Synthesis and Structure-Activity Relationship of Aminobenzophenones. A Novel Class of p38 MAP Kinase Inhibitors with High Antiinflammatory Activity

Article abstract of DOI:10.1021/jm030851s

We wish to report the synthesis and structure-activity relationship (SAR) of a series of 4-aminobenzophenones, as a novel compound class with high antiinflammatory activity. Our initial lead, {4-[(2-aminophenyl)amino]phenyl}(phenyl)methanone (3), was systematically optimized and resulted in compounds that potently inhibited the release of the proinflammatory cytokines IL-1β and TNF-α in human peripheral blood mononuclear cells stimulated by LPS. One of the most potent compounds, among others, was {4-[(2-aminophenyl)amino]-2-chlorophenyl}(2-methylphenyl)methanone (45) with IC₅₀ values of 14 and 6 nM for the inhibition of IL-1β and TNF-α, respectively. Furthermore, we found these types of compounds to be potent and selective p38 MAP kinase inhibitors, e.g. 45 had an IC₅₀ value of 10 nM. Molecular modeling was used to rationalize our SAR data and to propose a model for the interaction of compound 45 with the p38 MAP kinase. The model involved a favorable hydrogen bond between the carbonyl group of the benzophenone and the NH of Met-109, positioning ring A in the hydrophobic pocket I of the enzyme. Good antiinflammatory effects were demonstrated in two murine models of dermatitis after topical application (oxazolone and TPA model).

Full text of DOI:10.1021/jm030851s

J . Med. Chem. 2003, 46, 5651-5662
5651
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip of Am in oben zop h en on es. A
Novel Cla ss of p 38 MAP Kin a se In h ibitor s w ith High An tiin fla m m a tor y Activity
Erik Rytter Ottosen,*,† Morten Dahl Sørensen,† Fredrik Bjo¨rkling,† Tine Skak-Nielsen,‡
Marianne Scheel Fjording,‡ Helle Aaes,§ and Lise Binderup‡
Departments of Medicinal Chemistry, Biochemistry, and Pharmacology, LEO Pharma,
Industriparken 55, DK-2750 Ballerup, Denmark
Received March 25, 2003
We wish to report the synthesis and structure-activity relationship (SAR) of a series of
4-aminobenzophenones, as a novel compound class with high antiinflammatory activity. Our
initial lead, {4-[(2-aminophenyl)amino]phenyl}(phenyl)methanone (3), was systematically
optimized and resulted in compounds that potently inhibited the release of the proinflammatory
cytokines IL-1â and TNF-R in human peripheral blood mononuclear cells stimulated by LPS.
One of the most potent compounds, among others, was {4-[(2-aminophenyl)amino]-2-chlorophe-
nyl}(2-methylphenyl)methanone (45) with IC50 values of 14 and 6 nM for the inhibition of IL-
1â and TNF-R, respectively. Furthermore, we found these types of compounds to be potent
and selective p38 MAP kinase inhibitors, e.g. 45 had an IC50 value of 10 nM. Molecular modeling
was used to rationalize our SAR data and to propose a model for the interaction of compound
45 with the p38 MAP kinase. The model involved a favorable hydrogen bond between the
carbonyl group of the benzophenone and the NH of Met-109, positioning ring A in the
hydrophobic pocket I of the enzyme. Good antiinflammatory effects were demonstrated in two
murine models of dermatitis after topical application (oxazolone and TPA model).
In tr od u ction
signaling cascades coupled to the TNF-R and IL-1â
receptors.8,21 Thus, regulation of TNF-R and IL-1â via
inhibition of p38 MAP kinase is considered an attractive
target for antiinflammatory therapy.14,22
It is well-established that the proinflammatory cy-
tokines interleukin-1â (IL-1â) and tumor necrosis fac-
tor-R (TNF-R) play an important role in the patho-
genesis of various inflammatory diseases such as rheu-
matoid arthritis (RA), inflammatory bowel disease,
osteoarthritis, psoriasis, endotoxemia, and/or toxic shock
syndrome.1-9 Under normal conditions, the regulated
synthesis of TNF-R and IL-1â serves as a protection of
the body from infectious agents, tumors, or tissue
damage. But in autoimmune diseases, high production
of TNF-R and IL-1â may have deleterious effects on
various tissues, e.g. severe degradation of cartilage and
bone in RA. Therefore, agents that down-regulate the
release of TNF-R and IL-1â from inflammatory cells and
reestablish a basal level are expected to have beneficial
effects in the treatment of inflammatory diseases.10,11
Several approaches to inhibit the production of TNF-R
and IL-1â have been reported, e.g. the use of soluble
TNF receptors, anti-TNF antibodies, TNF-R converting
enzyme inhibitors, protein tyrosine kinase inhibitors,1,11
and IL-1 receptor antagonists.12,13
The prototypical, low-molecular-weight p38 MAP ki-
nase inhibitor triarylimidazole 1 (SB203580) is known
to reduce levels of TNF-R and IL-1â both in vitro and
in vivo.15,16,23-25 Recently, several reports and reviews
covering new p38 MAP kinase inhibitors have been
published.22,26-32 For example, some of the most ad-
vanced compounds, 2 (VX-745, now discontinued due to
adverse neurological effects), BIRB-796, and SCIO-469,
have entered phase II clinical trial for the treatment of
rheumatoid arthritis.33,34
Our interest in this promising therapeutic principle
was initiated a few years ago by the discovery of a new
structural class of antiinflammatory compounds. Our
starting compound 3 (Figure 1) in this work was found,
in a cell-based screen, to inhibit the release of TNF-R
(IC50 ) 159 nM) and IL-1â (IC50 ) 226 nM) from human
peripheral blood mononuclear cells stimulated by LPS.
An additional new target for the treatment of inflam-
matory disorders is the mitogen-activated protein (MAP)
kinase p38.14-18 The p38 MAP kinase (p38R) is part of
the stress-activated transduction cascade responsible for
cytokine production. Accordingly, p38 MAP kinase is
involved in the lipopolysaccharide (LPS) stimulated
release of TNF-R and IL-1â from monocytes.18-20 Fur-
thermore, p38 MAP kinase is known to regulate the
We now report the structure-activity optimization of
these novel compounds (see the generic formula 4 in
Table 1). Furthermore, it is demonstrated that ana-
logues of this new structural class of antiinflammatory
compounds are specific inhibitors of the p38R and p38â
MAP kinase isoforms. To rationalize our SAR data, a
molecular modeling study was performed and we pro-
pose a possible model of the interaction between our
most active compounds and the p38R MAP kinase
enzyme. In our animal models, the most active com-
pounds effectively inhibited both acute and chronic
dermatological inflammation.
* To whom correspondence should be addressed. Tel: +45 44 92 38
00. Fax: +45 44 94 55 10. E-mail: erik.ottosen@leo-pharma.com.
† Department of Medicinal Chemistry.
‡ Department of Biochemistry.
§ Department of Pharmacology.
10.1021/jm030851s CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/11/2003

5652 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Ottosen et al.
In our hands, the protection step35 of readily available
bromoanilines as the corresponding N,N-bis(trimethyl-
silyl) analogues was unsatisfactory because of low yields
and problems with product stability.
In a second approach, which allowed us to readily
vary the substituents of ring A and B, a Negishi cross-
coupling reaction was used as the key step.37-39 Bromo-
lithium exchange of, for example, the aryl bromide 10
gave the corresponding organolithium compound, which
then was transmetalated with ZnCl2. The resulting
organozinc compound was coupled with, for example,
the acid chloride 13 in the presence of tetrakis(tri-
phenylphosphine)palladium(0), which gave the desired
benzophenone 17. Subsequently, reduction of the ni-
trobenzophenone 17 with stannous chloride dihydrate40
gave the aminobenzophenone 25. The aminobenzophe-
nones 23, 24, 26, and 27 were synthesized using the
same procedure. The yields were in the range 46-86%
for the Negishi reactions and 66-98% for the reductions.
F igu r e 1.
Ta ble 1. Structure Activity Data
A related cross-coupling reaction was used for the
preparation of the aminobenzophenones 22 and 28
(Scheme 2). The organozinc bromide obtained from 5
was readily cross-coupled with 2-bromobenzoyl chloride
(55), as described above to give 22. Similarly, the
organozinc bromide of 2-bromotoluene (10) and 4-bromo-
2-methylbenzoyl chloride yielded the benzophenone 53.
Interestingly, in either case no concomitant biphenyl
side products were observed. This demonstrated that
compared with aryl bromides, aroyl chlorides were
significantly more reactive in this type of reactions.
Palladium-catalyzed amination41 of the 4-bromben-
zophenone derivative 53 with benzophenone imine gave
the imine 54, which was then hydrolyzed under acidic
conditions to yield 28 (Scheme 2).
Coupling42 of 4-aminobenzophenone and the ami-
nobenzophenones 20-28 with 2-fluornitrobenzene (50)
and of the aminobenzophenone 25 with 5-bromo-2-
fluoronitrobenzene (51) in the presence of potassium
tert-butoxide gave the diarylamines 29 and 30-39 in
moderate to good yields (Scheme 1). Reduction of the
nitro group in 29 with hydrazine hydrate in the presence
of a catalytic amount of palladium on carbon gave the
desired compound 3. The analogues 30-39 were re-
duced with stannous chloride dihydrate, which gave the
desired compounds 40-49 (Table 1). Both methods
produced good to excellent yields.
structure
R2
IC50 (nM)a
compd no. R1
R3
IL-1â TNF-R
anal.
3
35
40
41
42
43
44
45
46
47
48
49
56
57
58
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
H
H
2-NH2
226
427
67
159 NDb
382 C,H,N,Cl
47 C,H,N
2-Me 2-Cl 2-NO2
2-Me
3-Me
2-Br
2-Ph
H
H
H
H
H
2-NH2
2-NH2
2-NH2
2-NH2
580
45
483
437
14
467 ND
34 C,cH,N
444 C,cH,N
214 C,H,N,Cl
6 C,H,N,Cl
127 ND
2-Cl 2-NH2
2-Me 2-Cl 2-NH2
2-Me 3-Me 2-NH2
2-OMe 2-Cl 2-NH2
2-Me 2-Me 2-NH2
2-Me 2-Cl 2-NH2, 4-Br
2-Me 2-Cl 2-NHAc
343
28
20 C,H,N,Cl
8 ND
5 C,H,N
22
30
21
9
16
70
119
26
17
10 C,H,N,Cl
3 C,H,N,Cl
6 C,H,N,Cl
44 Cc,H,N,Clc
24 C,H,N,Clc
14 C,H,N,Cl
4 C,H,N,Cl
1349 ND
2-Me 2-Cl 2-NHCONHEt
2-Me 2-Cl 2-NHCOOEt
60 4a 2-Me 2-Cl 2-NH2
62
63
64
65
4
4
4
4
2-Me 2-Cl
H
2-Me 2-Cl 2-Me
2-Me 2-Cl 2-Me, 4-F
H
H
H
175
70 4b 2-Me 2-Cl 2-NH2
71 4c 2-Me 2-Cl 2-Me, 4-F
>5000 >10000 ND
>10000 >10000 ND
n ) 3, SD (mean) ) 63%. Not determined. c Anal. not within
a
b
(0.4%.
Ch em istr y
We developed a general and useful synthesis of
analogues of 3 with a wide variation of substituents in
the three rings (Scheme 1). In short, this procedure
comprised of two coupling reactions, one that gave the
benzophenone and one that gave the diarylamine. We
found the most efficient method in all cases was to first
synthesize the benzophenone part and then the diaryl-
amine part to finalize the molecule.
Substituted 4-aminobenzophenones were in general
prepared by two different routes. In the first, the anion
obtained by a bromo-lithium exchange reaction of the
protected bromoaniline 535 (commercially available,
Aldrich) with n-butyllithium was condensed with the
Weinreb amides36 6 and 7, and the resulting protected
benzophenones were deprotected during the aqueous
workup to the aminobenzophenones 20 and 21. Our
efforts to use this strategy for the introduction of
substituents in ring B were unsuccessful due to prob-
lems with the synthesis of substituted derivatives of 5.
The derivatives 56-58 were synthesized in excellent
yields by standard procedures from 45 by treatment
with acetic anhydride, ethyl isocyanate, or ethyl chlo-
roformate. Deprotonation of the diarylamine 35 with
potassium hydride, alkylation with iodomethane, and
reduction of the nitro group gave the N-methylamino
derivative 60 (Scheme 3).
Preparation of diarylamines by nucleophilc aromatic
substitution of activated fluoroarenes was efficient
(Scheme 1). However, we found that substitution of
unactivated fluoroarenes was sluggish, even at elevated
temperatures, and alternative routes for the synthesis
of this type of compound were sought (Scheme 4).
Palladium-catalyzed amination of bromides proved to
be an efficient method.43 Thus, coupling of aminoben-
zophenone 25 with the appropriate bromide e.g. 9 using
rac-BINAP as the ligand in the presence of either

Synthesis and SAR of Aminobenzophenones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5653
Sch em e 1a
a
Reagents: (a) (i) ArBr, n-BuLi, Et2O, 0 °C; (ii) Ar′CON(OMe)Me, THF, -78 to 20 °C, 24 h; (b) (i) ArBr, n-BuLi, THF, -78 °C, 30 min;
(ii) ZnCl2, -78 °C to rt; (iii) Ar′COCl, Pd(PPh3)4, rt, 18 h; (c) SnCl2‚2H2O, EtOH, 70 °C, 2 h; (d) t-BuOK, DMSO, 18 h; (e) NH2NH2‚H2O,
10% Pd/C, rt, 24 h. bSee Scheme 2.
Sch em e 2a
Sch em e 3a
a
Reagents: (a) Ac2O, glacial acetic acid, rt, 45 min; (b) ethyl
isocyanate, pyridine, rt, 4 h; (c) ethyl chloroformate, K2CO3, DCM,
0 °C, then 27 h at rt; (d) NaH, THF, 15 min, then MeI, reflux, 2
h; (e) SnCl2‚2H2O, EtOH, 70 °C, 2 h.
a
Reagents: (a) (i) ArBr, n-BuLi, THF, -78 °C, 30 min; (ii)
ZnCl2, -78 °C to rt; (iii) Ar′COCl, Pd(PPh3)4, rt, 18 h; (b) Pd2(dba)3,
rac-BINAP, t-BuONa, toluene, 80 °C, 48 h; (c) 2 N HCl(aq), THF,
1 h.
borohydride in trifluoroacetic acid.45 A very low yield
was obtained due to the formation of a byproduct,
identified as compound 72.
cesium carbonate or sodium tert-butoxide gave 62 in
good yield. The analogues 63-65 were prepared simi-
larly.
Resu lts a n d Discu ssion
The modified analogues 70 and 71 were obtained as
described in Scheme 5. Treatment of 25 with acetonyl-
acetone under Dean-Stark conditions gave the pro-
tected derivative 66 in quantitative yield.44 Reaction of
66 with methyllithium led to the intermediary tertiary
alcohol, which eliminated water upon treatment with
acetic acid under reflux and gave the alkene 67. Cleav-
age of the pyrrole was then performed with a large
excess of hydroxylamine hydrochloride and triethyl-
amine. The obtained amine 68 was coupled with 2-bro-
monitrobenzene to give 69, which was reduced to the
desired derivative 70. The reduced analogue 71 was
obtained directly from 64 by reduction with sodium
In Vitr o SAR. Determination of the antiinflamma-
tory activity of our new benzophenone compounds was
made in vitro by measuring the inhibitory effect of
TNF-R and IL-1â release (results summarized in Figure
2). In this primary screen, human peripheral blood
mononuclear cells (PBMC’s) were stimulated with LPS
for release of TNF-R and IL-1â, with or without com-
pound. Our starting compound in this structure-
activity optimization, the aminobenzophenone 3, showed
an IC50 of 159 and 226 nM in TNF-R and IL-1â,
respectively (Table 1). Our first modification, introduc-
tion of simple substituents such as methyl, bromo,
chloro, fluoro, and methoxy (data not shown) in ring A

5654 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Ottosen et al.
Sch em e 4a
a
Reagents: Pd2(dba)3, rac-BINAP, Cs2CO3, 1,4-dioxane, 100 °C,
F igu r e 2. An overview of structure-activity relationships
(SAR) of aminobenzophenones.
72 h.
Sch em e 5a
respectively, a 25-fold increase of activity compared to
our starting compound 3.
We speculated that these two lipophilic ortho sub-
stituents could force the benzophenone system into a
more favorable conformation due to hindered rotation
around the ketone bridge and initiated a modeling study
(see Computational Chemistry). Introduction of substit-
uents in the 3-position in ring B resulted in less active
compounds (compare 46 and 40). Further substitution
in ring A and B did not improve the in vitro activity.
We therefore focused on ring C. Introduction of simple
substituents in the 3-6-positions in ring C gave com-
pounds with altered activities. The most interesting
compounds in this series were found to be the 4-sub-
stituted analogues 49 and 64, both with similar activity
in vitro as 45. Replacement of the primary amino group
with a nitro group resulted in almost complete loss of
activity (compare 45 with 35), indicating the importance
of the primary amino group for activity. However,
further modifications showed that the amino group was
not essential. For example the unsubstituted analogue
62 was found to be moderately active (also, compare 65
with 3), and the 2-methyl analogues 63 and 64 were
equipotent to compound 45. Simple derivatives of 45
(amide 56, urea 57, and carbamate 58) were also very
active in vitro, e.g. the urea compound 57 showed an
IC50 of 3 nM in the TNF-R assay.
a
Reagents: (a) Acetonylacetone, cat. TsOH‚H2O, toluene, Dean-
To investigate the importance of the ketone connect-
ing rings A and B and the secondary amine connecting
rings B and C, these were modified. Thus, the reduced
analogue 71 and the alkene analogue 70 were both
found to be inactive, indicating the importance of the
ketone. Alkylation of the secondary amine was tolerated
to some extent but generally gave less active compounds
(e.g. 60). Replacement of the secondary amino group in
the optimized analogue 45 with, for example, oxygen
or sulfur gave inactive compounds (data not shown).
F u r th er Testin g. To investigate this new structural
class in more detail, some of the potent compounds were
tested for their ability to inhibit a series of cytokines in
whole cell assays. With the objective of getting informa-
tion about the mechanism of action of our compounds,
a number of known reference compounds were included.
Stark reflux 2.5 h; (b) (i) MeLi, THF, -78 °C, then warm to 10 °C
(2.5 h), extraction; (ii) glacial acetic acid, reflux, 3 h; (c) HONH2‚HCl,
Et3N, EtOH/H2O, approximately 100 °C, 24 h; (d) 2-bromoni-
trobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, 1,4-dioxane, 100 °C,
72 h; (e) SnCl2‚2H2O, EtOH, 70 °C, 2 h; (f) CF3COOH, NaBH4, 0
°C, then 64 in DCM added slowly (30 min), rt, 22 h.
showed the importance of a substituent in the 2-position
of ring A (e.g. 40-42). Small substituents such as
methyl and bromo were the most efficient, with a 3-fold
increase of activity (compare 40 and 42 with 3, and 45
with 47). Larger substituents such as phenyl (e.g. 43)
in any position on ring A led to inactive or considerably
less active compounds as compared with 3.
Next, simple substituents were introduced in ring B.
Monosubstituted analogues derived from our initial lead
3 did not result in improved activity (e.g. 44). However,
when the same set of analogues in a series related to
compound 40 were prepared, increased activity was
obtained (e.g. 45, 46, and 48). Thus, the best compound
in this series was 45, having a 2-chloro substituent in
ring B and a 2-methyl substituent in ring A. This
compound was found to inhibit the TNF-R production
and the IL-1â production with an IC50 of 6 and 14 nM,
The results of compounds 45, 49, 64, and HEP689,27
a
p38 MAP kinase inhibitor structurally related to 2, are
shown in Table 2. Our compounds inhibited TNF-R (IC50
4-6 nM) more potently than IL-1â (IC50 ) 14-30 nM).
Similarly, but less potently, HEP689 inhibited TNF-R
(IC50 ) 90 nM) and IL-1â (IC50 ) 272 nM). The four
compounds had a similar profile with respect to the
inhibition of IL-6 and IL-8. Compound 49 inhibited IL-6

Synthesis and SAR of Aminobenzophenones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5655
Ta ble 2. Further in Vitro (IC50, nM; n g 3) Activity of Selected
Compounds
compd
assay
HEP689
45
49
64
IL-1â
TNF-R
IL-2
IL-6
IL-8
IL-10
IL-12
IFN-γ
272 ( 37
90 ( 59
>1000
125a
14 ( 6
6 ( 1
30 ( 21
5 ( 0
17 ( 15
4 ( 3
>1000b
18 ( 1
6a
>1000b
17 ( 7
4 ( 1
>1000
35 ( 28
79 ( 104
51 ( 32
>1000a
>1000
50a
71 ( 24
>1000
>1000
12 ( 8
>1000
>1000b
74 ( 74
>1000a
>1000
a
b
n ) 1. n ) 2.
Ta ble 3. p38R MAP Kinase Activity Data
compd
IC50 (nM); n g 3
compd
IC50 (nM); n g 3
HEP 689
VX-745
3
35
40
41
42
43
44
38 ( 13
29 ( 8
145 ( 62
437 ( 14
29 ( 9
574 ( 16
54 ( 17
811 ( 410
139 ( 9
10 ( 3
143 ( 26
5 ( 2
48
49
56
57
58
60
62
63
64
65
70
71
13 ( 3
39 ( 9
63 ( 8
21 ( 8
42 ( 15
101 ( 46
7 ( 1
12 ( 4
F igu r e 3. The carbonyl torsion angles for all conformations
of 3, 40, 44, and 45 within 3 kcal/mol of the global minimum.
Angle 1 and 2 are defined by the atoms corresponding to 1-2-
3-4 and 4-3-5-6 in 45, respectively.
4 ( 0.2
193 ( 54
1550 ( 190
>100000
45
46
47
kinase activity of ERK1/2 or J NK1 when tested at 1 or
10 µM (Table 4). Furthermore, compound 49 was
evaluated externally (upstate, UK) against a panel of
60 different kinases. Only three kinases (p38R, p38â,
and MKK6) were inhibited by this compound at 1 µM.
Thus, this new compound class was found to be highly
potent and specific p38 MAP kinase inhibitors.
Ta ble 4. Kinase Selectivity of Selected Compounds (n ) 2)
compd
assay
HEP689
45
49
64
p38Ra
73
59
0
8
0
88
93
0
8
20
NT
82
93
0
0
0
88
92
5
a
p38â2
p38γa
p38δa
4
0
0
Com p u ta tion a l Ch em istr y. We have performed an
energy analysis of the two carbonyl rotations in com-
pounds 3, 40, 44, and 45. Intuitively, one would expect
that introducing ortho-substituents in ring A and B of
compound 3 would restrict the rotation of these rings
due to steric interactions between the substituents and
the carbonyl group (Figure 3). It is immediately clear
that the conformational space, as expected, becomes
considerably restricted when ortho-substituents are
introduced.
ERK1/2b
J NK1b
9
0
a
b
Percent inhibition at 1 µM. Percent inhibition at 10 µM.
(IC50 ) 17 nM) and IL-8 (IC50 ) 4 nM) potently and
was approximately 10 times more potent than HEP689
in both assays. In addition, all four compounds inhibited
IL-10. None of the compounds including the literature
reference compound HEP689 inhibited IL-2, IFN-γ, and
IL-12 (IC50 > 1000 nM).
The higher p38R kinase activities of compounds 40
(29 nM) and 45 (10 nM) could therefore be explained
by a lower entropy loss upon protein binding. This has
earlier been shown for the restriction of the intramo-
lecular rotation of a biaryl bond leading to a decreased
loss of entropy upon ligand binding and thus to higher
affinity.48 Obviously, other factors may also contribute,
as evidenced by compound 44. Thus, despite the rota-
tional restriction, compound 44 (139 nM) is equipotent
to the original lead compound 3 (145 nM).
Due to a cytokine inhibitory profile similar to that of
the p38 MAP kinase inhibitor HEP689, our compounds
and the reference compounds HEP689 and VX-74533
were tested in a p38R MAP kinase assay (Table 3). The
lead compound, 45, was found to be a potent inhibitor
of p38R kinase activity (IC50 ) 10 nM). Likewise,
compound 64 (IC50 ) 4 nM) and compound 49 (IC50 )39
nM) inhibited p38R MAP kinase. In comparison, HEP689
and VX-745 gave IC50 of 38 and 29 nM, respectively.
The compounds were evaluated for their in vitro
kinase selectivity. Kinase assays for the four isoforms
of p38 were performed using cellular transfection of the
cDNA containing the respective FLAG-p38 isoform into
COS-1 cells.46,47 The kinase activity of p38R and p38â2
was inhibited by the test compounds at 1 µM, whereas
p38γ and p38δ were unaffected (Table 4). In addition,
the compounds were tested in an ERK1/2 and J NK1
kinase assay using MCF-7 cells stimulated with EGF
or UV light, respectively. The assays were performed
using a modified cell signaling ERK1/2 kinase kit and
a J NK1 kinase kit. The compounds did not inhibit the
Examination of the low-energy conformations of com-
pounds 44 and 45 reveals a difference in the preferred
orientations of the carbonyl group and the chloro
substituent. In compound 45 low-energy minima con-
formations exist where the carbonyl group is positioned
either opposite to (e.g. in the global minimum conforma-
tion) or on the same side as the chloro substituent
(conformations from 1.4 kcal/mol above the global
minimum). In contrast, no low-energy minima confor-
mations exist for compound 44, where the carbonyl
group and the chloro substituent are positioned on the

5656 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Ottosen et al.
Ta ble 5. Inhibition of Ear Swelling and MPO Activity of
Selected Compounds in Oxazolone-Induced Murine Allergic
Contact Dermatitisa
inhibn of ear swelling (%)
postchallenge
dose
inhibn of MPO
activity (%)
compd
(mg/ear) 24 h
48 h
72 h
HCS-17-bu t.
0.001
0.5
0.5
43 ( 3 55 ( 5 55 ( 4
43 ( 4 46 ( 2 46 ( 5
35 ( 4 38 ( 6 46 ( 10
40 ( 7 46 ( 7 54 ( 7
41 ( 6
56 ( 8
56 ( 10
57 ( 5
45
49
64
0.5
a
Values represent the mean inhibition (%) ( SEM of four or
five experiments.
Ta ble 6. Inhibition of Ear Swelling, Ear Weight, and MPO
Activity of Selected Compounds in TPA-Induced Murine
Chronic Irritative Contact Dermatitisa
inhibition (%)
dose
ear
ear
MPO
compd
(mg/ear)
swelling
weight
activity
BMS-17-va l.
0.001
0.5
0.5
41 ( 2
37 ( 5
27 ( 2
26 ( 4
38 ( 2
27 ( 6
34 ( 2
26 ( 2
67 ( 7
73 ( 14
79 ( 8
60 ( 9
45
49
64
0.5
F igu r e 4. Model of 45 (atom colors) bound in the ATP-binding
site of p38 MAP kinase. The important residues Tyr35,
Thr106, and Met109 are shown in yellow. Also shown in
magenta is the position of SB220025.
a
Values represent the mean inhibition (%) ( SEM of three to
five experiments
kinase49 as a template (Figure 4). The most important
features are the placement of ring A in the hydrophobic
pocket and a hydrogen bond between the carbonyl group
and the amide proton of Met109 (p38R numbering).
Replacement of the carbonyl group, as in compound 70
and 71, gave significantly lower p38R kinase activity
(Table 3). This indicated that the hydrogen bond to the
carbonyl group is very important.
The lower activity of compound 60 and others without
the secondary amino group (vide supra) suggests that
the amino group connecting rings B and C are involved
in a hydrogen bond. However, the model does not clearly
indicate the potential partner in such a hydrogen bond.
The aromatic ring C is essential for activity (data not
shown). Unfortunately, the position of ring C in the
present model is somewhat arbitrary, due to the lack
of consistent SAR data. However, it has been shown that
the glycine-rich loop of kinases can undergo significant
conformational changes upon ligand binding49,55
same side. For compound 44, forcing these groups into
such a position requires 2.6 kcal/mol.
The observations described above together with the
activities of 44 compared to 3 and of 45 compared to 40
indicate that the best protein-ligand interactions are
obtained when the carbonyl group and the chloro
substituent are on the same side. The higher energy
penalty for 44 possibly explains why this compound does
not have increased activity compared to 3.
p38 MAP Kin ase-Ligan d In ter action . Recent works
have described the crystal structures of p38R MAP
kinase complexed with pyridinyl/pyrimidinyl imidazole
inhibitors, e.g. SB220025 and HEP689. These studies
have shown that the pyridinyl/pyrimidinyl imidazole
inhibitors are ATP-competitive inhibitors.49-53 Specifi-
cally, residue 106 (p38R numbering) has been pin-
pointed as the most important residue for inhibitor
specificity. Thus, potent inhibitors of p38R and p38â
MAP kinases but inactive against the isoforms p38γ and
p38δ are known. This is explained by the presence of
the relatively small threonine in position 106 of p38R
and p38â compared to the larger methionine in p38γ
and p38δ.54 The side chain of metheonine blocks access
to the hydrophobic region/pocket I in p38γ and p38δ,
which in p38R and p38â may be occupied, in the case of
SB220025 and HEP689, by the p-flourophenyl ring.
We found the benzophenone compounds to be highly
potent inhibitors of p38R and p38â isoforms, but they
were inactive against the p38γ and p38δ isoforms. This
suggests that part of the benzophenone compounds
occupy the hydrophobic pocket I and consequently that
they bind in the ATP-binding cleft.
and one
might speculate that a conformational change can
facilitate favorable interactions with Tyr35 (p38R num-
bering) which is positioned in the glycine-rich loop.
In Vivo. To evaluate our compounds in vivo, com-
pound 45, 49, and 64 were tested in an acute and a
chronic murine model of skin inflammation (Tables 5
and 6). Both models are characterized by high levels of
IL-1â, which is rapidly synthesized after challenge.
Other inflammatory mediators including TNF-R and
IL-2 are also up-regulated, but at lower levels compared
to the IL-1â level.56 The compounds were applied
topically in acetone, and steroids with clinical relevance
were included as positive controls. In the oxazolone
model57 (acute model), inhibition of ear swelling and the
myeloperoxidase (MPO) activity (72 h postchallenge)
was in the range of 46-54% and 56-57%, respectively
(Table 5). In the 12-O-tetradecanoylphobol-13-acetate
(TPA) model58 (chronic model) inhibition of ear swelling
and the myeloperoxidase (MPO) activity was in the
range of 26-37% and 60-79%, respectively (Table 6).
Thus, the in vivo antiinflammatory effects of compounds
As a result of the relatively large and diverse sub-
stituents allowed in ring C, we speculated that ring A
was most likely to be positioned in the hydrophobic
pocket I. To obtain a model of the benzophenone
compounds bound to p38R MAP kinase, we docked
compound 45 in the ATP-binding cleft using the crystal
structure of SB220025 complexed with p38R MAP

Synthesis and SAR of Aminobenzophenones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5657
132.8, 131.4, 129.5, 128.1, 127.7, 127.3, 126.7, 125.8, 119.1,
116.3, 113.1.
45, 49, and 64 were almost identical in both models and
correlated well with the similar potency of the com-
pounds in the inhibition of IL-1â and TNF-R in vitro
(Table 1). The included steroids, hydrocortisone-17-
butyrate (HCS-17-but.) and betamethasone-17-valerate
(BMS-17-val.), gave similar effects in both models
compared to our compounds. The chosen dose (0.001 mg/
ear) of the steroids was known not to give toxic side
effects on spleen and thymus and is clinically relevant
in humans.
N-Meth oxy-2,N-d im eth ylben za m id e (6). To a stirred
suspension of 2-methylbenzoic acid (1.36 g, 10.0 mmol) in
toluene (5.0 mL) were added thionyl chloride (0.8 mL, 11
mmol) and DMF (0.08 mL, 1 mmol). The resulting mixture
was refluxed for 2 h and then cooled to ambient temperature
and concentrated in vacuo to give the crude acid chloride. The
acid chloride was used immediately without any further
purification by dissolving it in ethanol-free DCM (20 mL)
followed by the addition of N,O-dimethylhydroxylamine hy-
drochloride (1.07 g, 11.0 mmol). The solution was cooled to 0
°C, and pyridine (1.8 mL, 22 mmol) was added. A white solid
was rapidly formed (pyridinium hydrochloride). The reaction
mixture was stirred at ambient temperature overnight and
then poured into a mixture of DCM/ether 1:1 (50 mL). The
organic phase was separated, washed with brine (50 mL), dried
(Na2SO4), and filtered, and the solvent was evaporated in
vacuo to leave a residue, which was purified by flash chroma-
tography eluting with petroleum ether/EtOAc 2:1 to yield 6
(1.68 g, 94%) as an oil: 1H NMR (CDCl3) δ 7.27 (m, 2H), 7.19
(m, 2H), 3.52 (bs, 3H), 3.30 (bs, 3H), 2.33 (s, 3H); 13C NMR
(CDCl3) δ 135.3, 134.8, 130.1, 129.2, 126.2, 125.4, 61.0, 32.9,
19.1.
Con clu sion
A novel structural class of aminobenzophenones were
optimized as inhibitors of the proinflammatory cyto-
kines TNF-R and IL-1â. The best compounds were
potent inhibitors of TNF-R (IC50 ) 3-8 nM) and IL-1â
(IC50 ) 9-30 nM) in human PBMC’s stimulated by LPS.
In addition, the compounds 45, 49, and 64 were found
to be specific inhibitors of the p38R MAP kinase with
IC50 values of 10, 39, and 4 nM, respectively. A computer
model of the interaction of compound 45 with the p38R
MAP kinase in the ATP binding pocket was proposed.
The model involved a favorable hydrogen bond between
the carbonyl group of the benzophenone and the NH of
Met-109, positioning ring A in the hydrophobic pocket
I of the enzyme. Furthermore, an interaction of ring C
and the aromatic ring of Tyr-35 was suggested.
Finally, compound 45, 49, and 64 showed antiinflam-
matory activity in two murine skin models of acute and
chronic dermatitis. However, further studies are needed
to examine the potential toxicity of these compounds.
These and other studies, including the evaluation of our
compounds for the treatment of systemic disorders such
as RA, are in progress.
A similar procedure was used to prepare 7.
(4-Am in op h en yl)(2-m eth ylp h en yl)m eth a n on e (20). n-
Butyllithium (5.85 mL, 1.68 M in hexane, 9.84 mmol) was
added slowly (5 min) to a solution of 4-bromo-N,N-bis(tri-
methylsilyl)aniline (5, Aldrich, 3.11 g, 9.84 mmol) in Et2O (15
mL) at 0 °C. After stirring for 1 h, the solution was transferred
via a syringe to a precooled (-78 °C) solution of 6 (1.68 g, 9.37
mmol) in THF (10 mL) under stirring, and the resulting
reaction mixture was allowed to warm to room temperature
overnight. The yellow solution was poured into 1 N HCl (60
mL) and stirred for 15 min. The aqueous phase was neutral-
ized with saturated NaHCO3 and the organic phase separated.
The aqueous phase was extracted with DCM/ether (1:1), and
the combined extracts were dried over MgSO4 and concen-
trated in vacuo. The residue was purified by flash chromatog-
raphy using petroleum ether/EtOAc 2:1 to yield 20 as a yellow
sticky solid (1.47 g, 74%): 1H NMR (CDCl3) δ 7.64 (m, 2H),
7.34 (m, 1H), 7.28-7.18 (m, 3H), 6.61 (m, 2H), 4.22 (bs, 2H),
2.28 (s, 3H); 13C NMR (CDCl3) δ 197.1, 151.5, 139.8, 135.8,
132.8, 130.6, 129.4, 127.8, 127.6, 125.1, 113.7, 19.7.
Exp er im en ta l Section
Gen er a l P r oced u r es. Commercial-grade reagents and
solvents were used without further purification except as
indicated. THF (Merck Secosolv, max 0.0075% H2O), 1,4-
dioxane, dichloromethane (DCM), and toluene were dried by
sequential treatment with two portions of 4 Å molecular sieves
and then stored in a closed bottle over 4 Å molecular sieves.
All reactions were carried out under an inert atmosphere of
dry argon. Flash column chromatography was carried out on
Merck silica gel 60 (0.040-0.063 mm). Thin-layer chromatog-
raphy (TLC) analyses were performed on Merck silica gel 60-
F254 plates and visualized using UV light (254 nm) and/or a
solution consisting of ceric ammonium sulfate and hexaam-
monium heptamolybdate in 10% H2SO4. Melting points were
determined in open glass capillaries using a Buchi melting
point apparatus and are uncorrected. 1H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded on a Bruker ARX-
300 spectrometer. Chemical shifts (δ) are reported in parts per
million (ppm) relative to TMS as internal standard. Elemen-
tary analyses were performed by the analytical department
at LEO on home-built combustion equipment. High-resolution
electron ionization mass spectra (EIMS) were obtained using
a Micromass Autospec spectrometer. No attempt was made
to optimize the reported yields.
{4-[(2-Am in op h e n yl)a m in o]p h e n yl}(p h e n yl)m e t h a -
n on e (3). To a stirred suspension of 29 (10.2 g, 32.0 mmol) in
EtOH (300 mL) was added hydrazine hydrate (3.0 mL, 60
mmol) and 10% Pd/C (3.0 g) at room temperature. The reaction
mixture was stirred for 24 h, filtered through Celite, and then
crystallized by the addition of water (1000 mL). The solid was
filtered, washed with water, and dried to yield 3 (8.62 g, 93%)
as a solid: mp 115-116 °C; 1H NMR (CDCl3) δ 7.77-7.67 (m,
4H), 7.52 (m, 1H), 7.48-7.38 (m, 2H), 7.14 (m, 1H), 7.08 (dt,
1H), 6.81 (m, 1H), 6.77 (dt, 1H), 6.69 (m, 2H), 5.77 (bs, 1H),
3.80 (bs, 2H); 13C NMR (CDCl3) δ 195.3, 150.0, 142.8, 138.8,
A similar procedure was used to prepare 21.
(2-C h lo r o -4-n it r o p h e n y l)(2-m e t h y lp h e n y l)m e t h a -
n on e (17). n-Butyllithium (13.42 mL, 1.49 M in hexane, 20.0
mmol) was added dropwise (10 min) to a solution of 1-bromo-
2-methylbenzene (10, 2.41 mL, 20.0 mmol) in THF (20 mL) at
-78 °C, and the resulting mixture was stirred for 20 min. A
THF solution of dry ZnCl2 (1.0 M, 25 mL) was added with a
syringe and the reaction mixture was allowed to warm to room
temperature. After 2 h the reaction mixture was cooled to 0
°C, and tetrakis(triphenylphosphine)palladium(0) (1.15 g, 1.00
mmol) was added followed by the addition of 2-chloro-4-
nitrobenzoyl chloride (13, prepared from 2-chloro-4-nitroben-
zoic acid (4.33 g, 21.5 mmol) by treatment with thionyl chloride
as described in the synthesis of 6 above) in THF (5 mL). The
stirring reaction mixture was allowed to warm to room
temperature overnight. The mixture was partitioned between
EtOAc (50 mL) and 1 N HCl (100 mL), and the aqueous phase
was extracted with EtOAc (50 mL). The combined organic
extracts were washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by flash
chromatography using petroleum ether/EtOAc 9:1 to yield 17
1
as a pale yellow solid (4.21 g, 76%): mp 78-80 °C; H NMR
(CDCl3) δ 8.31 (d, 1H), 8.22 (dd, 1H), 7.58 (d, 1H), 7.47 (dt,
1H), 7.36 (bd, 1H), 7.27 (dd, 1H), 7.22 (bt, 1H), 2.64 (s, 3H);
13C NMR (CDCl3) δ 195.1, 148.9, 145.5, 140.6, 135.0, 133.1,
132.7, 132.4, 131.9, 130.0, 125.9, 125.4, 121.9, 21.5.
A similar procedure was used to prepare 15, 16, 18, 19, 22,
and 53.
(4-Am in o-2-ch lor op h e n yl)(2-m e t h ylp h e n yl)m e t h a -
n on e (25). A solution of 17 (4.20 g, 15.2 mmol) in absolute

5658 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Ottosen et al.
EtOH (30 mL) was added to stannous chloride dihydrate (17.1
g 76.2 mmol) and the resulting mixture was stirred at 70 °C
for 2 h. The reaction mixture was cooled to room temperature
and poured into ice/water (50 mL), and the pH was made
strongly alkaline by the addition of saturated NaOH (100 mL)
before being extracted with EtOAc (2 × 100 mL). The organic
phases were combined and washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo to yield 25 as a light-yellow
solid (3.59 g, 96%): mp 94-96 °C; 1H NMR (CDCl3) δ 7.35
(dt, 1H), 7.30 (dd, 1H), 7.28 (d, 1H), 7.24 (bd, 1H), 7.18 (bt,
through a pad of silica gel. Silica gel was added to the filtrate,
and the solvents were removed in vacuo. The residue was
poured onto a column and eluted successively with petroleum
ether/DCM 5:1 and petroleum ether/DCM 1:1 to yield 54 as a
yellow oil (1.046 g, 78%): 1H NMR (CDCl3) δ 7.85-7.05 (m,
15H), 6.68 (d, 1H), 6.46 (dd, 1H), 2.39 (s, 3H), 2.26 (s, 3H); 13
C
NMR (CDCl3) δ 200.1, 168.7, 154.3, 140.2, 139.1, 137.1, 135.8,
133.1, 132.4, 131.0, 130.4, 129.4, 128.9, 128.3, 128.0, 125.4,
123.9, 117.4, 21.2, 20.3.
N-(2-{[3-Ch lor o-4-(2-m et h ylb en zoyl)p h en yl]a m in o}-
p h en yl)a ceta m id e (56). A mixture of 45 (1.50 g, 4.45 mmol)
and acetic anhydride (0.59 mL, 5.9 mmol) in glacial acetic acid
(10 mL) was stirred at room temperature for 45 min. Water
(15 mL) was added and the mixture was stirred for 10 min
before being extracted with EtOAc. The organic layer was
separated and concentrated in vacuo. The residue was co-
evaporated with cyclohexane (2 × 10 mL) to leave a syrup
which crystallized on the addition of petroleum ether/EtOAc
1:1 to yield 56 as a yellow solid (1.42 g, 84%): mp 150-152
1H), 6.66 (d, 1H), 6.49 (dd, 1H), 4.19 (bs, 2H), 2.40 (s, 3H); 13
C
NMR (CDCl3) δ 196.7, 150.5, 139.5, 137.6, 135.1, 133.9, 131.2,
130.7, 129.5, 127.5, 125.3, 116.0, 112.2, 20.3; HRMS (EI) m/z
245.0596 [(M+) calcd for C14H12ClNO 245.0607].
A similar procedure was used to prepare 23, 24, 26, 27, 40-
44, 46-49, 60, and 70.
(4-Am in o-2-m e t h ylp h e n yl)(2-m e t h ylp h e n yl)m e t h a -
n on e (28). A solution of 54 (1.04 g, 2.67 mmol) in THF (10
mL) was added to aqueous 2.0 N HCl (1.0 mL) and then the
reaction stirred for 1 h. The reaction mixture was partitioned
between aqueous 0.5 N HCl (30 mL) and 2:1 hexane/EtOAc
(30 mL). The aqueous layer was separated and made alkaline
with 2 N NaOH (20 mL) and then extracted with EtOAc (2 ×
30 mL). The organic phases were dried (MgSO4), filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography using petroleum ether/EtOAc 5:1 to yield 28
as an oil (0.55 g, 91%): 1H NMR (CDCl3) δ 7.32 (dt, 1H), 7.28-
7.14 (m, 4H), 6.52 (d, 1H), 6.37 (dd, 1H), 4.03 (bs, 2H), 2.54 (s,
3H), 2.30 (s, 3H); 13C NMR (CDCl3) δ 199.1, 149.9, 142.9, 141.2,
136.3, 135.4, 130.7, 129.6, 128.4, 127.7, 125.2, 117.4, 110.8,
22.0, 19.9.
1
°C; H NMR (CDCl3) δ 7.71 (s, 1H), 7.60 (dd, 1H), 7.40-7.10
(m, 8 H), 6.81 (d, 1H), 6.64 (dd, 1H), 6.59 (bs, 1H), 2.42 (s,
3H), 2.16 (s, 3H); 13C NMR (CDCl3) δ 196.7, 169.4, 148.7, 139.1,
137.8, 135.1, 133.6, 132.9, 131.7, 131.3, 130.9, 129.6, 128.8,
126.4, 125.8, 125.4, 124.8, 124.0, 116.2, 112.6, 24.1, 20.4;
HRMS (EI) m/z 378.1146 [(M+) calcd for C22H19ClN2O2
378.1135]. Anal. (C22H19ClN2O2): C, H, N, Cl.
N-(2-{[3-Ch lor o-4-(2-m et h ylb en zoyl)p h en yl]a m in o}-
p h en yl)-N′-eth ylu r ea (57). To a solution of 45 (6.50 g, 19.3
mmol) in dry pyridine (60 mL) was added ethyl isocyanate
(2.06 g, 29.0 mmol) dropwise under stirring. The reaction
mixture was stirred for 4 h, diluted with water (60 mL), and
cooled in ice-water. Collection of the crystals by filtration and
drying in vacuo overnight gave 57 as a yellow solid (7.63 g,
97%): mp 161-162 °C; 1H NMR (DMSO-d6) δ 8.34 (s, 1H),
8.04 (d, 1H), 7.79 (s, 1H), 7.42 (m, 1H), 7.10-7.34 (m, 6H),
6.96 (m, 1H), 6.67 (m, 2H), 6.57 (m, 1H), 3.07 (m, 2H), 2.29 (s,
3H), 1.02 (t, 3H); HRMS (EI) m/z 407.1418 [(M+) calcd for
{2-Ch lor o-4-[(2-n it r op h en yl)a m in o]p h en yl}(2-m et h -
ylp h en yl)m eth a n on e (35). A mixture of 1-fluoro-2-nitroben-
zene (50, 1.65 mL, 15.6 mmol) and 25 (4.00 g, 16.3 mmol) in
DMSO (25 mL) was cooled in an ice bath (0 °C). A solution of
KOtBu (3.88 g, 34.6 mmol) in DMSO (20 mL) was placed in a
dropping funnel and added dropwise over 5 min. The ice bath
was removed and the mixture was stirred for 18 h at room
temperature. Water (100 mL) was added and after 5 min the
reaction mixture was extracted with EtOAc (3 × 100 mL). The
combined organic extracts were washed with water and brine,
dried (MgSO4), filtered, and concentrated in vacuo. The residue
was purified by flash chromatography using petroleum ether/
EtOAc 9:1 to yield 35 as a orange solid (3.95 g, 69%): mp 150-
C
23H22ClN3O2 407.1401]. Anal. (C23H22ClN3O2): C, H, N, Cl.
Eth yl 2-{[3-Ch lor o-4-(2-m eth ylben zoyl)p h en yl]a m in o}-
p h en ylca r ba m a te (58). A mixture of 45 (337 mg, 1.00 mmol)
and K2CO3 (415 mg, 3.0 mmol) in DCM (8.0 mL) was stirred
at 0 °C for 10 min. Ethyl chloroformate (217 mg, 2.0 mmol)
was added with a syringe and after 1 h the temperature was
raised to room temperature. After 28 h the reaction mixture
was partitioned between water and DCM. The organic layer
was dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by chromatography eluting with DCM
followed by DCM/EtOAc 50:1 to give a pale yellow syrup which
was crystallized from a mixture of petroleum ether/ether to
yield 58 as white solid (302 mg, 74%): mp 114-115 °C; 1H
NMR (CDCl3) δ 7.82 (d, 1H), 7.40-7.16 (m, 7H), 7.13 (dt, 1H),
6.91 (bs, 1H), 6.75 (d, 1H), 6.60 (dd, 1H), 6.08 (bs, 1H), 4.22
(q, 2H), 2.44 (s, 3H), 1.30 (t, 3H); 13C NMR (CDCl3) δ 196.6,
154.1, 149.1, 139.1, 138.0, 135.0, 133.5, 133.4, 131.3, 130.9,
130.5, 129.7, 129.2, 126.9, 126.0, 125.4, 124.9, 121.7, 116.1,
112.5, 61.7, 20.5, 14.5; HRMS (EI) m/z 408.1238 [(M+) calcd
for C23H21ClN2O3 408.1241]. Anal. (C23H21ClN2O3): C, H, N,
Cl.
{2-Ch lor o-4-[m et h yl(2-n it r op h en yl)a m in o]p h en yl}(2-
m eth ylp h en yl)m eth a n on e (59). To a solution of 35 (1.00 g,
2.73 mmol) in THF (10 mL) was added NaH (72 mg, 3.0 mmol)
and then, after 15 min, iodomethane (426 mg, 2.94 mmol), and
the mixture was stirred for 2 h under reflux. The reaction
mixture was partitioned between EtOAc and saturated aque-
ous NaHCO3 (×3) and then the organic phases were dried
(MgSO4), filtered, and concentrated in vacuo. The residue was
purified by chromatography eluting with petroleum ether/
EtOAc 5:1 to yield 59 as an orange syrup (1.04 g, 100%): 1H
NMR (CDCl3) δ 7.99 (dd, 1H), 7.70 (dt, 1H), 7.49 (dt, 1H), 7.42
(dd, 1H), 7.39-7.28 (m, 3H), 7.24 (bd, 1H), 7.18 (bt, 1H), 6.61
(d, 1H), 6.42 (dd, 1H), 3.33 (s, 3H), 2.41 (s, 3H); 13C NMR
(CDCl3) δ 196.4, 150.9, 147.3, 139.9, 139.2, 137.8, 135.0, 134.7,
134.6, 133.3, 131.2, 131.2, 130.7, 129.6, 128.1, 126.0, 125.4,
114.8, 111.1, 40.1, 20.4.
1
152 °C; H NMR (CDCl3) δ 9.43 (s, 1H), 8.23 (dd, 1H), 7.55-
733 (m, 6H), 7.30 (bd, 1H), 7.22 (m, 2H), 6.95 (m, 1H), 2.53 (s,
3H); 13C NMR (CDCl3) δ 196.4, 142.7, 140.1, 139.0, 137.6,
135.8, 135.1, 134.7, 134.0, 132.2, 131.8, 131.7, 130.6, 126.9,
125.6, 123.0, 119.8, 119.7, 117.1, 20.9; HRMS (EI) m/z 366.0771
[(M+) calcd for C20H15ClN2O3 366.0771]. Anal. (C20H15ClN2O3):
C, H, N, Cl.
A similar procedure was used to prepare 29-34 and 36-
39.
{4-[(2-Am in op h en yl)a m in o]-2-ch lor op h en yl}(2-m et h -
ylp h en yl)m eth a n on e (45). This compound was prepared as
described in the synthesis of 25 from 35 (14.2 g, 38.8 mmol).
The residue was triturated in a mixture of Et2O/petroleum
ether (1:1), and the solid was collected by filtration and dried
in vacuo to yield 45 as yellow crystals (11.55 g, 89%): mp 113-
1
114 °C; H NMR (CDCl3) δ 7.40-7.05 (m, 7H), 6.85-6.74 (m,
2H), 6.69 (d, 1H), 6.55 (dd, 1H), 5.61 (bs, 1H), 3.79 (bs, 2H),
2.42 (s, 3H); 13C NMR (CDCl3) δ 196.5, 149.5, 142.9, 139.3,
137.7, 135.2, 133.7, 131.2, 130.7, 129.5, 128.2, 127.7, 126.9,
125.3, 119.2, 116.4, 115.3, 111.8, 20.4; HRMS (EI) m/z 336.1030
[(M+) calcd for C20H17ClN2O 336.1029]. Anal. (C20H17ClN2O):
C, H, N, Cl.
{2-Ch lor o-4-[(d ip h en ylm et h ylen e)a m in o]p h en yl}(2-
m eth ylp h en yl)m eth a n on e (54). An oven-dried Schlenk tube
was charged with Pd2(dba)3 (15.8 mg, 0.017 mmol) and rac-
BINAP (32.3 mg, 0.052 mmol) and purged with argon. To the
flask were added 53 (1.00 g, 3.46 mmol), benzophenoneimine
(0.87 mL, 5.2 mmol), NaOtBu (465 mg, 4.84 mmol), and toluene
(9.0 mL), and the mixture was heated to 80 °C and stirred for
48 h. The mixture was cooled to room temperature and filtered

Synthesis and SAR of Aminobenzophenones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5659
{2-Ch lor o-4-[(4-flu or o-2-m eth ylp h en yl)a m in o]p h en yl}-
(2-m eth ylp h en yl)m eth a n on e (64). A Schlenk tube was
charged with 25 (737 mg, 3.00 mmol) in 1,4-dioxane (30 mL),
1-bromo-4-fluoro-2-methylbenzene (61, 624 mg, 3.30 mmol),
Cs2CO3 (1.37 g, 4.20 mmol), Pd2(dba)3 (69 mg, 0.075 mmol),
and rac-BINAP (140 mg, 0.23 mmol). The tube was capped
with a rubber septum, flushed with argon for 5 min, and then
stirred at 100 °C for 72 h. The reaction mixture was allowed
to cool to room temperature and then poured into a mixture
of water and EtOAc. The aqueous phase was extracted with
more EtOAc (×2). The combined organic phases were washed
with brine, dried (MgSO4), filtered, and concentrated in vacuo.
The crude product was purified by chromatography eluting
with petroleum ether/ether 4:1 to yield an orange solid (910
mg). Trituration in a mixture of petroleum ether/ether 10:1,
filtration, and washing of the solid with petroleum ether/ether
10:1 gave 64 as a yellow solid (733 mg, 69%): mp 151-152
°C; 1H NMR (CDCl3) δ 7.39-7.29 (m, 3H), 7.27-7.14 (m, 3H),
6.99 (dd, 1H), 6.92 (dt, 1H), 6.66 (d, 1H), 6.53 (dd, 1H), 5.65
(s, 1H), 2.42 (s, 3H), 2.23 (s, 3H);13C NMR (CDCl3) δ 196.5,
160.5, 149.6, 139.3, 137.8, 136.3, 135.3, 133.7, 131.2, 130.7,
129.5, 128.2, 127.1, 125.3, 117.8, 115.2, 113.8, 111.6, 20.4, 18.1;
HRMS (EI) m/z 353.0997 [(M+) calcd for C21H17ClFNO
353.0983]. Anal. (C21H17ClFNO): C, H, N, Cl.
The aqueous phase was extracted with more EtOAc (×2). The
combined organic phases were washed with saturated NaHCO3
and brine and then dried (MgSO4), filtered, and concentrated
in vacuo. The residue was purified by chromatography eluting
with petroleum ether/DCM 2:1 to yield 68 as an oil (138 mg,
79%): 1H NMR (CDCl3) δ 7.22-7.08 (m, 4H), 6.96 (d, 1H), 6.67
(d, 1H), 6.48 (dd, 1H), 5.55 (d, 1H), 5.39 (d, 1H), 3.68 (bs, 2H),
2.14 (s, 3H); 13C NMR (CDCl3) δ 147.1, 146.4, 142.1, 135.8,
133.1, 131.8, 130.9, 130.3, 129.7, 127.2, 125.5, 119.4, 116.0,
113.3, 20.4.
N-[3-Ch lor o-4-(2-m eth ylben zyl)p h en yl]-N-(4-flu or o-2-
m eth ylp h en yl)a m in e (71). A Schlenk tube was charged with
CF3COOH (2.50 mL) and cooled to 0 °C. NaBH4 (114 mg, 3.0
mmol) was added in small portions over 5 min and the mixture
was stirred for 1 h. A solution of 64 (176 mg, 0.497 mmol) in
DCM (1.50 mL) was added over a period of 30 min, the mixture
was allowed to warm to room temperature, and stirring was
continued for 22 h. More NaBH4 (57 mg, 1.5 mmol) was added
to the reaction mixture and stirring was continued for an
additional 5 h. The reaction mixture was poured into water,
made basic by the addition of NaOH (pellets), and extracted
with ether (×2). The combined organic extracts were dried over
MgSO4, filtered, and concentrated to a brown solid (206 mg).
Flash chromatography using petroleum ether/EtOAc 8:1 gave
an almost inseparable mixture of 71 and 72 (approximately
170 mg). A pure sample (7.1 mg) of the undesired compound
72 was obtained. Purification of a small portion (ca 40 mg) of
the mixture by preparative gradient HPLC (10 × 250 mm RP
column, C-18; mobile phase 0 to 95% CH3CN in water with
0.01% CF3COOH) gave a pure sample of 71 (20 mg) as a
hygroscopic solid. 71: 1H NMR (CDCl3) δ 7.21-7.08 (m, 4H),
7.03-6.82 (m, 3H), 6.81 (d, 1H), 6.72 (d, 1H), 6.55 (dd, 1H),
5.19 (bs, 1H),3.96 (s, 2H), 2.25 (s, 3H), 2.22 (s, 3H).
A similar procedure was used to prepare 62, 63, 65, and
69.
[2-Ch lor o-4-(2,5-d im eth yl-1H-p yr r ol-1-yl)p h en yl](2-m e-
th ylp h en yl)m eth a n on e (66). A 100-mL round-bottom flask
was charged with 25 (2.01 g, 8.18 mmol), toluene (70 mL),
TsOH‚H2O (16 mg, 1 mol %), and acetonylacetone (1.20 g, 10.5
mmol). The solution was warmed to reflux under Dean-Stark
conditions for 2.5 h, at which point TLC analysis indicated
complete conversion to the pyrrole. The solution was cooled
to room temperature, washed with aqueous NaHCO3, dried
(MgSO4), filtered, and concentrated in vacuo. The crude
product was purified by chromatography eluting with petro-
leum ether/DCM 2:1 followed by DCM to yield 66 as a white
N-[3-Ch lor o-4-(2-m eth ylben zyl)p h en yl]-N-(4-flu or o-2-
m eth ylph en yl)-N-(2,2,2-tr iflu or oeth yl)am in e (72): 1H NMR
(CDCl3) δ 7.20-7.06 (m, 4H), 7.04-6.93 (m, 3H), 6.69 (d, 1H),
6.57 (d, 1H), 6.33 (dd, 1H), 4.13 (m, 2H), 3.94 (s, 2H), 2.23 (s,
3H), 2.08 (s, 3H).
1
solid (2.49 g, 94%): mp 99-100 °C; H NMR (CDCl3) δ 7.53
(d, 1H), 7.44 (dt, 1H), 7.38 (dd, 1H), 7.33 (m, 1H), 7.31 (d, 1H),
7.24 (bt, 1H), 7.20 (dd, 1H), 5.93 (s, 2H), 2.59 (s, 3H), 2.09 (s,
6H); 13C NMR (CDCl3) δ 196.6, 141.7, 139.6, 138.6, 136.7,
132.5, 132.3, 132.0, 131.2, 130.5, 129.9, 128.6, 126.6, 125.7,
106.8, 21.2, 13.1.
Molecu la r Mod elin g. All calculations were performed on
a Silicon Graphics O2 R10000 workstation. The conformational
and torsional rotational analyses were carried out using the
Monte Carlo (Mcrlo) and DRIVE routines of MacroModel 7.0
(Schro¨dinger Inc.), respectively. The structures were energy-
minimized with the Truncated Newton Conjugate Gradient
(TNCG) method using the MMFF94s force field, until the
default derivative convergence criterion of 0.05 kJ /(mol Å) were
met. The solvent was in all MacroModel calculations set to
water using the SLVNT command.
Docking of the conformations of compound 45 obtained from
a MacroModel conformational search in the ATP-binding
pocket of p38R MAP kinase was done using Sybyl 6.7 (Tripos
Inc.). We started with the crystal structure of p38R complexed
with SB22002548 (pdb code 1BL7). After removing the water
molecules in the PDB file and adding hydrogens, possible
identical pharmacophore elements of SB220025 and 45 were
superimposed using the Fit Atoms facility of Sybyl (Figure 5a).
A minor manual adjustment of the position of 45 was done
prior to CScore (as implemented in Sybyl 6.7) evaluation of
the different conformations. Five conformations had a CScore
value of 5, suggesting that these conformations had the best
interactions with p38R MAP kinase. These conformations were
subjected to further analysis.
1-{3-Ch lor o-4-[1-(2-m et h ylp h en yl)vin yl]p h en yl}-2,5-
d im eth yl-1H-p yr r ole (67). MeLi (2.82 mL, 1.07 M in THF,
3.02 mmol) was added dropwise (15 min) to a stirred solution
of 66 (0.956 g, 29.5 mmol) in THF (30 mL) at -78 °C. The
reaction was warmed to 10 °C over a period of 2.5 h and then
quenched with saturated aqueous NH4Cl. Extraction with
EtOAc (×2), collection of the organic phases, washing with
water and brine, and concentration in vacuo gave the crude
alcohol, which was used without any further purification. The
residue was dissolved in glacial acetic acid (60 mL) and
refluxed for 3 h. The cooled reaction mixture was partitioned
between water and EtOAc (×2), the organic phases were
combined, dried (MgSO4), filtered, and concentrated, and the
residue was purified by flash chromatography eluting with
petroleum ether/EtOAc 12:1 to yield 67 as a white solid (0.514
g, 54%): mp 139-141 °C; 1H NMR (CDCl3) δ 7.30 (d, 1H),
7.27-7.14 (5H), 7.06 (dd, 1H), 5.89 (bs, 2H), 5.71 (d, 1H), 5.59
(d, 1H), 2.17 (s, 3H), 2.05 (s, 6H); 13C NMR (CDCl3) δ 146.6,
141.1, 140.2, 138.8, 135.7, 132.8, 131.3, 130.6, 129.8, 128.7,
127.7, 126.5, 125.8, 121.4, 106.2, 20.5, 13.0.
3-Ch lor o-4-[1-(2-m eth ylph en yl)vin yl]ph en ylam in e (68).
A thick-walled vial (8 mL) was charged with 67 (233 mg, 0.72
mmol), Et3N (0.44 mL, 3.16 mmol), EtOH (2.5 mL), water (0.86
mL) CHCl3 (0.50 mL), and hydroxylamine hydrochloride (577
mg, 8.30 mmol) and then closed and warmed to 90 °C for 16
h. TLC indicated that there was unreacted starting material
left. More Et3N (0.44 mL, 3.16 mmol) and hydroxylamine
hydrochloride (550 mg, 7.91 mmol) were added to reaction
mixture, and stirring was continued at 100 °C for 8 h. The
reaction mixture was allowed to cool to room temperature and
then poured into a mixture of ice-cold 1 N HCl and EtOAc.
As described in the Results and Discussion section, the
activity of compound 44 indicates that that the best protein-
ligand interactions are obtained when the carbonyl group and
the chloro substituent are on the same side. Three of the five
conformations of 45 mentioned above have this geometry.
Visual inspection of these three conformations revealed that
a favorable interaction between ring C and Tyr35 was possible
for one conformation. Accordingly, this conformation was
chosen to represent the bound conformation of 45.
Refinement of the p38R-45 complex was carried out with
Sybyl 6.7 using the MMFF94s force field and MMFF94 charges
and the following protocol: 500-step Powell energy minimiza-

5660 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Ottosen et al.
against IL-12 p70 and biotinylated polyclonal antibody against
p40/p70 (Pharmingen, Becton Dickenson no. 555065 and no.
20512D, respectively); IFN-γ, monoclonal antibody no. MAB285
and biotinylated polyclonal antibody no. BAF285].
P la te Assa y for Estim a tin g th e Kin a se Activity of
Com p ou n d s for p 38 MAP Kin a se. The kinase activity of
inhibitors of human recombinant p38 MAP kinase was deter-
mined using a simple ELISA-based plate assay. The assay was
based upon the phosphorylation of the p38 MAP kinase
substrate, ATF-2, and was measured using a specific antibody
raised against the phosphorylated ATF-2. In brief, Nunc
Maxisorb plates were coated with GST-AFT-2 (SantaCruz,
sc-4114) (2,5 µg/mL) in 40 mM Tris/HCl pH 7.4, 10 mM
magnesium acetate overnight at 4 °C. The plates were blocked
with 1% BSA in PBS for 1 h at room temperature. Test
compounds were diluted in 25 mM HEPES, pH 7.5, 10 mM
magnesium acetate and placed in the coated 96-well microtiter
plate. Active p38R (Upstate Ltd. cat. no. 14-251) was diluted
in 25 mM HEPES, pH 7.5, 10 mM magnesium acetate and
added to the wells in a final concentration of 100 ng/mL. The
kinase reaction was allowed to react for 1 h at room temper-
ature. The plate was washed three times with wash buffer (1
× PBS, 0.1% Tween-20) and hereafter incubated with primary
antibody, anti-phospho-ATF-2 (F-1) (Santa Cruz, sc-8398),
followed by incubation with secondary antibody (rabbit-anti-
mouse-HRP, P-0260, DAKO, Denmark). The plates were
finally incubated with TMB, and the color reaction was read
in an ELISA-reader at A460. The assay was run in duplicate,
and the test compounds were diluted from 10-10 to 10-4M. The
DMSO concentration was 1% final in all concentrations. The
data points from three or more individual assays were
combined and analyzed by nonlinear regression (GraphPad
Prism version 3.00 for Windows, GraphPad software Inc. San
Diego, CA). The calculated IC50 value is the concentration of
the test compound that caused a 50% decrease in the maximal
inhibition of p38 MAP kinase activity. In all cases, standard
errors were within statistically acceptable limits. A standard
compound, SB203580, was included with each set of experi-
ments, and its IC50 was 53.3 ( 12.8 nm.
Oxa zolon e-In d u ced Mu r in e Aller gic Con ta ct Der m a -
titis. Six female inbreed BALB/cABOM mice per group
(weighing 18-21 g; M & B A/S) were sensitized to oxazolone
(Sigma Chemical Co.) by application to the clipped abdomen
7 days before challenge with the chemical to the right ear.
Twenty microliters of test compound, dissolved in acetone, or
vehicle were applied once 30 min after oxazolone challenge.
Ear swelling (right ear thickness - left ear thickness) was
measured 24, 48, and 72 h postchallenge using a Mitutyo
digimatic indicator, and MPO (myeloperoxidase) activity in ear
tissue was determined 72 h post challenge. Hydrocortisone-
17-butyrate (HCS-17-but.) (Sigma Chemical Co.) was tested
as reference compound.57
TP A-In d u ced Mu r in e Ch r on ic Ir r ita tive Con ta ct Der -
m a titis. Skin inflammation in groups of six female inbreed
BALB/cABOM mice (weighing 18-21 g; M & B A/S) was
induced by phorbol 12-myristate-13-acetate (PMA ) TPA from
Sigma Chemical Co.) applications to the right ear on days 1,
3, 5, 8, and 10. Twenty microliters to test compound, dissolved
in acetone, or vehicle were applied twice daily on days 8, 9,
and 10 and once on day 11. Ear swelling, ear weight, and MPO
activity in ear tissue were determined 5 h after the last test
compound application. Betamethasone-17-valerate (BMS-17-
val.) (Sicor) was tested as reference compound.58
F igu r e 5. (a) The pharmacophore elements of SB220025 and
45 used in the superposition of these compounds were (1) the
centroid of the p-flourophenyl ring and the centroid of ring A,
(2) the 4-pyridine nitrogen and the carbonyl oxygen, (3) the
2-pyridine carbon and carbon 1 of ring B, and (4) the 6-pyridine
carbon and carbon 1 of ring A. (b) The constraints used in the
refinement procedure for the H-bond between Met109 and 45
and for the interaction between the Tyr35 ring and ring C.
tion (Initial Optimization parameter set to Simplex) followed
by 1 ps molecular dynamics at 300 K and finally 1000-step
Powell energy minimization (Initial Optimization parameter
set to Simplex). During these calculations constraints were
added to maintain a hydrogen bond between the carbonyl
group and the amide proton of Met109 and an interaction
between ring C and Tyr35 (Figure 5b).
Biologica l Meth od s. The in vitro results are expressed as
IC50 values, where the IC50 value is defined as the concentra-
tion of compound required to reduce the cytokine levels (or
kinase activity) to 50% of control values. All determinations
were done in triplicate unless otherwise noted.
P r od u ction of cytok in es. Human peripheral blood mono-
nuclear cells (PBMC) were isolated from buffy coats. The blood
was mixed with saline at a ratio of 1:1, and the PBMC were
isolated by the use of Lymphoprep tubes (Nycomed, Norway).
The PBMC were suspended in RPMI1640 (no. 42401-018) and
supplemented with 2% fetal calf serum (FCS) (IL-1â, TNF-R,
IL-6, IL-8, IL-12) or 10% FCS (IL-2, IL-10, IFN-γ) and 2 mM
l-glutamine, 100 U penicillin, and 100 mg streptomycin/mL
at
a
concentration of 5 × 105 cells/mL. The cells were
preincubated for 30 min with the test compounds in 48- or
96-well tissue culture plates (Life Technologies) and treated
as follows:
IL-1â, TNF -R. The cells were stimulated with 1 mg/mL of
bacterial endotoxin (LPS) (Sigma L-2880) and incubated (at
37°C with 5% CO2) for 18 h.
IL-6, IL-8. The cells were stimulated with 1 mg/mL of LPS
and incubated for 6 h.
IL-12. The cells were stimulated with 95 ng/mL of IFN-γ
for 18 h followed by 1 mg/mL LPS for 24 h.
IL-2, IL-10, IF N-γ. Cells were stimulated with 13, 5, and 3
mg/mL of phytohemagglutinin (no. 3110-57, Becton-Dickin-
son), respectively, and incubated for 48 h. The levels of
cytokines were measured in the culture supernatant by
sandwich ELISA [IL-1â, monoclonal antibody no. MAB201 and
biotinylated polyclonal antibody no. AB-201-NA (R&D systems,
UK); TNF-R, monoclonal antibody no. MAB210 and biotiny-
lated polyclonal antibody no. AB-210-NA (R&D systems, UK);
IL-2, monoclonal antibody no. MAB202 and biotinylated
polyclonal antibody no. AB-202-NA (R&D systems, UK); IL-6
and IL-8, ELISA kits no. M-1916 and M-1918 (Kem-en-Tec,
DK); IL-10, monoclonal antibody no. MAB217 and biotinylated
polyclonal antibody no. BAF217; IL-12, monoclonal antibody
Det er m in a t ion of Myelop er oxid a se Act ivit y. Ear
punches were placed in a Teflon shaking flask with a steel
ball and frozen in liquid nitrogen. The frozen ear punch was
homogenized for 60 s in a Brown Mikrodismembrator U. The
obtained powder was transferred to a preweighed Eppendorf
tube and reweighed. Then 200 µL of HTAB solution (0.5% in
acetate buffer, pH 4.0) per mg of ear powder was added to each
tube, and the tubes were shaken on a whirly mixer and
incubated for 1 h in a 37° water bath. The tubes were
centrifugated at 2500g for 5 min and MPO activity was
assayed in the supernatant in quadruplicates as follows: 10

Synthesis and SAR of Aminobenzophenones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5661
(20) Beyaert, R.; Cuenda, A.; Vanden-Berghe, W.; Plaisance, S.; Lee,
J . C.; Haegeman, G.; Cohen, P.; Fiers, W. The p38/RK mitogen-
activated protein kinase pathway regulates interleukin-6 syn-
thesis in response to tumor necrosis factor. EMBO J . 1996, 15,
1914-1923.
µL of supernatant was mixed in the wells of a microtiter plate
with 250 µL of a solution of TMB (385 mg/mL in 0.025% H2O2).
After 5 min incubation, the absorbance was measured at 655
nm in a micro plate reader (Bio-Rad, model 450). MPO activity
was expressed in arbitrary units, one unit being defined as
the activity yielding 100 mE in 5 min under the above-
described conditions.
(21) Ono, K.; Han, J . The p38 signal transduction pathway: Activa-
tion and function. Cellular Signaling 2000, 12, 1-13.
(22) Herlaar, E.; Brown, Z. p38 MAPK signaling cascades in inflam-
matory disease. Mol. Med. Today 1999, 5, 439-447.
(23) Gallagher, T. F.; Seibel, G. L.; Kassis, S.; Laydon, J . T.;
Blumenthal, M. J .; Lee, J . C.; Lee, D.; Boehm, J . C.; Fier
Thompson, S. M.; Abt, J . W.; Soreson, M. E.; Smietana, J . M.;
Hall, R. F.; Garigipati, R. S.; Bender, P. E.; Erhard, K. F.; Krog,
A. J .; Hofmann, G. A.; Sheldrake, P. L.; McDonnell, P. C.;
Kumar, S.; Young, P. R.; Adams, J . L. Regulation of stress-
induced cytokine production by pyridinylimidazoles; inhibition
of CSBP kinase. Bioorg. Med. Chem. 1997, 5, 49-64.
(24) Badger, A. M.; Bradbeer, J . N.; votta, B.; Lee, J . C.; Adams, J .
L.; Griswold, D. E. Pharmacological profile of SB203580, a
selective inhibitor of cytokine suppressive binding protein(p38
kinase, in animal models of arthritis, bone resorption, endotoxin
shock and immune function. J . Pharm Exp. Ther. 1996, 279,
1453-1461.
(25) J ackson, J . R.; Bolognese, B.; Hillegass, L.; Kassis, S.; Adams,
J .; Griswold, D. E.; Winkler, J . D. Pharmacological effects of
SB220025, a selective inhibitor of p38 mitogen-activated protein
kinase, in angiogenesis and chronic inflammatory disease mod-
els. J . Pharmacol. Exp. Ther. 1998, 284, 687-692.
(26) Hanson, G. H. Inhibitors of p38 kinase. Exp. Opin. Ther. Patents
1997, 7, 729-733.
(27) Boehm, J . C.; Adams, J . L. New inhibitors of p38 kinase. Exp.
Opin. Ther. Patents 2000, 10, 25-37.
(28) Salituro F. G.; Germann, U. A.; Wilson, K. P.; Bemis, G. W.;
Fox, T.; Su, M. S-S. Inhibitors of p38 MAP kinase: Therapeutic
intervention in cytokine-mediated diseases. Curr. Med. Chem.
1999, 6, 807-823.
(29) Dumas, J .; Sibley, R.; Riedl, B.; Monahan, M. K.; Lee, W.;
Lowinger, T. B.; Redman, A. M.; J ohnson, J . S.; Kingery-wood,
J .; Scott, W. J .; Smith, R. A.; Bobko, M.; Schoenleber, R.; Ranges,
G. E.; Housley, T. J .; Bhargava, A.; Wilhelm, S. M.; Shrikhande,
A. Discovery of a new class of p38 kinase inhibitors. Bioorg. Med.
Chem. Lett. 2000, 10, 2047-2050.
(30) Redman, A. M.; J ohnson, J . S.; Dally, R.; Swartz, S.; Wild, H.;
Paulsen, H.; Caringal, Y.; Gunn, D.; Renick, J .; Osterhout, M.;
Kingery-Wood, J .; Smith, R. A.; Lee, W.; Dumas, J .; Wilhelm,
S. M.; Housley, T. J .; Bhargava, A.; Ranges, G. E.; Shrikhande,
A.; Young, D.; Bombara, M.; Scott, W. J . p38 kinase inhibitors
for the treatment of arthritis and osteoporosis: Thienyl, furyl
and pyrrolyl ureas. Bioorg. Med. Chem. Lett. 2001, 11, 9-12.
(31) Substituted pyrazoles as p38 kinase inhibitors. Exp. Opin. Ther.
Patents 1999, 9, 975-979.
(32) p38 inhibitors based on the pyridylurea and pyridylacetoamide
templates. Exp. Opin. Ther. Patents 2000, 10, 1151-1154.
(33) Ferraccioli, G. F. VX-745 Vertex Pharmacuetical. Curr. Opin.
Anti-inflam. Immunomod. Invest. Drugs 2000, 2, 74-77.
(34) Pargellis, C.; Regan, J . Inhibitors if p38 mitogen-activated
protein kinase for the treatment of rheumatoid arthritis. Curr.
Opin. Invest. Drugs 2003, 4, 566-571
(35) Pratt, J . R.; Massey, W. D.; Pinkerton, F. H.; Thames, S. F.
Organosilicon compounds. XX. Sythesis of aromatic diamines via
trimethylsilyl-protecting aniline intermediates. J . Org. Chem.
1975, 40, 1090-1094.
(36) Weinreb, M.; Nahm, S. N-Methoxy-N-methylamides as effective
acylating agents. Tetrahedron Lett. 1981, 22, 3815-3818.
(37) Negishi, E.-i.; Bagheri, V.; Chatterjee, S.; Luo, F.-T.; Miller, J .
A.; Stoll, A. T. Palladium-catalyzed acylation of organozincs and
other organometallics as a convenient route to ketones. Tetra-
hedron Lett. 1983, 24, 5181-5184.
(38) Evans, P. A.; Nelson, J . D.; Stanley, A. L. Directed lithiation/
transmetalation approach to palladium-catalyzed cross-coupling
acylation reactions. J . Org. Chem. 1995, 60, 2298-2301.
(39) Bumagin, N. A.; Ponomaryov, A. B. Palladium- and nickel-
catalyzed aryl- and acyl-demetalation of organometallic com-
pounds. J . Organomet. Chem. 1985, 291, 129-132.
(40) Bellamy, F. D.; Ou, K. Selective reduction of aromatic nitro
compounds with stannous chloride in non acidic and non aqueous
medium. Tetrahedron Lett. 1984, 25, 839-842.
Ack n ow led gm en t. We thank the Spectroscopic
Department for spectral data and elemental analyses.
Rita Christensen is acknowledged for her technical
assistance in the synthesis and Sophie Havez for the
synthesis of compound 71. Camilla Hauge is acknowl-
edged for testing most of the compounds in the p38 MAP
kinase assay. Schneur Rachlin is acknowledged for his
useful discussions and suggestions during the lead
optimizations.
Su p p or tin g In for m a tion Ava ila ble: The complete ex-
perimental procedures with spectroscopical and analytical data
for the compounds 7, 15, 16, 18, 19, 21-24, 26, 27, 29-34,
36-39, 40-44, 46-49, 53, 60, 62, 63, 65, 69, 70, and 71. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) Dinarello, C. A. Inflammatory cytokines: Interleukin-1 and
tumor necrosis factor as effector molecules in autoimmune
diseases. Curr. Opin. Imm. 1991, 3, 941-948.
(2) Arend, W. P.; Dayer, J .-M. Cytokines and cytokine inhibitors or
antagonists in rheumatoid arthritis. Arthitis Rheum. 1990, 33,
305-15.
(3) Dayer, J . M.; Demczuk, S. Cytokines and other mediators in
rheumatoid arthritis. Springer Semin. Immunopathol. 1984, 7,
387-413.
(4) Barnes, P. J .; Chung, K. F.; Page, C. P. Inflammatory mediators
of asthma: An update. Pharmacol. Rev. 1998, 50, 515-596.
(5) Loyau, G.; Punol, J . P. The role of cytokines in the development
of osteoarthritis. Scand. J . Rheumaol Suppl. 1990, 81, 8-12.
(6) Kirkham, B. Interluekin-1, Immune activation pathways and
different mechanisms in osteoarthitis and rheumatoid arthritis.
Ann. Rheum. Dis. 1991, 50, 395-400.
(7) Nickoloff, B. J . The cytokine network in psoriasis. Arch. Der-
matol. 1991, 135, 1104.
(8) Saklavala, J .; Davis, W.; Guesdon, F. Interleukin 1 (IL-1) and
tumor necrosis factor (TNF) signal transduction. Philos. Trans.
R. Soc. London B. 1996, 351, 151-157.
(9) Sacca, R.; Cuff, C. A.; Ruddle, N. H. Mediators of inflammation.
Curr. Opin. Imm. 1997, 9, 851-857.
(10) Lewis, A. J .; Manning, A. M. New targets for antiinflammatory
drugs. Curr. Opin. Chem. Biol. 1999, 3, 489-494.
(11) Sekut, L.; Connolly, K. M. Pathophysiology and regulation of
TNFa in inflammation. DN&P 1996, 9, 261-269.
(12) Elliott, M. J .; Maini, R. N. New directions for biological therapy
in rheumatoid arthritis. Int. Arch. Allergy. Immunol. 1994, 104,
112-115.
(13) Moreland, L. W.; Heck, L. W., J r.; Koopam, W. J . Biological
therapy in rheumatoid arthritis: Concepts and progress. Ar-
thritis Rheum. 1997, 40, 397-409.
(14) Lee, J . C.; Kumar, S.; Griswold, D. E.; Underwood, D. C.; Votta,
B. J .; Adams, J . L. Inhibition of p38 MAP kinase as a therapeutic
strategy. Immunopharmacol. 2000, 47, 185-201.
(15) Lee, J . C.; Laydon, J . T.; McDonnell, P. C.; Gallagher, T. F.;
Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J .; Heys, J .
R.; Landvatter, S. W.; Strickler, J . E.; McLaughlin, M. M.;
Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J . R.; Adams,
J .; Young, L.; Young, P. R. A protein kinase involved in the
regulation of inflammatory cytokine biosynthesis. Nature 1994,
372, 739-746.
(16) Young, P. R.; McDonnell, P.; Dunnington, D.; Hand, A.; Laydon,
J .; Lee, J . C. Pyridinyl imidazoles inhibit IL-1 and TNF produc-
tion at the protein level. Agents Actions 1993, 39, C67-C69.
(17) Han, J .; Lee, J . D.; Bibbs, L.; Ulevitch; R. J . A MAP kinase
targeted by endotoxin and hyperosmolarity in mammalian cells.
Science 1994, 265, 808-811.
(18) Lee, J . C.; Young, P. R. Role of CSBP/p38/RK stress response
kinase in LPS and cytokine signaling mechanisms. J . Leukoc.
Biol. 1996, 59, 152-157.
(19) Raingeaud, J .; Whitmarsh, A. J .; Barrett, T.; Derijard, B.; Davis,
R. J . MKK3- and MKK6-regulated gene expression is mediated
by the p38 mitogen-activated protein kinase signal transduction
pathway. Mol. Cell. Biol. 1996, 16, 1247-1255.
(41) Buchwald, S. L.; Wolfe, J . P.; A° hman, J .; Sadighi, J . P.; Singer,
R. A. An ammonia equivalent for the palladium-catalyzed
amination of aryl halides and triflates. Tetrahedron Lett. 1997,
38, 6367-6370.
(42) Gorvin, J . H. The synthesis of di- and tri-arylamines through
halogen displacement by base-activated arylamines: Compari-
son with the Ullmann condensation. J . Chem. Soc., Perkin Trans.
1 1988, 1331-1335.
(43) Buchwald, S. L.; Wolfe, J . P. Improved functional group compat-
ibility in the palladium-catalyzed amination of aryl bromides.
Tetrahedron Lett. 1997, 38, 6359-6362.

5662 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Ottosen et al.
(44) Ragan, J . A.; Makowski, T. W.; Castaldi, M. J .; Hill, P. D. 2,5-
Dimethylpyrrole protection facilitates cupper(I)-mediated meth-
oxylations of aryl iodides in the presence of anilines. Synthesis
1998, 11, 1599-1603.
istry 1998, 37, 16573-16581.
(52) Liu, Y.; Bishop, A.; Witucki, L.; Kraybill, B.; Shimizu, E.; Tsien,
J .; Ubersax, J .; Blethrow, J .; Morgan, D. O.; Shokat, K. M.
Structural Basis for Selective Inhibition of Src Family Kinases
by PP1. Chem. Biol. 1999, 6, 671-678.
(45) Gribble, G. W.; Kelly, W. J .; Sanford, E. Reactions of sodium
borohydride in acidic media; VII. Reduction of diaryl ketones in
trifluoroacetic acid. Synthesis 1978, 10, 763-765.
(53) Schindler, T.; Sicheri, F.; Pico, A.; Gazit, A.; Levitzki, A.;
Kuriyan, J . Crystal Structure of Hck in Complex with a Src
Family-Selective Tyrosine Kinase Inhibitor. Mol. Cell. 1999, 3,
639-648.
(54) Gum, R. J .; McLaughlin, M. M.; Kumar, S.; Wang, Z.; Bower,
M. J .; Lee, J . C.; Adams, J . L.; Livi, G. P.; Goldsmith, E. J .;
Young, P. R. Acquisition of Sensitivity of Stress-activated Protein
Kinases to the p38 Inhibitor, SB 203580, by Alteration of One
or More Amino Acids within the ATP Binding Pocket. J . Biol.
Chem. 1998, 273, 15605-15610.
(55) Toledo, L. M.; Lydon, N. B.; Elbaum, D. The Structure-Based
Design of ATP-site Directed Protein Kinase Inhibitors. Curr.
Med. Chem. 1999, 6, 775-805.
(56) Skak-Nielsen, T.; Tranholm, M.; Hansen, J . R. Unpublished
results.
(57) Grabbe, S.; Schwarts, T. Immunoregulatory mechanisms in-
volved in elicitation of allergic contact hypersensitivity. Rev.
Immunol. Today 1998, 19, 37-44.
(58) Alford, J . G.; Stanley, P. L.; Todderud, G.; Tramposch, K. M.
Temporal infiltration of leukocyte subsets into mice skin in-
flamed with phorbol ester. Agents Action 1992, 37, 260-267.
(46) J iang, Y.; Gram, H.; Zhao, M.; Gu, J .; Feng, L.; Padova, F. D.;
Ulevitch, R. J .; Han, J . Characterization of the structure and
function of the fourth member of the p38 group mitogen-
activated protein kinase, p38δ. J . Biol. Chem. 1997, 272, 30122-
30128.
(47) Fjording, M. S. Unpublished results
(48) Mene´ndez, M.; Laynez, J .; Medrano, F. J .; Andreu, J . M. A
Thermodynamic Study of the Interaction of Tubulin with Colchi-
cine Site Ligands. J . Biol. Chem. 1989, 264, 16367-16371.
(49) Wang, Z.; Canagarajah, B. J .; Boehm, J . C.; Kassisa`, S.; Cobb,
M. H.; Young, P. R.; Abdel-Meguid, S.; Adams, J . L.; Goldsmith,
E. J . Structural Basis of Inhibitor Selectivity in MAP Kinases.
Structure 1998, 6, 117-1128
(50) Wilson, K. P.; McCaffrey, P. G.; Hsiao, K.; Pazhanisamy, S.;
Galullo, V.; Bemis, G. W.; Fitzgibbon, M. J .; Caron, P. R.;
Murcko, M. A.; Su, M. S. S. The Structural Basis for the
Specificity of Pyridinylimidazole Inhibitors of p38 MAP Kinase.
Chem. Biol. 1997, 4, 423-431.
(51) Lisnock, J M.; Tebben, A.; Frantz, B.; O’Neill, E. A.; Croft,
G.; O’Keefe, S. J .; Li, B.; Hacker, C.; de Laszlo, S.; Smith,
A.; Libby, B.; Liverton, N.; Hermes, J .; LoGrasso, P. Molecu-
lar Basis for p38 Protein Kinase Inhibitor Specificity. Biochem-
J M030851S