The discovery of orally active triaminotriazine aniline amides as inhibitors of p38 MAP kinase

Article abstract of DOI:10.1021/jm049521d

A new structural class of triaminotriazine aniline amides possessing potent p38 enzyme activity has been discovered. The initial hit (compound 1a) was identified through screening the Pharmacopeia ECLiPS compound collection. SAR modification led to the identification of a short acting triaminotriazine aniline methoxyamide (compound 1m) possessing in vitro and in vivo oral activity in animal models of acute and chronic inflammatory disease. An X-ray crystal structure of compound 1m in this class, cocrystallized with unactivated p38a protein, indicates that these compounds bind to the ATP binding pocket and possess key H-bonding interactions within a deeper cleft. Hydrogen bonding between one of the triazine nitrogens and the backbone NH of the Met109 residue occurs through a water molecule. The methoxyamide NH and carbonyl oxygen are within H-bonding distance of Glu71 and Asp168.

Full text of DOI:10.1021/jm049521d

J. Med. Chem. 2004, 47, 6283-6291
6283
The Discovery of Orally Active Triaminotriazine Aniline Amides as Inhibitors
of p38 MAP Kinase
Katerina Leftheris,*,† Gulzar Ahmed,‡ Ran Chan,‡ Alaric J. Dyckman,† Zahid Hussain,‡ Kan Ho,‡
John Hynes, Jr.,† Jeffrey Letourneau,‡ Wei Li,‡ Shuqun Lin,† Axel Metzger,‡ Kevin J. Moriarty,‡ Chris Riviello,‡
Yvonne Shimshock,‡ James Wen,‡ John Wityak,† Stephen T. Wrobleski,† Hong Wu,† Junjun Wu,‡
Madhuri Desai,‡ Kathleen M. Gillooly,§ Tsung H. Lin,‡ Derek Loo,§ Kim W. McIntyre,§ Sidney Pitt,§
Ding Ren Shen,§ David J. Shuster,§ Rosemary Zhang,§ David Diller,‡ Arthur Doweyko,| John Sack,|
Jack Baldwin,‡ Joel Barrish,† John Dodd,† Ian Henderson,‡ Steve Kanner,§ Gary L. Schieven,§ and Maria Webb‡
Departments of Chemistry, Biology and Modeling, Pharmacopeia, Departments of Discovery Chemistry, Macromolecular
Structure, and Immunology, Bristol-Myers Squibb, PO Box 4000, Princeton, New Jersey 08543-4000
Received June 18, 2004
A new structural class of triaminotriazine aniline amides possessing potent p38 enzyme activity
has been discovered. The initial hit (compound 1a) was identified through screening the
Pharmacopeia ECLiPS compound collection. SAR modification led to the identification of a
short acting triaminotriazine aniline methoxyamide (compound 1m) possessing in vitro and
in vivo oral activity in animal models of acute and chronic inflammatory disease. An X-ray
crystal structure of compound 1m in this class, cocrystallized with unactivated p38R protein,
indicates that these compounds bind to the ATP binding pocket and possess key H-bonding
interactions within a deeper cleft. Hydrogen bonding between one of the triazine nitrogens
and the backbone NH of the Met109 residue occurs through a water molecule. The methoxy-
amide NH and carbonyl oxygen are within H-bonding distance of Glu71 and Asp168.
Chart 1
Introduction
Overproduction of cytokines such as IL-1 and TNF-R
are implicated in a wide variety of inflammatory dis-
eases, including rheumatoid arthritis (RA), psoriasis,
multiple sclerosis, inflammatory bowel disease, endot-
oxic shock, osteoporosis, Alzheimer’s disease, and con-
gestive heart failure, among others.1 There is significant
clinical proof of concept demonstrating that reduction
of systemic circulating levels of these key inflammatory
mediators is beneficial in the treatment of inflammatory
diseases such as rheumatoid arthritis, Crohn’s disease,
and psoriasis. Examples of these agents include the
soluble TNF-R receptor Fc fusion protein (etanercept),
anti-TNF antibody (infliximab), and the IL-1 receptor
antagonist (anakinra).2
The stress-activated signal transduction pathway
leading to inflammatory cytokine production in stimu-
lated immune cells is known to be regulated in part by
p38R mitogen activated protein (MAP) kinase. p38 MAP
kinase is a member of a Ser-Thr kinase superfamily that
includes p38, ERK2, and JNK.3 There are four known
isoforms of p38: p38R and p38â are widely expressed
in eukaryotic cells including endothelial and inflamma-
tory cells. Expression of p38γ is found in skeletal
muscle, and p38δ is predominantly expressed in the
small intestine, kidney, and lung tissue.4 p38R is part
of a stress-activated intracellular signaling cascade.
Activation of the cascade occurs through external stimuli
such as osmotic stress, inflammatory cytokines, heat,
and UV light. Once activated, p38R phosphorylates
intracellular substrates such as MAPKAP K2, which in
turn regulate the biosynthesis of TNFR and IL-1â at
the posttranscriptional level.
Targeting TNFR and IL-1â through inhibition of p38R
with an orally active small molecule inhibitor represents
an attractive alternative to current biologic therapy.
There is potential for lower cost of goods, oral vs
parenteral administration, lower patient costs, and
suppression of both TNFR and IL-1â with monotherapy.
Numerous companies have efforts in pursuing p38R
inhibitors for the treatment of inflammatory disease.
VX-745 (Chart 1) advanced to phase II trials and
reported a correlation between p38 inhibition and an
* To whom correspondence should be addressed: Bristol-Myers
Squibb, PO Box 4000, Princeton, NJ 08543-4000. Phone: (609)-252-
6188. Fax: (609)-252-7410. E-mail: katerina.leftheris@bms.com.
† Department of Discovery Chemistry.
‡ Departments of Chemistry, Biology and Modeling, Pharmacopeia.
§ Department of Immunology.
| Department of Macromolecular Structure.
10.1021/jm049521d CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/06/2004

6284 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Leftheris et al.
Scheme 1a
anilines compared to aliphatic amines, coupling of the
desired aniline to cyanuric chloride was selected first.
This was followed by subsequent couplings of the
desired amines under basic conditions to form the
corresponding triaminotriazines 1a-n. For amino-
pyrrolidine-containing derivatives, the aminopyrrolidine
was coupled as the Boc-protected amine (9, Scheme 2)
to avoid formation of the incorrect regioisomer. For
several members of this series, modification of the
aniline was desired while maintaining the pyrrolidine
and the neopentyl(methyl) amino side chains. Therefore,
the aniline was coupled as the methyl 3-amino-4-
methylbenzoate (5). Ester hydrolysis to the carboxylic
acid followed by amide coupling and Boc deprotection
of the pyrrolidine occurred after the triaminotriazine
was constructed.
a
Conditions: (a) cyanuric chloride, (iPr)2NEt, acetone, 0 °C;
(b) R2R3NH2+Cl-, (iPr)2NEt,THF, 0 °C to room temperature or
R2R3NH, THF, 0 °C to room temperature; (c) R4R5NH, N-
methylpyrrolidinone, 80 °C.
Discussion
Compounds were evaluated for their ability to inhibit
phosphorylation of substrate (myelin basic protein) by
recombinant human p38R. A THP-1 assay was used to
measure the ability of compounds to inhibit LPS-
induced TNF production in cells.
antiinflammatory effect in RA patients before it was
discontinued. VX-702 (structure not disclosed) is cur-
rently in clinical trials for acute coronary syndrome.5
BIRB-796 is currently in clinical trials for several
antiinflammatory indications.6 In designing inhibitors,
there has been an emphasis on identifying molecules
that bind at least in part to the ATP binding pocket of
p38 as well as accessing allosteric sites.5,7
Herein, we describe our initial efforts in developing
potent, selective 1,3,5-triaminotriazine aniline amides
as inhibitors of p38R MAP kinase through iterative SAR
studies. Our initial hit 1a was identified through
screening the Pharmacopeia ECLiPS compound collec-
tion.
The triaminotriazine inhibitor hit (1a) possesses three
potential sites for SAR modification (N1, N2, and N3).
We utilized an SAR strategy for identifying potent p38R
inhibitors that involved altering substituents at one site
while the remaining sites were held constant. As shown
in Table 1, initial efforts focused on exploring modifica-
tion of the 1,4-diazepan ring of 1a to determine if other
aliphatic ring systems containing a basic residue are
tolerated. Conversion to the methylated derivative 1b
did not alter enzyme activity. Replacement of the 1,4-
diazepan by NH2 (1c) led to a compound with a slight
reduction in enzyme inhibition. However, introduction
of the amino pyrrolidine ring (1d) improved enzyme
inhibitory potency by 25-fold. However, cell activity was
not significantly improved over that of the parent.
Chirality of the pyrrolidine ring does not appear to play
a role in enzyme inhibition or cell activity since the
corresponding (S)-pyrrolidine demonstrated similar po-
Design and Synthesis
As shown in Scheme 1, the desired aniline amide 2
was first coupled to cyanuric chloride through direct
chloride displacement to form 3. Order of addition of
substituents onto the triazine core can occur in several
ways. Due to the relatively poor nucleophilicity of
Scheme 2a
a Conditions: (a) cyanuric chloride, acetone, 0 °C, 98%; (b) 7, (iPr)2NEt, THF, 0 °C to room temperature, 98%; (c) (i) 9,
N-methylpyrrolidinone, 80-140 °C; (ii) aqueous NaOH, MeOH, 79%; (d) (i) EDCI, HOBt, R1R2NH, room temperature; (ii) HCl, dioxane,
room temperature 22-88%.

Inhibitors of p38 MAP Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6285
Table 3. Selected Triaminotriazine R3 Replacements 11a-h
Table 1. Selected Substituted Triaminotriazines 1a-d
p38R
TNFR (THP-1)
R1
IC50 (nM)a
IC50 (nM)b
p38R
TNFR (THP-1)
1a
1,4-diazepan-1-yl
1360
1420
5900
53
1700
nd
R3
IC50 (nM)a
IC50 (nM)b
1b 1,4-diazepan-1-yl(methyl)
1c
NH2
nd
11a
11b
11c
11d
11e
11f
CONHEt
31
264
64
346
100
8
140
680
85
>250
130
16
1d (R)- pyrrolidine-3-ylamino
1100
CONHCH2CH2OCH3
CONHBn
a n ) 4, variation in individual values, <20%. b n ) 3, variation
in individual values, <25%.
COOCH3
CONHcyclopentyl
CONHOCH3
CONHOH
Table 2. Selected Triaminotriazine R2 Replacements 1e-j
11g
11h
16
51
150
150
CONHCH(CH3)2
a n ) 4, variation in individual values, <20%. b n ) 3, variation
in individual values, <25%.
reduction in enzyme inhibition. Therefore, focus was
placed on conservative changes where positioning of the
2-methyl and the 5-amido were not altered. Simple alkyl
amides in place of CONH2 are well tolerated. As shown
for 11a, both enzyme and cell activity are equivalent
compared to 1i. Branching of the alkyl group (11h) is
also well tolerated. Introduction of longer chains includ-
ing an alkoxy group results in a 9-fold reduction in
enzyme inhibition (11b). Benzyl as well as cyclopentyl
groups are tolerated (11c, 11e). However, a methyl ester
results in greater than a 10-fold reduction in enzyme
potency. Introduction of a methoxyamide (11f) leads to
a 4-fold improvement in enzyme and >9-fold improve-
ment in cell activity vs 11a. The electron withdrawing
capability of the alkoxy group in alkoxyamides may
contribute to improving the H-bonding capability of the
amide NH. The hydroxamic acid 11g also displays
potent enzyme activity. However, cell activity is dimin-
ished compared to 11f potentially due to the impact this
group may have on the overall hydrophobicity of the
molecule. Other alkoxyamides are well tolerated. How-
ever, as seen for the alkyl amides, extension beyond an
ethyl group leads to compounds with diminished enzyme
activity (data not shown). Because of the improvement
in enzyme and cell activity, the 1-amino-2-methyl-5-(N-
methoxy)benzamide was chosen as a key functionality
for the N3 position.
Table 4 shows further modification of N1 where N3
contains the aniline methoxyamide. The optimal com-
pound in this series based on cell activity (1m) contains
a 1,4-diazepan-1-yl group where the terminal amine is
methylated.
The X-ray crystal structure of 1m bound to unphos-
phorylated p38R enzyme was examined for key binding
interactions. The p38-bound 1m ligand exhibits several
key hydrogen-bonding contacts in the ATP binding
pocket (Figure 1). A distinct contact occurs between the
triazine nitrogen and Met109 backbone NH through a
water molecule (NH-O, 1.84 Å; OH-N, 2.11 Å). In most
other kinase inhibitors accessing the ATP pocket,
contact with Met109 backbone NH occurs directly with
a ligand heteroatom. The pendant methoxyamide ester
NH and carbonyl oxygen are within hydrogen-bonding
distance of Glu71 (2.38 Å) and Asp168 (2.18 Å, backbone
p38R
TNFR (THP-1)
R
IC50 (nM)a
IC50 (nM)b
1e p-fluorophenethylamino
1f 2-phenylpropyl(methyl)amino
1g 2-methylpropylamino
1h 2-methylpropyl(methyl)amino
1i neopentylmethyl(methyl)amino
1j cyclohexylmethyl(methyl)amino
227
84
>5000
2600
>5000
325
167
1300
73
28
24
250
a n ) 4, variation in individual values, <20%. b n ) 3, variation
in individual values, <25%.
tency (data not shown). Therefore, the R configuration
of the pyrrolidine was chosen for subsequent SAR
studies.
As shown in Table 2, modification of the myrtanyl
group at N2 was explored while maintaining the (R)-
pyrrolidine at N1 and 3-amino-4-methylbenzamide at
N3. Introduction of aromatic substituents (1e) or ali-
phatic substituents (1g) led to potent inhibitors of the
p38 enzyme. However, cell activity remained modest.
Introduction of a methyl group in addition to the
hydrophobic substituents at N2 (thereby forming a
tertiary amine) gave a dramatic improvement in cell
activity. The increase in cell permeability on methyla-
tion may be due to the increase in overall hydrophobic-
ity. Comparison of 1g (without methyl) vs 1h (with
methyl) shows a 3-fold improvement in enzyme inhibi-
tion and >10-fold improvement in cell activity. Further
branching of the side chain leads to 1i, the most potent
compound in this series.
With N1 and N2 optimized ((R)-pyrrolidine and neo-
pentyl(methyl)amino, respectively), modification of N3
was undertaken (Table 3). Modifications of the 2-methyl
of the aniline to include conservative changes such as
F, Cl, and methoxy are tolerated (data not shown).
However, introduction of additional functional groups
to the aniline aromatic ring or any of the methyl
regioisomers led to a significant drop in enzyme inhibi-
tion. This was also true for the 5-amido substituent.
Modifications such as introduction of the amide in any
position other than the 5 position led to a >100-fold

6286 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Table 4. Optimization of R1 1k-n
Leftheris et al.
p38R
TNFR (THP-1)
R1
IC50 (nM)a
IC50 (nM)b
1k
1l
4-methylpiperazine-1-yl
piperazine-1-yl
57
18
44
52
158
120
85
Figure 2. LPS-induced TNF inhibition of several p38 inhibi-
tors in mouse. For oral dosing, compounds were prepared in a
solution of 100% polyethylene glycol (MW 300) and a dosing
volume of 0.2 mL per mouse (BALB\c female mice, 6-8 weeks
of age) was administered by gavage 30 min prior to LPS
injection (0.1 mL of LPS suspended at 10 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 TNF-R 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.
1m 4-methyl-1,4-diazepan-1-yl
1n 1,4-diazepan-1-yl
a n ) 4, variation in individual values, <20%. b n ) 3, variation
in individual values, <25%.
131
Figure 1. X-ray crystal structure of 1m cocrystallized with
p38 protein.
NH), respectively. In addition, the N-methyl portion of
the 1,4-diazepan ring is located near Asp168 (1.64 Å),
suggesting that the protonated amine may form a salt
bridge with the Asp168 anion. Two hydrophobic inter-
actions are evident as well. The angular aniline fits well
within the classic hydrophobic pocket located distal to
Thr106, while the N-neopentyl group lies along the
hinge region.
Figure 3. Rat adjuvant arthritis study using 1m. Lewis rats
(n ) 8/group) were immunized with complete Freund’s adju-
vant on day 0. On day 11, twice-daily oral treatment was
initiated with vehicle (Tween80:ethanol:water, 5:5:90) or 1m
(50 mg/kg/dose; circles). Dexamethasone (0.5 mg/kg; triangles)
given orally once daily was used as a positive control. Hind
paw volume increases were determined by plethysmometry on
the days indicated. * ) p < 0.05 vs vehicle, Student’s t test.
To determine the in vivo efficacy profile, several
compounds were screened for oral activity in an acute
model of TNFR inhibition (murine LPS/TNF model).
Mice were dosed with 3 compounds (11f, 1k, and 1m)
at 5 mg/kg (po) 30 min prior to LPS administration. TNF
levels were measured after 90 min. As shown in Figure
2, 1m showed near complete suppression of TNF levels.
Other compounds suppressed TNF levels but not to the
same extent. The ED50 for 1m in this model was
determined to be 3-10 mpk. A time course study was
undertaken where 1m was dosed at time intervals from
30 min to 10 h prior to LPS challenge at 10 mpk (data
not shown). Suppression of TNF diminished when the
compound was dosed 6 h prior to LPS challenge.
However, the compound was active at 30 min and 2 h
prior to LPS challenge. Further evaluation of 1m in a
model of chronic inflammation (adjuvant arthritis rat)
demonstrated 50% suppression of paw swelling when
dosed at 50 mg/kg, (b.i.d., po) (Figure 3).
Table 5. Kinase Selectivity Profile of Compound 1m
kinase assay
(IC50, µM)
kinase assay
(IC50, µM)
kinase
kinase
p38R
0.004 (Ki)
0.022
>10
PKA
LCK
SYK
Zap70
KDR
>10
>10
>10
>50
>10
p38â
IKK2â
MK2
JAK3
PKCR,δ
>10
>10
>10
liabilities. The Ki of 1m was determined to be 4.2 nM.
LPS-induced TNF production in human peripheral blood
mononuclear cells (hPBMC) gave an IC50 of 4.6 nM. A
difference in IC50 between hPBMC and THP-1 (trans-
formed cell line) may reflect a difference in dependence
on p38 in mediating TNF levels in different cell types.
A cytotoxicity assay using PHA blast cells demonstrated
minimal toxicity (70% of control when treated for 24 h
at 30 µM), and kinase profiling of 1m (Table 5) did not
identify significant off target kinase activity. However,
Further profiling of 1m was undertaken to determine
characteristics of the compound as well as potential

Inhibitors of p38 MAP Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6287
aqueous media were run under an ambient atmosphere unless
otherwise noted.
there is little kinase selectivity between p38R and p38â
for 1m.
General Procedure for the Synthesis of Compounds
3a and 3b. Example: 3-(4,6-Dichloro-1,3,5-triazin-2-
ylamino)-N-methoxy-4-methylbenzamide (3a). To a solu-
tion of 3-amino-N-methoxy-4-methylbenzamide (5.4 mmol, 2a)8
in acetone (20 mL) at 0 °C was added 1.0 g (5.4 mmol) of
cyanuric chloride, and the resulting mixture was stirred at 0
°C for 2 h and then at room temperature for an additional 2
h. Crushed ice (∼10 mL) was added, and the mixture was
allowed to warm to room temperature over 1 h. The solid was
collected by vacuum filtration and was washed with water (3
× 10 mL). Drying in vacuo afforded the title compound as a
white solid in 90% yield. 1H NMR (400 MHz, MeOD4): δ 7.75
(d, J ) 1.5 Hz, 1H), 7.65 (dd, J ) 7.7, 1.6 Hz, 1H), 7.41 (d, J
) 8.2 Hz, 1H), 3.82 (s, 3H), 2.32 (s, 3H). HPLC: tR ) 2.43 min,
95% purity.
The rate of metabolism of 1m in liver microsomes is
high for mouse (0.8 nmol/min/mg protein) and human
(0.2 nmol/min/mg protein). The major metabolite ob-
served in both species is the carboxylic acid formed from
the methoxyamide by hydrolysis. Consequently, the PK
profile for this compound in mouse shows a high
clearance (6.4 L/h‚kg), short T1/2 (0.7 h), and low
bioavailability (8% BA). Nonetheless, oral efficacy was
observed for 1m in in vivo rodent models of acute and
chronic inflammation. In vivo efficacy was shown not
to be due to formation of an active metabolite since the
corresponding carboxylic acid hydrolysis product showed
very low p38 enzyme activity (>1 µM).
3-(4,6-Dichloro-1,3,5-triazin-2-ylamino)-4-methylbenz-
amide (3b). This compound was prepared according to the
procedure for 3a starting with 4-methylbenzamide (2b).8
Yield: 89%, white solid. 1H NMR (400 MHz, DMSO-d6): δ
11.17 (s, 1H), 10.80 (s, 1H), 7.94-7.32 (m, 4H), 2.24 (s, 3H).
HPLC: tR ) 2.89 min, 92.3% purity. LCMS (EI): m/z calcd
for C11H9N5O [M + H]+ 298.10, found 298.05.
Conclusions
In conclusion, we have identified novel triaminotri-
azine aniline methoxyamides as potent, orally active p38
inhibitors. One member of this class (1m) demonstrates
efficacy in a chronic inflammatory disease model, shows
significant kinase selectivity, is not cytotoxic, and pos-
sesses a PK profile synonymous with a short acting
compound. X-ray crystallography demonstrates that this
compound accesses the ATP binding pocket of p38,
forming a hydrogen bond through a water molecule to
the backbone NH of Met109. The aniline methoxyamide
plays a key role in accessing a deeper cleft through
hydrogen-bonding interactions with Glu71 and Asp168.
The triaminotriazine aniline methoxyamides, as exem-
plified by 1m, represent an attractive starting point for
the design and synthesis of potent and selective p38
inhibitors with superior pharmacokinetic profiles. Given
the toxicity issues p38 inhibitors have had in develop-
ment, a highly potent, short acting p38 inhibitor may
provide the desired efficacy needed with minimal ad-
verse effects. However, appropriate pharmacokinetic
and pharmacodynamic studies will be required to fully
understand the profile needed to maximize in vivo
efficacy as well as therapeutic index.
3-(4-Chloro-6-(methyl(neopentyl)amino)-1,3,5-triazin-
2-ylamino)-N-methoxy-4-methylbenzamide (4a). To a so-
lution of 3a (3.1 mmol) in THF (10 mL) at 0 °C were
successively added 1.2 mL (6.7 mmol) of diisopropylethylamine
and neopentyl(methyl)amine hydrochloride (6.2 mmol). The
resulting mixture was stirred at 0 °C for 30 min and then at
room temperature for 30 min before quenching with a 50%
saturated aqueous solution of ammonium chloride (15 mL).
The organic solvent was removed in vacuo using a rotary
evaporator, and the resulting mixture was extracted with ethyl
acetate (3 × 20 mL). The combined extracts were washed with
brine (10 mL), dried over anhydrous sodium sulfate, filtered,
concentrated, and dried in vacuo to afford the title compound
as a white solid in 94% yield. 1H NMR (400 MHz, MeOD4,
appears as 2 rotamers): δ 8.20, 7.99 (br s, 1H), 7.55-7.49 (m,
1H), 7.37-7.34 (m, 1H), 3.81 (s, 3H), 3.52, 3.40 (s, 2H), 3.15,
3.14 (s, 3H), 2.36, 2.33 (s, 3H), 0.99, 0.81 (s, 9H). HPLC: tR
3.73 min, 92% purity.
)
3-(4-Chloro-6-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]-
heptan-2-yl)methylamino)-1,3,5-triazin-2-ylamino)-4-me-
thylbenzamide (4b). This compound was prepared according
to the procedure for 4a starting with 3b and (S)-myrtanyl-
amine. Yield: 72%, white solid. 1H NMR (400 MHz, CDCl3,
rotameric): δ 8.42 (s, 1H), 7.52 (d, J ) 7.8 Hz, 1H), 7.28 (m,
1H), 6.90 (s, 1H), 3.39 (m, 2H), 2.35 (s, 3H), 2.24 (m, 1H), 1.92
Experimental Section
Chemistry. Proton magnetic resonance (1H NMR) spectra
were recorded on a Bruker ARX-400 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 nm with the MS detection
performed with a Micromass Platform LC spectrometer. HPLC
analyses were performed using the following conditions: Bal-
listic YMC S5 ODS 4.6 × 50 mm column with a binary solvent
system where solvent A ) 10% methanol, 90% water, 0.2%
phosphoric acid and solvent B ) 90% methanol, 10% water,
and 0.2% phosphoric acid, flow rate ) 4 mL/min, linear
gradient time ) 4 min, start %B ) 0, final %B ) 100.
LCMS analyses were performed using the following condi-
tions: Waters Xterra 5 µM 4.6 × 30 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.
HPLC purification was performed using a YMC column (SH-
345-15, S-15, 120A ODS, 20 × 500 mm) with acetonitrile:water
gradients containing 0.1% trifluoroacetic acid. 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
(m, 4H), 1.60 (s, 6H), 1.52 (m, 1H), 1.25 (m, 3H). HPLC: tR
)
4.31 min, 98.7% purity. HRMS (EI): m/z calcd for C21H27N6-
ClO [M + H]+ 415.2013, found 415.2017.
(S)-tert-Butyl-3-(4-(5-carbamoyl-2-methylphenylamino)-
6-chloro-1,3,5-triazin-2-ylamino)pyrrolidine-1-carboxy-
late (4c). To a solution of 3b (100 mg, 0.34 mmol) in THF
(1.5 mL) at 0 °C was added DIPEA (65 µL, 0.37 mmol) followed
by (R)-3-amino-1-N-Boc-pyrrolidine (86 µL, 0.37 mmol), and
the solution was warmed to room temperature. The solution
was stirred for 30 min, filtered, and concentrated to a
semisolid. Water (2 mL) was added, and the resulting solids
were stirred for 2 h, filtered off, and washed with water to
give the chloro analogue 4c as a white solid (60 mg, 39% yield).
1H NMR (400 MHz, CDCl3, rotameric): δ 8.32 (m, 1H), 7.54
(m, 1H), 7.28 (m, 2H), 6.30 (m, 3H), 3.45 (m, 4H), 2.24 (m,
3H), 1.92 (m, 1H), 1.45 (s, 9H). HPLC: tR ) 3.76 min, 95.0%
purity. LCMS (EI): m/z calcd for C20H26N7ClO3 [M + H]+
448.92, found 448.32.
3-(4-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-
methylamino)-6-(4-methyl-1,4-diazepan-1-yl)-1,3,5-triazin-2-yl-
amino)-4-methylbenzamide Trifluoroacetate (1b). A solu-
tion of 4b (0.38 mmol) and 4-methyl-1,4-diazepan (1.9 mmol)
in NMP (0.3 mL) was heated at 80 °C for 30 min. The mixture
was cooled to room temperature and diluted with methanol
(1 mL). This solution was subjected to purification by reverse-

6288 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Leftheris et al.
phase HPLC chromatography, the HPLC fractions containing
the purified title compounds were concentrated on a rotary
evaporator to remove the methanol solvent, and the resulting
aqueous solutions were lyophilized to afford 1b as a tan solid.
Yield: 40%. 1H NMR (500 MHz, MeOD4, rotameric): δ 8.06-
8.18(m, 1H), 7.62-7.75(m, 1H), 7.33-7.43(m, 1H), 3.79-4.01-
(m, 3H), 3.30-3.60 (m, 5H), 2.94 (br s, 3H), 2.38-2.44 (m, 2H),
2.37 (br s, 3H), 2.15-2.26 (m, 2H), 1.88-2.05 (m, 7H), 1.52-
1.64 (m, 1H), 1.23 (br s, 3H), 1.09 (br s, 3H), 0.92-0.98 (m,
1H) ppm. HPLC: tR ) 2.36 min, 95% purity. LCMS: m/z calcd
for C27H40N8O [M + H]+ 493.34, found 493.31.
3-(4-Amino-6-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]hep-
tan-2-yl)methylamino)-1,3,5-triazin-2-ylamino)-4-methylbenz-
amide (1c). To a solution of 46 mg (0.1 mmol) of 4b in EtOH
(1.3 mL) was added 0.2 mL of concentrated NH4OH. The
resulting mixture was heated at 150 °C for 60 min in a sealed
tube and then cooled to room temperature. EtOH was removed,
water (1 mL) was added, and the resulting solids were stirred
for 24 h, filtered off, and rinsed with water. The solids were
purified via column chromatography (EtOAc, then 1% MeOH/
EtOAc) to afford 28.3 mg (65%) of 4d as a white solid. 1H NMR
(400 MHz, DMSO-d6): δ 8.17-7.87 (m, 3H), 7.58 (d, J ) 7.7
Hz, 1H), 7.28 (s, 2H), 6.69 (s, 1H), 6.23 (s, 1H), 6.11 (s, 1H),
3.19 (m, 2H), 2.61 (m, 2H), 2.56 (s, 3H), 2.28 (br m, 5H), 1.87-
0.86 (m, 8H). HPLC: tR ) 3.42 min, 99.0% purity. HRMS
(EI): m/z calcd for C21H27N7O [M + H]+ 396.2512, found
396.2513.
water (25 mL). The organic layer was dried over Na2SO4,
filtered, and concentrated to an oil, which was used without
further purification. 1H NMR (400 MHz, CDCl3): δ 7.19-7.34
(m, 5H), 4.40 (br s, 1H), 3.39 (m, 1H), 3.18 (ddd, J ) 5.1, 8.3,
13.5 Hz, 1H), 2.92 (m, 1H), 1.41 (s, 9H), 1.26 (d, J ) 7.0 Hz,
3H). HPLC: tR ) 3.96 min, 92.8% purity. LCMS: m/z calcd
for C14H21NO2 [M + H]+ 236.16, found 236.24. To a solution
of 0.4 g (1.7 mmol) of tert-butyloxycarbonyl-2-methylphenethy-
lamine in THF (5 mL) at 0 °C was added 0.1 g of NaH (3.4
mmol, 60% in oil) in one portion. The solution was stirred at
room temperature for 2 h followed by the addition of methyl
iodide 0.5 mL (8.5 mmol). After 1 h, DMF (2 mL) was added
and the reaction mixture stirred for 18 h. An additional
equivalent of NaH (70 mg) was added and the reaction mixture
stirred for an additional 3 h. The reaction was quenched with
NH4Cl (saturated aqueous, 1 mL), poured into EtOAc (25 mL),
and washed with water and brine. The organic layer was dried
over Na2SO4, filtered, and concentrated to an oil. To the oil
was added 4 N HCl in dioxane (3 mL), and the reaction
mixture was stirred for 30 min. The solvent was removed and
the crude solid dried under vacuum to give 2-methylphenethyl-
(methyl)amine hydrochloride as a white solid, which was used
1
without further purification. H NMR (400 MHz, MeOD4): δ
7.31-7.18 (m, 5H), 3.56 (s, 2H), 3.13 (m, 1H), 2.56 (s, 3H), 1.25
(d, J ) 6.9 Hz, 3H). HPLC: tR ) 0.87 min, 93.8% purity.
LCMS: m/z calcd for C10H15N [M + H]+ 150.20, found 150.16.
1f was prepared similarly to 1e starting from chloride 4c
(40 mg, 0.09 mmol) and 2-methylphenethyl(methyl)amine
hydrochloride (20 mg, 0.1 mmol). Yield of 1f as a white solid:
21 mg, 52% yield: 1H NMR (400 MHz, MeOD4, rotameric): δ
8.05 (br m, 1H), 7.55 (m, 1H), 7.20 (m, 9H), 4.88 (s, 1H), 3.88
(m, 1H), 3.68-3.04 (m, 4H), 2.88 (s, 3H), 2.37 (s, 3H), 2.24 (s,
1H), 1.91 (s, 1H), 1.31 (s, 3H), 1.29-0.88 (m, 3H). HPLC: tR
) 2.68 min, 97.8% purity. HRMS (EI): m/z calcd for C25H32N8O
[M + H]+ 461.2777, found 461.2781.
(S)-3-(4-(Isopropylamino)-6-(pyrrolidin-3-ylamino)-1,3,5-
triazin-2-ylamino)-4-methylbenzamide Trifluoroacetate
(1g). 1g was prepared similarly to 1e starting from chloride
4c (45 mg, 0.1 mmol) and 2-methylpropylamine (74 mg, 0.5
mmol). Yield: 40 mg, 75%. 1H NMR (400 MHz, MeOD4,
rotameric): δ 8.01 (m, 1H), 7.71 (m, 1H), 7.38 (m, 1H), 4.68
(m, 1H), 3.98 (s, 1H), 3.45 (m, 2H), 3.34 (s, 3H), 2.35 (s, 3H),
2.13-1.88 (m, 3H), 1.25-0.75 (m, 6H). HPLC: tR ) 2.03 min,
98.0% purity. HRMS (EI): m/z calcd for C19H28N8O [M + H]+
385.2464, found 385.2471.
3-(4-(((1S,2R,5S)-6,6-Dimethylbicyclo[3.1.1]heptan-2-
yl)methylamino)-6-((S)-pyrrolidin-3-ylamino)-1,3,5-triazin-
2-ylamino)-4-methylbenzamide Trifluoroacetate (1d). To
a solution of chloride 4b (45 mg, 0.1 mmol) in NMP (0.8 mL)
was added (R)-3-amino-1-N-Boc-pyrrolidine (0.03 mL, 0.11
mmol) and DIPEA (0.05 mL, 0.22 mmol), and the solution was
heated at 100 °C for 4 h. The solution was removed from
heating, and water (2 mL) was slowly added. The precipitated
solids were stirred for 1 h, filtered off, and rinsed with water
to afford the Boc-protected intermediate.
To the solids was added 4 N HCl in dioxane (1.5 mL), and
the solution was stirred for 2.5 h at room temperature. The
solvent was removed in vacuo, and the crude solids were
purified via preparative HPLC to afford the compound 1d (32
1
mg, 64% yield) as a white solid. H NMR (400 MHz, DMSO-
d6, rotameric): δ 7.95 (m, 3H), 7.45 (s, 1H), 7.15 (s, 2H), 6.86-
6.32 (m, 2H), 4.15 (m, 1H), 3.41-3.07 (m, 4H), 2.76 (m, 1H),
2.61-1.74 (m, 13 H), 1.47-0.74 (m, 5H). HPLC: tR ) 3.07 min,
99.2% purity. HRMS (EI): m/z calcd for C25H36N8O [M + H]+
464.3012, found 464.3156.
(S)-3-(4-(Isopropyl(methyl)amino)-6-(pyrrolidin-3-ylami-
no)-1,3,5-triazin-2-ylamino)-4-methylbenzamide Trifluo-
roacetate (1h). 1h was prepared similarly to 1e starting from
chloride 4c (50 mg, 0.2 mmol) and 1-methylpropyl(methyl)-
(S)-3-(4-(4-Fluorophenethylamino)-6-(pyrrolidin-3-yl-
amino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide Tri-
fluoroacetate (1e). To a solution of chloride 4c (50 mg, 0.1
mmol) in EtOAc (0.8 mL) were added 4-fluorophenethylamine
(69 mg, 0.5 mmol) and Na2CO3 (0.046 mL, 0.22 mmol), and
the solution was heated at 130 °C for 20 min. The solution
was removed from heating, and water (2 mL) was slowly
added. The product was extracted with CH2Cl2, dried over Na2-
SO4, filtered, and concentrated to an oil.
1
amine (74 mg, 0.5 mmol). Yield: 13 mg, 22%. H NMR (400
MHz, MeOD4, rotameric): δ 8.00 (m, 1H), 7.75 (s, 1H), 7.41
(d, J ) 7.7 Hz, 1H), 4.72 (m, 1H), 4.00 (s, 1H), 3.64-3.62 (m,
8H), 2.38-1.97 (m, 6H), 1.28-0.98 (m, 6H). HPLC: tR ) 2.28
min, 96.8% purity. HRMS (EI): m/z calcd for C20H30N8O [M +
H]+ 399.2621, found 399.2621.
(S)-4-Methyl-3-(4-(methyl(neopentyl)amino)-6-(pyrro-
lidin-3-ylamino)-1,3,5-triazin-2-ylamino)benzamide Tri-
fluoroacetate (1i). 1i was prepared similarly to 1e starting
from chloride 4c (45 mg, 0.1 mmol) and neopentyl(methyl)-
To the solids were then added CH2Cl2 (2 mL) and TfA (0.2
mL), and the solution was stirred for 3 h at room temperature.
The solvents were then removed in vacuo and the crude solids
purified via preparative HPLC to afford the compound 1e (30
1
1
amine (74 mg, 0.5 mmol). Yield: 27 mg, 45%. H NMR (400
mg, 64% yield) as a white solid. H NMR (400 MHz, MeOD4,
MHz, MeOD4, rotameric): δ 8.05 (m, 1H), 7.76 (m, 1H), 7.41
(m, 1H), 4.77 (m, 1H), 3.99 (s, 1H), 3.67-3.16 (m, 8H), 2.44
(m, 1H), 2.35 (s, 3H), 2.20 (m, 1H), 0.98-0.79 (m, 9H). HPLC:
tR ) 2.49 min, 99.4% purity. HRMS (EI): m/z calcd for
C21H32N8O [M + H]+ 413.2777, found 413.2773.
rotameric): δ 8.02 (m, 1H), 7.74 (m, 1H), 7.42 (s, 1H), 7.29 (s,
1H), 7.02 (m, 3H), 4.85 (m, 1H), 4.00 (s, 1H), 3.76-3.34 (m,
4H), 2.92 (br s, 2H), 2.41 (br s, 1H), 2.37 (s, 3H), 2.13 (br s,
1H). HPLC: tR ) 2.45 min, 95.3% purity. HRMS (EI): m/z
calcd for C23H27N8OF [M + H]+ 451.2370, found 451.2366.
(S)-3-(4-(Cyclohexyl(methyl)amino)-6-(pyrrolidin-3-
ylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide
Trifluoroacetate(1j). 1j was prepared similarly to 1e starting
from chloride 4c (45 mg, 0.1 mmol) and cyclohexyl(methyl)-
amine (74 mg, 0.5 mmol) Yield: 23 mg, 39%. 1H NMR (400
MHz, MeOD4, rotameric): δ 8.21 (m, 1H), 7.73 (s, 1H), 7.41
(s, 1H), 4.68 (m, 2H), 3.63-3.34 (m, 4H), 3.06 (m, 2H), 2.42 (s,
1H), 2.38 (s, 3H), 2.19 (br s, 1H). 1.88-1.22 (m, 11H). HPLC:
4-Methyl-3-(4-(methyl(2-phenylpropyl)amino)-6-((S)-
pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)benz-
amide Trifluoroacetate (1f). To a solution of 0.5 mL (3.4
mmol) of 2-methylphenethylamine in THF (15 mL) at 0 °C was
added 0.5 mL of TEA (3.4 mmol) followed by 0.75 mg of Boc2O
(3.4 mmol). The reaction mixture was stirred for 10 min and
then concentrated in vacuo to an oil. The residue was dissolved
in EtOAc (50 mL) and washed with 0.5 N HCl (20 mL) and

Inhibitors of p38 MAP Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6289
tR ) 2.02 min, 97.8% purity. HRMS (EI): m/z calcd for
C22H32N8O [M + H]+ 425.2777, found 425.2775.
to 140 °C for 4 h. The resulting mixture was cooled to room
temperature and added dropwise with stirring to a mixture
of an aqueous saturated solution of ammonium chloride (50
mL) diluted with water (50 mL). The resulting solid was
collected by vacuum filtration and washed with water (3 × 75
mL) and allowed to partially air-dry. The moist solid was
dissolved in methanol (50 mL), 3 N aqueous sodium hydroxide
(13 mL) was added, and the solution was heated to 60 °C for
45 min. After cooling to room temperature, the methanol was
removed using a rotary evaporator and the resulting aqueous
solution was diluted with additional water (∼100 mL). The
solution was acidified to pH 4 by addition of 10% aqueous citric
acid solution (∼100 mL), and the resulting gelatinous precipi-
tate was collected by vacuum filtration. The solid was washed
with water (2 × 50 mL) and ethyl acetate (2 × 50 mL) and
then dried in vacuo overnight to afford 3.2 g (79%) of 10 as a
white powder. 1H NMR (400 MHz, DMSO-d6, appears as 2
rotamers): δ 12.50 (br s, 1H), 8.17-8.09 (m, 2H), 7.57 (m, 1H),
7.28 (d, J ) 7.9 Hz, 1H), 7.05-6.94 (m, 1H), 4.36-4.26 (m,
1H), 3.57-3.23 (m, 6H), 3.05 (s, 3H), 2.27 (br s, 3H), 2.04 (br
s, 1H), 1.84 (br s, 1H), 1.39, 1.38 (s, 9H), 0.93, 0.79 (s, 9H).
HPLC: tR ) 3.83 min, 95% purity.
General Procedure for the Synthesis of Compounds
11a-i. A solution of 0.10 g (0.20 mmol) of 10, EDCI (0.25
mmol), and HOBt (0.23 mmol) in DMF (0.4 mL) was stirred
at room temperature for 2 h, and then DIPEA (0.58 mmol)
and the amine R1R2NH (0.58 mmol) or its hydrochloride salt
were added. After stirring at room temperature for 16 h, water
(5 mL) and saturated aqueous NaHCO3 solution (1 mL) were
successively added. The desired intermediate compounds were
isolated either by vacuum filtration and washing with water
(3 × 1 mL) or by extraction with ethyl acetate and concentra-
tion in vacuo. Further drying in vacuo afforded a solid, which
was dissolved in anhydrous dioxane (1 mL). To this solution
was added a 4 N solution of anhydrous HCl in dioxane (0.5
mL), and the resulting solution was stirred at room temper-
ature for 15 h. The solvent was removed in vacuo, and the
resulting semisolid was dissolved in methanol (∼1 mL) and
subjected to purification by reverse-phase HPLC chromatog-
raphy. The HPLC fractions containing the purified product
were concentrated on a rotary evaporator to remove the
methanol solvent, and the resulting aqueous solutions were
lyophilized to afford the title compounds 11a-h.
N-Methoxy-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-ylamino)benza-
mide Trifluoroacetate (1k). This compound was prepared
according to the procedure for 1b starting with 4a and
methylpiperazine. Yield: 65%, white solid. 1H NMR (400 MHz,
MeOD4, appears as 2 rotamers): δ 8.34, 8.14 (s, 1H), 7.33-
7.31 (m, 1H), 7.23-7.21 (m, 1H), 3.70 (s, 3H), 3.47-3.02 (m,
13H), 2.84 (s, 3H), 2.27, 2.23 (s, 3H), 0.91, 0.80 (s, 9H). HPLC:
tR ) 2.28 min, 98% purity. HRMS (EI): m/z calcd for C23H37N8O2
[M + H]+ 457.3039, found 457.3033.
N-Methoxy-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(piperazin-1-yl)-1,3,5-triazin-2-ylamino)benzamide Tri-
fluoroacetate (1l). This compound was prepared according
to the procedure for 1b starting with 4a and piperazine: white
1
solid in 46% yield. H NMR (400 MHz, MeOD4, appears as 2
rotamers): δ 8.37, 8.16 (s, 1H), 7.32-7.30 (m, 1H), 7.23-7.21
(m, 1H), 3.96 (app t, J ) 5.1 Hz, 4H), 3.70 (s, 3H), 3.47, 3.35
(s, 2H), 3.19 (app t, J ) 5.3 Hz, 4H), 3.12, 3.08 (s, 3H), 2.28,
2.24 (s, 3H), 0.91, 0.80 (s, 9H). HPLC: tR ) 2.27 min, 99%
purity. HRMS (EI): m/z calcd for C22H35N8O2 [M + H]+
443.2883, found 443.2894.
N-Methoxy-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(4-methyl-1,4-diazepan-1-yl)-1,3,5-triazin-2-ylamino)-
benzamide Trifluoroacetate (1m). This compound was
prepared according to the procedure for 1b starting with 4a
and 4-methyl-1,4-diazepan. Yield: 68% of a white solid. 1H
NMR (400 MHz, MeOD4, appears as 2 rotamers): δ 8.57, 8.40
(br s, 1H), 7.45-7.43 (m, 1H), 7.36-7.34 (m, 1H), 4.36-4.32
(m, 1H), 4.10-3.89 (m, 2H), 3.83 (s, 3H), 3.78-3.71 (m, 1H),
3.65-3.50 (m, 2H), 3.35-3.22 (m, 7H), 2.98 (s, 3H), 2.44-2.41
(m, 3H), 2.30 (br s, 2H), 1.05 (br s, 9H). HPLC: tR ) 2.09 min,
>99% purity. HRMS (EI): m/z calcd for C24H38N8O2 [M + H]+
471.3118, found 471.3192.
3-(4-(1,4-Diazepan-1-yl)-6-(methyl(neopentyl)amino)-
1,3,5-triazin-2-ylamino)-N-methoxy-4-methylbenz-
amide Trifluoroacetate (1n). This compound was prepared
according to the procedure for 1b starting with 4a and 1,4-
diazepan: 68%, white solid. 1H NMR (400 MHz, MeOD4,
appears as 2 rotamers): δ 8.53, 8.30 (br s, 1H), 7.46-7.45 (m,
1H), 7.37-7.35 (m, 1H), 4.15-3.70 (m, 4H), 3.61-3.35 (m, 4H),
3.27-3.24 (m, 3H), 2.41-2.38 (m, 3H), 2.18 (m, 2H), 1.05, 0.93
(s, 9H). HPLC: tR ) 2.28 min, >99% purity. HRMS (EI): m/z
calcd for C23H37N8O2 [M + H]+ 457.3039, found 457.3027.
Methyl 3-(4,6-Dichloro-1,3,5-triazin-2-ylamino)-4-me-
thylbenzoate (6). To a solution of 5.7 g (33 mmol) of aniline
5 in acetone (120 mL) at 0 °C was added 6.0 g (32.5 mmol) of
cyanuric chloride. The resulting mixture was allowed to warm
to room temperature giving, a thick suspension, which was
stirred for 1 h before addition of crushed ice (∼100 mL). The
mixture was stirred for 20 min, then water (100 mL) was
added, and the solid was collected by vacuum filtration. The
solid was washed with additional water (3 × 100 mL), and
the solid was dried in vacuo to afford 9.95 g (98%) of 6 as a
(S)-N-Ethyl-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)benza-
mide Trifluoroacetate (11a). Yield: 69%. 1H NMR (400
MHz, MeOD4, appears as 2 rotamers): δ 8.12, 7.96 (s, 1H),
7.69-7.65 (m, 1H), 7.41-7.39 (m, 1H), 4.75 (br s, 1H), 3.63-
3.40 (m, 8H), 3.25-3.20 (m, 3H), 2.39-2.35 (m, 4H), 2.18 (m,
1H), 1.24 (t, J ) 7.2 Hz, 3H), 1.04, 0.82 (s, 9H). HPLC: tR
)
2.26 min, >99% purity. HRMS (EI): m/z calcd for C23H37N8O
[M + H]+ 441.3090, found 441.3076.
(S)-N-(2-Methoxyethyl)-4-methyl-3-(4-(methyl(neo-
pentyl)amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-yl-
amino)benzamide Trifluoroacetate (11b). Yield: 23%. 1H
NMR (400 MHz, MeOD4, appears as 2 rotamers): δ 8.03, 7.86
(br s, 1H), 7.57-7.52 (m, 1H), 7.28 (m, 1H), 4.77-4.74 (m, 1H),
3.51-3.25 (m, 13H), 3.12-3.08 (m, 3H), 2.27-2.23 (m, 4H),
2.06 (m, 1H), 0.92, 0.71 (s, 9H). HPLC: tR ) 2.55 min, >99%
purity. HRMS (EI): m/z calcd for C24H39N8O2 [M + H]+
471.3196, found 471.3201.
(S)-N-Benzyl-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)benza-
mide Trifluoroacetate (11c). Yield: 57%, 1H NMR (400 MHz,
MeOD4, appears as 2 rotamers): δ 8.19, 8.00 (br s, 1H), 7.74-
7.69 (m, 1H), 7.41-7.25 (m, 6H), 4.73, (m, 1H), 4.59 (s, 3H),
3.62-3.38 (m, 6H), 3.23-3.15 (m, 3H), 2.39-2.35 (m, 4H), 2.17
(m, 1H), 1.03, 0.78 (s, 9H). HPLC: tR ) 2.77 min, >99% purity.
HRMS (EI): m/z calcd for C28H39N8O [M + H]+ 503.3247, found
503.3242.
1
white solid. H NMR (400 MHz, DMSO-d6): δ 10.80 (s, 1H),
7.91 (d, J ) 1.3 Hz, 1H), 7.81 (dd, J ) 7.9, 1.6 Hz, 1H), 7.46
(d, J ) 8.0 Hz, 1H), 3.85 (s, 3H), 2.28 (s, 3H). HPLC: tR
)
3.15 min, 99% purity. HRMS (EI): m/z calcd for C12H11N4O2-
Cl2 [M + H]+ 313.0259, found 313.0253.
Methyl 3-(4-Chloro-6-(methyl(neopentyl)amino)-1,3,5-
triazin-2-ylamino)-4-methylbenzoate (8). This compound
was prepared using the general procedure described for
compound 3a to afford the title compound as a white solid:
98% yield. 1H NMR (400 MHz, MeOD4): δ 8.23 (s, 1H), 7.78-
7.74 (m, 1H), 7.38-7.34 (m, 1H), 3.90 (s, 3H), 3.52, 3.41 (s,
2H), 3.16, 3.14 (s, 3H), 2.37, 2.35 (s, 3H), 0.99, 0.81 (s, 9H).
HPLC: tR ) 4.08 min, >99% purity. HRMS (EI): m/z calcd
for C18H25 N5O2Cl [M + H]+ 378.1697, found 378.1700.
(R)-3-(4-(1-(tert-Butoxycarbonyl)pyrrolidin-3-ylamino)-
6-(methyl(neopentyl)amino)-1,3,5-triazin-2-ylamino)-4-
methylbenzoic acid (10). A solution of 3.0 g (8.0 mmol) of
8, 2.1 mL (12 mmol) of DIPEA, and 1.8 g (9.5 mmol) of (R)-
3-amino-1-N-Boc-pyrrolidine (9) in NMP (10 mL) was heated
(S)-Methyl-4-methyl-3-(4-(methyl(neopentyl)amino)-6-
(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)ben-
zoate Trifluoroacetate (11d). Yield: 27%, off-white solid. 1H
NMR (400 MHz, DMSO-d6, appears as 2 rotamers): δ 8.60,

6290 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Leftheris et al.
8.37 (br s, 1H), 7.54 (m, 1H), 7.20-7.17 (m, 1H), 4.38 (m, 1H),
3.77 (s, 3H), 3.45-3.34 (m, 2H), 3.20-2.96 (m, 7H), 2.24 (s,
well flat-bottomed plates. Tested compounds (10 µL) or vehicle
(1% DMSO final) was added to cells. Subsequently, LPS
(Sigma, No. L7261; 10 µL/well) was added to the cells for a
final concentration of 1 µg/mL. Plates were incubated over-
night at 37 °C and 5% CO2. Supernatant (50 µL/well) was
harvested for an ELISA assay. TNF was captured by an anti-
human TNF antibody (R&D, No. MAB610) which was preab-
sorbed in high binding EIA plates (Costar, No. 3590). Captured
TNF was recognized by a biotinylated anti-human TNF
polyclonal antibody (R&D, No. BAF210). Streptavidin conju-
gated with peroxidase was added to each well, and the activity
of peroxidase was quantitated by a peroxide substrate kit
(Pierce, No. 34062 and No. 34006).
LPS-Induced TNF Production in Human PBMC. Hu-
man 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)
and 10 µg/mL polymyxin (Sigma), 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 were 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 appropriate wells and incubated with cells for 30 min.
Cells were then stimulated by the addition of lipopolysaccha-
ride (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 TNF-R by ELISA
(R&D Systems).
3H), 2.13, 1.80 (br s, 2H), 0.83, 0.75 (br s, 9H). HPLC: tR
)
2.72 min, >99% purity. HRMS (EI): m/z calcd for C22H33N7O2
[M + H]+ 428.2767, found 428.2774.
(S)-N-Cyclopentyl-4-methyl-3-(4-(methyl(neopentyl)-
amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)-
benzamide Trifluoroacetate (11e). Yield: 62%. 1H NMR
(400 MHz, MeOD4, appears as 2 rotamers): δ 8.00, 7.85 (br s,
1H), 7.56-7.54 (m, 1H), 7.28-7.26 (m, 1H), 4.74-4.62 (m, 1H),
4.24-4.19 (m, 1H), 3.51-3.23 (m, 6H), 3.12-3.03 (m, 3H),
2.26-2.22 (m, 4H), 2.05 (m, 1H), 2.05-1.88 (m, 2H), 1.68-
1.66 (m, 2H), 1.58-1.44 (m, 4H), 0.92, 0.71 (s, 9H). HPLC: tR
) 2.71 min, >99% purity. HRMS (EI): m/z calcd for C14H18-
NO5 [M + H]+ 481.3403, found 481.3395.
(S)-N-Methoxy-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)benza-
mide Trifluoroacetate (11f). Yield: 85%. 1H NMR (400 MHz,
MeOD4, appears as 2 rotamers): δ 8.15, 7.97 (br s, 1H), 7.58
(m, 1H), 7.42-7.40 (m, 1H), 4.69 (m, 1H), 3.82 (s, 3H), 3.66-
3.44 (m, 6H), 3.24 (m, 3H), 2.39-2.36 (m, 4H), 2.18 (m, 1H),
1.04, 0.83 (s, 9H). HPLC: tR ) 2.20 min, 98% purity. HRMS
(EI): m/z calcd for C22H35N8O2 [M + H]+ 443.2883, found
443.2881.
(S)-N-Hydroxy-4-methyl-3-(4-(methyl(neopentyl)amino)-
6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)benza-
mide Trifluoroacetate (11g). Yield: 55%. 1H NMR (400
MHz, MeOD4, appears as 2 rotamers): δ 8.01, 7.80 (br s, 1H),
7.47-7.27 (m, 2H), 4.56 (br s, 1H), 3.52-3.31 (m, 6H), 3.12-
3.09 (m, 3H), 2.27-2.23 (m, 4H), 2.06 (br s, 1H), 0.92, 0.71 (s,
9H). HPLC: tR ) 1.89 min, >99% purity. HRMS (EI): m/z
calcd for C21H33N8O2 [M + H]+ 429.2726, found 429.2720.
(S)-N-Isopropyl-4-methyl-3-(4-(methyl(neopentyl)-
amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)-
benzamide Trifluoroacetate (11h). Yield: 88%. 1H NMR
(400 MHz, MeOD4, appears as 2 rotamers): δ 7.95, 7.70 (s,
1H), 7.66 (m, 1H), 7.40 (m, 1H), 4.76 (m, 1H), 4.22 (m, 1H),
3.64-3.40 (m, 6H), 3.28-3.19 (m, 3H), 2.38-2.34 (m, 4H),
2.22-2.18 (m, 1H), 1.26 (d, J ) 6.6 Hz, 6H), 1.04, 0.81 (s, 9H).
HPLC: tR ) 2.38 min, >99% purity. HRMS (EI): m/z calcd
for C24H39N8O [M + H]+ 455.3247, found 455.3236.
Generation of p38 kinases. cDNAs of human p38 R, â
isozymes were cloned using PCR technology. These cDNAs
were subcloned in the pGEX expression vector (Pharmacia).
GST-p38 fusion protein was expressed in Escherichia coli and
purified from bacterial pellets by affinity chromatography
using gluthathione agarose. p38 fusion protein was activated
by incubating with constitutively active MKK6. Active p38 was
separated from MKK6 by affinity chromatography. Phosphor-
ylated MKK6 was generated according to the literature.9
p38 Kinase Assay. The assay were performed in V-
bottomed 96-well plates. For both assays, 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 1 mM DTT). Bacterially expressed,
activated p38 was preincubated 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
prewetted filtermat using a Skatron Micro96 cell harvester
(Skatron) and then 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%.
LPS-Induced TNF Production in THP-1 Cells. Human
monocytic THP-1 cells, obtained from ATCC, were maintained
in RPMI 1640 medium supplemented with 10% fetal bovine
serum. Cells (40000 cells in 80 µL) were added to wells of 96-
Cytotoxicity Assay. T cells in 1.25 × 106/mL human
peripheral blood mononuclear cells (PBMC) isolated from
normal volunteers were stimulated with 5 µg/mL phytohe-
magglutinin (PHA) for 4 days to generate dividing T cell blasts.
The PHA blast cells at 1.5 × 106 per mL were incubated with
30 µM compound in RPMI media (Gibco) with 10% fetal bovine
serum and a final dimethyl sulfoxide concentration of 0.003%
for 24 h at 37 °C, 5% CO2. Cytotoxicity was determined by the
detection of metabolic activity using the AlamarBlue assay
10
(Biosource).
Inhibition of TNF-r Release in Mice. BALB\c female
mice, 6-8 weeks of age, were obtained from Harlan Labora-
tories 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 compounds were prepared in a solution of 100% polyeth-
ylene glycol (MW 300) and a dosing volume of 0.2 mL per
mouse was administered by gavage 30 min prior to LPS
injection (0.1 mL of LPS suspended at 10 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 TNF-R 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 Animal 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 Mycobac-
terium butyricum. Seven days later, baseline (predisease)
measurements of hind paw volume were determined by volume
displacement plethysmometry (Ugo Basile, Italy). Compound
was administered orally at 1 mL b.i.d. beginning on day 11.
Paw volume measurements were repeated 3×/week for the
remainder of the study. Data are presented as the summed
increase in volume (expressed in milliliters) above baseline for
each rat’s two hind paws.
Crystal Structure of p38 MAP Kinase. His-p38 MAP
kinase was expressed in E. coli (BL21 DE3) and purified by
sequential anion exchange, nickel chelate affinity, and size
exclusion chromatography. The protein at 2 mg/mL, in 25 mM
Tris (pH 7.4), 50 mM NaCl, 5% glycerol, 2mM DTT, was
complexed with a 5-fold molar excess of 1m and then concen-
trated to 17 mg/mL. Crystals of p38 MAP kinase were grown

Inhibitors of p38 MAP Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6291
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necrosis factor antibodies in rheumatoid arthritis. J. Interferon
Res. 1994, 14 (5), 299-300.
Table 6. Crystallographic Data Collection, Reduction, and
Refinement
Data Collection
temperature
detector
100 °K
Rigaku R-AXIS IV area detector
Rigaku RU-2000 rotating anode
2.2 Å
source
(3) (a) Cobb, M. H.; Goldsmith, E. J. How MAP kinases are
regulated. J. Biol. Chem. 1995, 270 (25), 14843-14846. (b) Gille,
H.; Sharrocks, A. D.; Shaw, P. E. Phosphorylation of transcrip-
tion factor p62TCF by MAP kinase stimulates ternary complex
formation at c-fos promoter. Nature 1992, 358 (6385), 414-417.
(4) Lee, J. C.; Kassis, S.; Kumar, S.; Badger, A.; Adams, J. L. p38
mitogen-activated protein kinase inhibitorssmechanisms and
therapeutic potentials. Pharmacol. Ther. 1999, 82 (2-3), 389-
397.
(5) Behr, T. M.; Berova, M.; Doe, C. P.; Ju, H.; Angermann, C.e E.;
Boehm, J.; Willette, R. N. p38 mitogen-activated protein kinase
inhibitors for the treatment of chronic cardiovascular disease.
Curr. Opin. Invest. Drugs (PharmaPress Ltd.) 2003, 4 (9), 1059-
1064 and references therein.
(6) (a) Regan, J.; Breitfelder, S.; Cirillo, P.; Gilmore, T.; Graham,
A. G.; Hickey, E.; Klaus, B.; Madwed, J.; Moriak, M.; Moss, N.;
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From Lead Compound to Clinical Candidate. J. Med. Chem.
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Weijer, S.; Madwed, J.; Bos, C. L.; Gupta, A.; Yong, C.; Polmar,
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Peppelenbosch, M. P.; Van der Poll, T. Anti-inflammatory effects
of a p38 mitogen-activated protein kinase inhibitor during
human endotoxemia. J. Immunol. 2002, 168 (8), 4070-4077. (c)
Reference 5.
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W.; Veal, J.; Kuyper, L. F. Binding mode of the 4-anilino-
quinazoline class of protein kinase inhibitor: X-ray crystal-
lographic studies of 4-anilinoquinazolines bound to cyclin-
dependent kinase 2 and p38 kinase. J. Med. Chem. 2000, 43
(1), 133-138. (b) Henry, J. R.; Cavender, D. E.; Wadsworth, D.
A. p38 Mitogen Activated Protein Kinase as a Target for Drug
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G.; Germann, U. A.; Wilson, K. P.; Bemis, G. W.; Fox, T.; Su, M.
S. Inhibitors of p38 MAP kinase: therapeutic intervention in
cytokine-mediated diseases. Curr. Med. Chem. 1999, 6, 807-
823. Reference 6a.
(8) Moriarty, K.; Shimshock, Y.; Ahmed, G.; Wu, J.; Wen, J.; Li,
W.; Erickson, S.; Letourneau, J.; McDonald, E.; Leftheris, K.;
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pyrimidines for pharmaceutical use as cytokine, especially TNF-
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R. J. MKK3- and MKK6-regulated gene expression is mediated
by the p38 mitogen-activated protein kinase signal transduction
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(10) (a) Fields, R. D.; Lancaster, M. V. Dual attribute continuous
monitoring of cell proliferation/cytotoxicity. Am. Biotechnol. Lab.
1993, 11 (4), 48-50. (b) Ahmed, S. A.; Gogal, R. M., Jr.; Walsh,
J. E. A new rapid and simple nonradioactive assay to monitor
and determine the proliferation of lymphocytes: an alternative
to H3-thymidine incorporation assay. J. Immunol. Methods 1994,
170, 211-224.
max obsd resolution
Data Reduction (10.0-2.2 A)
data reduction program
completeness
Rsymm (on intensity)
reflections collected
no. of unique reflections
average I(/σI)
data redundancy
real space cell a
real space cell b
HKL (Denzo)
99.7%
10.2%
63975
19798
10.7
3.2
65.81 Å
74.45 Å
78.26 Å
P212121
real space cell c
space group
Refinement Procedure
refinement program
completeness
no. of reflections used
R value
CNX 2000
99.3%
19748
29.9%
35.1%
2732
free R value
no. of atoms used
protein atoms
inhibitor atoms
solvent molecules
estimate B value (Wilson plot)
mean B value
ESD coordinate error (Luzzati plot)
RMS deviations from ideal values
bond lengths
2653
34
45
42.7 Å2
41.3 Å2
0.38 Å
0.007 Å
1.3°
22.8°
0.8
bond angles
dihedral angles
improper angles
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 Å 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. The crystallographic data collection, reduction, and
refinement are summarized in Table 6.
References
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tumor necrosis factor as effector molecules in autoimmune
diseases. Curr. Opin. Immunol. 1991, 3, 941-948. (b) Arend,
W. P.; Dayer, J. M. Cytokines and cytokine inhibitors or
antagonists in rheumatoid arthritis. Arthritis Rheum. 1990, 33,
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mediators in rheumatoid arthritis. Springer Semin. Immuno-
pathol. 1984, 7, 387-413.
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experience in patients with rheumatoid arthritis. Pharmaco-
Economics 2004, 22 (2, Suppl.), 39-53. (b) Bresnihan, B.
Interleukin-1 receptor antagonist treatment in rheumatoid
JM049521D