Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase

Article abstract of DOI:10.1016/S0960-894X(02)00752-7

The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38α MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38α inhibitor reported by Merck Research Laboratories and VX-745. O

Full text of DOI:10.1016/S0960-894X(02)00752-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 277–280
Design and Synthesis of Potent, Orally Bioavailable
Dihydroquinazolinone Inhibitors of p38 MAP Kinase
John E. Stelmach,a,* Luping Liu,a Sangita B. Patel,a James V. Pivnichny,a
Giovanna Scapin,a Suresh Singh,a Cornelis E. C. A. Hop,b Zhen Wang,b
John R. Strauss,c Patricia M. Cameron,d Elizabeth A. Nichols,d Stephen J. O’Keefe,d
Edward A. O’Neill,d Dennis M. Schmatz,d Cheryl D. Schwartz,d Chris M. Thompson,d
Dennis M. Zallerd and James B. Dohertya
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Comparative Medicine, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
dDepartment of Inflammation/Rheumatology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 30 July 2002; accepted 22 August 2002
Abstract—The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38a MAP
kinase is described. These analogues are hybrids of a pyridinylimidazole p38a inhibitor reported by Merck Research Laboratories
and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excel-
lent suppression of TNF-a production in LPS-stimulated whole blood (IC50=10 nM) and good oral exposure in rats (F=68%,
AUCn PO=0.58 mM h).
# 2002 Elsevier Science Ltd. All rights reserved.
The biological agents infliximab and etanercept are
TNF-a sequestrants and are effective for the treatment
of rheumatoid arthritis (RA).1 Inhibitors of p38a MAP
kinase suppress the production of proinflammatory
cytokines TNF-a and IL1-b and have excellent potential
for the treatment of inflammatory disorders.2 Many of
the published p38a inhibitors possess a vicinal aryl/4-
pyridinyl-heterocycle arrangement exemplified by the
pyridinylimidazole SB-203580 and compound 1.3 Crys-
tal structures of p38-inhibitor complex have shown that
this class of compounds utilizes two key binding inter-
actions.4 The pyridine nitrogen forms a hydrogen bond
with the main chain N–H of Met 109 and the aryl sub-
stituent penetrates into a hydrophobic pocket not
accessed by ATP. Recently, a new class of inhibitors has
emerged that utilize similar interactions to bind to p38a
but which possess a carbonyl hydrogen bond acceptor
instead of pyridine nitrogen.3 Of particular interest
Vertex has reported the development of VX-745, which
was recently in phase II clinical trials for the treatment
of RA.5 VX-745 was reported to be more than 1000-fold
selective for p38a over the kinases ERK2, JNK1, JNK3,
lck, Src, and MAPKAP-2.6 Selectivity for p38a over
other MAP kinases has been an issue with the
pyridinylimidazole inhibitors. This new class of
p38a inhibitors is of interest because of its inherent
selectivity, novel structure, unique binding and potential
for improved pharmacology. We have described the
*Corresponding author. Fax: +1-732-594-8080; e-mail: john_stelmach@
merck.com
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00752-7

278
J. E. Stelmach et al. / Bioorg. Med. Chem. Lett. 13 (2003) 277–280
development of compound 2 which is a reduced-iso-
meric analogue of VX-745.7 In addition, we have shown
that polar amine substituents at C-7 (e.g., 3) improves
the potency and physical properties.
refluxing in TFA and the C-5 phenyl substituents were
attached by Suzuki coupling.
In the pyridinylimidazole series of p38 inhibitors aryl
substituents bearing small alkyl or halide groups were
optimal for the p38 hydrophobic pocket. Molecular
modeling suggested that the C-5 phenyl substituent in
compound 9 penetrates deeper into the p38a hydro-
phobic pocket than the 3-trifluoromethylphenyl sub-
stituent in compound 1. The p38a inhibition of
compounds 9 and 10 bearing a series of C-5 phenyl
substituents is shown in Table 1. Consistent with our
p38a binding model, compounds 9a and 9b, which lack
the critical hydrophobic substituent, were inactive. In
contrast, compound 9c which has a C-5 phenyl sub-
stituent was very potent against p38a. Substitution with
a fluoride or chloride at the 3-position resulted in a
2-fold decrease in potency (9e and 9h, respectively).
Substitution at the 3-position with larger substituents
gave an even greater attenuation in potency. Fluoride,
The rationale for the design of hybrid compound 3 was
based on molecular modeling and by analogy to the
potent pyridinylimidazole 1 reported by Merck.8 This
analysis also suggested that the p38a hydrophobic
pocket, which is accessed by the C-6 thioether in 2,
could also be accessed by a C-5 phenyl substituent. We
report in this paper the preparation, p38a inhibitory
activity and rat pharmokinetic (PK) properties of
dihydroquinazolinones 4.
In order to confirm the predicted binding mode for the
dihydroquinazolinone compounds the structure of p38a
in complex with 14e was solved to 2.4 A resolution.9 The
binding orientation of 14e relative to the binding orien-
tation of 1 is shown in Figure. 1. The structure of 1 is
taken from its crystallographic complex with p38a.10
The urea carbonyl oxygen in 14e and the pyrimidine
ring nitrogen in 1 form hydrogen bond(s) interactions
with the backbone of the hinge region. In contrast to 1,
which forms a single hydrogen bond with Met109, 14e
forms three hydrogen bond interactions (Leu107,
Met109 and Gly110). The 2,6-dichlorophenyl ring of
14e superimposes with the phenyl ring of the a-methyl-
benzyl ring of 1. The 3-trifluoromethylphenyl ring in 1,
the critical hydrophobic substituent, overlays quite well
with the 2,4-difluorophenyl ring in 14e.
The preparation of quinazolinones 9 and 10 is sum-
marized in Scheme 1. Urea 7 (R=CO2Me) was pre-
pared in three steps beginning with the benzylic
bromination of methyl 3,5-dibromo-4-methylbenzoate.
Alkylation of the product benzyl bromide with 4-methoxy
benzyl amine gave 6, which was then treated with
2,6-dichloroisocyanate. Initial attempts to cyclize com-
pounds similar to 7 using palladium mediated condi-
tions resulted in extended reaction times and the
isolation of considerable amounts of debrominated
starting material and debrominated cyclized product.11
In contrast, cyclization of urea 7 using conventional
Ullmann coupling conditions cleanly gave compound
8.12 The PMB protecting group was removed by
Scheme 1. Reagents and conditions: (a) NBS, Bz2O2; (b) PMBNH2;
(c) 2,6-diCl-PhNCO; (d) CuI, K2CO3, 150 ꢀC DMF, 15 min; (e) TFA
reflux; (f) ArB(OH)2, Pd(PPh3)4.
Table 1. p38a inhibition of 9 and 10
Compd
Ar
p38a
IC50 (nM)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
Br
H
Phenyl
2-Fl-Phenyl
3-Fl-Phenyl
4-Fl-Phenyl
2-Cl-Phenyl
3-Cl-Phenyl
4-Cl-Phenyl
0% @ 1000 nM
0% @ 1000 nM
30
7
79
22
1
67
47
50% @ 890 nM
2-CF3-Phenyl
3-CF3-Phenyl
4-CF3-Phenyl
2-CH3-Phenyl
3-CH3-Phenyl
4-CH3-Phenyl
2,4-di Fl-Phenyl
2,6-di Fl-Phenyl
2-CH3-4-Fl-Phenyl
-S-2,4-di Fl-Phenyl
2-Cl-4-Fl-Phenyl
2,4-di Fl-Phenyl
2-Cl-Phenyl
170
130
11
320
44
7
44
11
48% @ 1000
9s
10a
10b
10c
3
11
6
Figure 1. Relative binding orientations of 1 (yellow) and 14e (green).

J. E. Stelmach et al. / Bioorg. Med. Chem. Lett. 13 (2003) 277–280
279
chloride and methyl substituents at the 2-position were
optimal. Compound 9g, which possesses a 2-chloro-
phenyl substituent, was 10-fold more potent than the
corresponding unsubstituted phenyl. A fluoride at the
4-position gave a slight increase in potency. Other
groups at the 4-position attenuated potency. Compound
9s, which has the VX-745 hydrophobic substituent (i.e.,
2,4-difluorothiophenyl ether), had modest activity.13
While we were satisfied with the achieved p38a enzyme
inhibition we found that none of the compounds in
Table 1 gave satisfactory inhibition of TNF-a produc-
tion in LPS-stimulated monocyte or THP-1 cells. For
example, at 2 mM concentration, 9g (IC50=1 nM p38a)
inhibited the production of TNF-a in monocytes by a
modest 33%. We attributed the poor cellular activity to
the low aqueous solubility, high lipophilicity and
anticipated high protein binding of these compounds.
cates that the C-7 position is the optimal attachment
point for a piperidinyl-like group. The preparation of
analogues possessing a directly attached 4-piperidine is
summarized in Scheme 2. The C-7 methyl ether of 10
was cleaved with boron tribromide and phenol product
11 converted to the triflate 12. Stille coupling of 12 with
the vinyl tin reagent 13 gave the desired coupled
product. The trisubstituted double bond was hydro-
genated and the BOC group removed by treatment with
acid to give analogues 14a–c.
In addition, analogues containing a C-7 piperidine or
piperazine moiety directly attached to the dihydro-
quinazolinone or separated by one atom were also pre-
pared (see Table 2). Analogues 14d, e, h, and i were
prepared by Buchwald–Hartwig coupling with triflate
12 and the appropriate amine. Analogues 14f and 14g
were prepared by Mitsunobu coupling with phenol 11.
Compounds 14j–14n were derived from 9 by reductive
amination or by carbodiimide mediated carboxylic acid/
amine coupling. Piperidine and piperazine C-7 sub-
stituents were well tolerated in the p38a enzyme assay.
In most cases sub-nanomolar inhibition of p38a was
observed. More importantly, these compounds are
excellent inhibitors of TNF-a production in LPS-stimu-
lated monocytes and THP-1 cells. Consistent with the
anticipated selectivity of these p38a inhibitors, com-
pound 14b had insignificant activity against ERK1,
JNK3, FYN, JAK1, JAK2, JAK3, lck, lyn, tyk2, and
src kinases (i.e., IC50 >10 mM).
The piperidinyl moiety of compound 1 confers aqueous
solubility and increased p38a inhibition. The increased
potency is presumed to arise from a salt bridge interac-
tion between the protonated piperidine nitrogen and
Asp168. This is a binding interaction not attained in
VX-745. The superposition of 1 and 14e (Fig. 1) indi-
We evaluated the rat PK properties of these promising
inhibitors (Table 3). Unfortunately, this series of com-
pounds had poor rat PK properties characterized by
high clearance and low oral bioavailability. Only com-
pound 14d had a modest PK profile. HPLC/MS/MS
analysis of 14c incubated with rat liver microsomes
indicated extensive oxidation of the piperidine moiety.
We reasoned that the metabolism, whether occurring by
electron transfer from the piperidine nitrogen or by a
hydrogen atom abstraction proximal to the nitrogen,
would be suppressed by bulky nitrogen substituents.
Scheme 2. Reagents and conditions: (a) BBr3; (b) PhN(Tf)2; (c)
Pd(PPh3)4, LiCl, 13; (d) H2, PtO2; (e) TFA.
Table 2. p38a inhibition of 14
Analogues of 14c containing alkyl substituents on the
piperidine nitrogen were investigated (Table 4). With
the exception of 15i, all compounds were prepared by
standard alkylation or reductive amination of 14c with
the appropriate alkyl halide or aldehyde.14 Piperidine
nitrogen alkyl substituents were well tolerated in the
p38a enzyme assay. These analogues also had excellent
inhibition of TNF-a production in LPS-stimulated
Compd
Ar
X
Y
p38a
IC50 (nM)
TNF-a Cell
IC50 (nM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
2-Cl-Phenyl
2,4-di Fl-Phenyl
2-Cl-4-Fl-Phenyl
2-Cl-Phenyl
2,4-di Fl-Phenyl
2-Cl-Phenyl
2-Cl-4-Fl-Phenyl
2-Cl-Phenyl
2,4-di Fl-Phenyl
2-Cl-Phenyl
—
—
—
—
—
O
O
NH
NH
CH2
CH
CH
CH
N
0.9
0.2
0.1
1.4
2.6
0.2
0.1
0.5
0.6
0.1
0.13
1.5
2.4
1.1
2.0a
1.3a
1.0a
2.9a
7.8a
1.6a
0.7b
4.4b
4.5a
1.4a
0.7a
11.4a
26.8a
3.8a
N
Table 3. Rat pharmokinetic data for compound 14
CH
CH
CH
CH
N
N
N
N
N
Compd
IV
t1/2 (h)
Vd
(L/kg)
Clp
(mL/min/kg)
AUCn PO
(mM h)
F
(%)
14a
14b
14c
14d
14f
2.5
3.3
2.0
1.6
3.0
2.8
2.3
21.4
13.6
7.8
181.0
58.6
72.3
0
0.02
0
0.22
0.02
0.04
0
0
4.3
0
22.7
6.3
15.7
1.5
2-Cl-4-Fl-Phenyl CH2
2-Cl-Phenyl
2,4-di Fl-Phenyl
2-Cl-4-Fl-Phenyl
CO
CO
CO
4.3
34.4
21.0
18.6
12.8
115.5
139.5
94.7
14k
14m
aMonocyte cells.
bTHP-1 cells.

280
J. E. Stelmach et al. / Bioorg. Med. Chem. Lett. 13 (2003) 277–280
Table 4. p38a inhibition and rat pharmokinetic data for compound 15
Compd
R
p38a
IC50 (nM)
TNF-a cell
IC50 (nM)
TNF-a WB
IC50 (nM)
IV
t1/2 (h)
Vd
(L/kg)
Clp
(mL/min/kg)
AUCn PO
(mM h)
F
(%)
15a
15b
15c
15d
15e
15f
15g
15h
15i
Methyl
Ethyl
i-Propyl
0.5
1.2
0.6
1.1
0.5
0.3
0.4
0.6
0.2
1.3a
0.7b
0.2b
16.6a
0.3b
—
—
4.0
5.6
—
7.6
7.0
12.2
27.1
10.1
1.5
2.2
1.9
2.5
1.9
2.0
1.7
1.6
2.2
5.3
7.5
12.2
8.1
6.3
4.7
6.6
2.5
6.0
49.8
56.3
84.8
51.7
50.9
31.1
53.7
24.2
35.2
0.06
0.11
0.06
0.39
0.15
0.36
0.14
0.14
0.58
9.2
20.0
16.0
59.3
22.6
38.0
24.7
11.6
67.7
Cyclopropyl
Methyl Cyclopropyl
Ethyl 1-Cyclopropyl
Cyclobutyl
Methyl Cyclobutyl
t-butyl
0.5b
—
0.6b
aMonocyte cells.
bTHP-1 cells.
5. (a) Salituro, F. G. 11th RSC-SCI Medicinal Chemistry
Symposium, Churchill College, Cambridge, UK, Sept 9–12,
2001. (b) Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Cochran,
J. E.; Harrington, E. M.; Murcko, M. A.; Wilson, K. P.; Su,
M.; Galullo, V. P. Intl. Patent WO 98/27098, 1998.
6. Salituro, F. G. 27th National Medicinal Chemistry Sym-
posium, Kansas City, MO, June 13–17, 2000.
7. Natarajan, S. R.; Wisnoski, D. D.; Singh, S. B.; Stelmach,
J. E.; O’Neill, E. A.; Schwartz, C. D.; Thompson, C. M.;
Fitzgerald, C. E.; O’Keefe, S. J.; Kumar, S.; Hop, C. E. C. A;
Zaller, D. M.; Schmatz, D.M.; Doherty, J. B. Biorg. Med.
Chem. Lett. 2003, 13, 273
8. Liverton, N. J.; Butcher, J. W.; Claiborne, D. A.; Libby,
B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Selnick, H. G.;
Smith, G. R.; Tebben, A.; Vacca, J. P.; Varga, S. L.; Agarwal,
L.; Dancheck, K.; Forsyth, A. J.; Fletcher, D. S.; Frantz, B.;
Hanlon, W. A.; Harper, C. F.; Hofsess, S. J.; Kostura, M.;
Lin, J.; Luell, S.; O’Neill, E. A.; Orevillo, C. J.; Pang, M.;
Parsons, J.; Rolando, A.; Sahly, Y.; Visco, D. M.; O’Keefe,
S. J. J. Med. Chem. 1999, 42, 2180.
monocytes, THP-1 cells and human whole blood (WB).
Small N-alkyl substituents (i.e., methyl, ethyl, i-propyl,
15a–c) gave a modest improvement in rat PK.15 In
contrast, a bulky N-t-butyl substituent (15i) markedly
improved the rat PK (F=68%).
In summary, we have described the design and optimi-
zation of dihydroquinazolinone p38a inhibitors, which
can be considered hybrids of the pyridinylimidazole 1
and VX-745. Dihydroquinazolinone analogues posses-
sing a C-7 piperidine or piperazine moiety effectively
suppressed the production of TNF-a in monocyte cells,
THP-1 cells and whole blood. These analogues had high
clearance and low oral bioavailability in rats. However,
substitution of the piperidine nitrogen with a bulky
t-butyl substituent dramatically improved the clearance
and oral exposure in rats.
9. Human non activated p38a was purified and crystallized as
described by Wilson, K. P.; Fitzgibbon, M. J.; Caron, P. R.;
Griffith, J. P.; Chen, W.; McCaffrey, P. G.; Chambers, S. P.;
Su, M. J. Biol. Chem. 1996, 271, 27696. The complex was
obtained by soaking an apo crystal in 200 mM compound 14e
for 6 h. Additional details including coordinates can be found
at the Protein Data Bank, accession code 1M7Q.
References and Notes
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14. Analogue 15i was prepared via the Stille coupling of 12c
with the corresponding N-t-butyl analogue of 13.
15. HPLC/MS/MS analysis of 15c incubated with rat hepato-
cytes indicated oxidative N-dealkylation of the piperidine and
oxidation/conjugation of the C-5 phenyl and the C-4 benzylic
carbon proximal to the urea moiety.
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