 39 10 nM) with limited po-
tency in a cell-based functional assay measuring TNFa
biosynthesis (human PBMC IC50 ꢀ 1.5 lM).9 This com-
pound was originally derived from a program directed
towards identifying inhibitors of the tyrosine kinase
p56Lck (Lck IC50 140 nM for 2).10 We envisioned that
the structural diversity of this series relative to the previ-
ously studied pyrrolo[2,1-f][1,2,4]triazines would pro-
duce divergent in vitro and in vivo profiles.
Subsequently, we undertook an SAR effort aimed at
increasing p38a inhibition, cellular potency, and kinase
selectivity, with the goal of producing efficacious com-
pounds in multiple in vivo models of inflammation.
We have previously reported the in vitro and in vivo
evaluation of pyrrolo[2,1-f][1,2,4]triazine-based p38a
inhibitors (1, Fig. 1).8 In this letter, we report the discov-
The compounds were synthesized according to the proce-
dures outlined in Schemes 1 and 2. Briefly, 2-bromothia-
zole-5-carboxylic acid (4) was coupled to a variety of aryl
amines to afford 5. Nucleophilic displacement of the C2
halogen with various amines was accomplished at
Keywords: p38; MAP kinase; Aminothiazole; TNF.
*
Corresponding author.Tel.: +1 609 252 4431; fax: +1 609 252 3993;
e-mail: john.hynes@bms.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.031

J. Hynes, Jr. et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1762–1767
1763
H
N
Cl
HN
OMe
N
H
N
2
NH
5
O
3
O
N
6
S
N
O
NH
N
1
1
2
Figure 1. BMS p38a inhibitors.
H
N
Ar
O
Ar
OH
HN
HN
3a–e, Ar =
3a, R1 = ethyl
6a–l, Ar =
R2
a
b
Cl
O
O
O
6a, R1 = n-propyl, R2 = H
6b, R1 = i-propyl, R2 = H
6c, R1 = n-propyl, R2 = Me
6d, R1 = i-propyl, R2 = Me
N
S
N
N
3b, R1 = n-propyl
3c, R1 = i-propyl
3d, R1 = phenyl
3e, R1 = cyc-butyl
S
S
R1
NH
Br
Br
4
5
3a–e, 6a–l
6e, R1 = (R)-sec-butyl, R2 = Me
6f, R1 = (S)-sec-butyl, R2 = Me
6g, R1 = MeOCH2CH2, R2 = Me
6h, R1 = n-propyl, R2 = Et
6i, R1 = i-propyl, R2 = Et
6j, R1 = i-propyl, R2 = 5-pyrazole
6k, R1 = i-propyl, R2 = N-Me-5-pyrazole
6l, R1 = i-propyl, R2 = 3-isoxazole
Scheme 1. Reagents and conditions: (a) Ar–NH2, EDC, HOBt, DIPEA, DMF, rt; (b) R1-NH2, EtOH, 150 ꢁC.
O
2
R
N
H
Br
NH
13a, R1 = n-propyl, R2 = Me
13b, R1 = i-propyl, R2 = Me
OEt
OEt
OEt
H
N
13c, R1 = cyc-pentyl, R2 = Me
13d, R1 = n-propyl, R2 = Et
13e, R1 = i-propyl, R2 = Et
13f, R1 = cyc-pentyl, R2 = Et
a
b
c, d
e
O
O
O
N
N
N
N
N
S
S
S
R'
Br
S
O
O
Br
Br
Br
7
8
9a, R2 = Me
9b, R2 = Et
10a, R2 = Me
10b, R2 = Et
H
N
H
N
H2N
H2N
O
O
11
12
Scheme 2. Reagents and conditions: (a) NBS, AIBN, CCl4, D, 54%; (b) 11 or 12, K2CO3, DMF, rt, 80–83%; (c) 1 N NaOH, THF, MeOH, 70–87%;
(d) EDC, CH2Cl2, 0 ꢁC, 91–98%; (e) R1-NH2, EtOH, 150 ꢁC, 50–80%.
high temperature to afford analogs 3a–e and 6a–l. The
fused bicyclic aminothiazoles (13a–f) were prepared via
the bromination of thiazole 7 to give 8. Dibromide 8
was reacted with 11 or 12 to give 9a or 9b. Hydrolysis
of the ester was followed by EDC-mediated lactam
formation to afford the intermediate bromides 10a and
10b. These were reacted with a variety of amines to pro-
vide analogs 13a–f.
Table 1 summarizes the in vitro potency of the C2 amino
variants of 2. Branched acyclic and cyclic C2 substitu-
tion was well tolerated in the p38a enzyme inhibition
assay. The aryl amine 3d had reduced enzyme inhibition
relative to 2, as did the C2 ethylamine analog 3a. In the
PBMC functional assay, 3c was potent, whereas 3b was
inactive in this assay.
The inability to significantly enhance p38a and TNFa
inhibition via C2 modification, combined with the lack
of Lck selectivity (i.e., 3e), prompted us to modify our
compound design strategy. Based upon our previous
studies,8 we hypothesized that the replacement of the
aniline of 2 with the meta-substituted carboxamide ani-
lines found in 1 would enhance p38a kinase inhibition,
cellular potency, and selectivity for p38a against Lck.
The synthesized C2-aminothiazole analogs were
screened and optimized for inhibition of human p38a
MAP kinase. Compounds with significant enzyme inhi-
bition (IC50 < 50 nM) were then evaluated in a human
lipopolysaccharide (LPS)-induced TNFa biosynthesis
assay in human peripheral blood mononuclear cells
(PBMCs).

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J. Hynes, Jr. et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1762–1767
Table 1. Preliminary C2 SAR
In general, selectivity against Lck (>1000-fold) was ob-
served, with the exception of the amino-isoxazole 6l
(Lck IC50 380 nM).
N
R1
H
N
N
H
2
S
O
The conformationally restricted fused lactams (13a–f)
had decreased p38a inhibition relative to the C4 unsub-
stituted analogs and, when tested, were weak inhibitors
in the PBMC functional assay (Table 3). C4 methyl
substituted analogs (14a,b, Table 3) had decreased
p38a enzyme inhibition and were not pursued further.
Cl
Compound
R1
IC50 (nM)
p38aa
TNFab
Lckc
140
2
cyc-Propyl
Ethyl
39
140
28
13
62
36
1500
ntd
3a
3b
3c
3d
3e
n-Propyl
i-Propyl
Phenyl
>2000
160
ntd
ntd
Advanced compounds were evaluated in a murine model
of acute inflammation (mouse LPS-TNFa). Criteria for
evaluation in this model included potent cell inhibition
of LPS-induced TNFa (IC50 < 15 nM) and reasonable
Caco-2 permeability (>75 nm/s).11 Several of the C50
methyl and ethyl amides met the above criteria and pro-
gressed into this model. Compounds were initially
screened at 5 mg/kg dosed orally, using a 4 h pre-dosing
interval to LPS challenge. A secondary screen using a
24 h pre-dosing interval was used to identify long-acting
potent inhibitors (Table 4). Compound 6e was effica-
cious in the 24 h pre-dosing protocol in mice and pro-
gressed into a dose–response study in rats, whereby an
oral ED50 of 2 mg/kg was obtained (Fig. 2).
cyc-Butyl
3310
a Compounds were assayed in triplicate. Variability around the mean
value was <50%.
b Compounds were typically assayed in duplicate.
c n = 1.
d nt, not tested.
As shown in Table 2, >3-fold increases in p38a enzyme
and human PBMC potency were observed when
2-methyl-5-carboxamido anilines were substituted in
the place of 2-chloro-6-methyl aniline. A trend towards
an increased cellular potency was observed when the C2
substituent contained a branched aliphatic group (e.g.,
6c vs 6e and 6f). Ether incorporation within the C2
chain (6g) decreased enzyme and cellular potency. C50
heterocyclic amides (6j–l) were tolerated in both assays.
Compound 6e was then subjected to advanced in vitro
pharmacological and profiling assays, some of which
are highlighted in Table 5. In addition to TNFa block-
ade, 6e inhibited LPS-induced IL-1b production in
Table 2. Effect of the C2 and C50-substituents on enzymatic and cellular activity
O
N
R2
H
R1
5'
N
N
H
N
H
2
S
O
Compound
R1
R2
IC50 (nM)
p38aa
TNFab
Lckc
6a
6b
6c
6d
6e
6f
n-Propyl
i-Propyl
H
4.1
3.2
4.5
3.5
3.5
2.3
22
23
35
ntd
H
>50,000
4800
n-Propyl
i-Propyl
Me
Me
Me
Me
Me
Et
42
8.7
2.9
4.1
>250
2.7
11
>50,000
22,000
>50,000
(R)-sec-butyl
(S)-sec-butyl
MeOCH2CH2
n-Propyl
6g
6h
6i
2.0
3.4
1900
>50,000
i-Propyl
Et
ntd
N
6j
i-Propyl
4.2
7.9
N
H
N
6k
6l
i-Propyl
i-Propyl
5.4
3.8
6.4
1.0
5800
380
N
O
N
a Compounds were assayed in triplicate. Assay variability was measured using a standard control, <40% (n = 46).
b Compounds were typically assayed in duplicate. Assay variability was measured using a standard control, <80% (n = 25).
c n = 1.
d nt = not tested.

J. Hynes, Jr. et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1762–1767
Table 3. C4 substituent SAR and fused ring thiazoles Table 5. In vitro profiling data for compound 6e
1765
Parameter
Result
4
O
N
R2
H
N
R1
5'
LPS-IL-1b IC50
WB LPS-TNFa IC50
WBLPS-IL-1b IC50
Metabolisma
CYP inhibitionc
hERGd
99 36 nM
54 44 nM
N
H
H
N
N
N
N
H
R1
S
R2
N
H
600 397 nM
HLM, RLM, MLM 0.00b
O
S
O
O
13
14
>40 lM 1A2, 2C9,2C19, 2D6, 3A4
7% inhib at 30 lM
>1000-fold selective
1.5, > 30,000, > 30,000
Human-82%; Rat-89%
83 nm/s
Compound
R1
R2
IC50 (nM)
Kinases (ꢀ64)
p38aa
TNFab
p38b, c, dIC50 (nM)
Protein binding
Caco-2 permeability
Aq. sol., pH 6.5
13a
13b
13c
13d
13e
13f
14a
14b
n-Propyl
i-Propyl
Me
Me
Me
Et
270
110
110
160
34
0.022 mg/mL
cyc-Pentyl
n-Propyl
i-Propyl
cyc-Pentyl
Ethyl
a HLM, human liver microsomes; RLM, rat liver microsomes; MLM,
mouse liver microsomes.
Et
Et
>250
240
b nmol/min/mg.
53
c CYP, cytochrome P450.
Et
Et
330
130
d Patch clamp assay.
n-Propyl
a Compounds were assayed in triplicate. Assay variability was mea-
sured using a standard control, <40% (n = 46).
multiple species, 6e had minimal liver microsomal turn-
over. Minimal hERG channel or Cyp isoform inhibition
was observed. When screened against a panel of kinases,
6e was shown to be highly selective for p38a. Among the
four p38 isoforms, only p38a and p38b were potently
inhibited (IC50 1.5 nM). The physiochemical properties
of 6e (protein binding, Caco-2 permeability, and aque-
ous solubility) are also summarized.
b Compounds were typically assayed in duplicate. Assay variability
was measured using a standard control, <80% (n = 25).
Table 4. Inhibition of circulating TNFa in LPS-challenged mice dosed
orally with 5 mg/kg of compound
Entry Compound Dosing
interval (h)
% Inhiba Drug serum
Conc. (nM)
When dosed in rats, compound 6e was orally bioavail-
able (%F = 75), had moderate clearance, and a reason-
able half-life (Table 6). High levels of serum drug
levels were obtained (Cmax and AUC0ꢁ24h) through the
course of the study.
1
2
3
4
5
6d
6f
6i
ꢁ4b
62 11c
69 8.9c
68 13c
nad
400 190
250 46
ꢁ4b
ꢁ4b
ꢁ24
ꢁ24
330 110
Not measured
Not measured
6h
6e
46 31e
a Percent reduction in circulating TNFa, mean SD, n = 8 animals.
b Not active at 24 h pre-dosing interval.
c p < 0.001 versus vehicle, Student’s T-test.
d na, not active.
Table 6. Pharmacokinetic properties of 6e in rats
Parameter
Valuea
iv dose (1 mg/kg)
t1/2 (h)
e p < 0.05 versus vehicle, Student’s T-test.
1.8
CL (mL/min/kg)
Vss (L/kg)
14.1
1.8
po dose (10 mg/kg)
Cmax (lM)
AUC0–24h (lM-h)
C24h (nM)
%F
8.7 2.2
25 2.3
7.6 3.5
75
80,000
0.16
70,000
vehicle
60,000
a Vehicle: PEG300. n = 3 animals.
0.63
2.5*
50,000
The favorable acute efficacy in rodents, in vitro profiling
data, and aforementioned PK properties supported the
advancement of 6e into the advanced disease models
of chronic inflammation. In the rat adjuvant-induced
arthritis model, 6e significantly inhibited disease pro-
gression at 1 and 3 mg/kg, (po, qd) as measured by
paw swelling (Fig. 3). A minimally efficacious dose of
0.3 mg/kg, (po, qd) for 6e was obtained (46% inhibition,
data not shown). Micro computed tomography (CT)
imaging of isolated rat hocks revealed that drug-treated
animals (0.3 mg/kg, po, qd) had minimal disease pro-
gression or bone degeneration.13
40,000
30,000
20,000
10*
10,000
0
Dose (mg/kg)
Figure 2. Inhibition of LPS-induced TNFa in rats by compound 6e.12
Compound 6e was co-crystallized with p38a MAP ki-
nase and the structure of the complex was determined
via X-ray diffraction (Fig. 4). Consistent with previous
PBMCs (IC50 99 nM). Furthermore, in human whole
blood, LPS-induced TNFa and IL-1b were inhibited
with IC50 values of 54 and 600 nM, respectively. Across

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J. Hynes, Jr. et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1762–1767
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
In summary, a novel series of potent and selective C2-
alkylaminothiazole p38a MAP kinase inhibitors has
been identified. Structure–activity studies led to the dis-
covery of (R)-2-(sec-butylamino)-N-(2-methyl-5-(meth-
ylcarbamoyl)phenyl) thiazole-5-carboxamide 6e (BMS-
640994), which was orally efficacious in acute and
chronic models of inflammation. The molecular basis
for p38a kinase interactions for this class of inhibitors
has been established via X-ray crystallography.
Vehicle
1 mg/kg
3 mg/kg
*
*
*
*
*
*
Acknowledgment
10 11 12 13 14 15 16 17 18 19 20 21
Study Day
The authors thank Georgia Cornelius of the Depart-
ment of Bioanalytical Research for obtaining serum
concentrations for the in vivo studies.
Figure 3. Efficacy of Compound 6e in the Rat Adjuvant Arthritis
Model (qd, po). Terminal serum concentrations (18 h after final dose)
for 6e at 1 and 3 mg/kg were 8.1 and 16 nM, respectively.
References and notes
1. (a) Choy, E. H. S.; Panayi, G. S. N. Eng. J. Med. 2001,
344, 907; (b) Dinarello, C. A. Curr. Opin. Immunol. 1991,
3, 941.
2. (a) Palladino, M. A.; Bahjat, F. R.; Theodorakis, E. A.;
Moldawer, L. L. Nat. Rev. Drug Disc. 2003, 2, 736; (b)
Braddock, M.; Quinn, A. Nat. Rev. Drug Disc. 2004, 3, 1.
3. (a) Salituro, F. G.; Germann, U. A.; Wilson, K. P.; Bemis,
G. W.; Fox, T.; Su, M. S. Curr. Med. Chem. 1999, 6, 807;
(b) Foster, M. L.; Halley, F.; Souness, J. E. Drug News
Perspect. 2000, 13, 488; (c) Kumar, S.; Boehm, J.; Lee, J.
C. Nat. Rev. Drug Disc. 2003, 2, 717; (d) Saklatvala, J.
Curr. Opin. Pharmacol. 2004, 4, 372; (e) Kumar, S.; Blake,
S. M. Handb. Exp. Pharmacol. 2005, 167, 65.
4. Westra, J.; Limburg, P. C. Mini-Rev. Med. Chem. 2006, 6,
867.
5. For reviews detailing the current small molecule p38
kinase inhibitor field see: (a) Hynes, J.; Leftheris, K. Curr.
Top. Med. Chem. 2005, 5, 967; (b) Wagner, G.; Laufer, S.
Med. Res. Rev. 2006, 1, 1; (c) Lee, M. R.; Dominguez, C.
Curr. Med. Chem. 2005, 12, 2979.
Figure 4. X-ray structure of 6e co-crystallized with p38a.16
6. (a) Fijen, J. W.; Zijlstra, J. G.; De Boer, P.; Spanjersberg,
R.; Cohen Tervaert, J. W.; van der Werf, T. S.; Lighten-
berg, J. J. M.; Tulleken, J. E. Clin. Exp. Immunol. 2001,
124, 16; (b) Branger, J.; van den Blink, B.; Weijer, S.;
Madwed, J.; Bos, C. L.; Gupta, A.; Yong, C.-L.; Polmar,
S. H.; Olszyna, D. P.; Hack, E. C.; van Deventer, S. J. H.;
Peppelenbosch, M. P.; van der Poll, T. J. Immunol. 2002,
168, 4070.
7. (a) Hale, K. K.; Trollinger, D.; Rihanek, M.; Manthey, C.
L. J. Immunol. 1999, 162, 4246; (b) Allen, M.; Svensson,
L.; Roach, M.; Hambor, J.; McNeish, J.; Gabel, C. A.
J. Exp. Med. 2000, 191, 859; (c) Fearns, C.; Kline, L.;
Gram, H.; Di Padova, F.; Zurini, M.; Han, J.; Ulevitch, R.
J. J. Leukocyte Biol. 2000, 67, 705.
8. Hynes, J., Jr.; Dyckman, A.; Lin, S.; Wrobleski, S. T.; Wu,
H.; Gillooly, K. M.; Kanner, S. B.; Lonial, H.; Loo, D.;
McIntyre, K. W.; Pitt, S.; Shen, D. R.; Shuster, D. J.;
Yang, X.; Zhang, R.; Behnia, K.; Zhang, H.; Marathe, P.
H.; Doweyko, A.; Tokarski, J. S.; Sack, J. S.; Pokross, M.;
Kiefer, S. E.; Newitt, J. A.; Barrish, J. C.; Dodd, J.;
Schieven, G. L.; Leftheris, K. J. Med. Chem. 2008, 51, 4.
9. Peripheral blood mononuclear cells (PBMCs) are purified
from human whole blood by Ficoll–Hypaque density
gradient centrifugation and resuspended at a concentra-
tion of 5 · 106/mL in assay medium (RPMI medium
containing 10% fetal bovine serum). 50 lL of cell suspen-
sion is incubated with 50 lL of test compound (4·
p38a inhibitors containing a meta-substituted carbox-
amide aniline, the C50 carboxamide NH and CO form
˚
hydrogen bonds with Glu71 (2.1 A) and Asp168
˚
(1.8 A), respectively. The thiazole ring nitrogen forms
˚
a hydrogen bond (2.0 A) with the backbone NH of
Met109. An additional hydrogen bond interaction is
gained between the Met109 carbonyl and the C2 amine
14
˚
NH (1.8 A). The observed deleterious effects of C4
substitution (i.e., compounds 13–14) can be rationalized
from this structure due to the projection of the C4 sub-
stituent towards the His107-Thr106 region surface and
disruption of a Thr106 hydrogen bond interaction with
the pendant C5 amide NH. Examination of recently
published Lck X-ray structures reveals a significant dif-
ference in the conformation of the inner region of the
activation loop (168–172 in p38 corresponding to 382–
386 in Lck).15 In the case of p38, the C50 benzamide car-
bonyl oxygen can engage in a H-bond to the backbone
NH at Asp168, while the trajectory of Glu71 is aimed
away from this region. In the case of Lck, the corre-
sponding Glu (Glu288) appears to be H-bonding to
the backbone NH at Phe383 (Phe169 in p38), which
may block a benzamide approach to the adjacent NH
(at Asp168).

J. Hynes, Jr. et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1762–1767
1767
concentration in assay medium containing 0.2% DMSO)
in 96-well tissue culture plates for 5 min at RT. 100 lL of
LPS (200 ng/mL stock) is then added to the cell suspension
and the plate is incubated for 6 h at 37 ꢁC. Following
incubation, the culture medium is collected and stored at
ꢁ20 ꢁC. TNFa concentration in the medium is quantified
using a standard ELISA kit (Pharmingen-San Diego, CA).
Concentrations of TNFa and IC50 values for test com-
pounds (concentration of compound that inhibited LPS-
stimulated TNFa production by 50%) are calculated by
linear regression analysis.
13. Krishnan, B.; Kukral, D., Bristol-Myers Squibb, unpub-
lished results.
14. Fitzgerald, C. E.; Patel, S. B.; Becker, J. W.; Cameron, P.
M.; Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin, G.
N. N. Nat. Struct. Biol. 2003, 10, 764.
15. (a) Martin, M. W.; Newcomb, J.; Nunes, J. J.; McGo-
wan, D. C.; Armistead, D. M.; Boucher, C.; Buchanan,
J. L.; Buckner, W.; Chai, L.; Elbaum, D.; Epstein, L.
F.; Faust, T.; Flynn, S.; Gallant, P.; Gore, A.; Gu, Y.;
Hseih, F.; Huang, X.; Lee, J. H.; Metz, D.; Middleton,
S.; Mohn, D.; Morgenstern, K.; Morrison, M. J.;
Novak, P. M.; Oliveira-dos-Santos, A.; Powers, D.;
Rose, P.; Schneider, S.; Sell, S.; Tudor, Y.; Turci, S. M.;
Welcher, A. A.; White, R. D.; Zack, D.; Zhao, H.; Zhu,
L.; Ghiron, C.; Amouzegh, P.; Ermann, M.; Jenkins, J.;
Johnston, D.; Napier, S.; Power, E. J. Med. Chem.
2006, 49, 4981; (b) Martin, M. W.; Newcomb, J.;
Nunes, J. J.; Bemis, J. E.; McGowan, D. C.; White, R.
D.; Buchanan, J. L.; DiMauro, E. F.; Boucher, C.;
Faust, T.; Hseih, F.; Huang, X.; Lee, J. H.; Schneider,
S.; Turci, S. M.; Zhu, Z. Bioorg. Med. Chem. Lett. 2007,
17, 2299.
10. Wityak, J.; Das, J.; Moquin, R. V.; Shen, Z.; Lin, J.; Chen,
P.; Doweyko, A. M.; Pitt, S.; Pang, S.; Shen, D. R.; Fang,
Q.; de Fex, H. F.; Schieven, G. L.; Kanner, S. B.; Barrish,
J. C. Bioorg. Med. Chem. Lett. 2003, 13, 4007.
11. The primary amides 6a and 6b had poor in vitro perme-
ability and were not evaluated further.
12. Male Lewis rats (Harlan labs, n = 5/group) were dosed
orally with compound solubilized in PEG300. Two hours
after dosing, LPS (10 lg, Escherichia coli O11:B4 strain
from Sigma) was suspended in PBS (1 mL) and dosed IP.
Ninety minutes after LPS challenge, the rats were bled and
serum TNFa determined by Elisa (R&D Systems),
according to the manufacturer’s instructions. *p < 0.05
versus vehicle, Student’s T-test. Three out of 5 rats at the
10 mg/kg dose had undetectable levels of serum TNFa.
16. For details of the X-ray collection method, see Ref. 8. The
coordinates for the structure in Figure 4 have been
deposited with the RCSB Protein Data Bank, PDB ID
code 3BX5.