5-Cyanopyrimidine derivatives as a novel class of potent, selective, and orally active inhibitors of p38a MAP kinase

Article abstract of DOI:10.1021/jm0503594

A novel class of 5-cyanopyrimidine-based inhibitors of p38a MAP kinase has been investigated. Analogues optimized through SAR iterations display low nanomolar enzymatic and cellular activity. The in vivo efficacy of this class of p38 inhibitors was demonstrated by 3a and 3b (>50% reduction in TNF levels when orally dosed at 5 mg/kg, 5 h prior to LPS administration in an acute murine model of inflammation). For 3a and 3b, the previously identified N-methoxybenzamide moiety (1) was replaced with N-(isoxazol-3-yl)benzamide, thereby providing increased metabolic stability. Cyanopyrimidine 3a demonstrated 100% oral bioavailability in mouse. High p38 kinase selectivity versus over 20 kinases was observed for analogue 3b. Direct hydrogen bonding of the cyano nitrogen of the 5-cyanopyrimidine core to the backbone NH of Met109 was confirmed by X-ray crystallographic analysis of 3a bound to p38α.

Full text of DOI:10.1021/jm0503594

J. Med. Chem. 2005, 48, 6261-6270
6261
5-Cyanopyrimidine Derivatives as a Novel Class of Potent, Selective, and Orally
Active Inhibitors of p38r MAP Kinase
Chunjian Liu,*,† Stephen T. Wrobleski,† James Lin,† Gulzar Ahmed, Axel Metzger, John Wityak,†
Kathleen M. Gillooly,† David J. Shuster,† Kim W. McIntyre,† Sidney Pitt,† Ding Ren Shen,† Rosemary F. Zhang,†
Hongjian Zhang,† Arthur M. Doweyko,† David Diller, Ian Henderson, Joel C. Barrish,† John H. Dodd,†
Gary L. Schieven,† and Katerina Leftheris*,†
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, New Jersey 08543-4000, and
Pharmacopeia, Inc., CN5350, Princeton, New Jersey 08543-5350
Received April 15, 2005
A novel class of 5-cyanopyrimidine-based inhibitors of p38R MAP kinase has been investigated.
Analogues optimized through SAR iterations display low nanomolar enzymatic and cellular
activity. The in vivo efficacy of this class of p38 inhibitors was demonstrated by 3a and 3b
(>50% reduction in TNF levels when orally dosed at 5 mg/kg, 5 h prior to LPS administration
in an acute murine model of inflammation). For 3a and 3b, the previously identified
N-methoxybenzamide moiety (1) was replaced with N-(isoxazol-3-yl)benzamide, thereby
providing increased metabolic stability. Cyanopyrimidine 3a demonstrated 100% oral bioavail-
ability in mouse. High p38 kinase selectivity versus over 20 kinases was observed for analogue
3b. Direct hydrogen bonding of the cyano nitrogen of the 5-cyanopyrimidine core to the backbone
NH of Met109 was confirmed by X-ray crystallographic analysis of 3a bound to p38R.
Introduction
We recently disclosed the discovery of a class of
triaminotriazine aniline amides as novel p38 MAP
kinase inhibitors7 SAR modification led to the identifi-
cation of 1 as a potent inhibitor of p38R (Figure 1).
Triazine-based 1 was orally active in vivo and demon-
strated unique H-bonding interactions relative to known
p38 kinase inhibitors at the time. However, this com-
pound is metabolically unstable, resulting in a poor
pharmacokinetic (PK) profile. The pendant methoxy-
amide moiety proved to be the major cause of the in vivo
metabolic instability.
X-ray crystallography revealed that the binding in-
teractions of 1 and the p38R enzyme include three key
hydrogen bonds and two hydrophobic interactions, as
depicted in Figure 1. Two of the three hydrogen bonding
interactions arise from the methoxyamide moiety of the
inhibitor. There is a hydrogen bond between the meth-
oxyamide carbonyl oxygen and the backbone NH of
Asp168 and a hydrogen bond between the methoxy-
amide NH and the side chain carboxylate of Glu71. The
third important hydrogen bond occurs between a tria-
zine ring nitrogen and the backbone NH of Met109,
through a bridged water molecule.
We were interested in modifying 1 so that the
through-water hydrogen bond was replaced by a direct
interaction between a cyano-substiututed pyrimidine
group (e.g. 2) and the Met109 backbone N-H. Success-
ful displacement of active-site water molecules with a
cyano functionality has been previously demonstrated
with inhibitors of scytalone dehydratase8 and epidermal
growth factor receptor kinase.9 In this paper, we present
the synthesis and SAR of 5-cyanopyrimidine derivatives
as inhibitors of p38R MAP kinase. Inhibitors such as
3a and 3b identified from this study not only showed
efficacy in an acute murine model of inflammation, but
also demonstrated appropriate metabolic and pharma-
cokinetic profiles for an orally active drug.
p38 mitogen-activated protein kinase (MAPK) is a
member of a serine-threonine protein kinase family that
also includes extracellular-regulated protein kinase
(ERK) and c-Jun NH2-terminal kinase (JNK). Members
of this family have a homologous threonine-X-tyrosine
amino acid sequence motif with X equal to glycine for
p38, glutamic acid for ERK, and proline for JNK. A
common feature of this family is that the activation of
its members requires dual phosphorylation of threonine
and tyrosine residues.1 Four isoforms of p38 are
known: p38R and p38â are widely expressed in eukary-
otic cells including endothelial and inflammatory cells.
Expression of p38γ is found in skeletal muscle, and p38δ
is predominantly expressed in the small intestine,
kidney, and lung tissue.2 p38 MAP kinase plays a
crucial role in regulating the biosynthesis of many
inflammatory cytokines including tumor necrosis factor
alpha (TNFR) and interleukin-1 (IL-1â).3 Excessive
production of TNFR and IL-1â is implicated in many
inflammatory diseases.4 The blockade of TNFR function
by biological agents such as etanercept (Enbrel), a
soluble TNFR receptor, and infliximab (Remicade), a
TNFR antibody, is clinically proven to be effective in the
treatment of rheumatoid arthritis, Crohn’s disease, and
psoriasis.5 As a result, discovery and development of
small molecule p38 MAP kinase inhibitors as orally
active therapeutic agents for the treatment of inflam-
matory diseases has been a goal of numerous pharma-
ceutical research groups, and many classes of p38 MAP
kinase inhibitors have been reported.6
* To whom correspondence should be addressed. For C.L., phone:
609-252-3682; fax: 609-252-6601; e-mail: Chunjian.liu@bms.com.
For K.L., phone: 609-252-6188; fax: 609-252-6804; e-mail:
Katerina.leftheris@bms.com.
† Briston-Myers Squibb Pharmaceutical Research Institute.
Pharmacopeia, Inc.
10.1021/jm0503594 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/13/2005

6262 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Liu et al.
Scheme 2a
Figure 1.
a Reagent and conditions: (a) formamidine hydrochloride, NEt3,
EtOH; (b) POCl3, reflux; (c) N-methoxy-3-amino-4-methylbenz-
amide hydrochloride, (i-Pr)2NEt, DMF, 65 °C; (d) Oxone, MeOH/
H2O, 0 °C; (e) appropriate amines, 1,4-dioxane, rt.
Scheme 1a
N-methoxy-3-amino-4-methylbenzamide hydrochloride7
in the presence of Hu¨nigs base at room temperature to
afford 5. Treatment of 5 with N-methylneopentylamine
at room temperature provided 6. Reaction of 6 with
Oxone gave sulfone 7, which served as a common
precursor to compounds 2 and 8a-c by either heating
7 with the corresponding amines (2, 8a,b) or reduction
with superhydride (LiBHEt3) at -78 °C (8c).
Scheme 2 outlines the synthesis of 14a-d. The
cyanopyrimidine ring system 10 was constructed from
commercially available methyl 3,3-bis(methylthio)-2-
cyanoacrylate (9) and formamidine hydrochloride with
triethylamine in refluxing ethanol.11 Phosphorus oxy-
chloride converted 10 into chlorocyanopyrimidine 11.
Heating 11 with N-methoxy-3-amino-4-methylbenz-
amide hydrochloride in the presence of Hu¨nigs base in
DMF supplied 12. Oxidation of 12 using Oxone at 0 °C
over 5 min resulted in the formation of sulfoxide 13.
However, if the oxidation was allowed to proceed for a
longer period of time, 13 was further oxidized to the
corresponding sulfone. This proved to be too reactive and
unstable to be isolated. Compounds 14a-d were readily
prepared by reacting 13 with the corresponding amines
at room temperature.
For the preparation of 3a,b and 17a,b, chlorocyano-
pyrimidine 11 was heated with 3-amino-4-methylben-
zoic acid in DMF to give 15 (Scheme 3). The conversion
of 15 to 16a required generation of the acid chloride
followed by treatment of 3-aminoisoxazole in the pres-
ence of pyridine. Amide 16b could be obtained via a
direct coupling reaction of the carboxylic acid 15 with
5-amino-1-ethylpyrazole in the presence of benzotria-
zole-1-yloxytris(dimethylamino)phosphonium hexafluo-
rophosphate (BOP reagent). Conversion to the corre-
sponding sulfones was carried out by treatment of 16a
and 16b with m-chloroperbenzoic acid (mCPBA) in THF.
a Reagent and conditions: (a) N-methoxy-3-amino-4-methyl-
benzamide hydrochloride, (i-Pr)2NEt, DMF, rt; (b) N-methylneo-
pentylamine hydrochloride, (i-Pr)2NEt, DMF, rt; (c) Oxone, H2O/
MeOH, rt; (d) appropriate amines, 50-70 °C; (e) LiBHEt3, THF,
-78 °C.
Chemistry
The synthesis of 5-cyanopyrimidine derivative 2 and
its analogues 8a-c are shown in Scheme 1. 5-Cyano-
4,6-dichloro-2-methylthiopyrimidine (4), prepared ac-
cording to a literature procedure,10 was reacted with

5-Cyanopyrimidine Inhibitors of p38R MAP Kinase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6263
Scheme 3a
Scheme 4a
a Reagent and conditions: (a) 3-amino-4-methylbenzoic acid,
DMF, 55 °C; (b) i. thionyl chloride, ii. 3-aminoisoxazole or 5-amino-
1-phenylpyrazole, pyridine; (c), 5-amino-1-ethylpyrazole, BOP
reagent, 1-methylmorpholine; (d) mCPBA, THF; (e) isopropylamine
or cyclopentylamine, 1,4-dioxane, 150 °C, microwave.
Subsequent reaction with isopropylamine or cyclopen-
tylamine in 1,4-dioxane at 150 °C under microwave
conditions completed the syntheses of 3a,b and 17a,b.
In the process of synthesizing 25a-d (Scheme 4), the
key intermediate 20 was prepared from commercially
available 9 via an analogous sequence to that used for
the synthesis of 12, as previously described in Scheme
2. This intermediate contains both a 2-ethylthio and a
4-methylthio on the pyrimidine ring. The ethylthio
group could be selectively oxidized to ethylthionyl with
1 equiv of mCPBA at 0 °C within 1 h.12 The product 21
was heated with 2 N ammonia in isopropyl alcohol to
provide 22. The methyl sulfide 22 was then oxidized
with excess mCPBA to the corresponding methyl sulfone
and was subsequently converted, under microwave
heated conditions, to 23a and 23b with isopropylamine
and cyclopentylamine, respectively. Hydrolysis of esters
23a and 23b to the corresponding acids 24a and 24b
was effected with LiOH at room temperature, with the
cyano group remaining intact. The conversion of 24a
and 24b to 25a-d was accomplished either through the
use of the BOP reagent or via an acid chloride inter-
mediate in a manner similar to the transformation of
15 to 16a,b in Scheme 3.
a Reagent and conditions: (a) 2-ethyl-2-thiopseudourea hydro-
chloride, (i-Pr)2NEt, EtOH, reflux; (b) POCl3, reflux; (c) methyl
3-amino-4-methylbenzoate, DMF, 65 °C; (d) mCPBA (1.15 equiv),
THF, 0 °C; (e) NH3/i-PrOH, 65 °C; (f) mCPBA (excess), THF, rt;
(g) isopropylamine or cyclopentylamine, 1,4-dioxane, microwave,
150 °C; (h) LiOH, THF/MeOH/H2O, rt; (i) i. thionyl chloride, ii.
3-aminoisoxazole, pyridine; (j), 5-amino-1-ethylpyrazole, BOP
reagent, 1-methylmorpholine, DMF.
after 24 h of treatment at 30 µM), and therefore the
cellular activity of 3.3 nM was questionable. Also,
similar to triaminotriazine 1, cyanopyrimidine 2 was not
stable to liver microsomes. To address these issues, an
SAR study of 2, focused on the replacement of the
N-methyl-1-homopiperazino (R1), N-methylneopentyl-
amino (R2), and methoxy (R3), was systematically un-
dertaken.
The possibility of replacing the N-methyl-1-homo-
piperazino group in 2 was first explored while main-
taining the N-methylneopentylamino and N-methoxy-
benzamide functional groups. An important finding with
the cyanopyrimidine scaffold was that the 1-homopip-
erazino group could be replaced with small amino
groups such as methylamino and amino without sig-
nificantly altering the p38R enzyme activity. Unlike in
the triaminotriazine series, where an analogous modi-
fication resulted in a 4-fold reduction in enzyme activity,
8a and 8b displayed essentially the same p38 enzyme
activity as 2, as seen in Table 1. It was further
discovered that the 1-homopiperazino group could be
completely removed without a substantial loss of p38R
Results and Discussion
Compounds were evaluated for their ability to inhibit
phosphorylation of substrate (myelin basic protein) by
recombinant human p38R. A peripheral blood mono-
nuclear cell assay (hPBMC) was used to measure the
ability of compounds to inhibit LPS-induced TNFR
production in human primary cells. Compound 2 was
prepared to test the concept of replacing the water
involved in the hydrogen bond to Met109 with the
nitrogen of a nitrile. It was found that 2 displayed a Ki
value of 0.057 nM versus 3.7 for 1 (60-fold difference)
The cellular activity (TNFR IC50) for 2 was 3.3 nM.
However, 2 was cytotoxic to PHA blast cells (0% control

6264 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Table 1. In Vitro Activity of 2 and 8a-c
Liu et al.
Table 3. In Vitro Activity of 3a,b, 17c,d, and 25a-d versus
that of 14c,d
a n ) 4, variation in individual values, <20%. b n ) 3, variation
in individual values, <25%. c Cytotoxic @ 30 micromolar.
Table 2. In Vitro Activity of 14a-d versus that of 8c
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%.
was not the case with our previous triaminotriazine
series, where a tertiary amino of R2 was essential for
cellular activity.
activity to afford 8c (p38R Ki ) 0.15 nM). The IC50
values of 8a, 8b, and 8c in inhibiting LPS induced
TNFR production in cells were 64, 17, and 42 nM,
respectively. Furthermore, 8a, 8b, and 8c were found
not to be cytotoxic (g73% control after 24 h of treatment
at 30 µM). It appeared that the observed cytotoxicity
seen with 2 was due to the presence of the basic
1-homopiperazino group.
Table 2 contains SAR results for R2 while maintaining
R1 as hydrogen and R3 as methoxy. The N-methylneo-
pentylamino group on 2 is expected to occupy a hydro-
phobic pocket. Replacement of this group with a much
smaller, less hydrophobic methylamino group led to a
100 fold less potent p38R inhibitor 14a. The cellular IC50
of 14a was greater than 250 nM. The use of n-
propylamino in place of methylamino improved the p38R
Ki to 0.97 nM (14b). The cellular activity for this
compound was also improved (IC50 ) 158 nM) compared
to 14a. R-Branched alkyl or cycloalkylamines such as
i-propylamino (14c) and cyclopentylamino (14d) re-
sulted in good enzymatic (Ki ) 0.61 nM and 0.40 nM,
respectively) and cellular (IC50 ) 4.6 nM and 4.1 nM,
respectively) potencies. These analogues not only re-
duced the overall hydrophobicity and molecular weight
but also improved the cellular potency by 8-9-fold. This
After identifying the appropriate replacements for R1
and R2, a study to replace the N-methoxybenzamide
moiety with more stable functionalities was undertaken.
For this SAR study, R1 was fixed as hydrogen or amino,
and R2 was selected from either isopropylamino or
cyclopentylamino. As with our previous triaminotriazine
series,7 the replacement of N-methoxybenzamide with
N-alkylbenzamide in the cyanopyrimidine series caused
significant losses in both p38 enzyme and cell activity
(data not shown). However, N-(isoxazol-3-yl)benzamide
analogues were found to be highly potent p38R inhibi-
tors and demonstrated very good cell activity. As seen
in Table 3, 3a was comparable to 14c in both the p38R
enzyme and cellular assays, with the p38R Ki ) 0.41
nM and the TNFR IC50 ) 8.7 nM. Similarly, 3b had
enzymatic and cellular activities (p38R Ki ) 1.6 nM;
TNFR IC50 ) 3.5 nM) comparable to that of 14d.
Compounds 25a and 25b differ from 3a and 3b due to
the introduction of an amino group at R1. As expected,
25a and 25b were very potent p38 inhibitors (p38R Ki
) 0.057 nM and 0.16 nM, respectively) and were
effective in cells (TNFR IC50 ) 18 nM and 1.5 nM,
respectively). 1-Ethyl-5-pyrazolyl was found not to be

5-Cyanopyrimidine Inhibitors of p38R MAP Kinase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6265
Figure 3. LPS-induced TNF inhibition by 3a and 3b in
mouse. BALB/c female mice (Harlan), 6-8 weeks of age, were
used. Compound was dissolved in poly(ethylene glycol) (MW
) 300; PEG300) and administered to mice (n ) 8/treatment)
by oral gavage in a volume of 0.1 mL. Control mice received
PEG300 alone (“Vehicle”). Five hours later, mice were injected
intraperitoneally with 50 µg/kg lipopolysacchride (LPS; E. coli
O111:B4; Sigma). Blood samples were collected 90 min after
LPS injection. Serum was separated and analyzed for the level
of TNF-R by commercial ELISA assay (BioSource) according
to the manufacturer’s instructions. Data shown are mean (
SD.
Figure 2. Binding interactions between 3a and unphospho-
rylated p38R based on X-ray crystallographic analysis. Hy-
drogen bond distances are given in angstroms. The green
sphere denotes the location of the water molecule found in the
p38-bound triaminotriazine 1.
as good as 3-isoxazolyl for the replacement of methoxy,
as judged by the activities of 17c (p38R Ki ) 15 nM;
TNFR IC50 ) 130 nM) and 17d (p38R Ki ) 14 nM; TNFR
IC50 ) 72 nM), which were 24- and 34-fold less active
in the enzyme assay, and 28- and 18-fold less active in
the cellular assay, respectively, compared to 14c and
14d. However, the p38R enzyme activity of 17c and 17d
could be improved by changing R1 from hydrogen to
amino, as shown by 25c (p38R IC50 ) 1.5 nM; TNFR
IC50 ) 67 nM) and 25d (p38R IC50 ) 1.9 nM; TNFR IC50
) 21 nM). It is noticed that generally, when R1 is amino,
analogues containing cyclopentylamino as the R2 group
demonstrate improved cell activity compared to ana-
logues where R2 is i-propylamine, even though the
enzyme activity for both compounds is similar. This is
true for 25d versus 25c, and 25b vs 25a. This is not as
pronounced when R1 is hydrogen (17c vs 17d and 14c
vs 14d).
Thus far we have described the identification and
SAR of a novel series of in vitro potent, 5-cyanopyrimi-
dine-based p38 inhibitors. To confirm our originally
proposed binding mode for this series, an X-ray crystal-
lographic study of 3a bound to purified, unphosphory-
lated p38R was completed.13 The binding interactions
between 3a and the p38R enzyme are illustrated in
Figure 2. The proposed hydrogen bond between the
cyano nitrogen of 3a and the Met109 backbone NH of
p38 is evident (1.76 Å). A secondary hydrogen bond
between the i-propylamino NH and the Met109 carbonyl
oxygen is observed (2.15 Å). The remaining interactions
of 3a with p38R remain similar to that of 1 as follows:
The pendant amido group provides two more H-bonds
with Asp168 (1.99 Å) and Glu71 (1.87 Å). Hydrophobic
interactions include the angular methylaniline group of
3a seated in a deep hydrophobic pocket, while the
pendant i-propyl participates in a weaker hydrophobic
interaction at the mouth of the binding site.
Table 4. Kinase Selectivity of 3b
kinase
IC50 (µM)
kinase
IC50 (µM)
p38R
p38â
0.0041
<0.1
>30
IKK
JAK3
Lck
MEK
Met
>10
>50
>50
>25
>50
>50
>50
>30
>50
>30
>50
>50
p38γ
p38δ
Akt
>30
>50
>50
CaMKII
cdk2
MK2
PKC
Raf
>100
>50
Itk
ERK
>5
Syk
FGF
>50
KDR
HER1
PKA
GSK3
HER2
IGF-IR
>50
>50
>50
orally active, inhibiting the production of TNFR by 60%
and 83%, respectively.
To determine kinase selectivity, 3b was tested for
inhibitory activity versus over 20 varied kinases. The
results are reported in Table 4. Compound 3b did not
show significant activity against any of the tested
kinases except p38R and p38â. In most cases, the IC50
values were greater than 30-50 µM, indicating a
selectivity of over 7000-fold. ERK is closely related to
p38, and it was inhibited by 3b with an IC50 of greater
than 5 µM. This is over 1200-fold compared to its
activity against p38R.
Additional profiling of 3a indicates that it is not
cytotoxic to PHA blast cells (82% of control after 24 h
of treatment at 30 µM) and has excellent Caco-2
permeability (239 nm/s). Compound 3a is stable in liver
microsomes with a metabolic rate of 0.022 nmol/min‚
mg-protein in rats and 0.012 nmol/min‚mg-protein in
humans. Pharmacokinetic evaluation in mice revealed
that 3a has a t1/2 of 1.7 h and a clearance of 1.2 L/h‚kg
after intravenous dosing. The oral bioavailability of 3a
was approximately 100% after administration of a
solution dose in PEG400.
The in vivo activity of 5-cyanopyrimidine-based p38
inhibitors was demonstrated by assessment of 3a and
3b in an acute murine model of TNFR inhibition. Mice
were dosed orally with 3a and 3b at 5 mg/kg, 5 h prior
to LPS administration. The TNFR levels were measured
90 min later. As shown in Figure 3, 3a and 3b are both
Conclusions
In conclusion, we have developed a series of 5-cyano-
4,6-diaminopyrimidine derivatives as a novel class of
p38R MAP kinase inhibitors. Optimized analogues such

6266 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Liu et al.
sulfate, the solution was filtered and concentrated in vacuo to
afford 6 (0.34 g, 93% yield) as a pale yellow solid; 96% purity
by HPLC; LCMS (EI) m/z Calcd for C21H29N6O2S [M + H]+
429.20. Found: 429.56; 1H NMR (400 MHz, CDCl3): δ 8.69
(br s, 1H), 8.43 (s, 1H), 7.46 (d, J ) 7.9 Hz, 1H), 7.28 (s, 1H),
7.12 (s, 1H), 3.89 (s, 3H), 3.72 (s, 2H), 3.47 (s, 3H), 2.46 (s,
3H), 2.36 (s, 3H), 0.99 (s, 9H).
as 3a and 3b possess potent in vitro and in vivo
activities and display appropriate metabolic and phar-
macokinetic profiles for an orally active drug. A repre-
sentative example (3b) displays high kinase selectivity
versus over 20 kinases. Finally, X-ray crystallographic
analysis of 3a bound to unphosphorylated p38R has
confirmed the proposed binding interactions of these
novel inhibitors.
3-(5-Cyano-6-(methyl(neopentyl)amino)-2-(methylsul-
fonyl)pyrimidin-4-ylamino)-N-methoxy-4-methylbenz-
amide (7). To a solution of 6 (70 mg, 0.16 mmol) in methanol
(1.75 mL) at room temperature was added a slurry of Oxone
(0.40 g, 0.65 mmol) in water (0.75 mL), and the resulting
mixture was stirred at room temperature for 16h, then
concentrated in vacuo. The resulting residue was suspended
in water (5 mL), and the solid was collected by vacuum
filtration. After washing with additional water (3 × 3 mL),
the solid was dried in vacuo to afford 7 (62 mg, 83% yield) of
the title compound as a white solid; 88% purity by HPLC;
LCMS (EI) m/z Calcd for C21H29N6O4S [M + H]+ 461.90.
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 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 × 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 conditions: Phenomenex 5 µm
C18 4.6 × 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 following
conditions: Ballistic YMC S5 ODS 20 × 100 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. Fractions containing the product were concentrated in
vacuo to remove the methanol and neutralized with aqueous
sodium bicarbonate. The products were collected by suction
filtration or extracted with ethyl acetate and subsequent
concentration.
1
Found: 461.33. H NMR (400 MHz, MeOD4): δ 7.85 (d, J )
1.6 Hz, 1H), 7.62 (dd, J ) 7.9, 1.6 Hz, 1H), 7.42 (d, J ) 7.9 Hz,
1H), 3.83 (s, 2H), 3.82 (s, 3H), 3.56 (s, 3H), 3.08 (s, 3H), 2.34
(s, 3H), 1.04 (s, 9H).
3-(5-Cyano-6-(methyl(neopentyl)amino)-2-(4-methyl-
1,4-diazepan-1-yl)pyrimidin-4-ylamino)-N-methoxy-4-meth-
ylbenzamide Trifluoroacetic Acid Salt (2). A solution of
7 (51 mg, 0.11 mmol) and N-methylhomopiperazine (0.04 mL,
0.33 mmol) in N-methylpyrrolidinone (0.25 mL) was heated
at 70 °C for 3 h. After cooling to room temperature, the mixture
was purified by reverse-phase preparative HPLC to afford the
title compound (16 mg, 29%) as a pale yellow solid; 97% purity
by HPLC; HRMS (ESI) m/z Calcd for C26H37N8O2 [M - H]+
1
493.3039. Found: 493.3052. H NMR (400 MHz, MeOD4): δ
8.26 (s, 1H), 7.32 (dd, J ) 7.9, 1.7 Hz, 1H), 7.21 (d, J ) 7.9
Hz, 1H), 3.76-3.58 (m, 4H), 3.70 (overlapping s, 3H), 3.60
(overlapping s, 2H), 3.31 (s, 3H), 2.64-2.49 (m, 4H), 2.28 (s,
3H), 2.24 (s, 3H), 1.91-1.79 (m, 2H), 0.89 (s, 9H).
3-(5-Cyano-6-(methyl(neopentyl)amino)-2-(methylami-
no)pyrimidin-4-ylamino)-N-methoxy-4-methylbenzam-
ide (8a). A solution of 7 (50.0 mg, 0.109 mmol) in 2 M MeNH2/
THF (3.0 mL, 6.0 mmol) was heated at 50 °C in a sealed tube
for 30 min and then concentrated under vacuum. The residue
was subjected to preparative HPLC to afford 8a (30.9 mg, 69%
yield) as a white solid. 100% purity by HPLC; HRMS (ESI)
m/z Calcd for C21H29N7O2 [M + H]+ 412.2583. Found: 412.2455.
1H NMR (500 MHz, DMSO-d6, 80 °C): δ 11.3 (s, 1H), 7.87 (s,
1H), 7.46 (d, J ) 7.7 Hz, 1H), 7.28 (d, J ) 7.7 Hz, 1H), 6.65 (s,
br., 1H), 3.70 (s, 3H), 3.67 (s, 2H), 3.33 (s, 3H), 2.70 (d, J )
4.4 Hz, 3H), 2.24 (s, 3H), 0.95 (s, 9H).
3-(2-Amino-5-cyano-6-(methyl(neopentyl)amino)pyri-
midin-4-ylamino)-N-methoxy-4-methylbenzamide (8b). A
solution of 7 (50.0 mg, 0.109 mmol) in 2 M NH3/i-PrOH (3.0
mL, 6.0 mmol) was heated at 65 °C in a sealed tube for 30
min and then concentrated under vacuum. The residue was
subjected to preparative HPLC to afford 8b (29.5 mg, 68%
yield) as a white solid. 100% purity by HPLC; HRMS (ESI)
m/z Calcd for C20H27N7O2 [M + H]+ 398.2526. Found: 398.2303.
1H NMR (500 MHz, DMSO-d6): δ 11.6 (s, 1H), 8.29 (s, 1H),
7.68 (s, 1H), 7.48 (d, J ) 8.2 Hz, 1H), 7.28 (d, J ) 8.2 Hz, 1H),
6.46 (s, 2H), 3.68 (s, 3H), 3.62 (s, 2H), 3.28 (s, 3H), 2.19 (s,
3H), 0.91 (s, 9H); 13C NMR (500 MHz, DMSO-d6): δ 164.5,
164.4, 163.4, 161.3, 137.8, 137.6, 129.9, 129.8, 125.2, 123.7,
119.2, 62.8, 61.2, 59.9, 42.0, 34.0, 27.7, 17.7.
3-(5-Cyano-6-(methyl(neopentyl)amino)pyrimidin-4-
ylamino)-N-methoxy-4-methylbenzamide (8c). To a solu-
tion of 7 (60.0 mg, 0.130 mmol) in THF (4 mL) at -78 °C was
added super hydride (LiBHEt3) (1.0 M in THF, 0.65 mL, 0.65
mmol, -78 °C) over 2 min. The mixture was stirred at -78 °C
for 30 min before it was poured into water (40 mL). The
resulting mixture was stirred at room temperature for 30 min
and then extracted with AcOEt (3 × 30 mL). The combined
extract was washed with brine and concentrated under
vacuum. The residue was subjected to preparative HPLC to
afford 8c (31.7 mg, 64% yield) as a white solid. 100% purity
All reagents were purchased from commercial sources and
used without further purification unless otherwise noted. All
reactions were performed under an inert atmosphere. Reac-
tions run in aqueous media were run under an ambient
atmosphere unless otherwise noted.
3-(6-Chloro-5-cyano-2-(methylthio)pyrimidin-4-ylami-
no)-N-methoxy-4-methylbenzamide (5). To a solution of
N-methoxy-3-amino-4-methylbenzamide hydrochloride (0.27 g,
1.4 mmol) in DMF (2.4 mL) at room temperature was added
diisopropylethylamine (0.19 mL, 1.1 mmol), and the mixture
was stirred until homogeneous, then 4,6-dichloro-2-(meth-
ylthio)pyrimidine-5-carbonitrile (4) (0.30 g, 1.4 mmol), pre-
pared according to a literature procedure,10 was added. After
18 h at room temperature, the mixture was diluted with water
(10 mL) and the solid was collected by vacuum filtration. The
solid was washed with additional water and was dried in vacuo
to afford 5 (0.37 g, 72% yield) as an off-white solid; 88% purity
by HPLC; LCMS (EI) m/z Calcd for C15H15ClN5O2S [M + H]+
364.06. Found: 364.32. 1H NMR (400 MHz, DMSO-d6): δ 11.75
(s, 1H), 10.11 (s, 1H), 7.71 (s, 1H), 7.63 (d, J ) 1.4 Hz, 1H),
7.61 (d, J ) 1.4 Hz, 1H), 3.70 (s, 3H), 2.24 (s, 3H), 2.22 (s,
3H).
3-(5-Cyano-6-(methyl(neopentyl)amino)-2-(methylthio)-
pyrimidin-4-ylamino)-N-methoxy-4-methylbenzamide (6).
To a mixture of 5 (0.31 g, 0.85 mmol) and N-methylneopen-
tylamine hydrochloride (0.18 g, 1.3 mmol) in DMF (3 mL) at
room temperature was added diisopropylethylamine (0.45 mL,
2.6 mmol). After stirring for 4 h at room temperature, the
mixture was diluted with water (10 mL) and extracted with
ethyl acetate (3 × 30 mL). The combined extracts were diluted
with hexanes (60 mL) and washed with water (3 × 10 mL)
and brine (20 mL). After drying over anhydrous sodium

5-Cyanopyrimidine Inhibitors of p38R MAP Kinase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6267
by HPLC; HRMS (ESI) m/z Calcd for C20H26N6O2 [M + H]+
3-(5-Cyano-6-(propylamino)pyrimidin-4-ylamino)-N-
methoxy-4-methylbenzamide (14b). A solution of 13 (50
mg, 60%, 0.087 mmol) and n-propylamine (36 µL, 0.44 mmol)
in 1,4-dioxane (1.5 mL) was stirred at room temperature for
10 min and then concentrated under vacuum. The residue was
subjected to preparative HPLC to provide 14b (19 mg, 23%
yield from 12) as a white solid. 100% pure by HPLC. HRMS
(ESI) m/z Calcd for C17H20N6O2 [M + H]+ 341.1848. Found:
1
383.2372. Found: 383.2194. H NMR (500 MHz, DMSO-d6):
δ 11.7 (s, 1H), 9.00 (s, 1H), 8.01 (s, 1H), 7.64 (s, 1H), 7.54 (d,
J ) 8.2 Hz, 1H), 7.33 (d, J ) 8.2 Hz, 1H), 3.72 (s, 2H), 3.68 (s,
3H), 3.38 (s, 3H), 2.18 (s, 3H), 0.92 (s, 9H); 13C NMR (500 MHz,
DMSO-d6): δ 163.8, 163.2, 163.0, 157.6, 138.5, 137.1, 130.0,
129.9, 125.9, 124.4, 117.0, 69.1, 63.0, 60.4, 42.4, 34.0, 27.7, 17.6.
4-(Methylthio)-6-oxo-1,6-dihydropyrimidine-5-carbo-
nitrile (10). A mixture of 3,3-bis(methylthio)-2-cyanoacrylate
(9) (10.1 g, 49.8 mmol), formamidine hydrochloride (4.40 g, 54.6
mmol), and triethylamine (11.4 mL, 81.8 mmol) in ethanol (300
mL) was heated at reflux for 5 h and then concentrated under
vacuum to dryness. To the residue was added water (30 mL),
and the mixture was acidified with 6 N HCl to pH 1. The
precipitating material was collected by suction filtration,
washed with water, and dried over drierite under vacuum to
afford 10 (7.64 g) as a white solid. This product was 85% pure
by HPLC, but it was used in the next step without further
purification.
4-Chloro-6-(methylthio)pyrimidine-5-carbonitrile (11).
An initially heterogeneous mixture of 10 (7.64 g, 85%, 35.0
mmol) and POCl3 (50 mL) was heated at reflux for 7 h, and
then the excess POCl3 was removed under vacuum. The
residue was diluted with AcOEt and washed twice with cold
water, saturated NaHCO3 solution, and brine. The solution
was dried over anhydrous MgSO4 and concentrated under
vacuum to dryness to afford 11 (7.74 g) as a yellow solid. This
product was 85% pure by HPLC, but it was used in the next
step without further purification.
1
341.1716. H NMR (400 MHz, DMSO-d6) δ 11.7 (s, 1H), 9.10
(s, 1H), 8.04 (s, 1H), 7.60 (s, 1H), 7.59-7.55 (m, 2H), 7.34 (d,
J ) 7.9 Hz, 1H), 3.69 (s, 3H), 3.34 (m, 3H), 2.18 (s, 3H), 1.54
(m, 2H), 0.86 (t, J ) 7.4 Hz, 3H); 13C NMR (400 MHz, DMSO-
d6): δ 163.8, 163.1, 162.5, 159.9, 139.5, 137.5, 130.7, 130.5,
126.9, 125.2, 115.6, 69.0, 63.6, 22.7, 18.3, 11.6.
3-(5-Cyano-6-(isopropylamino)pyrimidin-4-ylamino)-
N-methoxy-4-methylbenzamide (14c). 14c was prepared in
exactly the same manner as 14b. White solid; 21% yield from
12; 100% pure by HPLC. HRMS (ESI) m/z Calcd for C17H20N6O2
[M + H]+ 341.1845. Found: 341.1713. 1H NMR (500 MHz,
DMSO-d6, 80 °C) δ 11.4 (s, 1H), 8.05 (s, 1H), 7.64 (s, 1H), 7.55
(d, J ) 7.7 Hz, 1H), 7.31 (d, J ) 7.7 Hz, 1H), 6.83, (s, 1H),
4.36 (m, 1H), 3.70 (s, 3H), 2.20 (s, 3H), 1.20 (d, J ) 6.6 Hz,
6H). 13C NMR (500 MHz, DMSO-d6): δ 163.3, 162.1, 161.7,
159.3, 138.9, 137.0, 130.1, 130.0, 126.3, 124.6, 115.0, 68.6, 63.0,
42.6, 21.9, 17.7.
3-(5-Cyano-6-(cyclopentylamino)pyrimidin-4-ylamino)-
N-methoxy-4-methylbenzamide (14d). 14d was prepared
in exactly the same manner as 14b. White solid; 22% yield
from 12; 100% pure by HPLC. HRMS (ESI) m/z Calcd for
C19H22N6O2 [M + H]+ 367.2004. Found: 367.1874. 1H NMR
(500 MHz, DMSO-d6) δ 11.7 (s, 1H), 9.05 (s, 1H), 8.03 (s, 1H),
7.58 (s, 1H), 7.54 (d, J ) 7.7, 1H), 7.32 (s, J ) 7.7 Hz, 1H),
4.41 (m, 1H), 3.67 (s, 3H), 2.16 (s, 3H), 1.90-1.86 (m, 2H),
1.68-1.62 (m, 2H), 1.58-1.49 (m, 4H).
3-(5-Cyano-6-(methylthio)pyrimidin-4-ylamino)-N-meth-
oxy-4-methylbenzamide (12). A mixture of 11 (1.90 g, 90%,
9.21 mmol), N-methoxy-3-amino-4-methylbenzamide hydro-
chloride (2.87 g, 13.2 mmol), and N,N-diisopropylethylamine
(2.3 mL, 13.2 mmol) in DMF (15 mL) was heated at 65 °C for
5 h. After cooling to room temperature the solution was poured
into water (150 mL). The resulting mixture was basified with
saturated NaHCO3 solution to pH 9 and was extracted with
AcOEt (4 × 80 mL). The combined extract was washed with
water and brine, dried over anhydrous MgSO4, and concen-
trated under vacuum. The reside was subjected to silica gel
chromatography (85% AcOEt/hexane) to afford 12 (2.15 g, 53%
3-(5-Cyano-6-(methylthio)pyrimidin-4-ylamino)-4-meth-
ylbenzoic Acid (15). A solution of 11 (5.00 g, 90%, 24.2 mmol)
and 3-amino-4-methylbenzoic acid (4.57 g, 30.2 mmol) in DMF
(35 mL) was heated at 65 °C for 20 h. After cooling to room
temperature, the solution was poured in water (300 mL), and
the resulting mixture was neutralized with saturated NaHCO3
solution to pH 5. The precipitating material was collected by
suction filtration, washed with water, and dried over drierite
under vacuum to provide 15 (5.89 g, 60% yield from 9) as a
beige solid. 98% purity by HPLC; LCMS (EI) m/z Calcd for
1
yield from 9) as a pale solid. 100% purity by HPLC; H NMR
(400 MHz, DMSO-d6) δ 11.7 (s, 1H), 9.75 (s, 1H), 8.44 (s, 1H),
7.62 (s, 1H), 7.61 (d, J ) 8.4, 1H), 7.38 (d, J ) 8.4 Hz, 1H),
3.70 (s, 3H), 2.59 (s, 3H), 2.18 (s, 3H); 13C NMR (400 MHz,
DMSO-d6): δ 173.8, 163.6, 160.6, 158.8, 139.7, 136.6, 130.9,
130.8, 127.0, 125.9, 114.1, 86.2, 63.6, 18.2, 12.7.
1
C14H12N4O2S [M + H]+ 301.07. Found: 301.05; H NMR (500
MHz, DMSO-d6) δ 12.9 (s, br., 1H), 9.71 (s, 1H), 8.43 (s, 1H),
7.77 (d, J ) 7.7, 1H), 7.76 (s, 1H), 7.40 (d, J ) 7.7 Hz, 1H),
2.58 (s, 3H), 2.19 (s, 3H).
3-(5-Cyano-6-(methylsulfinyl)pyrimidin-4-ylamino)-N-
methoxy-4-methylbenzamide (13). To a slurry of 12 (0.500
g, 1.52 mmol) in MeOH (15 mL) was added a solution of Oxone
(3.74 g, 6.08 mmol) in water (15 mL) at 0 °C over 3 min. The
mixture was stirred at 0 °C for 5 min and then neutralized
with saturated NaHCO3 to pH 8. The mixture was diluted with
water (30 mL) and extracted with AcOEt (4 × 30 mL). The
combined extract was washed with brine, dried over anhydrous
MgSO4, and concentrated under vacuum to give crude 13
(0.310 g) as a pale yellow solid. This crude product was 60%
pure by HPLC, but it was used in the next step without further
purification. LCMS (EI) m/z Calcd. for C15H15N5O3S [M + H]+
346.09. Found: 346.38.
3-(5-Cyano-6-(methylamino)pyrimidin-4-ylamino)-N-
methoxy-4-methylbenzamide (14a). A solution of 13 (40
mg, 60%, 0.070 mmol) and 2 N methylamine/THF (0.17 mL,
0.34 mmol) in 1,4-dioxane (1.5 mL) was stirred at room
temperature for 10 min and then concentrated under vacuum.
The residue was subjected to preparative HPLC to provide 14a
(16 mg, 26% yield from 12) as a white solid. 100% pure by
HPLC. HRMS (ESI) m/z Calcd for C15H16N6O2 [M + H]+
313.1535. Found: 313.1411. 1H NMR (400 MHz, DMSO-d6) δ
11.7 (s, 1H), 9.12 (s, 1H), 8.07 (s, 1H), 7.60 (s, 1H), 7.57 (d, J
) 7.8, 1H), 7.50 (d, J ) 4.2 Hz, 1H), 7.34 (d, J ) 7.8 Hz, 1H),
3.69 (s, 3H), 2.87 (d, J ) 4.2 Hz, 3H), 2.18 (s, 3H); 13C NMR
(400 MHz, DMSO-d6): δ 163.8, 163.5, 162.3, 160.0, 139.6,
137.5, 130.7, 130.5, 126.9, 125.2, 115.6, 69.2, 63.6, 28.3, 18.3.
3-(5-Cyano-6-(methylthio)pyrimidin-4-ylamino)-N-(isox-
azol-3-yl)-4-methylbenzamide (16a). An initially heteroge-
neous mixture of 15 (2.50 g, 8.32 mmol) and thionyl chloride
(50 mL) was stirred at room temperature for 1 h and then the
excess thionyl chloride was evaporated under vacuum. The
trace amount of thionyl chloride was removed by adding
toluene (5 mL) to the residue and repeating the evaporation
process. The residue thus obtained was dissolved in CH2Cl2
(100 mL), and to this solution was added 3-aminoisoxazole
(1.23 mL, 16.6 mmol) and pyridine (1.68 mL, 20.8 mmol). The
mixture was heated at reflux for 1 h, during which period a
large amount of precipitate formed. The solvent was removed
under vacuum, and to the residue was added water (60 mL).
The insoluble material was collected by suction filtration,
washed with water, and dried over drierite under vacuum to
provide 16a (2.34 g, 77% yield) as a pale yellow solid; 94%
purity by HPLC; LCMS (EI) m/z Calcd for C17H14N6O2S [M +
H]+ 367.09. Found: 367.11.
3-(5-Cyano-6-(methylthio)pyrimidin-4-ylamino)-N-(1-
ethyl-1H-pyrazol-5-yl)-4-methylbenzamide (16b). A mix-
ture of 15 (300 mg, 1.00 mmol), 5-amino-1-ethylpyrazole (333
mg, 3.00 mmol), BOP reagent (663 mg, 1.50 mmol), and
N-methylmorpholine (0.49 mL, 4.46 mmol) in DMF (3 mL) was
heated at 60 °C for 16 h. After cooling to room temperature,
the mixture was poured into water (30 mL), and the resulting
mixture was extracted with AcOEt (3 × 30 mL). The combined

6268 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Liu et al.
extract was washed with water, 0.5 N AcOH aqueous solution,
saturated NaHCO3 solution, and brine. The solution was dried
over MgSO4, and concentrated to dryness under vacuum to
provide 16b (420 mg) as a yellow powder; 80% pure by HPLC;
LCMS (EI) m/z Calcd for C19H19N7OS [M + H]+ 394.14.
Found: 394.04.
3-(5-Cyano-6-(isopropylamino)pyrimidin-4-ylamino)-
N-(isoxazol-3-yl)-4-methylbenzamide (3a). To a solution of
16a (0.300 g, 0.819 mmol) in THF (20 mL) was added mCPBA
(57-86%, 0.650 g, 2.15-3.24 mmol) at room temperature in
one portion. The mixture was stirred at room temperature for
6 h and concentrated under vacuum. The residue was stirred
with 5% Na2S2O3 (30 mL) at room temperature for 10 min,
and the aqueous solution was removed by decantation. This
process was repeated once. To the residue thus obtained was
added saturated NaHCO3 solution, and the insoluble product
(sulfone intermediate) (0.389 g) was collected by suction
filtration, washed with water, and dried over drierite under
vacuum.
A mixture of the above obtained sulfone intermediate (0.120
g) and isopropylamine (0.11 mL) in 1,4-dioxane (1.8 mL) was
heated at 150 °C under microwave for 15 min, and then
concentrated under vacuum. The residue was subjected to
preparative HPLC to afford 3a (33.2 mg, 35% yield from 16a)
as a pale solid; 100% purity by HPLC; HRMS (ESI) m/z Calcd
for C19H19N7O2 [M + H]+ 368.1800. Found: 367.1661. 1H NMR
(400 MHz, DMSO-d6) δ 11.4 (s, 1H), 9.11 (s, 1H), 8.85 (s, 1H),
8.06 (s, 1H), 7.93 (s, 1H), 7.87 (d, J ) 8.1 Hz, 1H), 7.41 (d, J
) 8.1 Hz, 1H), 7.22 (d, J ) 8.1 Hz, 1H), 7.05 (s, 1H), 4.38 (m,
1H), 2.23 (s, 3H), 1.18 (d, J ) 6.6 Hz, 6H); 13C NMR (400 MHz,
DMSO-d6) δ 165.0, 162.7, 162.2, 160.4, 158.4, 140.4, 137.6,
131.3, 130.7, 128.0, 126.3, 115.6, 100.2, 69.2, 42.7, 22.4, 18.4.
3-(5-Cyano-6-(cyclopentylamino)pyrimidin-4-ylamino)-
N-(isoxazol-3-yl)-4-methylbenzamide (3b). 3b was pre-
pared in the same manner as 3a; Beige solid; 32% yield from
16a 99% purity by HPLC; HRMS (ESI) m/z Calcd for
C21H21N7O2 [M + H]+ 404.1957. Found: 404.1815. 1H NMR
(500 MHz, DMSO-d6) δ 11.3 (s, 1H), 9.08 (s, 1H), 8.83 (s, 1H),
8.04 (s, 1H), 7.92 (s, 1H), 7.85 (d, J ) 8.2 Hz, 1H), 7.39 (d, J
) 8.2 Hz, 1H), 7.31 (d, J ) 7.1 Hz, 1H), 4.41 (m, 1H), 2.21 (s,
3H), 1.88 (m, 2H), 1.68 (m, 2H), 1.58-1.49 (m, 4H).
(400 MHz, CDCl3) δ 175.5, 175.3, 161.1, 113.0, 100.6, 26.4,
25.6, 14.4, 13.7.
Methyl 3-(5-Cyano-2-(ethylthio)-6-(methylthio)pyrimi-
din-4-ylamino)-4-methylbenzoate (20). A mixture of 19
(1.78 g, 7.24 mmol) and methyl 3-amino-4-methylbenzoate
(1.50 g, 8.90 mmol) in DMF (15 mL) was heated at 65 °C for
2 days and then diluted with AcOEt (300 mL). The resulting
solution was washed with saturated NaHCO3 solution and
brine, dried over anhydrous MgSO4, and concentrated under
vacuum. Column chromatography (silica gel, 15% AcOEt/
hexane) of the residue provided 20 (1.90 g, 70% yield) as a
white solid; 98% purity by HPLC; 1H NMR (400 MHz, DMSO-
d6) δ 9.68 (s, 1H), 7.85 (s, 1H), 7.80(d, J ) 8.0 Hz, 1H), 7.44
(d, J ) 8.0 Hz, 1H), 3.84 (s, 3H), 2.84 (q, J ) 7.3 Hz, 2H), 2.56
(s, 3H), 2.23 (s, 3H), 1.04 (t, J ) 7.3 Hz, 3H).
Methyl 3-(5-Cyano-2-(methylsulfinyl)-6-(methylthio)-
pyrimidin-4-ylamino)-4-methylbenzoate (21). To a solu-
tion of 20 (1.65 g, 4.41 mmol) in THF was added mCPBA (1.14
g, e77%, e5.09 mmol) at 0 °C in portions over 3 min. The
resulting mixture was stirred at 0 °C for 1 h and then
concentrated under vacuum to the half of its original volume.
The residue was diluted with AcOEt (200 mL) and washed
with saturated NaHCO3 solution (twice) and brine. The
solution was dried over anhydrous MgSO4 and concentrated
under vacuum to dryness to give crude 21 (1.78 g) as a white
solid; This product was 60% pure by HPLC, but it was used
in the next step without further purification.
Methyl 3-(2-Amino-5-cyano-6-(methylthio)pyrimidin-
4-ylamino)-4-methylbenzoate (22). A mixture of the above
obtained crude 21 (1.78 g, 60%) and 2 N NH3/i-PrOH (20 mL)
was heated at 65 °C in a sealed tube for 1 h. After cooling to
room temperature, product precipitated from the solution, and
collected by suction filtration to afford the first crop of 22 (0.500
g, 34% yield) as a beige solid. The filtrate was concentrated
and the residue was subjected to preparative HPLC to provide
the second crop of 22 (0.156 g, 11% yield); 97% purity by HPLC;
LCMS (EI) m/z Calcd for C15H15N5O2S [M + H]+ 330.09.
Found: 330.05; 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H),
7.77 (s, 1H), 7.75 (d, J ) 7.8 Hz, 1H), 7.40 (d, J ) 7.8 Hz, 1H),
7.13 (s, br., 2H), 3.84 (s, 3H), 2.50 (s, 3H), 2.21 (s, 3H).
Methyl 3-(2-Amino-5-cyano-6-(isopropylamino)pyrimi-
din-4-ylamino)-4-methylbenzoate (23a). To a solution of 22
(0.500 g, 1.52 mmol) in THF (15 mL) was added mCPBA
(e77%, 0.740 g, e3.30 mmol) at room temperature in one
portion. The resulting mixture was stirred at room tempera-
ture for 5 h before it was diluted with AcOEt (200 mL) and
washed with saturated NaHCO3 solution (twice) and brine.
The solution was dried over anhydrous MgSO4 and concen-
trated under vacuum to dryness. To the residue (sulfone
intermediate) were added 1,4-dioxane (5 mL) and isopropyl-
amine (0.53 mL, 6.22 mmol). The mixture was heated at 150
°C under microwave for 15 min. It was then diluted with
AcOEt, washed with water (three times) and brine, and dried
over anhydrous MgSO4. The evaporation of solvent under
vacuum provided 23a (0.520 g, 100% yield) as a pale solid;
93% purity by HPLC; LCMS (EI) m/z Calcd for C17H20N6O2
[M + H]+ 341.16. Found: 341.10; 1H NMR (400 MHz, DMSO-
d6) δ 8.46 (s, 1H), 7.79 (s, 1H), 7.69 (d, J ) 8.0 Hz, 1H), 7.36
(d, J ) 8.0 Hz, 1H), 6.48 (s, 2H), 6.44 (d, J ) 8.3 Hz, 1H), 4.33
(m, 1H), 3.84 (s, 3H), 2.22 (s, 3H), 1.15 (d, J ) 6.6 Hz, 6H).
3-(5-Cyano-6-(isopropylamino)pyrimidin-4-ylamino)-
N-(1-ethyl-1H-pyrazol-5-yl)-4-methylbenzamide (17a). 17a
was prepared in the same manner as 3a; Pale yellow solid;
6% yield from 16b 100% purity by HPLC; HRMS (ESI) m/z
Calcd for C21H24N8O2 [M + H]+ 405.2273. Found: 367.2137.
3-(5-Cyano-6-(cyclopentylamino)pyrimidin-4-ylamino)-
N-(1-ethyl-1H-pyrazol-5-yl)-4-methylbenzamide (17b). 17b
was prepared in the same manner as 3a; Pale solid; 8% yield
from 16b 100% purity by HPLC; HRMS (ESI) m/z Calcd for
C23H26N8O [M + H]+ 431.2430. Found: 431.2312.
2-(Ethylthio)-4-(methylthio)-6-oxo-1,6-dihydropyrimi-
dine-5-carbonitrile (18). A mixture of 9 (10.0 g, 49.2 mmol),
2-ethyl-2-thiopseudourea hydrobromide (10.0 g, 54.0 mmol),
and N,N-diisopropylethylamine (14.0 mL, 80.4 mmol) in etha-
nol (300 mL) was heated at reflux for 24 h and then concen-
trated under vacuum. To the residue was added water (60 mL),
and the resulting mixture was acidified with 1 N HCl to pH
1. The insoluble material was collected by suction filtration,
washed with water, and dried over drierite under vacuum to
provide 18 (10.4 g) as a beige solid. This product was 65%
purity by HPLC, but it was used in the next step without
further purification.
4-Chloro-2-(ethylthio)-6-(methylthio)pyrimidine-5-car-
bonitrile (19). A mixture of 18 (3.50 g, 65%, 10.0 mmol) and
POCl3 (35 mL) was heated at reflux for 1.5 h and then
concentrated under vacuum. The residue was dissolved in
AcOEt (200 mL) and washed with cold water, saturated
NaHCO3 solution, water, and brine. The solution was dried
over MgSO4 and concentrated under vacuum. The residue was
subjected to column chromatography (silica gel, 3% AcOEt/
hexane) to afford 19 (1.72 g, 42% yield from 9) as a yellow
solid. 98% purity by HPLC; 1H NMR (400 MHz, CDCl3) δ 3.12
(q, J ) 7.4 Hz, 2H), 2.58 (s, 3H), 1.35 (t, J ) 7.4 Hz); 13C NMR
Methyl 3-(2-Amino-5-cyano-6-(cyclopentylamino)py-
rimidin-4-ylamino)-4-methylbenzoate (23b). 23b was pre-
pared in exactly the same manner as 23a; white solid; 65%
yield; 99% purity by HPLC; LCMS (EI) m/z Calcd for
1
C19H22N6O2 [M + H]+ 367.18. Found: 367.31; H NMR (500
MHz, DMSO-d6) δ 8.43 (s, 1H), 7.77 (s, 1H), 7.68 (d, J ) 8.2
Hz, 1H), 7.34 (d, J ) 8.2 Hz, 1H), 6.52 (d, J ) 7.7 Hz, 1H),
6.46 (s, 2H), 4.38 (m, 1H), 3.82 (s, 3H), 2.20 (s, 3H), 1.86 (m,
2H), 1.67 (m, 2H), 1.53-1.47 (m, 4H).
3-(2-Amino-5-cyano-6-(isopropylamino)pyrimidin-4-
ylamino)-4-methylbenzoic Acid (24a). To a solution of 23a
(100 mg, 0.294 mmol) in THF (5 mL) and MeOH (2 mL) was
added a solution of LiOH (25.0 mg, 1.05 mmol) in water (2
mL) at room temperature. The mixture was stirred at room

5-Cyanopyrimidine Inhibitors of p38R MAP Kinase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6269
temperature for 4 h and then concentrated under vacuum. The
residue was diluted with water (2 mL) and neutralized to pH
6 with 1 N HCl. The resulting precipitate was collected by
suction filtration, washed with water, and dried over drierite
under vacuum to provide 24a (80.0 mg, 83% yield) as a white
solid; 94% purity by HPLC; LCMS (EI) m/z Calcd for C16H18N6O2
[M + H]+ 327.15. Found: 327.31; 1H NMR (500 MHz, DMSO-
d6) δ 12.8 (s, 1H), 8.41 (s, 1H), 7.72 (s, 1H), 7.66 (d, J ) 7.7
Hz, 1H), 7.31 (d, J ) 7.7 Hz, 1H), 6.45 (s, 2H), 6.39 (d, J ) 8.3
Hz, 1H), 4.31 (m, 1H), 2.19 (s, 3H), 1.14 (d, J ) 6.6 Hz, 6H).
(d, J ) 1.6 Hz, 1H), 4.39 (m, 1H), 3.98 (q, J ) 7.2 Hz, 2H),
2.22 (s, 3H), 1.86 (m, 2H), 1.67 (m, 2H), 1.53-1.46 (m, 4H).
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 E. 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. Phospho-
rylated MKK6 was generated according to the literature.13
p38 Kinase Assay. The assay was performed in V-bottomed
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 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 prewet filtermat using a Skatron
Micro96 Cell Harvester (Skatron), 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%. This assay was used for initial
determination of IC50 values for the compounds.
Alternate Enzyme Assay for Tight Binding Com-
pounds. For Ki determinations, the p38 enzyme assay was
modified to permit analysis of more potent compounds. Each
reaction mixture consisted of a total volume of 240 µL and
contained 1.07 ng/mL of the bacterially expressed recombinant
p38-GST fusion protein, 66.7 µg/mL of myelin basic protein, 1
µM of ATP, and 2.4 µCi of [γ-33P]ATP (NEN). The mixtures
also contained 20 mM HEPES, pH 7.4, 15 mM MgCl2, 25 mM
â-glycerophosphate, 0.1 mg/mL bovine serum albumin, and 1
mM DTT. The reaction mixtures were incubated at room
temperature for 21 h, and kinase activity was determined by
quantitation of the amount of radioactive phosphate trans-
ferred to myelin basic protein. The reactions were terminated
by the addition of 60 µL of a solution of 50% trichloroacetic
acid/250 mM sodium pyrophosphate. The samples were incu-
bated on ice for 60 min to allow precipitation of the labeled
substrate. The precipitated substrate was harvested using a
Packard Harvester with a Unifilter-96, GF/C Filter Plate
(Perkin-Elmer). Bound radiolabeled phosphate was quanti-
tated using a TopCount 96-well liquid scintillation counter
(Packard Instrument Co.). The concentration of the p38-GST
fusion protein, 1.07 ng/mL, corresponds to a concentration of
15.9 pM of p38 enzyme. This concentration fell within the
linear range of the enzyme assay. The enzyme assay run under
these conditions maintained linearity over a range of 3.97 pM
to 509 pM of p38. The p38 enzyme preparation assayed under
these conditions retained full activity through the 21 h
incubation time.
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),
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 mL/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 lipopolysaccharide (LPS, BioWhittaker), with a
final concentration of 25 ng/mL, and incubated for 6 h at 37
3-(2-Amino-5-cyano-6-(cyclopentylamino)pyrimidin-4-
ylamino)-4-methylbenzoic acid (24b). 24b was prepared
in exactly the same way as 24a; White solid; 100% purity by
HPLC; LCMS (EI) m/z Calcd for C18H20N6O2 [M + H]+ 353.16.
1
Found: 353.31; H NMR (500 MHz, DMSO-d6) δ 12.9 (s, br.,
1H), 8.58 (s, br, 1H), 7.74 (s, 1H), 7.70 (d, J ) 7.8 Hz, 1H),
7.35 (d, J ) 7.8 Hz, 1H), 6.65 (s, br., 2H), 4.39 (m, 1H), 2.21
(s, 3H), 1.89 (m, 2H), 1.69 (m, 2H), 1.57-1.48 (m, 4H).
3-(2-Amino-5-cyano-6-(isopropylamino)pyrimidin-4-
ylamino)-N-(isoxazol-3-yl)-4-methybenzamide (25a). Thio-
nyl chloride (3 mL) was cooled to 0 °C and then added to 24a
(60 mg, 0.18 mmol). The resulting mixture was stirred at room
temperature for 1 h. The excess amount of thionyl chloride
was evaporated under vacuum. The trace amount of thionyl
chloride was removed by adding toluene (1 mL) to the residue
and repeating the evaporation process. The residue thus
obtained was dissolved in CH2Cl2 (5 mL), and to this solution
was added at 3-aminoisoxazole (0.040 mL, 0.59 mmol) and
pyridine (0.037 mL, 0.46 mmol). The whole was heated at
reflux for 1 h and then concentrated under vacuum. The
residue was subjected to preparative HPLC to afford 25a (51
mg, 71%yield) as a white solid; 100% purity by HPLC; HRMS
(ESI) m/z Calcd for C19H20N8O2 [M + H]+ 393.1909. Found:
393.1772.
3-(2-Amino-5-cyano-6-(cyclopentylamino)pyrimidin-4-
ylamino)-N-(isoxazol-3-yl)-4-methylbenzamide (25b). 25b
was prepared in the same manner as 25a; White solid; 79%
yield; 100% purity by HPLC; HRMS (ESI) m/z Calcd for
C21H22N8O2 [M + H]+ 419.2066. Found: 419.1955. 1H NMR
(500 MHz, DMSO-d6) δ 11.3 (s, 1H), 8.79 (s, 1H), 8.36 (s, 1H),
7.87 (s, 1H), 7.74 (d, J ) 7.7 Hz, 1H), 7.30 (d, J ) 7.7 Hz, 1H),
6.99 (s, 1H), 6.47 (d, J ) 7.7 Hz, 1H), 6.43 (s, 2H), 4.35 (m,
1H), 2.17 (s, 3H), 1.83 (m, 2H), 1.63 (m, 2H), 1.50-1.43 (m,
4H); 13C NMR (500 MHz, DMSO-d6) δ 164.6, 163.1, 163.0,
162.6, 159.7, 157.8, 139.4, 137.7, 130.6, 129.9, 127.0, 124.9,
117.2, 99.5, 60.7, 51.3, 31.8, 23.1, 17.9.
3-(2-Amino-5-cyano-6-(isopropylamino)pyrimidin-4-
ylamino)-N-(1-ethyl-1H-pyrazol-5-yl)-4-methylbenzam-
ide (25c). A mixture of 24a (30 mg, 0.092 mmol), 5-amino-1-
ethylpyrazole (35 mg, 0.32 mmol), BOP reagent (61 mg, 0.14
mmol), and N-methylmorpholine (0.045 mL, 0.41 mmol) in
DMF (0.5 mL) was heated at 65 °C for 16 h. The mixture was
diluted with AcOEt (40 mL), washed with water (twice) and
brine, and dried over anhydrous MgSO4. Purification by flash
chromatography (silica gel, 80% AcOEt/hexane) provided 25c
(17 mg, 45% yield) as a white solid; 95% purity by HPLC;
HRMS (ESI) m/z Calcd for C21H25N9O2 [M + H]+ 420.2382.
Found: 420.2262. 1H NMR (500 MHz, DMSO-d6) δ 10.1 (s,
1H), 8.42 (s, 1H), 7.84 (s, 1H), 7.71 (d, J ) 7.7 Hz, 1H), 7.41
(d, J ) 1.6 Hz, 1H), 7.36 (d, J ) 7.7 Hz, 1H), 6.47 (s, 2H), 6.40
(d, J ) 8.2 Hz, 1H), 6.18 (d, J ) 1.6 Hz, 1H), 4.31 (m, 1H),
3.99 (q, J ) 7.1 Hz, 2H), 2.21 (s, 3H), 1.29 (t, J ) 7.1 Hz, 3H),
1.14 (d, J ) 6.7 Hz, 6H); 13C NMR (500 MHz, DMSO-d6) δ
165.0, 162.9, 162.6, 162.5, 139.0, 137.6, 137.2, 135.0, 130.8,
129.9, 126.5, 124.6, 117.1, 100.7, 60.5, 42.4, 41.0, 22.0, 17.8,
14.5.
3-(2-Amino-5-cyano-6-(cyclopentylamino)pyrimidin-4-
ylamino)-N-(1-ethyl-1H-pyrazol-5-yl)-4-methylbenzam-
ide (25d). 25d was prepared in the same manner as 25c;
White solid; 45% yield; 96% purity by HPLC; HRMS (ESI) m/z
Calcd for C23H27N9O [M + H]+ 446.2539. Found: 446.2422.
1H NMR (500 MHz, DMSO-d6) δ 10.1 (s, 1H), 8.42 (s, 1H), 7.84
(s, 1H), 7.72 (d, J ) 8.2 Hz, 1H), 7.41 (d, J ) 1.6 Hz, 1H), 7.35
(d, J ) 8.2 Hz, 1H), 6.52 (d, J ) 7.7 Hz, 1H), 6.47 (s, 2H), 6.18

6270 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Liu et al.
(6) Selected articles and reviews: (a) Cumming, J. G.; McKenzie,
C. L.; Bowden, S. G.; Campbell, D.; Masters, D. J.; Breed, J.;
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several models of rheumatoid arthritis. Bioorg. Med. Chem. Lett.
2004, 14, 3595-3599. (d) Dombroski, M. A.; Letavic, M. A.;
McClure, K. F.; Barberia, J. T.; Carty, T. J.; Cortina, S. R.; Csiki,
C.; Dipesa, A. J.; Elliott, N. C.; Gabel, C. A.; Jordan, C. K.;
Labasi, J. M.; Martin, W. H.; Peese, K. M.; Stock, I. A.; Svensson,
L.; Sweeney, F. J.; Yu, C. H. Benzimidazolone p38 inhibitors.
Bioorg. Med. Chem. Lett. 2004, 14, 919-923. (e) Trejo, A.;
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Moar, J.; Lim, J.; Mclntosh, J.; Miller, J.; Papp, E.; Reuter, D.;
Roberts, R.; Sanpablo, F.; Saunders, J.; Song, K.; Villasenor, A.;
Warren, S. D.; Welch, M.; Weller, P.; Whiteley, P.; Zeng, L.;
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°C, 5% CO2. The cell supernatants were removed and assayed
for TNF-R by ELISA (R&D Systems).
Inhibition of TNFR 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% poly-
(ethylene glycol) (mw 300), and a dosing volume of 0.2 mL per
mouse was administered by average 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 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 Animal Care and Use Committee.
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 phytohema-
glutinin (PHA) for 4 d 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
(Biosource).14
Metabolic Stability in Liver Microsomes. Incubations
with rat and human liver microsomes (BD Gentest, Woburn,
MA) were conducted at 1 mg/mL protein concentration, 3 µM
compound in 56 mM phosphate buffer (pH 7.4), and 1 mM
â-nicotinamide adenine dinucleotide phosphase (â-NADPH) at
37 °C for 1 h. Aliquots of incubation mixtures (0.2 mL) were
taken at 0 and 15 min, and the reaction was quenched with
one volume of acetonitrile. The rate of metabolism was
calculated based on the fraction of parent disappearance by
comparing 15 min to 0 min time points.
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