Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N- propylpyrrolo[1,2-f ][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases

Article abstract of DOI:10.1021/jm100540x

The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp² character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.

Full text of DOI:10.1021/jm100540x

J. Med. Chem. 2010, 53, 6629–6639 6629
DOI: 10.1021/jm100540x
Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-
N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949),
a Clinical p38r MAP Kinase Inhibitor for the Treatment of Inflammatory Diseases†
Chunjian Liu,* James Lin, Stephen T. Wrobleski, Shuqun Lin, John Hynes, Jr., Hong Wu, Alaric J. Dyckman, Tianle Li,
John Wityak, Kathleen M. Gillooly, Sidney Pitt, Ding Ren Shen, Rosemary F. Zhang, Kim W. McIntyre, Luisa Salter-Cid,
David J. Shuster, Hongjian Zhang, Punit H. Marathe, Arthur M. Doweyko, John S. Sack, Susan E. Kiefer, Kevin F. Kish,
John A. Newitt, Murray McKinnon, John H. Dodd, Joel C. Barrish, Gary L. Schieven, and Katerina Leftheris
Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543-4000
Received May 4, 2010
The discovery and characterization of 7k (BMS-582949), a highly selective p38R MAP kinase inhibitor
that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to
the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported
clinical candidate p38R inhibitor. Unlike alkyl and other cycloalkyls, the sp2 character of the
cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide
NH. Inhibitor 7k is slightly less active than 1a in the p38R enzymatic assay but displays a superior
pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflamma-
tion and pseudoestablished rat AA model. The binding mode of 7k with p38R was confirmed by X-ray
crystallographic analysis.
Introduction
The identification and development of inhibitors of p38R
mitogen-activated protein (MAPa) kinase as orally active
therapeutic agents for the treatment of inflammatory diseases
has been one of the most active research areas in drug
discovery for the past decade. Because p38R MAP kinase
plays a critical role in regulating the biosynthesis of many
inflammatory cytokines, including tumor necrosis factor alpha
(TNF-R) and interleukin-1β (IL-1β), p38R inhibitors have
been considered a promising solution for the treatment of
inflammatory diseases.1 Numerous preclinical studies2 have
demonstrated that inhibition of p38R MAP kinase can effec-
tively inhibit TNF-R production both in vitro and in vivo.
Excessive production of TNF-R and IL-1β is believed to
underlie many inflammatory diseases.3 Blockage of TNF-R
function by biological agents such as etanercept, a soluble
TNF-R receptor, and infliximab, a TNF-R antibody, has
proven effective in the treatment of rheumatoid arthritis and
other autoimmune diseases.4 A number of reviews covering
the progress in this area have been published.5 Despite a
number of compounds having advanced into clinical trials,
including VX-702,6,7 Pamapimod,7,8 BIRB-796,9 Scio-469,7,10
and PH-797804 (aS)11 (Figure 1), proof of concept for the
efficacy and safety of a p38R inhibitor has yet to be realized.7
Figure 1. Examples of reported clinical p38R MAP kinase inhibi-
†PDB deposition number: 3MVL.
tors.
*To whom correspondence should be addressed. Phone: 609-252-
A recent paper from our group has disclosed pyrrolo[2,1-
f][1,2,4]triazine derivatives 1a and 1b (Figure 2) as potent
p38R MAP kinase inhibitors for consideration as clinical
candidates.12 However, compounds 1a and 1b suffered from
high clearance in vivo, which resulted in low exposures in
3682. Fax: 609-252-7410. E-mail: chunjian.liu@bms.com.
a Abbreviations: MAP, mitogen-activated protein; TNF-R, tumor
necrosis factor alpha; IL-1β, interleukin-1 beta; hPBMC, human peri-
pheral blood mononuclear cells; LPS, lipopolysaccharide; AA, adjuvant
arthritis; HHA, human hepatotoxicity assay; hERG, human ether-a-go-
go-related gene.
r
2010 American Chemical Society
Published on Web 08/30/2010
pubs.acs.org/jmc

6630 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Liu et al.
Scheme 1a
Figure 2. Our previously disclosed p38R MAP kinase inhibitors 1a
and 1b.
animals. Subsequent studies revealed that 1a was rapidly
converted to the corresponding carboxylic acid as the
major metabolite from the hydrolysis of N-methoxy-
benzamide. Naturally, efforts to modify 1a and 1b were
focused on eliminating the presence of the problematic
N-methoxybenzamide. In our previous disclosures,12,13 it
was shown that the N-methoxybenzamide functionality in
1a and 1b could be replaced with urea, carbamate, and
reverse amide moieties not containing the methoxylamino
group, resulting in a number of highly potent p38R MAP
kinase inhibitors. Herein, we describe an alternative ap-
proach to directly replace the N-methoxy group that
ultimately led to a superior p38R inhibitor, with overall
properties appropriate for clinical development, which is
currently in phase II clinical trials for the treatment of
rheumatoid arthritis.
Chemistry
Synthesis of analogues in which the N-methoxy group of 1a
and 1b was replaced was straightforward from previously
described advanced intermediates. Analogues 7a-c, 7j-l, and
7n-p were obtained according to Scheme 1. Treatment of
ethyl 4-chloro-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxy-
late (2)12 with 3-amino-N-ethyl-4-methylbenzamide (3a)14 at
room temperature provided 4a. Similarly, reaction of 2 with
3-amino-N-cyclopropyl-4-methylbenzamide (3b), which was
prepared from 3-amino-4-methylbenzoic acid (5) and cyclo-
propylamine, afforded 4b. Hydrolysis of 4a-c12 supplied
acids 6a-c. Standard coupling reaction conditions using
6a-c with appropriate amines gave rise to the target amides
7a-c, 7j-l, and 7n-p.
a Reagent and conditions: (a) N-ethyl-3-amino-4-methylbenzamide
(3a) or N-cyclopropyl-3-amino-4-methylbenzamide (3b), DMF, rt, over-
night, 80% yield; (b) NaOH, THF, 50 °C to reflux, 94-100% yield; (c)
R1NH2, coupling reagents such as EDC, HOBT, and BOP.
Scheme 2a
For the preparation of analogues 7d-i, 7m, 7q-v (Scheme 2),
we derivatized the previously described compounds 1a-c.12 N-
Methoxybenzamides 1a-c were treated with methanol in the
presence of hydrogen chloride in 1,4-dioxane to yield the corres-
ponding methyl benzoates. The benzoates were then hydrolyzed
to give acids 8a-c. Alternatively, 1a-c could be directly con-
verted to acids 8a-c with 1 N hydrochloric acid. Analogues
7d-i, 7m, and 7q-v were obtained from coupling reactions of
8a-c with the desired amines.
Results and Discussion
We initially investigated the replacement of the methyl
hydroxamate with alkyl amides (Table 1). Replacing the N-
methoxy group in 1a with N-ethyl resulted in over 35-fold loss
in p38R activity, as 7a inhibited p38R with an IC50 of 110 nM
versus 3.1 nM for 1a. Similarly, the ethyl counterpart of 1b
(7b, p38R IC50 = 64.9 nM) was nearly 30-fold less potent than
1b (p38R IC50 = 2.2 nM). In human peripheral blood mono-
nuclear cells (hPBMC), 7b inhibited LPS-induced TNFR
production with an IC50 of 380 nM, which was 38-fold less
a Reagent and conditions: (a) (i) MeOH, 4 N HCl in 1,4-dioxane,
rt, 16 h, (ii) KOH, 50 °C, 3 h, 71-99% yields; (b) 1N HCl, reflux, 6.5 h,
95% yield; (c) R2NH2, coupling reagents such as EDC, HATU, and BOP.
effective than 1b. Nevertheless, 7a and 7b appeared to be the
best N-alkyl analogues evaluated. The methyl, n-propyl, and
i-propyl amides 7c-e displayed p38R IC50 values of 397, 166,
and 259 nM, respectively, while the n-butyl and methoxyethyl

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6631
Table 3. in Vitro Activity of 7h-p
Table 1. in Vitro Activity of 7a-g vs 1a and 1b
p38R IC 50 hPBMC TNFR
compd
R1
R2
(nM)a
IC50 (nM)b
1a
1b
Et
OMe
3.1
2.2
61
10
(S)-R-methyl- OMe
benzyl
7a
7b
Et
Et
110
65
(S)-R-methyl- Et
benzyl
380
7c
7d
7e
7f
Et
Me
397
166
n-Pr
Et
n-Pr
i-Pr
259
>1000
>1000
Et
Et
n-Bu
CH2CH2OMe
7g
a n = 4, variation in individual values, <20%. b n = 3, variation in
individual values, <25%.
a n = 4, variation in individual values, <20%. b n = 3, variation in
individual values, <25%.
Table 2. Heat of Formation of Hydrogen Bonding
between N-cyclopropylacetamide NH and acetone carbo-
nyl oxygen was -5.06 kcal/mol (Table 2). This implied
that the activity of the N-cyclopropylbenzamide ana-
logues might lie between that observed for the N-methoxy-
benzamide and the N-ethylbenzamide analogues. It was
hoped that the N-cyclopropylbenzamide analogues might
also show improved pharmacokinetic properties, result-
ing in similar or better efficacy relative to 1a.
We were encouraged that the N-cyclopropylbenzamide 7h
displayed a p38R IC50 value of 18 nM (Table 3). Thus, as
predicted, it was ∼6-fold less potent than the N-methoxybenz-
amide and ∼5.5-fold more potent than the N-ethylbenzamide.
Compound 7h also inhibited LPS-induced TNFR production
in hPBMC with an IC50 of 191 nM. In contrast, the closely
related N-cyclobutylbenzamide analogue 7i was determined to
be 12-fold less active against p38R relative to 7h, with an IC50
value of 222 nM.
X
MeO
-6.72b
Et
c-Pr
heat of formation (kcal/mol)a
-4.86
-5.06
a Acetone and N-substituted acetamide were first minimized using
SAM1, and then the minimized complex were subjected to geometry
optimization in vacuo using DFT B3YLP/6-31þg(d,p) as implemented
within Jaguar. Hydrogen bond heats of formation were calculated as the
difference between components (acetone and N-substituted acetamide
and the complex. b Model complex includes an additional interaction
between the acetone methyl hydrogens and methoxy oxygen.
analogues 7f and 7g both inhibited p38R with IC50 values of
greater than 1000 nM.
In considering the suboptimal potency of the N-ethylbenz-
amide 7a relative to the N-methoxybenzamide 1a, we specu-
lated that the N-methoxy oxygen of 1a could make the amide
NH an excellent hydrogen bond donor due to its electron
withdrawing nature. Previously, X-ray crystallographic ana-
lysis of 1a bound to p38R had revealed that the N-methoxy-
benzamide NH in 1a forms a key hydrogen bond with Glu 71
of p38R.12 Computations using N-substituted acetamide as a
hydrogen bond donor and acetone as a hydrogen bond
acceptor indicated that the heat of formation of hydrogen
bonding between N-methoxyacetamide NH and acetone car-
bonyl oxygen was -6.72 kcal/mol. The corresponding hydro-
gen bond between N-ethyl acetamide and acetone had a heat of
formation of -4.86 kcal/mol (Table 2). Assuming that the
interaction between the acetone methyl hydrogens and methoxy
oxygen is negligible relative to the H-bonding component,
these results suggested that the N-methoxybenzamide NH in
1a could form a much stronger hydrogen bond with Glu 71 of
p38R than the N-ethylbenzamide NH in 7a. As a result of this
analysis, replacement of N-methoxy in 1a with N-cyclopropyl
was specifically proposed, rationalizing that the sp2 character of
the cyclopropyl carbons would make the benzamide NH a
stronger hydrogen bond donor. Similar computational analysis
determined that the heat of formation of hydrogen bonding
With N-cyclopropyl being identified as a suitable re-
placement for N-methoxy in 1a, our attention was then
focused on optimization of the 6-carboxamide substitution
in the N-cyclopropyl amide series (Table 3). The use of 6-N-
methyl amide in place of N-ethyl resulted in a slight drop in
both enzymatic and cellular activities, as 7j inhibited p38R
with an IC50 of 24 nM and TNFR production in hPBMC with
an IC50 of 225 nM. However, when the 6-N-ethyl group in 7h
was replaced with certain more hydrophobic alkyl groups, the
resulting analogues displayed improved activities in both the
enzymatic and cellular assays. For example, the N-propyl
6-carboxamide analogue 7k displayed a p38R IC50 of 13 nM
and a cellular TNFR IC50 of 50 nM. Similarly, the enzymatic
and cellular IC50 values for the N-butyl 6-carboxamide ana-
logue 7l were determined to be 9.7 and 81 nM, respectively.
Also, the N-(S)-R-methylbenzyl 6-carboxamide analogue 7m
(p38R IC50 = 12 nM, TNFR IC50 = 64 nM), the N-cyclo-
propylbenzamide counterpart of 1b, was a potent p38R
inhibitor and very effective in cells. The N-(S)-sec-butyl
6-carboxamide analogue 7n appeared to be less potent against
p38R (IC50 = 27 nM) than 7m, but its cellular potency (TNFR
IC50 = 57 nM) was very comparable to what was observed
with 7m. In contrast, introducing polar functionality in the

6632 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Liu et al.
Table 4. in Vitro Activity of 7q-v
Figure 3. X-ray crystallographic structure of 7k bound to p38R.
6-carboxamide moiety generally led to decreased activities in
the enzymatic and cellular assays, as demonstrated by 7o
(p38R IC50 = 31 nM, TNFR IC50 = 188 nM) and 7p (p38R
IC50 = 41 nM, TNFR IC50 = 543 nM). Notably, even though
7k-m are 3-9-fold less potent against p38R compared to 1a,
their cellular activities arecomparabletothat of1a. This result
may be related to the improved permeability of analogues
7k-m compared to 1a. Consistent with that assumption, the
Caco-2 transcellular permeability was found to be in a range
of 91-134 nm/s for 7k versus only 5 nm/s for 1a.
a n = 4, variation in individual values, <20%. b n = 3, variation in
individual values, <25%.
As anticipated, and shown by the X-ray cocrystal structure
of 7k with p38R enzyme (Figure 3), the binding mode of the
N-cyclopropylbenzamide is similar to that observed with 1a.
The benzamide moiety provides two key hydrogen bond
interactions with p38R: the carbonyl oxygen interaction with
˚
˚
Asp168 (2.86 A) and the NH interaction with Glu 71 (2.26 A).
Another important hydrogen bond interaction occurs be-
˚
tween the 6-carboxamide oxygen and the Met 109 (2.49 A)
at the hinge region. Hydrophobic interactions include the
angular methylaniline moiety being positioned within a hydro-
phobic pocket and the pendant N-propyl interacting with a
hydrophobic site characteristically found at the outer rim of
the hinge region.
Figure 4. X-ray crystallographic structure of 7v bound to p38R.
We also examined the replacement of the methyl hydroxa-
mate with aryl amides (Table 4). The N-phenyl amide 7q exhi-
bited a p38R IC50 value of 50 nM, which was less potent than N-
methyoxybenzamide 1b or N-cyclopropylbenzamide 7m and
essentially equipotent with N-ethylbenzamide 7b. Our modeling
studies suggested that the N-phenyl group was a little too large
to fit into the binding pocket. This also explained the p38R
activity (IC50 = 72 nM) for N-2-thiazolylbenzamide 7r because
the actual dimensions of phenyl and thiazolyl are very similar.
The cellular TNFR IC50 values of 7q and 7r were both deter-
mined to be greater than 1000 nM. When a methyl was added to
thethiazoleringin7r, the resulting analogue 7s displayed a p38R
IC50 value of over 1000 nM. Analogue 7t containing a smaller
N-oxazolyl group in the benzamide moiety provided an im-
proved p38R IC50 value of 35 nM. However, the N-3-isoxazo-
lylbenzamides were found to be highly potent against p38R, as
7u and 7v inhibited p38R with IC50 values of 7.1 and 3.9 nM,
respectively. These two inhibitors were also extremely active at
inhibiting TNFR production in hPBMC, exhibiting IC50 values
of 9.7 and 6.0 nM, respectively. The significant improvement in
binding affinity for 7u and 7v compared to 7t was surprising.
A cocrystal of 7v with p38R was obtained,15 and the X-ray crys-
tallographic analysis showed that 7v bound to p38R in a similar
manner as inhibitor 7k (Figure 3) except that an active water
molecule was engaged with 7v, Asp168, and Lys 53 (Figure 4).
On the basis of cellular activity and other profiling data (e.g.,
liver microsomal stability and P450 inhibition), selected com-
pounds were evaluated in a murine model of acute inflamma-
tion. In this study, compounds were administrated to mice
prior to challenging with LPS. Ninety minutes postdose,
plasma samples were taken and analyzed for TNFR levels.
In addition to a vehicle group, 1a was included as a comparator
and positive control in each study. Compounds were consid-
ered active if they reduced the LPS induced TNFR production
by 50%. As seen in Table 5, when dosed at 5 mg/kg, 2 h prior to
LPS challenge, compounds 7k, 7l, 7u, and 7v all showed
improved efficacy relative to 1a, inhibiting the TNFR levels
by 77-92% (versus 65-66% inhibition with 1a). The weakest
compound studied in this protocol was 7o (41% reduction of
TNFR). To differentiate these active compounds, they were
further examined under a more stringent protocol, wherein the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6633
Table 5. Inhibition of LPS Induced TNFR Production in Micea
dose
dosing interval
percent
percent
cmpd (mg/kg)a prior to LPS (hr) inhibitionb inhibition for 1a
7k
7l
5
5
5
5
5
5
5
5
5
2
2
2
2
2
6
6
6
6
89
77
41
92
78
78
27
89
83
66
66
65
65
66
50
50
63
50
7o
7u
7v
7k
7l
7u
7v
a PEG 300 was used as the vehicle, 6-8 mice per group. b Percent
inhibition was calculated relative to vehicle group.
Figure 6. Compound 7k in the rat adjuvant arthritis model (bid
dosing). Vehicle: PEG 300.
Figure 5. Compound 7k in the rat adjuvant arthritis model (qd
dosing). Vehicle: PEG 300.
dosing interval was extended to 6 h prior to LPS challenge.
Under this protocol, 7k, 7u, and 7v were active, reducing
TNFR production by 78%, 89%, and 83%, respectively, with
1a giving 50-63% reduction of TNFR.
Figure 7. Compound 7k vs 1a in the rat adjuvant arthritis model
(qd dosing). Vehicle: PEG 300.
Table 6. Pharmacokinetic Properties of 7k in Micea and Ratsb
On the basis of the above characterization, inhibitors 7k and
7u were chosen to be evaluated in a pseudoestablished rat adju-
vant arthritis (rat AA) model. In this study, complete Freund’s
adjuvant was given to rats at day one and the rats’ immune
responses were allowed to develop for 10 days. At day 11, the
rats started to receive compounds orally and the paw swelling
was measured periodically. Though 7u was very active in the
acute murine model of inflammation even when dosed at 5 mpk
6 h prior to LPS challenge, it was not active in the rat AA model
when dosed once daily at 10 mg/kg. It was found in this study
that 7u initially provided high drug concentration, but the drug
concentration dropped dramatically (by 80%) after 6 h. How-
ever, inhibitor 7k displayed dose-dependent reduction in paw
swelling with qd dosing, with efficacy observed at doses of
10 and 100 mg/kg (Figure 5). With bid dosing, 7k showed
improved efficacy, achieving marked reduction in paw
swelling at doses of 1 and 5 mg/kg (Figure 6). Statistically
significant reduction in paw swelling was also observed at
doses as low as 0.3 mg/kg bid. In addition, it was demon-
strated that 7k was more effective than 1a in the rat AA
model at a dose of 10 mg/kg qd (Figure 7).
mouse
rat
60
% Fpo
Cmax (μM)
Tmax (h)
T1/2 (h)
90
15.3
1.0
2.6
3.3
4.4
0.9
75.5
7.0
1.5
4.0
3.4
5.4
1.1
MRT (h)
CL (mL/min/kg)
V
ss (L/kg)
AUC0-8 h (μM h)
AUC0-24 h (μM h)
3
45.4
3
a Vehicle: 20% NMP, 35% PEG 400, 10% PG, and 35% water for iv,
and PEG 400 for po; dose: 5 mg/kg for iv, and 10 mg/kg for po; 3 mice
per group. b Vehicle: 25% NMP, 33% PEG 400, 9% PG, and 33% water
for iv, and PEG 400 for po; dose: 2.5 mg/kg for iv, and 10 mg/kg for po;
3 mice per group.
4.4 mL/min/kg versus 93 mL/min/kg for 1a. And, at an oral
dose of 10 mg/kg, the mouse AUC0-8 h for 7k was 75.5 μM h
versus 3.6 μM h. Compound 7k exhibited oral bioavailability
3
3
values of 90% and 60% in mice and rats, respectively.
Table 7 contains the in vitro profile of 7k. The compound had
a low rate of oxidative metabolism in mouse (0.011 nmol/min/
mg), rat (0.008 nmol/min/mg), and human liver microsomes
(0.013 nmol/min/mg). The metabolic rate in hepatocytes was
also low in these species. Compound 7k did not significantly
inhibit cytochrome P450 isozymes 1A2, 2C9, 2C19, and 2D6
with IC50 values >40 μM. It was a weak inhibitor of CYP3A4,
with an IC50 value ranging from 18 to 40 μM based in multiple
tests. As mentioned earlier, the Caco-2 permeability of 7k was
Inhibitor 7k is approximately 4-fold less potent against p38R
than 1a, but 7k is more effective than 1a in both the acute and
chronic disease models. This is likely due to the significantly
improved pharmacokinetic profile of 7k (Table 6) relative to 1a.
The most significant improvement was the dramatically
reduced clearance rate which resulted in significantly increased
exposure. For example, the mouse clearance rate for 7k was

6634 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Table 7. In Vitro Profile of 7k
Liu et al.
7k is currently in phase II clinical trials for the treatment of
rheumatoid arthritis, the results of which will be reported in
due course.
profiling assays
results
mouse: 0.011
rat: 0.008
liver microsome metabolic rate
(nmol/min/mg)
human: 0.013
Experimental Section
Chemistry. Proton and carbon magnetic resonance (1H and
13C NMR) spectra were recorded either on a Bruker Avance 400
or a JEOL Eclipse 500 spectrometer and are reported in ppm
relative to the reference solvent of the sample in which they were
run. HPLC and LCMS analyses were conducted using a Shimadzu
LC-10AS liquid chromatograph and a SPD UV-vis detector at 220
or 254 nm with the MS detection performed with a Micromass
Platform LC spectrometer. HPLC analyses were performed using
the following conditions: Ballistic YMC S5 ODS 4.6 mm ꢀ 50 mm
column with a binary solvent system where solvent A=10% metha-
nol, 90% water, 0.2% phosphoric acid, and solvent B=90% metha-
nol, 10% water, and 0.2% phosphoric acid, flow rate=4 mL/min,
linear gradient time=4 min, start %B=0, final %B=100. All final
compounds had an HPLC purity of g95% unless specifically
mentioned. LCMS analyses were performed using the following
conditions: Phenomenex 5 μm C18 4.6 mm ꢀ 50 mm column with a
binary solvent system where solvent A=10% methanol, 90% water,
0.1% trifluoroacetic acid, and solvent B = 90% methanol, 10%
water, and 0.1% trifluoroacetic acid, flow rate=4 mL/min, linear
gradient time=2 min, start %B=0, final %B=100. Preparative
reverse-phase HPLC purifications were performed using the follow-
ing conditions: Ballistic YMC S5 ODS 20 mmꢀ100 mm column
with a binary solvent system where solvent A=10% methanol, 90%
water, 0.1% trifluoroacetic acid, and solvent B=90% methanol,
10% water, and 0.1% trifluoroacetic acid, flow rate=20 mL/min,
linear gradient time=10 min, start %B=20, final %B=100.
All reagents were purchased from commercial sources and
used without further purification unless otherwise noted. All
reactions were performed under an inert atmosphere. Reactions
run in aqueous media were run under an ambient atmosphere
unless otherwise noted.
3-Amino-N-cyclopropyl-4-methylbenzamide (3b). To a solu-
tion of 3-amino-4-methylbenzoic acid (5) (5.12 g, 33.9 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) (9.97 g, 52.0 mol), and 4-(dimethylamino)pyridine (0.89 g,
7.3 mol) in DMF (100 mL) at 0 °C was added cyclopropyl-
amine (4.0 mL, 57.7 mol) dropwise. After stirring for 15 min, the
cold bath was removed and the reaction mixture was stirred at
room temperature overnight. Volatiles were removed under
vacuum, and the residue was diluted with water and extracted
with dichloromethane (3 times). The organic layers were com-
bined, dried over sodium sulfate, and concentrated under
vacuum. The residue was subjected to silica gel chromatography
using dichloromethane/methanol (20:1) as eluent to afford the
title compound (6.98 g, 108% yield) as a yellow oil. This product
was contaminated with dimethylformamide but used in the next
step without further purification. LCMS (EI) m/z Calcd for
C11H14N2O (M þ H)þ = 191.11. Found: 192.09.
hepatocyte metabolic rate
(nmol/min/million cells)
mouse: 0.006
rat: 0.015
human: 0.015
P450 IC50 (μM)
>40 for 1A2, 2C9,
2C19, and 2D6
18-40 for 3A4
Caco-2 permeability (nm/s)
serum protein binding (%)
121-134
mouse: 86.3
rat: 89.7
human: 81.5
Ames
negative in T98 and
T100 ( S9 activation
SOS chromotest
HHA IC50 (μM)
hERG inhibition
kinase selectivity
negative
>138
16% at 30 μM
>2000 fold over 57
diverse kinases
450 fold over Jnk2
190 fold over Raf
5 fold over p38β
determined to be in a range of 91-134 nm/s. The serum protein
binding for 7k at 10 μM was 86%, 90%, and 82% in fresh sera
isolated from mice, rats, and humans, respectively. The com-
pound was negative in the Ames reverse-mutation and SOS
chromotest assays. Also, 7k was not cytotoxic in the human
hepatotoxicity assay (HHA) (IC50 >138 μM for all isoforms),
suggesting a decreased likelihood for clinical hepatic liabilities.
In addition, 7k only minimally inhibited hERG current in a
patch clamp assay (16% at 30 μM). The compound proved to be
highly selective over other kinases tested. It displayed >2000-
fold selectivity for p38R over a diverse panel of 57 kinases that
include serine kinases, nonreceptor tyrosine kinases, receptor
tyrosine kinases, and the p38γ and δ isoforms. Compound 7k
was also 450-fold selective over Jnk2, a MAP kinase involved in
inflammation, and 190-fold selective over Raf. However, 7k
showed only 5-fold selectivity over p38β. On the basis of its
safety profile in preclinical safety studies, compound 7k, also
designated as BMS-582949, was nominated for further devel-
opment and clinical studies in inflammatory disease.
In summary, to improve the pharmacokinetic properties
of our previously reported p38R MAP kinase inhibitors, the
N-methoxy moiety in 1a and 1b was replaced with an N-cyclo-
propyl group. This specific modification was based on our
hypothesis that the π-character of the cyclopropyl moiety
would enhance the hydrogen bonding ability of the benzamide
NH in comparison to the less potent N-alkyl analogues. This
proposal ultimately led to the discovery of 7k, a highly selective
p38R inhibitor. Compared to 1a, 7k displayed a significantly
improved pharmacokinetic profile and was more effective in
both the acute murine model of inflammation and rat adjuvant
arthritis model despite its slightly reduced potency. Compound
Ethyl 4-(5-(Ethylcarbamoyl)-2-methylphenylamino)-5-methyl-
pyrrolo[1,2-f][1,2,4]triazine-6-carboxylate (4a). To a mixture of
ethyl 4-chloro-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxy-
late (2) (4.83 g, 20.2 mmol) and the HCl salt of 3-amino-N-
ethyl-4-methylbenzamide (3a)12 (4.33 g, 20.2 mmol) in DMF
(70 mL) was added diisopropylethylamine (2.80 mL, 16.2 mmol).
The resulting mixture was heated at 60 °C for 5 h, then cooled to rt
and slowly added to a mixture of crushed ice (100 mL) and
saturated aqueous NaHCO3 solution (200 mL). After stirring
overnight, the solid was collected by suction filtration and rinsed
with water (200 mL) to afford a pale-yellow solid. Recrystallization
from ethanol/water (1:1) afforded the title compound (5.50 g,
71%) as white crystals. LCMS (EI) m/z Calcd for C20H23N5O3
(M þ H)þ = 382.28. Found: 382.30.
Ethyl 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxylate (4b). A solution

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6635
of ethyl 4-chloro-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxy-
late (2) (1.30 g, 5.40 mmol) and 3-amino-N-cyclopropyl-4-methyl-
benzamide (3b) (1.60 g, 8.40 mmol) in DMF (13 mL) was stirred at
room temperature overnight. Water was added, and the precipi-
tated product (1.70 g, 80% yield) was collected as an off-white solid
by filtration, followed by trituration with diethyl ether. LCMS (EI)
m/z Calcd for C21H23N5O3 (M þ H)þ=394.18. Found: 394.31.
4-(5-(Ethylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo-
[1,2-f][1,2,4]triazine-6-carboxylic Acid (6a). To a solution of ethyl
4-(5-(ethylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo-
[1,2-f][1,2,4]triazine-6-carboxylate (4a) (1.81 g, 4.74 mmol) in
THF/MeOH (1:1 v/v, 7 mL) at rt was added aqueous 3 N
KOH solution (4.7 mL, 14.2 mmol). The reaction mixture was
stirred at 60 °C for 6 h, then concentrated under vacuum to
remove THF and methanol. The residue was cooled at 0 °C, and
the pH value was adjusted to ∼3 by addition of aqueous 6 N HCl.
The resulting solid was collected by filtration, washed with water
(5 mL ꢀ 3), and dried under vacuum to afford the title compound
(1.64 g, 90% yield) as a white solid. LCMS (EI) m/z Calcd for
C18H19N5O3 (M þ H)þ = 354.15. Found: 354.30.
yield 71%. 1H NMR (MeOD) δ 8.54 (br s, 1H), 8.12 (br s, 1H),
8.02 (br s, 1H), 7.88 (br s, 1H), 7.69 (br s, 1H), 7.41-7.44 (m,
3H), 7.32-7.36 (m, 2H), 7.22-7.26 (m, 1H), 5.19-5.27 (m, 1H),
2.81 (s, 3H), 2.34 (s, 3H), 1.56 (d, J = 7.1 Hz, 3H).
4-Methyl-3-(5-methyl-6-(propylcarbamoyl)pyrrolo[1,2-f][1,2,4]-
triazin-4-ylamino)benzoic Acid (8c). A solution of N-n-propyl-
4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo-
[2,1-f][1,2,4]triazine-6-carboxamide (1c) (2.28 g) in aqueous 1 N
HCl (30 mL) was heated at 85 °C for 6.5 h. Upon cooling to rt, the
mixture was neutralized with aqueous 1 N NaOH solution to
pH 6. The precipitating product (1.60 g, 95% yield) was collected
by suction filtration and dried. LCMS (EI) m/z Calcd for C19-
H21N5O3 (M þ H)þ = 368.10. Found: 368.17. NMR (400 MHz,
MeOD) δ 8.20 (br s, 1H), 8.12 (br s, 1H), 7.92-7.89 (m, 2H), 7.69
(br s, 1H), 7.45 (d, J =8.0Hz, 1H), 3.35 (m, 2H), 2.86 (s, 3H), 2.36
(s, 3H), 1.67 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H).
N-Ethyl-4-(5-(ethylcarbamoyl)-2-methylphenylamino)-5-methyl-
pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7a). To a solution of
4-(5-(ethylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo-
[1,2-f][1,2,4]triazine-6-carboxylic acid (6a) (20.0 mg, 0.057
mmol) in DMF (0.30 mL) were added HOBt (9.1 mg, 0.068
mmol) and EDC (13.0 mg, 0.068 mmol). The resulting solution
was stirred at rt for 1 h before ethylamine hydrochloride (12.6
mg, 0.068 mmol) and diisopropylethylamine (24 μL, 0.14 mmol)
were added. The mixture was stirred at rt for 16 h, then subjected
to preparative HPLC to afford a TFA salt of the title compound
(10.0 mg, 46% yield) as a white solid; 97% purity by HPLC.
LCMS (EI) m/z Calcd for C20H24N6O2 (M þ H)þ = 381.20.
Found: 381.10. 1H NMR (400 MHz, MeOD) δ 7.85 (s, 1H), 7.76
(s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.65 (s, 1H), 7.33 (d, J = 8.0
Hz, 1H), 3.32-3.26 (m, 4H), 2.74 (s, 3H), 2.23 (s, 3H), 1.13 (t,
J = 7.2 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H).
(S)-4-(5-(Ethylcarbamoyl)-2-methylphenylamino)-5-methyl-N-
(1-phenylethyl)pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7b).
To a solution of 4-(5-(ethylcarbamoyl)-2-methylphenylamino)-
5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxylic acid (6a)
(20 mg, 0.056 mmol) in DMF (0.3 mL) were successively added
HOBt (8.4 mg, 0.062 mmol) and EDC (12 mg, 0.062 mmol). The
resulting solution was stirred at rt for 1 h before (S)-R-methyl-
benzylamine (14 μL, 0.112 mmol) was added. After stirring at rt
for 16 h, the mixture was subjected to preparative HPLC to
obtain a TFA salt of the title compound (13.0 mg, 52% yield) as
a white solid; 99% purity by HPLC. LCMS (EI) m/z Calcd for
C26H28N6O2 (M þ H)þ = 457.23. Found: 457.30. 1H NMR (400
MHz, MeOD) δ 8.12 (s, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.0 Hz,
1H), 7.71 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.44-7.43 (m, 2H),
7.38-7.35 (m, 2H), 7.26 (m, 1H), 5.25 (q, J = 7.0 Hz, 1H), 3.43
(q, J = 7.3 Hz, 2H), 2.84 (s, 3H), 2.36 (s, 3H), 1.58 (d, J = 7.0
Hz, 3H), 1.24 (t, J = 7.3 Hz, 3H).
N-Ethyl-5-methyl-4-(2-methyl-5-(methylcarbamoyl)phenylamino)-
pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7c). To a solution of
5-methyl-4-(2-methyl-5-(methylcarbamoyl)phenylamino)pyr-
rolo[1,2-f][1,2,4]triazine-6-carboxylic acid (6c) (0.15 g, 0.442 mmol)
in DMF (2.5 mL) was added BOP (0.215 g, 0.486 mmol). In a
second vial, ethylamine hydrochloride (0.072 g, 0.884 mmol) was
dissolved in DMF (0.5 mL) and DIPEA (0.15 mL, 0.884 mmol),
and 0.50 mL of the acid (6c)/BOP solution was introduced. The
reaction was agitated at room temperature overnight and then
diluted slowly with water (5 mL) with vigorous stirring and cooling
in an ice bath. The resulting solid was collected by filtration and air-
dried to afford the title compound (0.0267 g, 82% yield) as an off-
4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-
pyrrolo[1,2-f][1,2,4]triazine-6-carboxylic Acid (6b). A mixture
of ethyl 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxylate (4b) (5.00 g,
12.7 mmol) and 1 N NaOH solution (51 mL, 51 mmol) in THF
(30 mL) was heated at reflux for 16 h, then concentrated under
vacuum. The residue was extracted with ethyl acetate. The
aqueous solution was neutralized with aqueous 6 N HCl solution
to pH 6. The precipitating product (4.35 g, 94% yield) was
collected as a white solid by suction filtration, washed with water,
1
and dried over Drierite under vacuum. H NMR (400 MHz,
DMSO-d6/D2O) δ 8.46 (s, 1H), 8.02 (s, 1H), 7.84 (s, 1H), 7.78 (s,
1H), 7.67 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 2.83 (m,
1H), 2.81 (s, 3H), 2.22 (s, 3H), 0.69 (m, 2H), 0.55 (m, 2H).
5-Methyl-4-(2-methyl-5-(methylcarbamoyl)phenylamino)pyrrolo-
[1,2-f][1,2,4]triazine-6-carboxylic Acid (6c). To a solution of methyl
5-methyl-4-(2-methyl-5 (methylcarbamoyl)phenylamino)pyrrolo-
[1,2-f][1,2,4]triazine-6-carboxylate (4c) (0.17 g, 0.48 mmol) in
THF (1 mL) was added aqueous 1 N NaOH solution (1.0 mL,
1.0 mmol). The homogeneous solution was stirred at 50 °C. Upon
complete conversion by HPLC analysis, aqueous 1 N HCl was
slowly added with vigorous stirring to give a precipitate. The
mixture was agitated for 2 h, then the precipitate was collected by
filtration to afford the title compound as a tan solid in quantitative
yield (92% purity by HPLC analysis). The material was used
directly for the next step without further purification.
3-(6-(Ethylcarbamoyl)-5-methylpyrrolo[1,2-f][1,2,4]triazin-4-yl-
amino)-4-methylbenzoic Acid (8a). To a slurry of N-ethyl-4-(5-
(methoxycarbamoyl)-2-methylphenylamino)-5-methylpyrrolo-
[1,2-f][1,2,4]triazine-6-carboxamide (1a)12 (2.0 g, 4.20 mmol) in
anhydrous methanol (12 mL) was added a solution of HCl in
dioxane (4 N, 18 mL, 72 mmol) at rt. The resulting clear solution
was stirred at rt for 16 h, then concentrated under vacuum. The
resulting oil was dissolved in aqueous 1.5 N potassium hydroxide
solution (16 mL) and heated to 50 °C for 3 h. After cooling to rt,
the mixturewas dilutedwithwater(50mL) and10% aqueous HCl
was added until pH was approximately 3 or 4. The precipitating
product was collected by vacuum filtration, washed with water
(50 mL), and dried under vacuum to afford the title product (1.47 g,
99% yield) as a white solid. An analytical sample was obtained
after recrystallization from 10% aqueous acetonitrile. LCMS (EI)
m/z Calcd for C18H19N5O3 (M þ H)þ = 354.15. Found: 354.20.
1H NMR (400 MHz, CD3OD) δ 8.21 (br s, 1H), 8.11 (br s, 1H),
7.89-7.91 (m, 2H), 7.67 (br s, 1H), 7.44 (d, 1H), 3.40 (q, 2H), 2.86
(s, 3H), 2.36 (s, 3H), 1.25 (s, 3H).
white solid. LCMS (EI) m/z Calcd for C19H22N6O2 (M þ H)þ
=
367.18. Found: 367.10. NMR (400 MHz, MeOD) δ 8.09 (s, 1H),
7.89 (d, J = 1.8 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.54
(d, J = 8.0 Hz, 1H), 3.40 (q, J = 7.3 Hz, 2H), 2.93 (s, 3H), 2.87 (s,
3H), 2.37 (s, 3H), 1.24 (t, J = 7.3 Hz, 3H).
(S)-4-Methyl-3-(5-methyl-6-(1-phenylethylcarbamoyl)pyrrolo-
[1,2-f][1,2,4]triazin-4-ylamino)benzoic Acid (8b). This compound
was prepared from (S)-4-(5-(methoxycarbamoyl)-2-methylphe-
nylamino)-5-methyl-N-(1-phenylethyl)pyrrolo[1,2-f][1,2,4]tri-
azine-6-carboxamide (1b) in the same way as 8a was from 1a;
5-Methyl-4-(2-methyl-5-(propylcarbamoyl)phenylamino)-N-
propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7d). To a solu-
tion of 4-methyl-3-(5-methyl-6-(propylcarbamoyl)pyrrolo[1,2-f]-
[1,2,4]triazin-4-ylamino)benzoic acid (8c) (47.5 mg, 0.13 mmol)

6636 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Liu et al.
in DMF (0.4 mL) were added HOBt (21 mg, 0.16 mmol) and
EDC (30 mg, 0.16 mmol), and the solution was stirred for 2 h.
n-Propylamine (21.4 μL, 0.26 mmol) was added, and the mixture
was stirred for 15 h. Water (0.4 mL) was added and the pre-
cipitated solids were collected by filtration, washed with water,
and dried under vacuum to afford the title compound (45 mg,
85% yield) as a white solid; 96% purity by HPLC. LCMS (EI)
m/z Calcd for C22H28N6O2, (M þ H)þ = 409.10. Found 409.20.
1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.25 (br m, 1H),
7.93 (m, 2H), 7.75 (s, 1H), 7.61 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H),
7.20 (d, J = 7.9 Hz, 1H), 7.15 (m, 1H), 3.03 (m, 4H), 2.60 (s, 3H),
2.06 (s, 3H), 1.35 (m, 4H), 0.72 (m, 6H).
N-Ethyl-4-(5-(isopropylcarbamoyl)-2-methylphenylamino)-5-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7e). This com-
pound was prepared as a TFA salt in a similar way as 7l was yield
20%, 98% purity by HPLC. LCMS (EI) m/z Calcd for C21H26-
N6O2 (M þ H)þ = 395.21. Found: 395.21. 1H NMR (400 MHz,
MeOD) δ 7.91 (s, 1H), 7.77 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.59
(s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 4.11 (m, 1H), 3.30 (q, J = 7.3
Hz, 2H), 2.76 (s, 3H), 2.25 (s, 3H), 1.15 (d, J = 6.7 Hz, 6H), 1.14
(t, J = 7.3 Hz, 3H).
4-(5-(Butylcarbamoyl)-2-methylphenylamino)-N-ethyl-5-methyl-
pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7f). To a solution of
4-(5-(ethylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo-
[1,2-f][1,2,4]triazine-6-carboxylic acid (8a) (80 mg, 0.225 mmol)
and n-butylamine (44 μL, 0.45 mmol) in DMF (0.7 mL) was
added HATU (68 mg, 0.27 mmol), and the resulting solution
was stirred for 2 h. At this time, additional HATU (68 mg,
0.27 mmol) was added, and the mixture was allowed to stir at rt
for an additional 1 h. The resulting mixture was then added
dropwise into water (2.0 mL) with stirring. The solids were
dispersed by sonicating for 1 min, and the suspension was stirred
at rt for 1 h. The solid was collected by suction filtration and
dried to afford the title compound (70 mg, 76% yield) as a white
powder; 98% purity by HPLC. LCMS (EI) m/z Calcd for
C22H28N6O2 (M þ H)þ = 409.23. Found: 409.28. 1H NMR
(500 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.40 (t, J = 5.6 Hz, 1H),
8.11 (s, 1H), 8.09 (m, 1H), 7.94 (s, 1H), 7.79 (s, 1H), 7.72 (d, J =
8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 3.30-3.24 (m, 4H), 2.83 (s,
3H), 2.25 (s, 3H), 1.51 (m, 2H), 1.33 (m, 2H), 1.13 (t, J = 7.2 Hz,
3H), 0.90 (t, J = 7.4 Hz, 3H).
N-Ethyl-4-(5-(2-methoxyethylcarbamoyl)-2-methylphenylamino)-
5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7g). To a
solution of 3-(6-(ethylcarbamoyl)-5-methylpyrrolo[1,2-f][1,2,4]-
triazin-4-ylamino)-4-methylbenzoic acid (8a) (19 mg, 0.054 mmol)
in DMF (0.4 mL) was added HOBt (8 mg, 0.06 mmol) and EDC
(11.5 mg, 0.06 mmol). The mixture was stirred at rt for 1 h before
2-methoxyethanamine (4.5 mg, 0.06 mmol) and DIPEA (14 mg,
0.11 mmol) were added. After stirring at rt for 16 h, the mixture was
subjected to reverse-phase preparative HPLC to obtain the TFA
salt of the title compound (14.0 mg, 49%) as a white solid; 98%
purity by HPLC. LCMS (EI) m/z Calcd for C21H26N6O3 (M þ
H)þ=411.21. Found: 211.20. 1H NMR (500 MHz, MeOD) δ 8.28
(s, 1H), 7.97 (d, J=7.7 Hz, 1H), 7.95 (s, 1H), 7.84 (s, 1H), 7.63 (d,
J=7.7 Hz, 1H), 3.57 (s, 3H), 3.41 (q, J=7.3 Hz, 2H), 3.30 (m, 2H),
2.91 (s, 3H), 2.70 (br s, 2H), 2.42 (s, 3H), 1.24 (t, J=7.3 Hz, 3H).
4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-N-ethyl-
5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7h). A mix-
ture of 3-(6-(ethylcarbamoyl)-5-methylpyrrolo[1,2-f][1,2,4]tri-
azin-4-ylamino)-4-methylbenzoic acid (8a) (0.936 g, 2.65 mmol),
cyclopropylamine (0.360 mL, 5.20 mmol), EDC (0.635 g, 3.31
mmol), and 4-(dimethylamino)pyridine (65 mg, 0.532 mmol) in
DMF (7 mL) was stirred at 45 °C for 20 h. The mixture was
diluted with ethyl acetate (120 mL), washed sequentially with
water (2 ꢀ 25 mL), 10% aqueous Na2CO3 solution (25 mL), and
brine (25 mL). The organic solution was dried over anhydrous
MgSO4 and concentrated under vacuum. The residue was
subjected to flash chromatography (silica gel, 10% MeOH/
CHCl3) to provide the title product (0.448 g, 43% yield) as a
white solid; 100% purity by HPLC. LCMS (EI) m/z Calcd for
C21H24N6O2 (M þ H)þ = 393.20. Found: 393.33. 1H NMR (500
MHz, DMSO-d6) δ 8.31 (s, 1H), 8.23 (s, 1H), 7.93 (s, 1H), 7.92 (s,
1H), 7.70 (s, 1H), 7.57-7.54 (m, 2H), 7.28 (d, J = 7.8 Hz, 1H),
3.25 (m, 2H), 2.85 (m, 1H), 2.78 (s, 3H), 2.18 (s, 3H), 1.12 (t, J =
7.2 Hz, 3H), 0.68 (m, 2H), 0.57 (m, 2H).
4-(5-(Cyclobutylcarbamoyl)-2-methylphenylamino)-N-ethyl-
5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7i). This
compound was prepared as a TFA salt in a similar way as 7l
was; Yield: 6.4%; 100% purity by HPLC. LCMS (EI) m/z Calcd
for C22H26N6O2 (M þ H)þ = 407.21. Found: 407.19. 1H NMR
(500 MHz, DMSO-d6) δ 8.76 (br s, 1H), 8.62 (br s, 1H), 8.18-
8.08 (m, 2H), 7.98 (br s, 1H), 7.87-7.73 (m, 2H), 7.43 (d, J = 7.5
Hz, 1H), 4.48 (m, 1H), 3.31 (m, 2H), 2.86 (s, 3H), 2.28 (s, 3H),
2.27-2.24 (m, 2H), 2.14 (m, 2H), 1.71 (m, 2H), 1.18 (t, J = 7.2
Hz, 3H).
4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-N,5-di-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7j). A mix-
ture of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxylic acid (6b) (0.026 g,
0.071 mmol), EDC (0.021 g, 0.11 mmol), HOBt (0.015 g, 0.11
mmol), methylamine hydrochloride (8.0 mg,, 0.12 mmol), and
diisopropylethylamine (0.040 mL, 0.23 mmol) in dimethylfor-
mamide (0.20 mL) was mechanically shaken at rt overnight.
Water (1 mL) was added, and the precipitated material was
collected by filtration, washed with water, and dried to give the
title compound (0.019 g, 72% yield) as a white solid; 100%
purity by HPLC. LCMS (EI) m/z Calcd for C20H22N6O2 (M þ
H)þ = 379.18. Found: 379.17. 1H NMR (500 MHz, DMSO-d6)
δ 8.72 (s, 1H), 8.44 (d, J = 4.4 Hz, 1H), 8.12-8.11 (m, 2H), 7.96
(s, 1H), 7.84 (s, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.0 Hz,
1H), 2.92 (m, 1H), 2.87 (s, 3H), 2.81 (d, J=4.4 Hz, 3H), 2.29
(s, 3H), 0.75 (m, 2H), 0.63 (m, 2H).
4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-
N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7k). A mix-
ture of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxylic acid (6b) (2.16 g,
5.91 mmol), n-propylamine (1.0 mL, 12.2 mmol), BOP (3.40 g,
7.69 mmol), and N-methylmorpholine (2.5 mL, 22.7 mmol) in
DMF (10 mL) was stirred at 50 °C for 3 h. The mixture was poured
into a mixture prepared from saturated NaHCO3 solution (60 mL)
and water (60 mL). The precipitating product was collected by
suction filtration was washed with water. This crude product was
suspended into ethyl acetate (100 mL) and stirred at 70 °C for 1 h.
Upon cooling to rt, the title compound (2.07 g, 86% yield) was
collected as a white solid by suction filtration; 98% purity by HPLC.
LCMS (EI) m/z Calcd for C22H26N6O2 (M þ H)þ = 407.21.
1
Found: 407.22. H NMR (500 MHz, DMSO-d6) δ 8.49 (d, J =
3.6 Hz, 1H), 8.23 (s, 1H), 8.21 (s, 1H), 7.86 (s, 1H), 7.80 (s, 1H), 7.77
(d, J=7.8 Hz, 1H), 7.42 (d, J=7.8 Hz, 1H), 3.20 (m, 2H), 2.87 (m,
1H), 2.82 (s, 3H), 2.25 (s, 3H), 1.54 (m, 2H), 0.91 (t, J = 7.4 Hz,
3H), 0.68 (m, 2H), 0.59 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ
167.3, 164.45, 155.3, 148.7, 138.8, 137.1, 133.0, 130.6, 127.2, 125.8,
119.6, 118.8, 114.4, 113.3, 41.0, 23.6, 23.1, 18.5, 12.1, 12.0, 6.2.
N-Butyl-4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-
5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7l). A mix-
ture of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-
methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxylic acid (6b) (0.026 g,
0.071 mmol), EDC (0.021 g, 0.11 mmol), HOBt (0.015 g, 0.11
mmol), n-butylamine (0.015 mL, 0.15 mmol), and diisopropylethyl-
amine (0.040 mL, 0.23 mmol) in dimethylformamide (0.20 mL)
was mechanically shaken at rt overnight. Water (1 mL) was added,
and the precipitated material was collected by filtration, washed
with water, and dried to give the title compound (0.021 g, 70%
yield) as a white solid; 98% purity by HPLC. LCMS (EI) m/z
Calcd for C23H28N6O2 (M þ H)þ = 421.23. Found: 421.18. 1H
NMR (500 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.46 (d, J = 4.2 Hz,
1H), 8.16 (s, 1H), 8.11 (t, J= 5.7 Hz, 1H), 7.96 (s, 1H), 7.84 (s, 1H),
7.75 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 3.28 (m, 2H),
2.92 (m, 1H), 2.87 (s, 3H), 2.29 (s, 3H), 1.56 (m, 2H), 1.41 (m, 2H),
0.98 (t, J = 7.4 Hz, 3H), 0.74 (m, 2H), 0.63 (m, 2H).

Article
(S)-4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-me-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6637
The residue was subjected to preparative HPLC to provide the
desired product (36.4 mg, 30% yield) as an off-white solid; 100%
purity by HPLC. LCMS (EI) m/z Calcd for C21H21N7O2S (M þ
H)þ = 436.15. Found: 436.36. 1H NMR (500 MHz, DMSO-d6) δ
12.52 (s, 1H), 8.59 (s, 1H), 8.17 (s, 1H), 8.04 (s, 1H), 8.02 (t, J = 5.6
Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.48 (d, J = 3.2 Hz,
1H), 7.39 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 3.2 Hz, 1H), 3.18 (m,
2H), 2.76 (s, 3H), 2.22 (s, 3H), 1.04 (t, J = 7.2 Hz, 3H).
N-Ethyl-5-methyl-4-(2-methyl-5-(5-methylthiazol-2-ylcarba-
moyl)phenylamino)pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide
(7s). This compound was prepared in the same way as 7r; yield
71%; 100% purity by HPLC. LCMS (EI) m/z Calcd for C22H23-
N7O2S (M þ H)þ = 450.16. Found: 450.19. 1H NMR (500
MHz, DMSO-d6/D2O) δ ppm 8.12 (s, 1H)), 8.02 (s, 1H), 7.87 (d,
J = 7.2 Hz, 1H), 7.75 (s, 1H), 7.42 (d, J = 8.05 Hz, 1H), 7.15 (s,
1H), 3.20 (q, J = 7.2 Hz, 2H), 2.76 (s, 3H), 2.31 (s, 3H), 2.23 (s,
3H), 1.07 (t, J = 7.21 Hz, 3H).
thyl-N-(1-phenylethyl)pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide
(7m). A mixture of (S)-4-methyl-3-(5-methyl-6-(1-phenylethyl-
carbamoyl)pyrrolo[1,2-f][1,2,4]triazin-4-ylamino)benzoic acid (8b)
(34.4 mg, 0.080 mmol), cyclopropylamine (0.010 mL, 0.144 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) (20.1 mg, 0.104 mmol), and N,N-dimethylpyridin-4-amine
(DMAP) (2.4 mg, 0.020 mmol) in DMF (0.5 mL) was stirred at rt
for 16 h. The reaction mixture was diluted with methanol (1.5 mL)
and injected to prep. HPLC to afford the title compound (30.2 mg,
81% yield) as a white solid; 100% purity by HPLC. LCMS (EI) m/z
1
Calcd for C27H28N6O2 (M þ H)þ = 469.23. Found: 469.35. H
NMR (500 MHz, DMSO-d6) δ 8.59 (s, 1H), 5.06 (m, 1H), 8.37 (d,
J = 8.0 Hz, 1H), 8.32 (d, J = 4.2 Hz, 1H), 8.20 (s, 1H), 7.82 (s, 1H),
7.72 (s, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.32-7.23 (m, 5H), 7.14 (m,
1H), 2.76 (m, 1H), 2.70 (s, 3H), 2.14 (s, 3H), 1.38 (d, J = 7.2 Hz,
3H), 0.59 (m, 2H), 0.48 (m, 2H).
N-Ethyl-5-methyl-4-(2-methyl-5-(oxazol-2-ylcarbamoyl)phenyl-
amino)pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7t). A mix-
ture of 3-(6-(ethylcarbamoyl)-5-methylpyrrolo[1,2-f][1,2,4]tria-
zin-4-ylamino)-4-methylbenzoic acid (8a) (120 mg, 0.306 mmol),
2-aminoxazole (103 mg, 1.22 mmol), BOP (271 mg, 0.613 mmol),
and N-methylmorpholine (0.20 mL, 1.80 mmol) in DMF (1 mL)
was heated at 70 °C for 16 h. The mixture was diluted with ethyl
acetate (60 mL), and washed sequentially with water, saturated
NaHCO3 solution, and brine. The organic solution was dried over
anhydrous MgSO4 and concentrated under vacuum. The residue
was subjected to flash chromatography (silica gel, 8% MeOH/
CHCl3) to provide the desired product (53 mg, 41% yield) as beige
solid; 100% purity by HPLC. LCMS (EI) m/z Calcd for
C21H21N7O3 (M þ H)þ = 420.17. Found: 420.25. 1H NMR
(500 MHz, DMSO-d6/D2O) δ 8.22 (s, 1H), 8.15 (s, 1H), 8.00 (s,
1H), 7.91 (d, J = 7.4 Hz, 1H), 7.87 (s, 1H), 7.54 (d, J = 7.4 Hz,
1H), 7.25 (s, 1H), 3.33 (q, J = 7.2 Hz, 2H), 2.88 (s, 3H), 2.35 (s,
3H), 1.19 (t, J = 7.2 Hz, 3H).
(S)-N-sec-Butyl-4-(5-(cyclopropylcarbamoyl)-2-methylphenyl-
amino)-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7n).
This compound was prepared in the same way as 7l; Yield: 76%;
100% purity by HPLC. LCMS (EI) m/z Calcd for C23H28N6O2
(M þ H)þ = 421.23. Found: 421.22. 1H NMR (500 MHz,
DMSO-d6) δ 8.72 (s, 1H), 8.46 (s, 1H), 8.22 (s, 1H), 7.98 (s, 1H),
7.86 (s, 1H), 7.83 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.44 (d, J =
8.0 Hz, 1H), 3.97 (m, 1H), 2.93 (m, 1H), 2.88 (s, 3H), 2.30 (s, 3H),
1.56 (m, 2H), 1.20 (d, J = 6.7 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H),
0.76 (m, 2H), 0.64 (m, 2H).
4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-N-(2-me-
thoxyethyl)-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide
(7o). This compound was prepared in the same way as 7l; Yield:
73%; 100% purity by HPLC. LCMS (EI) m/z Calcd for
C22H26N6O2 (M þ H)þ = 423.21. Found: 423.17. 1H NMR
(500 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.46 (d, J = 4.4 Hz, 1H),
8.22 (t, J = 5.4 Hz, 1H), 8.20 (s, 1H), 7.96 (s, 1H), 7.85 (s, 1H),
7.75 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 3.51 (m, 2H),
3.46 (m, 2H), 3.38 (s, 3H), 2.92 (m, 1H), 2.88 (s, 3H), 2.29 (s, 3H),
0.75 (m, 2H), 0.63 (m, 2H).
(R)-4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-N-(2-
hydroxy-1-phenylethyl)-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-
carboxamide (7p). This compound was prepared in the same way
as 7l; Yield: 59%; 100% purity by HPLC. LCMS (EI) m/z Calcd
for C27H28N6O3 (M þ H)þ = 485.22. Found: 485.92. 1H NMR
(500 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.46 (d, J = 4.4 Hz, 1H),
8.41 (d, J = 8.2 Hz, 1H), 8.39 (s, 1H), 7.97 (s, 1H), 7.87 (s, 1H),
7.75 (d, J = 7.7 Hz), 7.47-7.37 (m, 5H), 7.30 (m, 1H), 5.11 (m,
1H), 5.00 (m, 1H), 3.72 (m, 2H), 2.92 (m, 1H), 2.85 (s, 3H), 2.29
(s, 3H), 0.74 (m, 2H), 0.63 (m, 2H).
(S)-5-Methyl-4-(2-methyl-5-(phenylcarbamoyl)phenylamino)-
N-(1-phenylethyl)-pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7q).
To a solution of (S)-4-methyl-3-(5-methyl-6-(1-phenylethylcarba-
moyl)pyrrolo[1,2-f][1,2,4]triazin-4-ylamino)benzoic acid (8b)
(50 mg, 0.116 mmol) in DMF (0.3 mL) were successively added
diisopropylethylamine (61 μL, 0.348 mmol) and HATU (66 mg,
0.174 mmol). The resulting solution was stirred at rt for 20 min
before aniline (21 μL, 0.232 mmol) was added. The mixture was
heated at 70 °C for 15 h, then subjected to preparative HPLC to
afford the title compound (2.0 mg, 3% yield) as a tan solid; 98%
purity by HPLC. LCMS (EI) m/z Calcd for C30H28N6O2 (M þ
H)þ = 505.23. Found: 505.30.
N-Ethyl-4-(5-(isoxazol-3-ylcarbamoyl)-2-methylphenylamino)-
5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7u). A mix-
ture of 3-(6-(ethylcarbamoyl)-5-methylpyrrolo[1,2-f][1,2,4]tria-
zin-4-ylamino)-4-methylbenzoic acid (8a) (12.0 g, 34.0 mmol),
3-aminoisoxazole (12.5 mL, 169 mmol), BOP (30.1 g, 68.0 mmol),
and N-methylmorpholine (22.4 mL, 204 mmol) in DMF (100 mL)
was heated at 65 °C for 48 h. The mixture was concentrated under
vacuum to approximately two-thirds of its original volume and
then poured into saturated NaHCO3 solution (900 mL). The
precipitating product was collected by suction filtration and dried
over Drierite under vacuum. The crude product was subjected to
flash chromatography (silica gel, 5% MeOH/CHCl3). The correct
fractions were combined and concentrated to a volume of approxi-
mately 100 mL. The insoluble product (3.38 g, 24%) was collected
as a white solid by suction filtration; 100% purity by HPLC.
LCMS (EI) m/z Calcd for C21H21N7O3 (M þ H)þ = 420.17.
1
Found: 420.20. H NMR (500 MHz, DMSO-d6) δ 11.4 (s, 1H),
8.86 (s, 1H), 8.69 (s, 1H), 8.18 (s, 1H), 8.12 (s, 1H), 8.11 (t, J = 5.5
Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.47 (d, J = 8.0 Hz,
1H), 7.06 (s, 1H), 3.27 (m, 2H), 2.84 (s, 3H), 2.30 (s, 3H), 1.13 (t,
J = 7.2 Hz, 3H).
4-(5-(Isoxazol-3-ylcarbamoyl)-2-methylphenylamino)-5-meth-
yl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7v). This
compound was prepared in a similar way from 8c as 7u was from
8a; yield 20%; 99% purity by HPLC. LCMS (EI) m/z Calcd for
C22H23N7O3 (M þ H)þ = 420.19. Found: 434.29. 1H NMR (500
MHz, DMSO-d6) δ 11.4 (s, 1H), 8.86 (s, 1H), 8.68 (s, 1H), 8.18 (s,
1H), 8.13 (s, 1H), 8.09 (t, J = 5.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H),
7.82 (s, 1H), 7.47 (d, J= 8.0 Hz,1H), 7.06 (s, 1H), 3.19 (m, 2H), 2.84
(s, 3H), 2.30 (s, 3H), 1.53 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H).
p38r Kinase Assay. The assays were performed in V-bot-
tomed 96-well plates. The final assay volume was 60 μL, which
was from three 20 μL additions of enzyme, substrates (myelin
basic protein (MBP) and ATP), and test compounds in assay
buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 50 mM NaCl, and
N-Ethyl-5-methyl-4-(2-methyl-5-(thiazol-2-ylcarbamoyl)phe-
nylamino)pyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (7r). A mix-
ture of 3-(6-(ethylcarbamoyl)-5-methylpyrrolo[1,2-f][1,2,4]triazin-
4-ylamino)-4-methylbenzoic acid (8a) (97.2 mg, 0.275 mmol),
2-aminothiazole (42.7 mg, 42.6 mmol), benzotriazole-1-yl-oxy-
tris(dimethylamino)phosphonium hexafluorophosphate (BOP)
(250 mg, 0.565 mmol), and N-methylmorpholine (0.15 mL, 1.36
mmol) in DMF (1 mL) was heated at 55 °C for 16 h. The mixture
was diluted with ethyl acetate, washed sequentially with water,
saturated NaHCO3 solution, and brine. The organic solution was
dried over anhydrous MgSO4 and concentrated under vacuum.

6638 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Liu et al.
1 mM DTT). Bacterially expressed, activated p38 was preincu-
bated with test compounds for 10 min prior to the initiation of
reaction by adding substrates. The plates were incubated at room
temperature for 45 min. The reaction was terminated by adding
5 μL of 0.5 M EDTA to each well. The reaction mixture was
aspirated onto a prewet filter mat using a Skatron Micro96 cell
harvester (Skatron) and washed with PBS. The filtermat was dried
in a microwave oven for 1 min, coated with a layer of MeltilLex A
scintillation wax (PerkinElmer), and counted on a Microbeta
scintillation counter (model 1450, PerkinElmer). The data were
analyzed using the Prizm nonlinear least-squares regression
(GraphPad Software). The final concentrations of reagents in the
assays were [ATP], 1 μM; [[γ-33P]ATP]], 3 nM; [MBP] (Sigma,
M1891), 2 μg/well; [p38], 15 ng/well; [DMSO], 0.3%.
Rat Pharmacokinetic Study of 7k. Three groups of male
Sprague-Dawley rats (250-300 g) were fasted overnight and
received compound 7k as an intravenous (IV) bolus dose
(2.5 mg/kg with 25% NMP, 33% PEG 400, 9% PG, and 33%
water as a vehicle) or by oral gavage (10 mg/kg with PEG 400 as
a vehicle). Blood samples (0.3 mL) were obtained retro-orbital
bleeding at 0.05, 0.25, 0.5, 1, 3, 6, 8, and 24 h post dose for the IV
group and at 0.25, 0.5, 1, 3, 6, 8, and 24 h post dose for the PO
dose group. At each time point, three mice were bled resulting in
a composite pharmacokinetic profile. The tubes were inverted
several times to ensure mixing and placed on ice. Plasma was
obtained following centrifugation at 4 °C (1500-2000g). Plasma
samples were stored at -20 °C until analysis by a nonchiral
LC/MS/MS assay.
LPS-Induced TNFr Production in Human PBMC. Human
PBMCs were isolated from whole blood collected from healthy
donors. Blood was diluted into RPMI 1640 (Life Technologies)
containing 2.5 mM EDTA (Life Technologies), 10 μg/mL
polymyxin (Sigma), and then underlaid with ficoll (Accurate
Scientific Co.) and centrifuged at 600g for 25 min. The interface
was collected and cells were washed twice and resuspended in
RPMI, 10% FBS. Cells are then distributed (200 μL/well) into
96-well tissue culture treated plates (Falcon) at 1 ꢀ 106 cells/mL
in RPMI, 10% FBS. Test compounds were added to appro-
priate wells and incubated with cells for 30 min. Cells were
then stimulated by the addition of lipopolysaccharide (LPS,
BioWhittaker), with a final concentration of 25 ng/mL, and
incubated for 6 h at 37 °C, 5% CO2. The cell supernatants were
removed and assayed for TNFR by ELISA (R&D Systems).
Inhibition of TNFr Release in Mice. BALB/c female mice,
6-8 weeks of age, were obtained from Harlan Laboratories and
maintained ad libitum on water and standard rodent chow
(Harlan Teklad). Mice were acclimated to ambient conditions
for at least one week prior to use. For oral dosing, the com-
pounds were prepared in a solution of 100% polyethylene glycol
(mw 300) and a dosing volume of 0.2 mL per mouse was
administered by gavage at various times (see Table 6) prior to
LPS injection (0.5 mL of LPS suspended at 2 μg/mL in PBS,
administered ip). Blood samples were obtained 90 min after LPS
injection. Serum was separated from clotted blood samples by
centrifugation (5 min, 5000g, room temperature) and analyzed
for levels of TNFR by ELISA assay (R&D Systems) according
to the manufacturer’s directions. Results are shown as mean (
SD of n = 8 mice per treatment group. All procedures involving
animals were reviewed and approved by the Institutional Ani-
mal Care and Use Committee.
Rat Adjuvant Arthritis. Male Lewis rats (Harlan, 175-200 g)
were immunized sc at the base of the tail with 0.1 mL complete
Freund’s adjuvant containing 10 mg/mL Mycobacterium butyr-
icum. Seven days later, baseline (predisease) measurements of
hind paw volume were determined by volume displacement
plethysmometry (Ugo Basile, Italy). Compounds were adminis-
tered orally in 1 mL PEG 300 beginning on day 11. Paw volume
mesurements were repeated 3 times/week for the remainder of
the study. Data are presented as the summed increase in volume
(expressed in mL) above baseline for each rat’s two hind paws.
Mouse Pharmacokinetic Study of 7k. Three groups of male
BALB/c mice (20-25 g) were fasted overnight and received
compound 7k as an intravenous (IV) bolus dose (5 mg/kg with
20% NMP, 35% PEG 400, 10% PG, and 35% water as a
vehicle) or by oral gavage (10 mg/kg with PEG 400 as a vehicle).
Blood samples (0.2 mL) were obtained retro-orbital bleeding
at 0.05, 0.25, 0.5, 1, 3, 6, 8, and 24 h post dose for the IV group
and at 0.25, 0.5, 1, 3, 6, 8, and 24 h post dose for the PO dose
group. At each time point, three mice were bled, resulting in
a composite pharmacokinetic profile. The tubes were inverted
several times to ensure mixing and placed on ice. Plasma was
obtained following centrifugation at 4 °C (1500-2000g). Plasma
samples were stored at -20 °C until analysis by a nonchiral
LC/MS/MS assay.
Co-Crystal Structure of 7k and 7v with p38 MAP Kinase. His-
p38 MAP kinase was expressed in Escherichia coli (BL21 DE3)
and purified by sequential anion exchange, nickel chelate affi-
nity, and size exclusion chromatography. The protein at 2 mg/
mL, in 25 mM Tris (pH 7.4), 50 mM NaCl, 5% glycerol, 2 mM
DTT, was complexed with a 5-fold molar excess of 7k or 7v and
then concentrated to 17 mg/mL. Co-crystals were grown by the
hanging drop method from solutions containing 30% PEG 10K,
5 mM MgSO4, 50 mM MES, pH 6.0. Data were collected to
˚
approximately 2.2 A resolution on the R-AXIS IV area detector,
reduced using the Denzo/HKL package, and refined by the
method of simulated annealing using program CNX.
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(15) PDB deposition number: 3MVM.