Structure-Activity Relationships of the p38α MAP Kinase Inhibitor 1-(5-tert-Butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy) naphthalen-1-yl]urea (BIRB 796)

Article abstract of DOI:10.1021/jm030121k

We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy) naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38α MAP kinase which has advanced into human clinical trials for the treatment of autoimmune diseases. Thermal denaturation was used to establish molecular binding affinities for this class of p38α inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic ring attached to N-2 of the pyrazole nucleus provides important π-CH ₂ interactions with the kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature of p38α by 16-17 °C translating into K_d values of 50-100 pM. Finally, we describe several compounds that potently inhibit TNF-α production when dosed orally in mice.

Full text of DOI:10.1021/jm030121k

4676
J . Med. Chem. 2003, 46, 4676-4686
Str u ctu r e-Activity Rela tion sh ip s of th e p 38r MAP Kin a se In h ibitor
1-(5-ter t-Bu tyl-2-p-tolyl-2H-p yr a zol-3-yl)-3-[4-(2-m or p h olin -4-yl-eth oxy)n a p h -
th a len -1-yl]u r ea (BIRB 796)
J ohn Regan,*,† Alison Capolino,† Pier F. Cirillo,† Thomas Gilmore,† Anne G. Graham,‡ Eugene Hickey,†
Rachel R. Kroe,‡ J effrey Madwed,§ Monica Moriak,† Richard Nelson,† Christopher A. Pargellis,‡ Alan Swinamer,†
Carol Torcellini,§ Michele Tsang,† and Neil Moss†
Departments of Medicinal Chemistry, Immunology and Inflammation, and Pharmacology, Boehringer Ingelheim
Pharmaceuticals, Research and Development Center, 900 Ridgebury Road, Ridgefield, Connecticut 06877
Received March 13, 2003
We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-
3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38R MAP
kinase which has advanced into human clinical trials for the treatment of autoimmune diseases.
Thermal denaturation was used to establish molecular binding affinities for this class of p38R
inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic
domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic
ring attached to N-2 of the pyrazole nucleus provides important π-CH2 interactions with the
kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene
and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen
bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature
of p38R by 16-17 °C translating into Kd values of 50-100 pM. Finally, we describe several
compounds that potently inhibit TNF-R production when dosed orally in mice.
In tr od u ction
of p38R and its use in several animal models of inflam-
mation validated this kinase as an important antiin-
flammatory therapeutic target.16-23 Analogues of 1,24,25
along with alternate structural types of compounds,26
continue to be developed as antiinflammatory agents.
We recently described N-pyrazole-N′-aryl urea 227 as
a modest inhibitor of p38R (Chart 1). Refining this lead
compound to include a tolyl ring at N-2 of the pyrazole
(i.e., 3), replacing the 4-chlorophenyl ring with naph-
thalene (i.e., 4), and attaching an ethoxy morpholine
group furnished 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-
yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]-
urea (BIRB 796, 5) as a potent and selective inhibitor
of p38R.28 Recently, 5 was shown to inhibit in vivo LPS-
stimulated TNF-R production in a human clinical trial.29
Compound 5 has advanced into human clinical trials
for the treatment of autoimmune diseases. One of the
unique characteristics of the pyrazole aryl urea class of
compounds is the binding kinetics to p38R: slow kon and
long koff rates.28 We can rationalize these results with
X-ray crystallographic observations (Figure 1a). For this
class of compounds to bind to p38R, the activation loop,
consisting in part of conserved residues Asp168-Phe169-
Gly170, must undergo a substantial conformational
change. It is our belief this conformational change of
the protein is likely the rate determining step in the
slow binding of this class of compounds. In the binding
conformation, the side chain phenyl ring of Phe169
moves about 10 Å (DFG-out conformation) and partici-
pates in a lipophilic interaction with the aryl portion of
the inhibitor. This interaction helps secure the inhibitor
to the kinase and also possibly contributes to the long
koff rates. The implementation of a molecular assay
employing exchange curve binding kinetics for deter-
The proinflammatory cytokines tumor necrosis fac-
tor-R (TNF-R) and interleukin-1â (IL-1â) contribute to
the regulation of the body’s response to infection and
cellular stress.1 However, chronic and excessive produc-
tion of TNF-R and IL-1â are believed to underlie the
progression of many autoimmune diseases such as
rheumatoid arthritis (RA),2 Crohn’s disease, and pso-
riasis.3 The chimeric TNF-R antibody, infliximab,4,5 is
approved for the treatment of RA and Crohn’s disease,
while the IL-1 receptor antagonist, anakinra,6 and the
soluble TNF-R receptor fusion protein, etanercept,7-10
are approved for the treatment of RA. These reports
confirm that cytokine regulation represents an impor-
tant advance in antiinflammatory therapeutics. An
alternate approach to controlling cytokine levels in-
volves disruption of the signal transduction pathway
leading to their release from stimulated-inflammatory
cells. A key element in this pathway is p38 mitogen
activated protein (MAP) kinase.11 Activation of this
kinase under a variety of cellular stresses results in bis-
phosphorylation on a Thr-Gly-Tyr motif located in the
activation loop. Once activated, p38 phosphorylates and
activates other kinases and transcription factors leading
to stabilized mRNA and an increase or decrease in the
expression of certain target genes.12-15
The discovery of pyridinyl imidazole 1 (SB 203580)11
(Chart 1) as a potent (Table 1) and selective inhibitor
* To whom correspondence should be addressed. Phone: (203) 798-
4768. Fax: (203) 791-6072. E-mail: jregan@rdg.boehringer-ingelhe-
im.com.
† Department of Medicinal Chemistry.
‡ Department of Immunology and Inflammation.
§ Department of Pharmacology.
10.1021/jm030121k CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/20/2003

p38R MAP Kinase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4677
Ch a r t 1
F igu r e 1. (a) Stereoscopic view of the X-ray crystallographic complex of 5 (yellow) and human p38-R. Amino acid residues behind
5 are shown in magenta and those in front are displayed in green. (b) Summary of binding interactions of 5 and human p38-R.
mining Kd values was used in the discovery of 5. This
assay provides a better ranking of compounds with
binding activities near the limit of our initial fluorescence-
binding assay.28 However, the labor intensity and ac-
curacy for very potent binding compounds prevent the
analysis of numerous target molecules using the ex-
change curve assay. For this reason, a method of
determining p38R binding affinity relying on thermal
denaturation was developed that has higher throughput
and better accuracy for potent inhibitors.30 In this case,
Kd values are calculated from the denaturation temper-
ature of the inhibitor/kinase complex. In this paper, we
describe the thermal denaturation-derived binding af-
finity of analogues of 5 and correlate them to the binding
mode observed from X-ray crystallography (Figure 1b).
The in vitro and in vivo TNF-R inhibition data for
selected compounds are also shown.
Ch em istr y Section
Target molecules replacing the tert-butyl group at the
5-position of the pyrazole were prepared according to
the procedure outlined in Scheme 1. Compound 11 was
chosen as a representative example from Table 1. To
affix a hydroxyl group to the tert-butyl moiety the
substituted â-keto nitrile 8 was required. Silyl ether
protection of methyl 2,2-dimethyl-3-hydroxypropionate
(6) affords 7 and acetonitrile anion addition to the ester
in hot toluene produces 8 with concomitant removal of
the silyl protecting group. Pyrazole formation to furnish
9 is accomplished by heating 8 and p-tolylhydrazine in

4678 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Regan et al.
Sch em e 1a
a
Reagents: (a) tert-butyldimethylchlorosilane, imidazole, DMF; (b) NaH,acetonitrile, toluene, reflux; (c) p-tolylhydrazine hydrochloride,
ethanol, reflux; (d) 10, COCl2, NaHCO3, then 9.
Sch em e 2a
chloroethane gives 2-chloroethoxy naphthalene 19.
Conversion of the chloroethoxy group to iodoethoxy 20
is accomplished with NaI, and subsequent treatment
of 20 with thiomorpholine furnishes 21. Oxidizing the
thioether of 21 with NaIO4 gives sulfoxide 22, which
upon exposure to TFA removes the Boc protecting group
and provides aminonaphthalene 23. Treatment of 23
with pyrazole-phenylcarbamate 24 in DMSO32 gives
target molecule 25.
Alternatively, nonnucleophilic groups can be ap-
pended to the naphthol using Mitsunobu coupling
conditions.33 The synthesis of an example from Table
3, compound 30, is shown in Scheme 4. Treatment of
2-pyridin-4-yl-ethanol (26) with naphthol 18 and triph-
enylphosphine and diethyl azodicarboxylate (DEADC)
gives naphthyl ether 27. Removal of the Boc protecting
group of 27 with HCl provides aminonaphthalene 28.
The urea is formed by exposure of aminopyrazole 29 to
phosgene and base and the subsequently produced
isocyanate reacts with 28 to give target molecule 30.
a
Reagents: (a) Diethyl malonate, sodium, toluene; (b) HCl,
heat; (c) H2, Pd on carbon, 50 psi; (d) HCl, NaNO2 then SnCl2; (e)
HCl, ethanol; (f) 10, COCl2, NaHCO3, then 16.
ethanol. The urea linkage is assembled from the phos-
gene-generated isocyanate of aminonaphthalene 10 and
coupling with amine 9 to produce target molecule 11.
Replacing the substitution at the N-2 position of the
pyrazole requires hydrazines for condensation with
â-keto-nitriles and subsequent urea formation as shown
above. The synthesis of 17 as a representative example
in Table 2 is outlined in Scheme 2. Treating 2-chloro-
5-nitropyridine (12) with the anion of diethyl malonate
followed by acid hydrolysis and decarboxylation provides
2-methyl-5-nitropyridine (13).31 Reducing the nitro group
of 13 with hydrogen and treating the amine with NaNO2
and then SnCl2 furnishes hydrazine 14. Acid-catalyzed
condensation of hydrazine 14 with â-keto-nitrile 15
gives amino pyrazole 16. Urea formation to produce 17
is accomplished as before by reacting amino-naphtha-
lene 10 with phosgene and base followed by 16.
We have shown that the morpholine attached to the
ethoxy linker makes an important contribution to the
potency of 5.27 To further explore this observation, two
routes were developed to produce analogues with other
functionality appended to the ethoxy linker. In the first,
amine-containing heterocycles are reacted with an iodo-
ethoxy-naphthyl intermediate (i.e., 20). As a representa-
tive example of Table 3, the synthesis of oxothiomor-
pholine analogue 25 is shown in Scheme 3. Base-
catalyzed alkylation of naphthol 18 with 1-bromo-2-
Resu lts a n d Discu ssion
X-ray crystallographic data of 5 with human p38R
(Figure 1a) reveals an important role for the tert-butyl
group at C-5 of the pyrazole nucleus. This group
occupies a lipophilic pocket (Phe169 pocket) between the
two lobes of the kinase which is exposed upon reorga-
nization of the activation loop (DFG-out conformation).
We previously demonstrated the important binding
contribution of lipophilic groups attached to C-5 of the
pyrazole ring of a N-pyrazole-N′-phenyl urea (i.e., 3).27
A similar set of modifications to an N-pyrazole-N′-
naphthyl urea (i.e., 33) was examined to verify the
essentiality of the tert-butyl group. Table 1 shows the
thermal denaturation temperature (Tm) of the inhibitor
and murine p38R complex34 along with the calculated
Kd value. The analogue lacking any lipophilic group at
C-5 (31) shows a Tm result with p38R near the apo-
protein value indicating a very weak binding compound.
Introducing an iso-propyl group (32) raises the Tm of
the ligand/kinase complex by nearly eight degrees or
nearly 900-fold in binding affinity compared to 31 and
is similar in binding potency to 1. Increasing the size
of the group at C-5 to a tert-butyl group (33) provides
an even higher Tm and hence stronger binding affinity.

p38R MAP Kinase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4679
Sch em e 3a
a
Reagents: (a) 1-bromo-2-chloroethane, K2CO3; (b) NaI, acetone; (c) thiomorpholine, DIPEA; (d) NaIO4, ethanol; (e) TFA; (f) 24, DMSO.
Ta ble 1. Substitution at C-5 of Pyrazole
Sch em e 4a
a
Reagents: (a) DEADC, PPh3; (b) HCl; (c) 29, COCl2; NaHCO3,
then 28.
However, the larger cyclohexyl analogue (34) loses three
degrees of thermal stability with p38R or nearly 10-fold
of binding affinity compared to 33 and may reflect a size
limitation in the protein for 33 vs 34. Introducing a
hydroxy group in this portion of the inhibitor (i.e., 11)
substantially reduces binding affinity consistent with
the lipophilic character of this domain in p38R.
a
b
Calculated from the equations in ref 30. Calculated from Tm
) 3.08(log Ka) + 31.2.
stable. The tolyl group (5) provides the best thermal
stability when complexed with p38R.
Substitution at N-2 of the pyrazole nucleus in the
N-pyrazole-N′-phenyl urea class of p38R inhibitors also
has an important role in defining biological activity. The
phenyl ring at N-2 of 5 interacts in a lipophilic π-CH2
mode with the alkyl side chain of Glu71 and may also
act as a water shield for the extensive hydrogen bond
network of the urea atoms with the protein (Figure 1b).
To further probe this particular region of the inhibitor,
a series of derivatives of 5 were prepared. As seen in
Table 2, replacing the phenyl ring in 33 with either
methyl (35), cyclohexyl (36), or benzyl (37) results in a
decrease of several degrees of thermal stability of the
ligand/kinase complex. Small polar substituents at-
tached to a phenyl ring (anilines 38 and 40) and
substituted pyridines (17 and 42) were generally toler-
ated compared to 33. However, acylation of anilines 38
and 40 produces compounds (39 and 41) whose enzyme
complexes are between 3 and 7 degrees less thermally
We previously demonstrated that attaching an ethoxy-
morpholine group to the naphthalene of 4, to furnish 5,
greatly improves binding affinity by forming a hydrogen
bond between the morpholine oxygen and the ATP-
binding domain of p38R (Figure 1a,b).27 Table 3 shows
the results of examining other hydrogen bond accepting
pharmacophores attached through an ethoxy linker to
the 4-position of the naphthalene that access the ATP-
binding domain. Support for the importance of this
hydrogen bond is seen by the substantial decrease in
thermal stability with piperidine (43), piperazine (44),
and thiomorpholine (47) replacements for morpholine.
Small lipophilic groups adjacent to the morpholine
oxygen can affect the Tm values. For example, cis-
dimethyl analogue 45 loses several degrees of thermal
stability compared to 5, indicating a potential steric
interaction with the protein backbone that is not

4680 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Ta ble 2. Substitution at N-2 of Pyrazole
Regan et al.
Con clu sion
We have described the SAR of 5, a potent inhibitor of
p38 that has advanced into human clinical trials. The
thermal stability of the ligand:kinase complex provided
the basis for determining the binding affinity for this
class of compounds. The tert-butyl group of 5 is a critical
binding element occupying a lipophilic domain in the
kinase. An aromatic ring attached to N-2 of the pyrazole
nucleus provides the best π-CH2 interactions with the
alkyl portion of the side chain of Glu71. Of those
examined, a tolyl group provides the optimum binding
affinity. We elucidated the role of a pharmacophore
attached through an ethoxy group to the 4-position of
the naphthalene and directed into the ATP-binding
domain. Groups lacking hydrogen bond potential do not
achieve any significant protein interactions. However,
morpholine, pyridine, and imidazole are effective hy-
drogen bond acceptors for this class of compounds and
facilitate very tight binding with the kinase. Finally,
several of the compounds potently inhibit TNF-R pro-
duction when dosed orally in an LPS-stimulated mouse.
comp no.
R
Tm (°C)
calc Kd (nM)
5
4-CH3-phenyl
2-CH3-pyridin-5-yl
phenyl
63.5
61.3
61.0
53.4
54.5
54.8
60.9
55.9
61.4
53.4
59.1
0.046 ( 0.025a
0.18 ( 0.03b
0.23 ( 0.11a
64 ( 10b
17
33
37
35
36
38
39
40
41
42
benzyl
methyl
26 ( 30a
cyclohexyl
23 ( 4b
3-NH2-phenyl
3-NHAc-phenyl
4-NH2-phenyl
4-NHAc-phenyl
2-CH3O-pyridin-5-yl
0.23 ( 0.04b
9.9 ( 1.6b
0.16 ( 0.03b
64 ( 10b
0.87 ( 0.14b
a
b
Calculated from the equations in ref 30. Calculated from Tm
) 3.08(log Ka) + 31.2.
observed with trans-dimethyl compound 46. Aromatic
hydrogen bond accepting moieties, such as 4-pyridine
(30) and imidazole (50), provide Tm values with p38R
comparable to morpholine (5). The location of the
hydrogen-accepting atom in the ring is also important.
Positional pyridine isomer 48 is 2.5 degrees less ther-
mally stable when complexed with p38R than 30 indi-
cating an erosion of the strength of the hydrogen bond.
Replacement of morpholine with other hydrogen bond
accepting pharmacophores,35 such as thiomorpholine
oxide and dimethoxyphenyl, produces analogues 25 and
49 with significantly lower thermal stability compared
to 5. These results, when taken together, emphasize the
requirement for a hydrogen bond accepting pharma-
cophore to achieve tight binding with this class of
compounds. Groups such as piperazine, piperidine, and
thiomorpholine, do not participate in any protein inter-
actions judging by their similar Tm values compared to
4. However, analogues with good hydrogen bond accept-
ing pharmacophores, such as morpholine (5), pyridine
(30), and imidazole (50), achieve very strong binding
affinities with p38R with Tm values between 62 and 63
°C, which translate into Kd values of 50-100 pM.
Selected compounds were further profiled for their
ability to inhibit TNF-R production in LPS-stimulated
THP-1 cells, human whole blood (HWB) and when orally
dosed in mice. The results are shown in Table 4. The
compounds inhibit TNF-R release in THP-1 cells with
EC50 values ranging from 10 to 65 nM and in HWB with
EC50 values of 440-3400 nM. The physicochemical
properties of this class of inhibitors can affect the
relative potency in the cellular assays. For example, 17
shows similar cellular inhibition compared to 5, despite
weaker binding affinity. The lower lipophilicity of 17
(log P ) 3.6) vs 5 (log P ) 4.6) likely reduces nonspecific
lipophilic binding interactions with cellular and plasma
proteins. In addition, THP-1 cellular potency for com-
pounds with strong binding affinities (i.e., 5, 30, and
50) may be underrepresented due to the potential limits
of this assay. Most of the compounds significantly
inhibit TNF-R production when administered orally to
mice at 30 mg/kg with the more lipophilic compounds
achieving high plasma concentrations. However, the less
lipophilic compounds (i.e., 17, 38, 40, and 50) tended
toward lower plasma levels compared to 5.
Exp er im en ta l Section
All solvents and reagents were obtained from commercial
sources and used without further purification unless indicated
otherwise. Melting points were obtained from a Mel-temp 3.0
or Fisher-J ohns melting point apparatus and are uncorrected.
1H NMR spectra were recorded on either a Bruker AC-F-270
spectrometer or Bruker Avance DPX 400 spectrometer. Chemi-
cal shifts are reported in parts per million (δ) from the
tetramethylsilane resonance in the indicated solvent. Mass
spectra were obtained from a Finnigan-SSQ7000 spectrometer.
Samples were generally introduced by particle beam and
ionized with NH4Cl. TLC analytical separations were con-
ducted with E. Merck silica gel F-254 plates of 0.25 mm
thickness and were visualized with UV or I2. Flash chromatog-
raphies were performed according to the procedure of Still et
al. (EM Science Kieselgel 60, 70-230 mesh). Elemental
analyses were performed at Quantitative Technologies, Inc.,
Whitehouse, NJ .
1-[5-(2-Hyd r oxy-1,1-d im eth yleth yl)-2-p-tolyl-2H-p yr a -
zol-3-yl]-3-[4-(2-m or p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]-
u r ea (11). A solution of methyl-2,2-dimethyl-3-hydroxypropi-
onate 6 (4.22 g, 31.9 mmol), imidazole (3.26 g; 47.8 mmol), and
tert-butyldimethylchlorosilane (5.77 g, 38.3 mmol) in anhy-
drous DMF (60 mL) was stirred under an inert atmosphere
for 12 h, quenched with water (200 mL), and extracted three
times with ether. The combined organic extracts were washed
with water and brine and dried (MgSO4). Removal of volatiles
in vacuo afforded 8.25 g of 7 as colorless oil (quantitative yield)
which was used without further purification.
To a suspension of NaH (60% in mineral oil, 0.55 g, 13.7
mmol) in anhydrous toluene (17 mL) at reflux was added
dropwise over 1 h a solution of ester 7 (2.44 g, 9.90 mmol) and
acetonitrile (0.72 mL, 13.9 mmol) in toluene (8 mL). The
mixture was heated at reflux for 5 h, cooled to ambient
temperature, and diluted with aqueous HCl to pH ∼ 4, and
extracted with ethyl acetate. The combined organic extracts
were washed with brine and dried (Na2SO4). Removal of the
volatiles in vacuo provided pale yellow oil 8 (1.10 g) which was
used without further purification.
A mixture of 8 (0.50 g, 3.5 mmol) and p-tolylhydrazine
hydrochloride (0.56 g, 3.5 mmol) in ethanol (20 mL) was
refluxed for 18 h, cooled to room temperature, and diluted with
saturated aqueous NaHCO3 solution and ethyl acetate. The
organic layer was washed with brine and dried (Na2SO4).
Removal of the volatiles in vacuo provided 9 as an orange oil
(0.69 g, 79%) which used without further purification.
To a mixture of 10 (0.26 g, 0.98 mmol) in CH2Cl2 (15 mL)
and saturated aqueous NaHCO3 solution (15 mL) at 0 °C was

p38R MAP Kinase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4681
Ta ble 3. Morpholine Analogues of Compound 5
a
b
Calculated from the equations in ref 30. Calculated from Tm ) 3.08(log Ka) + 31.2.
Ta ble 4. In Vitro and In Vivo Activity of Selected Inhibitors
inhibition (%)
of TNF-R in
LPS-stimulated
mice (n ) 6) at
plasma levels
(ng/mL)
90 min
post dose at
inhib. of TNF-R in
human whole blood
EC50 (nM)
inhib. of TNF-R in
THP-1 cells EC50 (nM)
comp. no. Tm (°C)
calc Kd (nM)
30 mg/kg 10 mg/kg 30 mg/kg 10 mg/kg
5
63.5
61.3
60.2
62.4
61.0
60.9
61.4
59.1
62.1
62.4
0.046 ( 0.025a
0.18 ( 0.03b
0.40 ( 0.06b
0.10 ( 0.05a
0.23 ( 0.11a
0.23 ( 0.04b
0.16 ( 0.03b
0.87 ( 0.14b
0.096 ( 0.016b
0.074 ( 0.012b
18
19
50
20
9
11
9
42
16
36
780
84
87
84
88
81
c
63
86
c
75000
54000
62800
30000
38000
c
30000
13000
17
25
30
33
38
40
42
46
50
530
1300
820
3400
1,600
440
c
57
NSd
NS
c
NS
c
5400
1400
NDe
c
c
>10000
40
50
81
80000
25000
7000
770
4200
1100
a
b
d
Calculated from the equations in ref 30. Calculated from Tm ) 3.08(log Ka) + 31.2. c Not determined. Not statistically significant.
e Not detected.
added phosgene (20% in toluene, 1.5 mL, 2.9 mmol). The
sodium (4.71 g, 0.20 mol) was slowly warmed to 90 °C, stirred
2 h, heated to 120 °C for 30 min and cooled to room
temperature. Toluene (200 mL) and 2-chloro-5-nitropyridine
(12, 25.0 g, 0.16 mmol) were added, and the slurry was heated
at 110 °C for 1.5 h, cooled to room temperature, and stirred
for 17 h. The volatiles were removed in vacuo, 6 N HCl (200
mL) was added, and the slurry heated to reflux for 4 h, cooled
to room temperature, and neutralized with solid sodium
carbonate. The mixture was extracted with ethyl acetate (6 ×
100 mL) and the combined organic extracts were dried
(MgSO4). Removal of the volatiles in vacuo provided a solid
which was purified by a plug of flash silica gel (2 × 2 in.) using
20% ethyl acetate in petroleum ether as the eluent. Concen-
tration in vacuo of the product-rich fractions afforded 2-methyl-
5-nitropyridine (13) as a tan solid (15.2 g, 70%). 1H NMR (400
MHz, CDCl3) δ 9.34 (s, 1H), 8.41 (m, 1H), 7.37 (m, 1H), 2.73
(s, 3H).
mixture was stirred 20 min and the organic layer dried (Na2-
SO4). Most of the volatiles were removed in vacuo and 9 (0.24
g, 0.98 mmol) in anhydrous THF (7 mL) was added. The
mixture was stirred at room temperature for 2 h. Removal of
the volatiles in vacuo provided a residue which was purified
by flash silica gel chromatography using 5% methanol in CH2-
Cl2 as the eluent. Concentration in vacuo of the product-rich
fractions provided a tan foam which was recrystallized from
1
ether and hexanes to afford 30 mg of 11. H NMR (400 MHz,
DMSO-d6): δ 1.21 (s, 6 H, gem-di-Me), 2.40 (s, 3 H, p-Me),
2.54-2.57 (br m; 4 H), 2.84-2.87 (m, 2 H), 3.43 (d, 2 H, J )
5.6 Hz, CH2-O), 3.58-3.61 (m, 4H), 4.25-4.28 (m, 2 H), 4.58
(t, 1 H, J ) 5.6 Hz, OH), 6.35 (s, 1 H, pyrazole), 6.96 (d, 1 H,
J ) 8.5 Hz, aromatic), 7.36 (d, 2 H, J ) 8 Hz, aromatic), 7.44
(d, 2 H, J ) 8 Hz, aromatic), 7.52-7.62 (m, 3 H, aromatic),
7.88 (d, 1 H, J ) 8 Hz, aromatic), 8.18 (d, 1 H, J ) 9 Hz,
aromatic), 8.57 (s, 1 H, urea), 8.74 (s, 1 H, urea); MS (ES+)
m/e 544 (MH+); Anal. (C31H37N5O4) C, H, N.
A mixture of 13 (13.0 g, 94.1 mmol) and 10% Pd/C (0.10 g)
in 1,4-dioxane (150 mL) was hydrogenated at 50 psi using a
Parr apparatus for 24 h and filtered over Celite. Removal of
the volatiles in vacuo afforded 5-amino-2-methylpyridine (9.90
g, 97%) as a tan solid. This material was dissolved immediately
1-[5-ter t-Bu tyl-2-(6-m eth yl-p yr id in -3-yl)-2H-p yr a zol-3-
yl]-3-[4-(2-m or p h olin -4-yl-e t h oxy)-n a p h t h a le n -1-yl]-
u r ea (17). A slurry of diethyl malonate (42 mL, 0.28 mol) and

4682 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Regan et al.
in 6 N HCl (100 mL), cooled to 0 °C, and vigorously stirred.
Sodium nitrite (6.32 g, 91.6 mmol) in water (50 mL) was added.
After 30 min, tin (II) chloride dihydrate (52.0 g, 230 mmol) in
6 N HCl (100 mL) was added, and the slurry was stirred at 0
°C for 3 h. The mixture was made basic with 40% aqueous
KOH solution to pH 14 and extracted with ethyl acetate (6 ×
250 mL). The combined organic extracts were dried (MgSO4)
and removal of the volatiles in vacuo afforded 5-hydrazino-2-
methylpyridine (14) as a tan solid (8.0 g, 71%). A solution of
14 (8.0 g, 65.0 mmol) and 4,4-dimethyl-3-oxopentanenitrile (15,
10.0 g, 79.9 mmol) in ethanol (200 mL) containing 6 N HCl (6
mL) was refluxed for 17 h, cooled to room temperature,
neutralized with NaHCO3, and filtered. Removal of the vola-
tiles in vacuo from the filtrate provided a residue which was
purified with flash silica gel chromatography using ethyl
acetate as the eluent. Concentration in vacuo of the product-
rich fractions afforded 5-amino-3-tert-butyl-1-(2-methylpyri-
yl)carbamic acid tert-butyl ester (21). 1H NMR (400 MHz,
CDCl3) δ 8.29 (d, 1H), 7.92 (d, 1H), 7.7-7.5 (m, 3H), 6.84 (d,
1H), 6.60 (bs, 1H), 4.41 (m, 2H), 3.08 (m, 6H), 2.78 (m, 4H),
1.58 (s, 9H).
To a 0-5 °C mixture of 21 (0.73 g, 1.87 mmol) in ethanol
(50 mL) was added NaIO4 (0.42 g, 1.95 mmol). The mixture
was slowly warmed to room temperature, stirred overnight,
and diluted with ethyl acetate and water. The organic layer
was washed with water and brine and dried (MgSO4). Removal
of the volatiles in vacuo provided a residue which was purified
by flash silica gel chromatography using 20% methanol in ethyl
acetate as the eluent. Concentration in vacuo of the product-
rich fractions provided 0.45 g (60%) of (4-(2-(1-oxo-thiomor-
pholin-4-yl)ethoxy)naphthalen-1-yl)carbamic acid tert-butyl
ester (22). 1H NMR (400 MHz, CDCl3): δ 8.28 (d, 1H), 7.92 (d,
1H), 7.7-7.5 (m, 3H), 6.82 (d, 1H), 6.55 (bs, 1H), 4.33 (m, 2H),
3.35 (m, 2H), 3.12 (M, 2H), 2.95 (m, 6H), 1.58 (s, 9H).
A mixture of 22 (0.45 g, 1.1 mmol) and trifluoroacetic acid
(0.63 g, 5.5 mmol) in CH2Cl2 (5 mL) was stirred at room
temperature overnight and quenched with K2CO3 (0.76 g, 5.5
mmol). The mixture was filtered, and the volatiles were
removed in vacuo to provide 1-amino-(4-(2-(1-oxo-thiomorpho-
lin-4-yl)ethoxy)naphthalene (23) which was used without
further purification.
1
dine-5-yl)pyrazole (16) as a tan solid (9.21 g, 62%). H NMR
(400 MHz, CDCl3) δ 8.51 (s, 1H), 8.17 (m, 1H), 7.71 (s, 1H),
6.39 (s, 1H), 2.93 (s, 2 H), 2.57 (s, 3H), 1.37 (s, 9H).
To a 0 °C solution of 10 (0.40 g, 1.47 mmol) in CH2Cl2 (20
mL) and saturated aqueous NaHCO3 (20 mL) was added
phosgene (1.93 M in toluene, 1.50 mL). The mixture was
stirred 15 min and the organic layer was dried (MgSO4), and
most of the volatiles were removed in vacuo. A solution of 16
(0.30 g, 1.30 mmol) in CH2Cl2 (10 mL) was added, and the
mixture was stirred for 17 h at ambient temperature. Removal
of the volatiles in vacuo provided a residue that was purified
by flash silica gel chromatography using ethyl acetate then
10% methanol in ethyl acetate as the eluents. Concentration
of the product-rich fractions afforded a colorless solid which
was recrystallized from warm tetrahydrofuran/petroleum ether
A mixture of 23 (0.08 g, 0.26 mmol) and [5-tert-butyl-2-(4-
methylphenyl)-2H-pyrazol-3-yl]-carbamic acid phenyl ester
(prepared from 29 and phenyl chloroformate in pyridine and
THF) (24, 0.14 g, 0.39 mmol) in anhydrous DMSO (1 mL) was
heated at 40 °C overnight, cooled to room temperature, and
diluted with ethyl acetate and water. The organic layer was
washed with 2 N NaOH, water, and brine and dried (MgSO4).
Removal of the volatiles in vacuo provided a residue which
was purified by flash silica gel chromatography using 20%
methanol in ethyl acetate as the eluent. Concentration in vacuo
of the product-rich fractions provided 0.05 g (34%) 25. mp
1
to give 17 as a colorless solid (0.65 g, 95%). mp 147-9°C. H
NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 8.25 (m, 1H), 7.78 (s,
1H), 7.52 (m, 3H), 7.37 (d, J ) 8.2 Hz, 1H), 7.05 (m, 3H), 6.70
(d, J ) 8.2 Hz, 1H), 6.45 (s, 1H), 4.28 (t, J ) 5.6 Hz, 2H), 3.78
(m, 4H), 2.99 (t, J ) 5.6 Hz, 2H), 2.70 (m, 4H), 2.54 (s, 3H),
1.34 (s, 9H). Anal. (C30H36N6O3) C, H, N.
1
182-4 °C. H NMR (400 MHz, CDCl3): δ 8.28 (m, 1H), 7.88
(m 1H), 7.55 (m, 1H), 7.41 (d, 1H), 7.12 (m, 2H), 7.02 (m, 2H),
6.79 (bs, 1H), 6.68 (d, 1H), 6.62 (bs, 1H), 6.36 (s, 1H), 4.28
(m,2H), 3.32 (m, 2H), 3.08 (m 2H), 2.90 (m, 6H), 2.33 (s, 3H),
1.36 (s, 9H). MS (CI): m/e 560 (MH+). Anal. (C31H37N5O3S*0.5
H2O); C, H, N.
1-[5-t er t -Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl]-3-[4-(2-(1-
oxoth iom or ph olin -4-yl)eth oxy)n aph th alen -1-yl]-u r ea (25).
A suspension of (4-hydroxy-naphthalen-1-yl)carbamic acid tert-
butyl ester (18, 7.0 g, 27 mmol), powdered K2CO3 (18.6 g, 139
mmol) and 1-bromo-2-chloroethane (7.74 g, 54 mmol) in
acetonitrile (120 mL) was heated at 80 °C overnight, cooled to
room temperature, and partitioned between ethyl acetate and
water. The organic layer was washed with brine, dried
(MgSO4), and the volatiles removed in vacuo. Purification of
the residue by flash chromatography using 25% ethyl acetate
in hexanes as the eluent and concentration of the product-
rich fractions in vacuo provided a solid which was triturated
with 25% ethyl acetate in hexanes to afford 3.30 g (38%) of
(4-(2-chloroethoxy)naphthalen-1-yl)carbamic acid tert-butyl
ester (19). 1H NMR (400 MHz, CDCl3): δ 8.38 (d, 1H), 7.88 (d,
1H), 7.7-7.5 (m, 3H), 6.83 (d, 1H), 6.62 (bs, 1H), 4.42 (m, 2H),
4.01 (m, 2H), 1.58 (s, 9H). MS (CI): m/e 322 (ΜΗ+).
A mixture of 19 (3.30 g, 10.2 mmol) and NaI (15 g, 102
mmol) in anhydrous acetone (30 mL) was heated at reflux for
64 h, cooled to room temperature, and diluted with CH2Cl2
and water. The organic layer was washed with water and brine
and dried (Na2SO4). Removal of the volatiles in vacuo provided
4.03 g (94%) of (4-(2-iodoethoxy)naphthalen-1-yl)carbamic acid
tert-butyl ester (20). 1H NMR (400 MHz, CDCl3): δ 8.41 (d,
1H), 7.93 (d, 1H), 7.7-7.5 (m, 3H), 6.81 (d, 1H), 6.55 (bs, 1H),
4.43 (m, 2H), 3.62 (m, 2H), 1.59 (s, 9H).
1-(5-ter t-Bu tyl-2-p-tolyl-2H-pyr azol-3-yl)-3-[4-(2-pyr idin -
4-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (30). To a solution of
18 (0.509 g, 1.96 mmol), triphenylphosphine (1.52 g, 5.81
mmol) and 4-(2-hydroxyethyl)pyridine (26, 0.710 g, 5.77 mmol)
in THF (10 mL) was added dropwise diethyl azodicarboxylate
(DEADC) (0.90 mL, 5.72 mmol). The mixture was stirred
overnight, and the volatiles were removed in vacuo. Purifica-
tion of the residue by flash silica gel chromatography using
ethyl acetate as the eluent and concentration in vacuo of the
product-rich fractions provided [4-(2-pyridin-4-yl-ethoxy)-
naphthalen-1-yl]-carbamic acid tert-butyl ester (27) contami-
nated with triphenylphosphine oxide. 1H NMR (400 MHz,
CDCl3): δ 8.58 (m, 2H), 8.20 (d, 1H), 7.90 (d, 1H), 7.7-7.4 (m,
3H), 7.35 (d,1H), 7.21 (s, 1H), 6.84 (d, 1H), 6.72 (bs, 1H), 4.43
(t, 2H), 3.29 (t, 2H), 1.57 (s, 9H). MS (EI): m/e 365 (MH+).
A solution of 27 (0.32 g) and HCl (5 mL of a 4.0 M solution
in dioxane) in THF (10 mL) was stirred 3 h. The solid was
filtered and dried in vacuo to provide 0.30 g (99%) of solid 4-(2-
pyridin-4-yl-ethoxy)-naphthalen-1-ylamine dihydrochloride (28).
MS (EI): m/e 265 (MH+).
To a 0-5 °C mixture of 1-(4-methylphenyl)-3-(1,1-dimeth-
ylethyl)-5-aminopyrazole (29, 0.202 g, 0.88 mmol) in 15 mL of
CH2Cl2 and 15 mL of saturated NaHCO3 was added phosgene
(0.5 mL of a 1.9 M solution in toluene; 0.97 mmol). The mixture
was stirred rapidly for 15 min and the organic layer dried (Na2-
SO4). Removal of most of the volatiles in vacuo provided a
residue which was added to a solution of 28 (0.280 g, 0.83
mmol) and DIPEA (0.52 mL) in THF (10 mL). The mixture
was stirred overnight and filtered. The filtrate was concen-
trated in vacuo, and the residue was purified by flash silica
gel chromatography using 25% hexanes in ethyl acetate as the
eluent. Concentration in vacuo of the product-rich fraction
provided a solid which was recrystallized with ethyl acetate
A mixture of 20 (1.0 g, 2.4 mmol), thiomorpholine (0.25 g,
2.4 mmol) and N,N-di-iso-propylethylamine (DIPEA) (0.42 mL,
2.4 mmol) in anhydrous DMF (5 mL) was heated at 40 °C
overnight, cooled to room temperature, and diluted with ether
and water. The organic layer was washed with water and brine
and dried (MgSO4). Removal of the volatiles in vacuo provided
a residue which was purified by flash silica gel chromatogra-
phy using 33% hexanes in ethyl acetate as the eluent.
Concentration in vacuo of the product-rich fractions provided
0.73 g (78%) of (4-(2-thiomorpholin-4-yl-ethoxy)naphthalen-1-

p38R MAP Kinase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4683
and methanol to provide 0.075 g (16%) of 30. mp 187-90 °C.
1H NMR (400 MHz, CDCl3): δ 8.53 (d, 2H), 8.13 (d, 1H), 7.82
(d, 1H), 7.48 (m, 4H), 7.36 (d, 1H), 7.13-6.93 (m, 5H), 6.82
(bs, 1H), 6.68 (d, 1H), 6.45 (s, 1H), 4.42 (t, 2H), 3.25 (t, 2H),
2.24 (s, 3H), 1.35 (s, 9H). MS (EI): m/e 520 (MH+). Anal.
(C32H33N5O2) C, H, N.
ethyl acetate and water. The organic layer was washed with
brine and dried. Removal of the volatiles in vacuo provided a
solid which was triturated with hot 10% ethyl acetate and
petroleum ether (80 mL). The solid was filtered and dried to
furnish 2.5 g (67%) of [5-tert-butyl-2-(3-nitro-phenyl)-2H-
1
pyrazol-3-yl]-carbamic acid phenyl ester. H NMR (270 MHz,
DMSO-d6) δ 8.38 (s, 1H), 8.24 (d, 1H), 8.10 (d, 1H), 7.85 (t,
1H), 7.40 (m, 2H), 7.25-7.1 (m, 4H), 6.48 (s, 1H), 1.36 (s, 9H).
A solution of [5-tert-butyl-2-(3-nitro-phenyl)-2H-pyrazol-3-
yl]-carbamic acid phenyl ester (2.49 g; 6.5 mmol), 10 (1.43 g;
5.2 mmol) and DIPEA (2.28 mL; 13.1 mmol) in anhydrous
DMSO (55 mL) was stirred at room temperature for 2 days
and diluted with ethyl acetate (10 mL) and brine (150 mL).
The solid was filtered and recrystallized with ethyl acetate,
THF and methanol to afford 1.87 g (64%) of 1-[2-(3-nitrophe-
nyl)-5-tert-butyl-2H-pyrazol-3-yl]-3-[4-(2-morpholin-4-yl-ethoxy)-
naphthalen-1-yl]-urea. mp 200-2 °C. 1H NMR (270 MHz,
DMSO-d6) δ 8.80 (m, 1H), 8.46 (s, 1H), 8.3-8.1 (m, 4H), 7.85
(m, 2H), 7.52 (m, 3H), 6.95 (d, 1H), 6.42 (s, 1H), 4.28 (m, 2H),
3.60 (m, 4H), 2.84 (m, 2H), 2.55 (m, 4H), 1.33 (s, 9H).
1-[4-(2-Mor p h olin -4-yl-et h oxy)-n a p h t h a len -1-yl]-3-(2-
p h en yl-2H-p yr a zol-3-yl)-u r ea (31). mp 173-4 °C; 1H NMR
(400 MHz, DMSO-d6): δ 2.54-2.56 (br m; 2 H), 2.84-2.87 (m,
2 H), 3.58-3.61 (m, 4H), 4.25-4.28 (m, 2 H), 6.46 (d, 1 H, J )
2 Hz), 6.96 (d, 1 H, J ) 8 Hz, aromatic), 7.46-7.49 (m, 1 H,
aromatic), 7.50-7.60 (m, 8 H, aromatic), 7.88 (d, 1 H, J ) 8
Hz, aromatic), 8.18 (d, 1 H, J ) 8 Hz, aromatic), 8.67 (s, 1 H,
urea), 8.76 (s, 1 H, urea); MS (NH3-CI) m/e 458 (MH+); Anal.
(C26H27N5O3*0.1C3H8O) C, H, N.
1-(5-iso-P r op yl-2-p h en yl-2H-p yr a zol-3-yl)-3-[4-(2-m or -
p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (32). mp 177-8
°C. 1H NMR (400 MHz, DMSO-d6): δ 1.23 (d, 6 H), 2.54-2.56
(br m; 4 H), 2.84-2.86 (m, 2 H), 2.87-2.94 (m, 1 H), 3.58-
3.61 (m, 4H), 4.25-4.28 (m, 2 H), 6.34 (s, 1 H, pyrazole), 6.96
(d, 1 H, J ) 8 Hz, aromatic), 7.42-7.47 (m, 1 H, aromatic),
7.52-7.61 (m, 7 H, aromatic), 7.88 (d, 1 H, J ) 8 Hz, aromatic),
8.18 (d, 1 H, J ) 8 Hz, aromatic), 8.64 (s, 1 H, urea), 8.77 (s,
1 H, urea); MS (PB-NH3CI) m/e 500 (MH+); Anal. (C29H33N5O3)
C, H, N.
To a refluxing solution of 1-[2-(3-nitrophenyl)-5-tert-butyl-
2H-pyrazol-3-yl]-3-[4-(2-morpholin-4-yl-ethoxy)-naphthalen-1-
yl]-urea (0.33 g) in ethanol (10 mL) was added 10% Pd on
carbon (0.33 g) and ammonium formate (0.23 g). The mixture
was heated for 1.5 h, cooled to room temperature and filtered
over Celite. The volatiles were removed in vacuo to provide
1-(5-ter t-Bu t yl-2-p h en yl-2H -p yr a zol-3-yl)-3-[4-(2-m or -
p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (33). mp 107-
10 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.28 (s, 9 H, tert-Bu),
2.54-2.56 (br m; 4 H), 2.84-2.87 (m, 2 H), 3.58-3.60 (m, 4H),
4.25-4.28 (m, 2 H), 6.38 (s, 1 H, pyrazole), 6.97 (d, 1 H, J ) 8
Hz, aromatic), 7.41-7.47 (m, 1 H, aromatic), 7.52-7.61 (m, 7
H, aromatic), 7.89 (d, 1 H, J ) 8 Hz, aromatic), 8.17 (d, 1 H,
J ) 9 Hz, aromatic), 8.63 (s, 1 H, urea), 8.77 (s, 1 H, urea);
MS (PB-NH3CI) m/e 514 (MH+); Anal. (C30H35N5O3) C, H, N.
1-[5-(1-Meth ylcycloh exyl)-2-p h en yl-2H-p yr a zol-3-yl]-3-
[4-(2-m or p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (34).
1
0.31 g (100%) of 38. H NMR (270 MHz, DMSO-d6) δ 8.86 (s,
1H), 8.62 (s, 1H), 8.19 (d, 1H), 7.93 (d, 1H), 7.66 (d, 1H), 7.55
(m, 2H), 7.18 (t, 1H), 6.97 (d, 1H), 6.73 (s, 1H), 6.64 (m, 2H),
6.35 (s, 1H), 5.44 (bs, 2H), 4.27 (t, 2H), 3.60 (m, 4H), 2.86 (m,
2H), 2.55 (m, 4H), 1.27 (s, 9H).
N-[3-(3-ter t-Bu t yl-5-{3-[4-(2-m or p h olin -4-yl-et h oxy)-
n a p h t h a le n -1-yl]-u r e id o}-p yr a zol-1-yl)-p h e n yl]a ce t a -
m id e (39). To a solution of acetyl chloride (0.25 µL, 0.3 mmol)
in THF (5 mL) was added a solution of 38 (0.15 g; 0.3 mmol)
and pyridine (28 µL, 0.3 mmol) in THF (2 mL). The mixture
was stirred overnight and diluted with ethyl acetate and water.
The organic layer was washed with brine and dried. Removal
of the volatiles in vacuo provided a residue which was purified
by flash silica gel chromatography using ethyl acetate as the
eluent. Concentration in vacuo of the product-rich fractions
provided 0.089 g (54%) of 39. 1H NMR (270 MHz, DMSO-d6) δ
10.2 (s, 1H), 8.79 (s, 1H), 8.69 (s, 1H), 8.20 (d, 1H), 7.93 (d,
1H), 7.81 (s, 1H), 7.65-7.48 (m, 4H), 7.45 (t, 1H), 7.23 (d, 1H),
6.97 (d, 1H), 6.36 (s, 1H), 4.28 (t, 2H), 3.60 (m, 4H), 2.85 (m,
2H), 2.56 (m, 4H), 2.08 (s, 3H), 1.28 (s, 9H). MS (CI) m/e 571
(MH+).
1-[2-(4-Am in op h en yl)-5-ter t-b u t yl-2H -p yr a zol-3-yl]-3-
[4-(2-m or p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]u r ea (40).
Prepared in a manner similar to 38. 1H NMR (270 MHz,
DMSO-d6) δ 8.83 (s, 1H), 8.45 (s, 1H), 8.19 (d, 1H), 7.90 (d,
1H), 7.65-7.4 (m, 5H), 7.13 (d, 1H), 6.96 (d, 1H), 6.70 (d, 1H),
6.30 (s, 1H), 5.41 (m, 2H), 4.28 (m, 2H), 3.61 (m, 4H), 2.86 (m
2H), 2.55 (m, 4H), 1.28 (s, 9H).
N-[4-(3-ter t-Bu t yl-5-{3-[4-(2-m or p h olin -4-yl-et h oxy)-
n a p h t h a le n -1-yl]-u r e id o}-p yr a zol-1-yl)-p h e n yl]a ce t a -
m id e (41). Prepared in a manner similar to 39. 1H NMR (270
MHz, DMSO-d6) δ 10.1 (s, 1H), 8.79 (s, 1H), 8.59 (s, 1H), 8.18
(d, 1H), 7.90 (d, 1H), 7.75 (s, 1H), 7.61-7.5 (m, 5H), 7.47 (t,
1H), 6.96 (d, 1H), 6.35 (s, 1H), 4.28 (t, 2H), 3.59 (m, 4H), 2.85
(m, 2H), 2.56 (m, 4H), 2.09 (s, 3H), 1.27 (s, 9H). MS (CI) m/e
571 (MH+).
1-[5-ter t-Bu tyl-2-(6-m eth oxyp yr id in -3-yl)-2H-p yr a zol-
3-yl]-3-[4-(2-m or p h olin -4-yl-et h oxy)-n a p h t h a len -1-yl]-
u r ea (42). Prepared in a manner similar to 17. mp 108-110
°C. 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 8.21 (m, 1H),
7.75 (s, 1H), 7.58 (m, 4H), 7.41 (d, J ) 8.1 Hz, 1H), 7.08 (d, J
) 8.1 Hz, 2H), 6.76 (d, J ) 8.1 Hz, 1H), 6.49 (s, 1H), 4.25 (t, J
) 5.6 Hz, 2H), 3.97 (s, 3H), 3.75 (m, 4H), 2.94 (t, J ) 5.6 Hz,
2H), 2.67 (m, 4H), 1.37 (s, 9H). Anal. (C30H36N6O4) C, H, N.
1-(5-ter t-Bu tyl-2-p-tolyl-2H-p yr a zol-3-yl)-3-[4-(2-p ip er i-
d in -1-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (43). A solution of
4-amino-1-naphthol (3.12 g, 16.0 mmol), di-tert-butyl dicar-
bonate (3.49 g, 16.0 mmol), triethylamine (6.7 mL, 48.0 mmol)
1
mp 147-149 °C. H NMR (400 MHz, DMSO-d6): δ 1.20 (s, 3
H), 1.44-1.50 (br m, 8 H), 1.98-2.00 (m, 2 H), 2.54-2.56 (br
m; 4 H), 2.84-2.87 (m, 2 H), 3.59-3.61 (m, 4H), 4.25-4.28
(m, 2 H), 6.36 (s, 1 H, pyrazole), 6.96 (d, 1 H, J ) 8 Hz,
aromatic), 7.42-7.45 (m, 1 H, aromatic), 7.52-7.61 (m, 7 H,
aromatic), 7.90 (d, 1 H, J ) 8 Hz, aromatic), 8.18 (d, 1 H, J )
8 Hz, aromatic), 8.62 (s, 1 H, urea), 8.76 (s, 1 H, urea); MS
(ES+) m/e 554 (MH+); Anal. (C33H39N5O3) C, H, N.
1-(5-ter t-Bu t yl-2-m et h yl-2H -p yr a zol-3-yl)-3-[4-(2-m or -
p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (35). mp 201-2
°C. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.89 (s, 1H),
8.21 (m, 2H), 7.99 (m, 1H), 7.75 (m, 2H), 7.67 (m, 1H), 6.11 (s,
1H), 3.67 (s, 3H), 3.46 (m, 4H), 2.51 (m, 2H), 2.39 (m, 4H),
1.72 (m, 2H), 1.45 (s, 9H). Anal. (C25H33N5O3) C, H, N.
1-(5-ter t-Bu t yl-2-cycloh exyl-2H -p yr a zol-3-yl)-3-[4-(2-
m or p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (36). mp
1
216-7 °C. H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.21
(m, 3H), 7.99 (m, 1H), 7.75 (m, 2H), 7.67 (m, 1H), 6.25 (s, 1H),
3.61 (m, 1H), 3.49 (m, 4H), 2.47 (m, 2H), 2.41 (m, 4H), 2.25-
1.72 (m, 12H), 1.45 (s, 9H). Anal. (C30H41N5O3) C, H, N.
1-(2-Ben zyl-5-ter t-b u t yl-2H -p yr a zol-3-yl)-3-[4-(2-m or -
ph olin -4-yl-eth oxy)-n aph th alen -1-yl]-u r ea (37). Mp: 180-1
°C;1H NMR (400 MHz, DMSO-d6): δ 1.23 (s, 9 H, tert-Bu),
2.54-2.57 (br m; 4 H), 2.84-2.87 (m, 2 H), 3.59-3.61 (m, 4H),
4.26-4.29 (m, 2 H), 5.24 (s, 1 H, pyrazole), 6.97 (d, 1 H, J ) 8
Hz, aromatic), 7.11 (d, 2 H, J ) 7 Hz, aromatic), 7.28 (d, 1 H,
J ) 7 Hz, aromatic), 7.36 (t, 2 H, J ) 2 Hz, aromatic), 7.52-
7.63 (m, 3 H, aromatic), 7.92 (d, 1 H, J ) 8 Hz, aromatic),
8.19 (d, 1 H, J ) 8 Hz, aromatic), 8.57 (s, 1 H, urea), 8.68 (s,
1 H, urea); MS (NH3-CI) m/e 528 (MH+); Anal. (C31H37N5O3)
C, H, N.
1-[2-(3-Am in op h en yl)-5-ter t-b u t yl-2H -p yr a zol-3-yl]-3-
[4-(2-m or p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]u r ea (38).
To a 0 °C solution of phenyl chloroformate (1.6 mL; 12 mmol)
in THF (30 mL) was added dropwise a solution of 2-(3-
nitrophenyl)-5-tert-butyl-2H-pyrazol-3-yl-amine (2.5 g; 9.6 mmol)
and pyridine (1.1 mL; 13 mmol) in THF (10 mL). The mixture
was stirred at room temperature overnight and diluted with

4684 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Regan et al.
and DMAP (0.3 g) in anhydrous THF (50 mL) was stirred at
room temperature for 2 days and diluted with ethyl acetate
and water. The organic layer was washed with saturated
NaHCO3 and brine and dried (MgSO4). Removal of the volatiles
in vacuo provided a residue which was purified by flash silica
gel chromatography using 33% ethyl acetate in hexanes as the
eluent. Concentration in vacuo of the product-rich fractions
provided 2.55 g (58%) of carbonic acid 4-amino-naphthalen-
1-yl ester tert-butyl ester. MS (CI): m/e 260 (MH+)
2.39 (s, 3H), 2.22 (m, 2H), 1.28 (s, 9H), 1.12 (d, 6H). MS (CI):
m/e 556 (ΜΗ+). Anal. (C33H41N5O3*H2O); C, H, N.
1-(5-ter t-Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-[4-(2-t h io-
m or p h olin -4-yl-eth oxy)-n a p h th a len -1-yl]u r ea (47). Pre-
1
pared in a manner similar to 25. mp 122-4 °C. H NMR (400
MHz, DMSO-d6): δ 8.75 (s, 1H), 8.53 (s, 1H), 8.18 (d, 1H), 7.88
(d, 1H), 7.6-7.3 (m, 7H), 6.95 (d, 1H), 6.35 (s, 1H), 4.24 (m
2H), 2.92 (m, 2H), 2.82 (m, 4H), 2.62 (m, 4H), 2.41 (s, 3H),
1.29 (s, 9H). MS (CI): m/e 544 (ΜΗ+). Anal. (C31H37N5O2S);
C, H, N.
To a 0-5 °C mixture of 29 (0.746 g, 3.24 mmol) in 60 mL of
CH2Cl2 and 60 mL of saturated NaHCO3 was added phosgene
(6.7 mL of a 1.9 M solution in toluene; 12.9 mmol). The mixture
was stirred rapidly for 15 min, and the organic layer was dried
(MgSO4). Removal of most of the volatiles in vacuo provided a
residue which was added to a solution of carbonic acid 4-amino-
naphthalen-1-yl ester tert-butyl ester (0.811 g, 2.95 mmol) in
25 mL THF. The mixture was stirred overnight and the
volatiles were removed in vacuo. The residue was purified by
flash silica gel chromatography using 25% ethyl acetate in
hexanes as the eluent. Concentration in vacuo of the product-
rich fractions provided 1.37 g (66%) of carbonic acid tert-butyl
ester 4-[3-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-ureido]-naph-
thalen-1-yl ester. MS (CI): m/e 515 (MH+), 415 (MH-BOC).
A solution of carbonic acid tert-butyl ester 4-[3-(5-tert-butyl-
2-p-tolyl-2H-pyrazol-3-yl)-ureido]-naphthalen-1-yl ester (0.56
g) in 7 mL of CH2Cl2 and 2 mL of trifluoroacetic acid (TFA)
was stirred at room-temperature overnight and the volatiles
removed in vacuo. The residue was diluted with ethyl acetate
and washed with saturated NaHCO3 and brine and dried
(MgSO4). Removal of the volatiles in vacuo provided 1-(5-tert-
butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-(4-hydroxy-naphthalen-1-yl)-
urea which was used without further purification. MS (CI):
m/e 415 (MH+).
1-(5-ter t-bu tyl-2-p-tolyl-2H-pyr azol-3-yl)-3-[4-(2-pyr idin -
3-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (48). Prepared in
a
manner similar to 30. mp 166-8 °C. 1H NMR (400 MHz,
CDCl3): δ 8.72 (s, 1H), 8.56 (d, 1H), 8.26 (d, 1H), 7.85 (d, 1H),
7.74 (d, 1H), 7.53 (m, 2H), 7.33 (m, 3H), 6.95-6.80 (m, 4H),
6.53 (bs, 1H), 6.42 (m, 2H), 4.38 (t, 2H), 3.32 (t, 2H), 2.23 (s,
3H), 1.33 (s, 9H). MS (EI): m/e 520 (MH+). Anal. (C32H33N5O2)
C, H, N.
1-(5-ter t-Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-{4-[2-(3,4-
dim eth oxyph en yl)-eth oxy]-n aph th alen -1-yl}u r ea (49). Pre-
1
pared in a manner similar to 30. mp 196-7 °C. H NMR (400
MHz, DMSO-d6): δ 8.75 (s, 1H), 8.58 (s, 1H), 8.20 (d, 1H), 7.88
(d, 1H), 7.65-7.35 (m, 7H), 7.03 (s, 1H), 6.95 (d, 1H), 6.88 (s,
2H), 6.35 (s, 1H), 4.32 (t, 2H), 3.73 (s, 3H), 3.70 (s, 3H), 3.12
(t, 2H), 2.39 (s, 3H), 1.28 (s, 9H). MS (CI): m/e 579 (ΜΗ+).
Anal. (C35H38N4O4*0.5H2O); C, H, N.
1-(5-ter t-bu tyl-2-p-tolyl-2H-p yr a zol-3-yl)-3-[4-(2-im id a -
zol-1-yl-eth oxy)-n a p h th a len -1-yl]-u r ea (50). To a solution
of 18 (0.341 g, 1.24 mmol), triphenylphosphine (0.975 g, 3.72
mmol) and 1-(2-hydroxyethyl)imidazole (0.417 g, 3.72 mmol)
in THF (6 mL) was added dropwise DEADC (0.59 mL, 3.72
mmol). The mixture was stirred overnight and the volatiles
were removed in vacuo. Purification of the residue with flash
silica gel chromatography using 9% methanol in ethyl acetate
as the eluent and concentration in vacuo of the product-rich
fractions provided a solid. Trituration of the solid with ether
and drying in vacuo provided 0.291 g (64%) of [4-(2-imidazol-
1-yl-ethoxy)-naphthalen-1-yl]-carbamic acid tert-butyl ester. 1H
NMR (270 MHz, CDCl3): δ 8.20 (d, 1H), 7.88 (d, 1H), 7.72-
7.5 (m, 6H), 7.13 (bs,1H), 6.74 (d, 1H), 4.50 (d, 2H), 4.42 (m,
2H), 1.54 (s, 9H). MS (EI): m/e 354 (MH+).
A solution of [4-(2-imidazol-1-yl-ethoxy)-naphthalen-1-yl]-
carbamic acid tert-butyl ester (0.283 g) in CH2Cl2 (6 mL) and
TFA (1.5 mL) was stirred at room temperature overnight.
Removal of the volatiles in vacuo provided a residue which
was diluted with ethyl acetate, washed with saturated NaH-
CO3 and brine, and dried (MgSO4). Removal of the volatiles
in vacuo provided 0.182 g (94%) of the solid 4-(2-imidazol-1-
yl-ethoxy)-naphthalen-1-ylamine which was used without
further purification.
A mixture of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-(4-
hydroxy-naphthalen-1-yl)-urea (0.123 g, 0.297 mmol), 1-(2-
chloroethyl)piperdine hydrochloride (0.060 g, 0/327 mmol),
NaOH (0.98 mL of a 1 M solution, 0.98 mmol) and N-benzyl-
tri-butylammonium chloride (0.03 mmol) in 3 mL of water and
2 mL of CH2Cl2 was heated at 50 °C for 8 h, cooled to room
temperature, diluted with ethyl acetate, washed with water
and brine. and dried (MgSO4). Removal of the volatiles in vacuo
provided a residue which was purified by flash silica gel
chromatography using 23% methanol in ethyl acetate as the
eluent. Concentration in vacuo of the product-rich fractions
provided 0.055 g (35%) of 43. mp 96-98 °C. 1H NMR (270 MHz,
CDCl3): δ 8.25 (dd, 1H), 7.79 (m, 1H), 7.44 (m, 2H), 7.25 (m,
1H), 7.0-6.5 (m, 7H), 6.40 (s, 1H), 4.25 (m, 2H), 2.95 (m, 2H),
2.64 (m, 4H), 2.27 (s, 3H), 1.68 (m, 4H), 1.47 (m, 2H), 1.28 (s,
9H). MS (EI): m/e 526 (MH+). Anal. (C32H39N5O2) C, H; N:
calcd, 13.32; found, 12.66.
To a 0-5 °C mixture of 29 (0.178 g; 0.778 mmol) in 7 mL of
CH2Cl2 and 7 mL of saturated NaHCO3 was added phosgene
(1.2 mL of a 1.9 M solution in toluene; 2.33 mmol). The mixture
was stirred rapidly for 20 min, and the organic layer was dried
(MgSO4). Removal of most of the volatiles in vacuo provided a
residue which was added to a solution of 4-(2-imidazol-1-yl-
ethoxy)-naphthalen-1-ylamine (0.179 g; 0.708 mmol) in 5 mL
THF. The mixture was stirred overnight and diluted with
ether. The solid was filtered and recrystallized from ethyl
1-(5-ter t-Bu tyl-2-p-tolyl-2H-p yr a zol-3-yl)-3-[4-(2-p ip er -
a zin -1-yl-eth oxy)-n a p h th a len -1-yl]u r ea Dih yd r och lor id e
(44). Prepared in a manner similar to 43. mp 190 °C (dec). 1H
NMR (400 MHz, DMSO-d6): δ 9.64 (bs, 2H), 9.07 (s, 1H), 8.89
(s, 1H), 8.32 (d, 1H), 8.02 (d, 1H), 7.68 (d, 1H), 7.56 (m, 2H),
7.45 (d, 2H), 7.34 (d, 2H), 7.00 (d, 2H), 6.38 (s, 1H), 4.60 (bs,
2H), 2.08 (s, 3H), 1.27 (s, 9H). MS (CI): m/e 527 (ΜΗ+). Anal.
(C33H41N5O3*2HCl*2H2O); C; H: calcd, 6.98; found, 6.49; N:
calcd, 13.22; found,12.73.
1
acetate to provide 0.10 g (28%) of 50. mp 201-2 °C. H NMR
1-(5-ter t-Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-{4-[2-((2S,-
6R )-2,6-d im et h ylm or p h olin -4-yl)-et h oxy]n a p h t h a len -1-
yl}u r ea (45). Prepared in a manner similar to 25. mp 153-4
°C. 1H NMR (400 MHz, DMSO-d6): δ 8.79 (s, 1H), 8.58 (s, 1H),
8.15 (d, 1H), 7.88 (d, 1H), 7.65-7.35 (m, 7H), 6.96 (d, 1H), 6.33
(s, 1H), 4.23 (m 2H), 3.58 (m, 2H), 2.85 (m, 4H), 2.37 (s, 3H),
1.80 (m, 2H), 1.29 (s, 9H), 1.05 (d, 6H). MS (CI): m/e 556
(ΜΗ+). Anal. (C33H41N5O3); C, H, N.
1-(5-ter t-Bu t yl-2-p -t olyl-2H -p yr a zol-3-yl)-3-{4-[2-((2S,-
6S)-2,6-d im et h ylm or p h olin -4-yl)-et h oxy]n a p h t h a len -1-
yl}u r ea (46). Prepared in a manner similar to 25. mp 137-9
°C. 1H NMR (400 MHz, DMSO-d6): δ 8.78 (s, 1H), 8.57 (s, 1H),
8.18 (d, 1H), 7.88 (d, 1H), 7.65-7.35 (m, 7H), 6.95 (d, 1H), 6.36
(s, 1H), 4.28 (m 2H), 3.92 (m, 2H), 2.79 (m, 2H), 2.58 (m 2H),
(270 MHz, CDCl3): δ 7.90 (d, 1H), 7.66 (s, 1H), 7.5-6.95 (m,
12H), 6.57 (d, 1H), 6.46 (s, 1H), 4.45 (m, 2H), 4.30 (m, 2H),
2.25 (s, 3H), 1.38 (s, 9H). MS (EI): m/e 507 (MH+). Anal.
(C30H32N6O2) C, H, N.
Biologica l Meth od s. 1. Th er m a l Den a tu r a tion Assa y.
The details of the thermal denaturation experiments is found
in the preceding article in this issue.30 Briefly, the UV thermal
melt experiments were carried out using a Perkin-Elmer
Lambda 40 spectrophotometer. For each measurement, a
quartz cuvette was loaded with 2.5 µM murine p38R MAP
kinase and 25 µM inhibitor in a pH 7.0 buffer containing 10
mM sodium phosphate, 100 mM NaCl, and 1mM TCEP.
Absorbance data at 230 nm was collected as the temperature
was scanned from 25 to 80 °C at a ramp rate of 0.2 °C/min.

p38R MAP Kinase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4685
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Weisman, M.; Smolen, J .; Emery, P.; Harriman, G.; Feldmann,
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Infliximab for the treatment of fistulas in patients with Crohn’s
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The melting temperature for each sample was calculated as
the maximum deflection point of the first derivative of the
melting transition using the Perkin-Elmer Templab software
(version 1.62). Generally, the reported Tm values have an
average standard error of ( 0.5 °C and are the average of at
least two experiments. The Tm values are converted into Kd
values using the equation Tm ) 3.08(log Ka) + 31.2, where Ka
) 1/Kd, unless otherwise noted. The error associated with Kd
calculations using this equation is ( 16%. A further description
of error propagation in this assay has been described.30
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recombinant human interleukin-1 receptor antagonist. BioDrugs
2001, 15, 87-97.
Procedures for the THP-1 cellular assay for inhibition of
LPS-stimulated TNF-R production, data analysis, and the in
vivo LPS challenge assay have been described.27 Briefly, THP-1
cells were preincubated in the presence and absence of test
compound for 30 min, 37 °C, 5% CO2. Cell mixture was
stimulated with LPS (Sigma L-2630, 1 µg/mL final) and
incubation continued overnight (18-24 h) as above. Superna-
tant was analyzed for human TNF-R by a commercially
available ELISA (Biosource KHC3012). Data from ng2 assays
were combined and analyzed by nonlinear regression using a
three parameter logistic model to obtain an EC50 value.
Compound 5 was analyzed in each experiment and the 95%
confidence intervals for the EC50 were between 16 and 22 nM.
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a review of its use in
rheumatoid arthritis. Drugs 1999, 57, 945-966.
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patients with rheumatoid arthritis. Ann. Rheum. Dis. 1999, 58
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Burge, D. J . Etanercept in the treatment of psoriatic arthritis
and psoriasis: a randomised trial. Lancet 2000, 356, 385-390.
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R.; Landvatter, S. W.; Strickler, J . E.; McLaughlin, M. M.;
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pathways. Cell. Mol. Life Sci. 1999, 55, 1230-1254.
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and function. Cell. Signalling 2000, 12, 1-13.
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Fox, T.; Su, M. S. Inhibitors of p38 MAP kinase: therapeutic
intervention in cytokine-mediated diseases. Curr. Med. Chem.
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2. Hu m a n Wh ole Blood TNF -r (HWB) Assa y. This assay,
performed in 96-well polypropylene plates, measures the
ability of test compounds to inhibit the elaboration of TNF-R
by human whole blood following stimulation ex vivo with LPS
(Escherichia coli lipopolysaccharide 0111:B4). Each test well
contained the following components: 15 µL of compound
serially diluted in human type AB serum, 135 µL of freshly
drawn heparinized human whole blood, and 15 µL of LPS
diluted in PBS containing 10% human serum for a final
concentration of 50 ng LPS/mL. The plates were incubated for
30 min at 37 °C prior to addition of LPS. Control wells on every
plate contained no test compound, with and without LPS.
Following addition of LPS to test and control wells, the plates
were placed on a rotary shaker for 30 s and then incubated
for 5 h at 37 °C. To harvest plasma, 100 µL of PBS was added
to all wells, the plates centrifuged to pellet cells, and the
diluted plasma removed for TNF-R quantitation by standard
ELISA, DELFIA, or electrochemiluminescence assay methods.
TNF-R in each test well was determined by signal interpolation
from a TNF-R standard curve run on the same plate. Test
compound EC50 values were determined by nonlinear curve
fitting of the data from cross-plate duplicate 11-point concen-
tration-response curves to a 4-parameter logistic equation.
Composite EC50 values were the geometric mean of EC50 values
determined in blood from at least three donors. The EC50 of a
standard inhibitor was determined on 239 independent test
days across 113 different blood donors and returned lower and
upper one-standard deviation values that were 29 and 37% of
the geometric mean EC50 value.
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(27) Regan, J .; Breitfelder, S.; Cirillo, P.; Gilmore, T.; Graham, A.
G.; Hickey, E. R.; Klaus, B.; Madwed, J .; Moriak, M.; Moss, N.;
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Ackn owledgm en t. The authors thank Steffen Breit-
felder for synthetic methodology development, Dorothy
Freeman for thermal denaturation measurements and
Peter Kinkade for sample concentration measurements
in the cellular assay. The Drug Metabolism and Phar-
macokinetics Department is thanked for determining
plasma concentrations.
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