2994
`J. Med. Chem.2002, 45,2994-3008
`Pyrazole Urea-Based Inhibitors of p38 MAP Kinase: From Lead Compound to
`Clinical Candidate
`John Regan,*,† Steffen Breitfelder,† Pier Cirillo,† Thomas Gilmore,† Anne G. Graham,‡ Eugene Hickey,†
`Bernhard Klaus,† Jeffrey Madwed,§ Monica Moriak,† Neil Moss,† Chris Pargellis,‡ Sue Pav,‡ Alfred Proto,‡
`Alan Swinamer,† Liang Tong,† and Carol Torcellini§
`Departments of Medicinal Chemistry, Biology, and Pharmacology, Boehringer Ingelheim Pharmaceuticals,
`Research and Development Center, 900 Ridgebury Road, Ridgefield, Connecticut 06877
`
`Received February 6, 2002
`
`We report on a series of N-pyrazole, N′-aryl ureas and their mode of binding to p38 mitogen
`activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine
`5′-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in
`part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen
`bonding interactions between this class of inhibitors and the protein. We describe the correlation
`of the structure-activity relationships and crystallographic structures of these inhibitors with
`p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond
`at the ATP binding site. This modification affords significant improvements in binding, cellular,
`and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the
`treatment of inflammatory diseases.
`In addition to the discovery of this important sig-
`Introduction
`nal
`transduction pathway, pyridinyl
`imidazole 1
`The proinflammatory cytokines tumor necrosis fac-
`(SB 203580)11 and analogues18-22 have been identified
`tor-R (TNF-R) and interleukin-1â (IL-1â) help regulate
`as potent and selective inhibitors of p38 MAP kinase.
`the body’s response to infections and cellular stresses.1
`Compound 1 was shown to be an effective orally active
`However, the pathophysiological consequences resulting
`agent in several animal models of acute and chronic
`from chronic and excessive production of TNF-R and IL-
`inflammation.23 Recently, an analogue of 1, compound
`1â are believed to underlie the progression of many
`2 (SB 242235), inhibited endotoxin-induced ex vivo
`inflammatory diseases such as rheumatoid arthritis
`production of TNF-R and IL-1â in human clinical
`(RA),2 Crohn’s disease, inflammatory bowel disease, and
`trials.24 The interest in p38 as a viable target for drug
`psoriasis.3 Recent data from clinical trials have secured
`intervention has escalated as a result of these early
`the continued use of the soluble TNF-R receptor fusion
`disclosures. In addition to a plethora of patent applica-
`protein, etanercept, or the chimeric TNF-R antibody,
`tions on imidazole-based compounds,25-27 several jour-
`infliximab, in the treatment of RA4-8 and Crohn’s
`nal papers have described strategies for the modification
`disease.9,10 The signal transduction pathway leading to
`of 1, by either the addition of other substituents to the
`the production of TNF-R from stimulated inflammatory
`imidazole or its replacement with different heterocycles.
`cells, while not fully understood, has been shown to be,
`in part, regulated by p38 mitogen activated protein
`These endeavors have produced imidazoles 328 and 4
`(MAP) kinase.11 p38 MAP kinase belongs to a group of
`(RPR200765A),29 a pyrrole analogue of 1,30 oxazole
`serine/threonine kinases that includes c-Jun NH2-
`5,31and pyrrolo[2,3-b]pyridine 6 (RWJ 68354).32 Imid-
`terminal kinase (JNK) and extracellular-regulated pro-
`azole 7 (RWJ 67657)33 was described to inhibit LPS-
`tein kinase (ERK).12 Upon extracellular stimulation by
`stimulated TNF-R production in human clinical
`a variety of conditions and agents,13 p38 is activated
`trials.34 Also, compounds with different structural fea-
`through bis-phosphorylation on a Thr-Gly-Tyr motif
`tures as compared to 1 have been reported as p38
`located in the activation loop. Activation is achieved by
`inhibitors. These include, among others, 8 (VX-745)35
`dual-specificity serine/threonine MAPK kinases, MKK3
`and N,N-diaryl urea 8a36 as well as pyrazole ketone 9
`and MKK6. Once activated, p38 can phosphorylate and
`(RO3201195)37 and pyrimido[4,5-d]pyrimidinone 10.38
`activate other kinases or transcription factors leading
`Indole amide 11 represents another group of p38 inhibi-
`to stabilized mRNA and an increase or decrease in the
`tors.39 A benzophenone class of p38 inhibitors, an
`expression of certain target genes.14-17
`example shown as 12 (EO1428), has recently been
`described.40 Diamides (13) are disclosed as p38 inhibi-
`* To whom correspondence
`should
`be
`tors41 (Chart 1).
`(203)798-4768.
`Fax:
`(203)791-6072.
`Our focus in cytokine-regulated approaches to inflam-
`rdg.boehringer-ingelheim.com.
`† Department of Medicinal Chemistry.
`matory diseases prompted us to evaluate the potential
`‡ Department of Biology.
`of p38 MAP kinase as a therapeutic target. Toward this
`§ Department of Pharmacology.
`10.1021/jm020057r CCC: $22.00 © 2002 American Chemical Society
`Published on Web 05/25/2002
`
`addressesd. Tel.:
`E-mail:
`jregan@
`
`FUSTIBAL Ex. 1010
`
`

`
`Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
`Chart 1. Structural Classes of p38 MAP Kinase Inhibitors
`
`
`
`Journal of Medicinal Chemistry, 2002, Vol. 45, No. 142995
`
`Table 1. Substitution at Pyrazole N-2
`
`Scheme 1a
`
`(a) Phenylhydrazine, toluene, reflux or aqueous
`aReagents:
`HCl, ethanol, reflux. (b) 4-Chlorophenyl
`isocyanate, THF or
`CH2Cl2, 25 °C.
`14, coupled with its distinction as a new structural type
`of inhibitor vs others (e.g., 1-13) prompted us to under-
`take a systematic evaluation of its pharmacophores. The
`structure-activity relationships (SAR) for this class of
`compounds and their correlation to structural data,
`which led to the discovery of the clinical candidate BIRB
`796,45 are the subjects of this paper.
`Chemistry
`Modifications to the 2-position of the pyrazole nucleus
`were prepared as shown in Scheme 1 using 16 as a
`representative example of the compounds in Table 1.
`The assembly of pyrazole nucleus 15 involved the
`condensation of phenylhydrazine and 4,4-dimethyl-3-
`oxopentanenitrile in either toluene or aqueous HCl in
`ethanol at reflux. Urea formation was accomplished
`with 15 and 4-chlorophenyl isocyanate to produce 16.
`For examples in Table 1 requiring noncommercially
`available aryl hydrazines, the method of Demers46 was
`used to convert aryl halides to aryl hydrazines. The
`cyclohexylhydrazine that was used in the synthesis of
`
`end, compound 14 was identified from high throughput
`screening. Reports on utilizing this compound as a lead
`have been disclosed.42-44 While 14 showed only a modest
`binding affinity for human p38 MAP kinase (Kd ) 350
`nM) (Table 1), our interest in this molecule further
`increased upon obtaining a cocrystal structure with
`recombinant human p38. The unique binding mode of
`
`

`
`2996 Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14
`Scheme 3a
`Table 2. Substitution at Pyrazole C-5
`
`Regan et al.
`
`Scheme 2a
`
`aReagents: (a) CNCH2CO2H, n-BuLi, THF, CH2Cl2, -70 °C and
`then 25 °C. (b) Phenylhydrazine, toluene, reflux. (c) THF, 25 °C.
`(d) LDA, THF, MeI, -78 °C. (e) NaH, CH3CN, THF, 75 °C.
`target compound 47 was obtained from the sodium
`cyanoborohydride-mediated reductive hydrazination of
`cyclohexanone with hydrazine.47
`Compound 46 was used as a frame of reference for
`probing the SAR at the 5-position of pyrazole by re-
`placing the t-butyl moiety (Table 2). This effort required
`the construction of a diverse set of oxopentanenitrile
`subunits. Scheme 2 outlines two general procedures to
`prepare oxopentanenitrile derivatives. Briefly, the ad-
`dition of the dianion of cyanoacetic acid48 to acid
`chlorides (e.g., 17) or the anion of acetonitrile49 to esters
`(e.g., 22) supplied the â-keto nitrile components. Fol-
`lowing the chemistry described in Scheme 1, pyrazole
`formation and urea couplings were completed for target
`20 as well as the other compounds in Table 2.
`To evaluate the role of the urea linkage to the binding
`of p38, the synthesis of several urea analogues was
`undertaken. The biological role of each of the urea N-H
`groups in 16 was examined by replacement with CH2
`(25 and 31) and N-methyl (34 and 37). Amides 25 and
`31 were prepared as shown in Scheme 3. EDC-mediated
`condensation of aminopyrazole 15 and 4-chlorophenyl-
`acetic acid (24) furnished amide 25. Amide 31, however,
`required the construction of pyrazole acetic acid 30.
`Thus, pyrazolidinone 26 was converted to its O-triflate
`derivative 27, which underwent Stille cross coupling
`with tributyl(vinyl)tin to give vinyl pyrazole 28. Regio-
`selective hydroboration of 28 produced alcohol 29, which
`was converted to the desired carboxylic acid 30 with
`Jones reagent. Amide bond formation between 30 and
`4-chloroaniline with DCC furnished 31. The syntheses
`of N-methyl urea analogues 34 and 37 were undertaken
`as follows. Exposure of aminopyrazole 15 to phosgene50
`
`aReagents: (a) EDC, CH2Cl2. (b) Tf2O, DTBMP, CH2Cl2, -78
`to 0 °C. (c) Tributyl(vinyl)tin, Pd[P(Ph)3]4, LiCl, dioxane, 100 °C.
`(d) (i) 9-BBN, THF, reflux. (ii) NaOH, H2O2. (e) Jones reagent. (f)
`4-Chloroaniline, DCC, DMAP, CH2Cl2. (g) COCl2, CH2Cl2, aqueous
`NaHCO3. (h) CH2Cl2, 25 °C. (i) HCO2H, reflux. (j) BH3-DMS, THF,
`25 °C. (k) 4-Chlorophenyl isocyanate, CH2Cl2, 25 °C. (l) 4-Chloro-
`phenyl isothiocyanate, CH2Cl2, 25 °C.
`produced pyrazole isocyanate 32, which was coupled
`with N-methyl-4-chloraniline (33) to provide N-methyl
`urea 34. Alternatively, aminopyrazole 15 was heated
`with formic acid to produce N-formyl aminopyrazole 35,
`which upon reduction with borane51 yielded N-methyl-
`aminopyrazole 36. Treatment of 36 with 4-chlorophenyl
`isocyanate produced N-methyl urea analogue 37. Thio-
`urea 38 served as a basis to understand the part that
`the O-atom plays in p38 binding, and its preparation
`was accomplished by treatment of aminopyrazole 15
`with 4-chlorophenyl isothiocyanate.
`The compounds designed to explore the region of the
`urea phenyl of 46 are summarized in Table 4. They were
`conveniently obtained by the treatment of pyrazole
`isocyanate 32 with aniline derivatives or alkylamines.
`For example, as shown in Scheme 4, exposure of
`isocyanate 32 to 2-aminoindan (39) furnished urea 40.
`Other target ureas were prepared according to Scheme
`1 wherein amine 15 was coupled to aryl isocyanates.
`To access target compounds with groups attached
`to the 4-position of the urea naphthalene, the route
`shown in Scheme 5 was utilized. Alkylation of N-Boc
`naphthol 41, prepared from 4-amino-1-naphthol with
`4-(2-chloroethyl)morpholine, gave ether 42. Removal of
`the Boc protecting group (43) and urea formation, as
`described above with the isocyanate derived from 44,
`gave 45.
`
`

`
`Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
`Table 3. Urea Modifications
`
`Scheme 4a
`
`
`
`Journal of Medicinal Chemistry, 2002, Vol. 45, No. 142997
`
`aReagents: (a) CH2Cl2, 25 °C.
`Gly170 (DFG) of the kinase is required for the observed
`binding mode of the diaryl urea inhibitor (Figure 2). In
`all of the currently known protein Ser/Thr kinase
`structures, the residues assume a conformation such
`that the Phe side chain is buried in a hydrophobic pocket
`in the groove between the two lobes of the kinase (DFG-
`in conformation). In the structure of the complex with
`compound 14, however, the Phe side chain has moved
`by about 10 Å to a new position (DFG-out conformation).
`In this position, one face of the Phe side chain and the
`urea phenyl ring are involved in hydrophobic interac-
`tions whereas the other face is exposed to solvent. This
`movement of the Phe side chain reveals a large hydro-
`phobic domain in the kinase, and the tert-butyl group
`of 14 inserts deep into this pocket (Figure 2). Neither
`nitrogen atom on the pyrazole ring participates in
`specific hydrogen-bonding interactions with the kinase.
`As shown in Figure 3, the urea of 14 establishes a
`bidentate hydrogen bond with the conserved side chain
`of Glu71.
`Most protein kinase inhibitors use the ATP binding
`pocket and inhibit the kinase by directly competing with
`the binding of ATP. In contrast, compound 14 does not
`compete directly with ATP binding, as it has no struc-
`tural overlap with the ATP molecule (Figure 2). How-
`ever, our structure shows that the DFG-out conforma-
`tion impedes ATP binding, as the side chain of the Phe
`residue would be sterically incompatible with the phos-
`phate groups of ATP (Figure 2). This is supported by
`our observation that compound 14 interferes with the
`inactivation of p38 MAP kinase activity by the fluores-
`cent ATP analogue 5′-p-fluorosulfonyl benzoyl adenosine
`(data not shown). Therefore, the diaryl urea compounds
`inhibit p38 MAP kinase by stabilizing a conformation
`of the kinase that is incompatible with ATP binding.
`The data in Tables 1 and 2 highlight the binding roles
`of the groups appended to the pyrazole nucleus of 14s
`methyl at N-2 and tert-butyl at C-5. Fortuitously, our
`first modification, replacement of the methyl of 14 with
`a phenyl group (16), improved binding potency 40-fold
`(Table 1) as measured in a fluorescent binding assay.
`The crystal structure of 16 and the recombinant human
`p38 complex (Figure 4) help rationalize this result. The
`phenyl ring at N-2 of the pyrazole participates in
`lipophilic interactions with the alkyl portion of the side
`chain of the Glu71 residue. In addition, the phenyl ring
`may serve as a water shield for the hydrogen bond
`network of the urea and the Glu71 carboxylate. The
`presence of the phenyl ring causes this Glu residue to
`adopt a side chain conformation that results in a
`monodentate hydrogen-bonding interaction with the
`urea moiety of the inhibitor. This alignment of Glu71
`is in contrast to the bidentate interactions in the
`complex with 14 (Figure 3).
`Further profiling of this key region in the inhibitor
`helped establish the preferred substitution at N-2 of the
`
`Table 4. Modification of Urea-Phenyl Ring
`
`Results and Discussion
`We recently reported the crystal structure of recom-
`binant human p38 MAP kinase in complex with com-
`pound 14 at 2.5 Å resolution (Figure 1).45 Interestingly,
`the crystal structure reveals that this compound utilizes
`binding interactions on the kinase that are spatially
`distinct from the adenosine 5′-triphosphate (ATP) pocket.
`There is no structural overlap between the atoms of
`compound 14 and the ATP (Figure 2). Similarly, there
`is only limited spatial overlap between 14 and an iodo
`analogue of SB203580 (1a)52 and this occurs in a
`lipophilic pocket commonly referred to in the kinase field
`as the specificity pocket (Figure 3). A large conforma-
`tional change for conserved residues Asp168-Phe169-
`
`

`
`2998 Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14
`Scheme 5a
`
`Regan et al.
`
`aReagents: (a) Di-tert-butyl dicarbonate, THF, 25 °C. (b) 4-(2-Chloroethyl)morpholine, K2CO3, acetonitrile, heat. (c) HCl, dioxane. (d)
`Compound 44, phosgene, THF.
`
`Figure 1. Crystal structure of human p38 MAP kinase and
`14 at 2.5 Å resolution.
`pyrazole and confirmed our hypothesis regarding bind-
`ing interactions at this domain. The diminished potency
`of saturated derivative 47 highlighted the necessity for
`an aromatic ring to achieve optimal hydrophobic inter-
`actions (Table 1). Addition of methyl groups to the 3-
`and 4-position of the phenyl ring of 46 provided modest
`improvements in binding (49-51). However, 2-methyl
`derivative 48 displayed a substantial loss of binding
`affinity possibly due to an increase in the torsional angle
`favored between the phenyl and the pyrazole rings
`beyond the observed angle of 54° for 16 (Figure 4). This
`position tolerates bulkier groups as judged by the
`binding potency of 2-naphthyl analogue 52. In addi-
`tion, heteroatoms (53-57) can be accommodated at this
`site. The close proximity of the 3- and 4-positions of
`the phenyl ring of 46 to solvent may explain these
`results.
`In Figure 4, the tert-butyl group at C-5 of pyrazole
`16 is embedded deep into a hydrophobic pocket formed
`by the reorganization of Phe169 in the DFG-out con-
`formation. In an effort to understand the binding role
`of the tert-butyl moiety in this class of compounds, we
`investigated the size and electronic requirements of this
`group. As can be seen in Table 2, removal of one methyl
`group lowered potency over 20-fold (cf. 46 vs 59).
`Further reduction to a methyl resulted in an inactive
`
`Figure 2. Overlap of 14 (blue) and ATP (red). The urea
`hydrogen atoms are shown for clarity. Phe169 is shown in red
`when occupying the DFG-in conformation (ATP bound) and
`in blue in the DFG-out conformation when 14 is bound to p38.
`compound (58). This lipophilic binding pocket tolerated
`bulkier tert-alkyl groups such as dimethylethyl (60) and
`methylcyclohexyl (20). However, the 50-fold loss of
`binding observed with dimethylbenzyl analogue 64 may
`indicate a size limitation for this domain. A comparison
`of cyclohexyl derivatives 62 and 20 further exemplifies
`the strong preference for a tertiary group. The relatively
`poor activity of compounds 61 and 63 as compared to
`60 and 20 suggest that lipophilic substitution at C-5 of
`the pyrazole is favored. Taken together, these results
`are rationalizable based on the crystal structure of 16
`and p38 (Figure 4) that indicate a lipophilic group at
`C-5 of the pyrazole has important hydrophobic binding
`interactions with the protein in the DFG-out conforma-
`tion. The tert-butyl group was incorporated into all
`subsequent target molecules since it offered the best
`balance of potency and physicochemical properties.
`The X-ray crystallographic structure of 16 with p38
`reveals a hydrogen bond network consisting of a urea
`hydrogen and the carboxylate oxygen of Glu71 and also
`the urea oxygen and N-H of Asp168. The data in Table
`3 highlight the significance of these interactions on
`binding affinity. Replacement of either N-H in the urea
`with a methylene group (compounds 25 and 31) or
`introduction of N-methyl (34 and 37) results in signifi-
`cant loss of activity. Likewise, the thiourea analogue 38
`shows a 60-fold decrease in binding potency to p38 as
`compared to 16. These findings underscore the crucial
`contribution that the urea makes to binding with p38
`through extensive hydrogen bonding and, also likely,
`to establishing the correct geometric relationships of the
`other pharmacophores of the inhibitor.
`
`

`
`Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
`
`
`
`Journal of Medicinal Chemistry, 2002, Vol. 45, No. 142999
`
`Figure 3. Two views of the overlap of 14 and 1a. The urea hydrogen atoms are shown for clarity. The urea phenyl group of 14
`occupies the same kinase specificity pocket in p38 as the phenyl ring of 1a. The bindentate hydrogen bond interaction between
`the urea hydrogen atoms and the carboxylate oxygens of Glu71 is shown.
`can also provide good binding affinity as seen with
`bicyclic indan derivatives (40 and 77). Thus, these data
`demonstrate that the kinase specificity pocket of p38
`favors lipophilic pharamacophores that are not limited
`to phenyl rings.
`We examined several compounds from this series for
`oral activity in a mouse model of LPS-stimulated TNF-R
`synthesis. Compound 50 (Table 5) furnished an inter-
`esting and important result. The tolyl group on 50
`provided a 100-fold increase in plasma concentration in
`the mouse vs 46, which lacks this substitution. The
`increased plasma levels in combination with a modest
`improvement in cellular activity provided our first orally
`active compound. Of the 4-methylphenyl derivatives
`examined in this model, analogue 78 showed the best
`in vivo profile. This compound, with even higher plasma
`concentrations than 50, suppressed TNF-R production
`by 90% when dosed at 100 mg/kg and also was active
`at 30 mg/kg (53% inhibition).
`Unfortunately, we were unable to improve the in vitro
`and in vivo activities of the phenyl-based urea inhibitors
`beyond that of compound 78. Despite available crystal-
`lographic data, obvious solutions to achieve additional
`binding interactions were not realized. A breakthrough
`arrived upon establishing a binding assay having more
`sensitivity for compounds whose binding activity was
`near the limit of the fluorescence assay.45 One observa-
`tion from this new assay suggested that 79 was more
`potent than initially thought. Table 6 shows selected
`examples of the Kd values from the fluorescence assay
`vs the exchange curve assay. Remarkably, naphthyl
`compound 79 binds 20-fold more tightly to p38 than
`phenyl analogue 50 despite similar cellular potencies.
`The overlap of the X-ray crystal structures of phenyl
`analogue 16 and naphthyl 75 (Figure 5) provides a
`possible explanation for this increased binding. The
`naphthyl moiety of 75 resides deep in the kinase
`specificity pocket and achieves substantial hydrophobic
`binding interactions with the protein that are not
`possible with the phenyl ring of 16. Thus, it seemed
`reasonable that the lack of improvement in cellular
`
`Figure 4. X-ray crystallographic structure of human p38 with
`16. The urea hydrogen atoms are shown for clarity. The
`hydrophobic effects of the pyrazole phenyl ring and the
`monodendate hydrogen bond of the urea N-H atoms with
`Glu71 are seen.
`As seen in Figure 3, the phenyl ring attached to the
`urea of 14 fits into the specificity pocket of p38 in a
`manner similar to the pyridinyl imidazole inhibitor 1a.52
`The importance of binding in this pocket to potency53
`and kinase specificity28 has been described for the
`imidazole-based group of inhibitors. Highlights of the
`prominent role that the phenyl ring plays in binding
`with this series of compounds are shown in Table 4.
`Removal of the urea phenyl ring results in complete loss
`of binding potency (cf. 46 and 65). Saturation of the
`phenyl ring (66) or separation of the ring from the urea
`by either one or two carbon atoms (73 and 74) resulted
`in decreased binding affinity. Incorporation of polar
`groups, through either pyridine (67-69) or aniline
`lowers potency. However,
`derivatives (70 and 71),
`lipophilic groups appended to the phenyl nucleus can
`improve potency (cf. 72 and 75). Other lipophilic groups
`
`

`
`3000 Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14
`Table 5. In Vivo Activity of Selected Pyrazole-Phenyl Ureas
`
`Regan et al.
`
`Table 6. Comparison of Phenyl vs Naphthyl Ureas with ATP
`Site Binding Pharmacophore
`
`Figure 5. Overlap of 16 (yellow) and 75 (green) with human
`p38 MAP kinase. The urea hydrogen atoms are shown for
`clarity. The naphthyl group of 75 fits deeper into the kinase
`specificity pocket as compared to the phenyl ring of 16. The
`4-position of the naphthalene ring points toward the hinge
`region and solvent. Phe169 is removed for clarity.
`activity of 79 might be a consequence of its higher
`lipophilicity as compared to 50.
`The crystal structure of 75, in addition to providing
`a rationale to the improved binding affinity of the
`naphthalene group, offered an opportunity to explore
`modifications from this platform that would be unavail-
`
`able from the phenyl ring of 16. That is, groups
`appended to the 4-position of the naphthalene could
`much more readily access the ATP binding region of p38
`than the 4-positon of the phenyl ring. Hence, pharma-
`cophores attached to the 4-position could have ad-
`ditional binding interactions or be used to improve
`physicochemical properties. The ethoxy morpholine
`group proved to be a very effective group for achieving
`both of these goals. This moiety improved binding
`potency 10-fold and, more significantly, increased cell
`activity over 20-fold. A crystal structure of 45 with
`recombinant human p38 (Figure 6a) provided an ex-
`planation for the enhanced potency.45 The gauche
`conformation of the ethoxy linker of 4554,55 effectively
`orients the morpholine group so that the morpholine
`oxygen can achieve a strong hydrogen bond with the
`N-H of Met109. This hydrogen bond is the same one
`used by the adenine base of ATP and the pyridine
`nitrogen of the pyridinyl imidazole class of compounds
`such as 1a.52 The favorable edge to π hydrophilic
`interactions of the phenyl ring of Phe169 and the
`naphthalene group of 45 is revealed in Figure 6b and
`likely further contributes to binding potency.
`To highlight the contribution of the naphthalene
`group to 45, we prepared phenyl derivative 80. Despite
`the inclusion of the ethoxy morpholine unit in phenyl
`analogue 80, it exhibited a dramatic loss in potency vs
`45 (Table 6). This result reinforces the importance of
`the naphthalene ring in providing better lipophilic
`interactions with the specificity pocket and properly
`aligning the ethoxy morpholine unit for productive
`binding with the ATP binding region.
`In addition to superior in vitro and cellular activities,
`compound 45 demonstrated enhanced in vivo potency.
`For example, in a mouse model of LPS-stimulated
`TNF-R synthesis, a 65% inhibition of TNF-R synthesis
`was observed when 45 was dosed orally at 10 mg/kg.
`In a 5 week model of established collagen-induced
`arthritis using B10.RIII mice, 45 produced a 63%
`inhibition of arthritis severity when dosed orally at 30
`mg/kg qd.56 Some pharmacokinetic data of 45 in mice
`and cynomolgous monkeys are summarized in Table 7.
`The selectivity profile against a panel of protein kinases
`for 45 was determined and is shown in Table 8.45 On
`the basis of these and other data, compound 45 (BIRB
`796) was selected for human clinical trials.
`
`

`
`Pyrazole Urea-Based Inhibitors of p38 MAP Kinase
`
`
`
`Journal of Medicinal Chemistry, 2002, Vol. 45, No. 143001
`
`(a) X-ray crystallographic complex of human p38 and 45. Phe169 is removed for clarity. (b) X-ray crystallographic
`Figure 6.
`complex of human p38 and 45 with Phe169.
`Table 7. Pharmacokinetic Properties of Compound 45
`
`Table 8. Selectivity Profile of Compound 45
`
`Conclusion
`We have shown that a series of N-pyrazole, N′-aryl
`ureas occupy a binding domain on p38 that is exposed
`when the conserved binding loop, consisting in part of
`Asp168-Phe169-Gly170, adopts a conformation (DFG-
`out) not previously noted in other protein Ser/Thr
`kinases. A 40-fold improvement in binding was achieved
`by the replacement of the methyl group in the original
`screening lead (14) with phenyl. The urea atoms, shown
`to be involved in an extensive hydrogen bond network
`with Glu71 and Asp168 (Figures 3 and 4), proved critical
`for binding activity. A toluene ring attached to the
`pyrazole nucleus was necessary to secure high plasma
`levels in the mouse. The naphthalene was a preferred
`pharmacophore as compared to phenyl to bind in the
`kinase specificity pocket. We added an ethoxy morpho-
`line pharmacophore, which successfully extended the
`binding of the inhibitor to also include a hydrogen-
`bonding interaction in the ATP binding region of p38.
`This modification afforded significant improvements in
`binding affinity, cellular activity, and in vivo reduction
`of TNF-R production and arthritis severity that resulted
`
`in the selection of 45 (BIRB 796) as a clinical candidate
`for the treatment of inflammatory diseases.45,57
`Experimental 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-Johns melting point apparatus and are uncorrected.
`1H nuclear magnetic resonance (NMR) spectrum were recorded
`on either a Bruker AC-F-270 spectrometer or Bruker Avance
`DPX 400 spectrometer. Chemical shifts are reported in parts
`per million (δ) from the tetramethylsilane resonance in the
`indicated solvent. Mass spectra were obtained from a Finni-
`gan-SSQ7000 spectrometer. Samples were generally intro-
`duced by particle beam and ionized with NH4Cl. Thin-layer
`chromatography (TLC) analytical separations were conducted
`with E. Merck silica gel F-254 plates of 0.25 mm thickness
`and were visualized with UV or I2. Flash chromatographies
`were performed according to the procedure of Still et al. (EM
`Science Kieselgel 60, 70-230 mesh). Elemental analysis were
`performed at Quantitative Technologies, Inc., Whitehouse, NJ.
`1-(5-tert-Butyl-2-phenyl-2H-pyrazol-3-yl)-3-(4-chloro-
`phenyl)urea (16). A solution of phenyl hydrazine (0.83 mL,
`8.39 mmol) and 4,4-dimethyl-3-oxo-pentanenitrile (1.0 g, 8.0
`mmol) in toluene (3 mL) was heated to reflux overnight.
`Removal of the volatiles in vacuo provided a residue, which
`was purified by silica gel chromatography using 50% ethyl
`acetate in hexanes as the eluent. Concentration in vacuo of
`the product-rich fractions provided 3-amino-5-tert-butyl-2-
`phenyl-2H-pyrazole (15) as a light orange solid (1.53 g, 89%).
`A solution of 15 (0.058 g 0.27 mmol) and 4-chlorophenyl
`isocyanate (0.038 g, 0.25 mmol) in CH2Cl2 (1 mL) was stirred
`overnight at room temperature under inert atmosphere.
`Removal of the volatiles in vacuo provided a residue, which
`was triturated with 50% dichloromethane in hexanes (2 mL).
`The urea (16) was filtered and dried in vacuo to afford 0.078
`g (85%) and was then recrystallized from methanol to afford
`analytically pure material; mp 202-203 °C. 1H NMR (400
`
`

`
`3002 Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14
`Regan et al.
`MHz, dimethyl sulfoxide (DMSO)-d6): δ 1.27 (s, 9H, tert-butyl),
`Trifluoromethanesulfonic Acid 5-tert-Butyl-2-phenyl-
`6.36 (s, 1H, pyrazole), 7.28-7.30 (m, 2H, aromatic), 7.39-7.43
`2H-pyrazol-3-yl Ester (27). To a solution of 5-tert-butyl-2-
`(m, 3H, aromatic), 7.50-7.52 (m, 4H, aromatic), 8.42 (s, 1H,
`phenyl-2,4-dihydro-pyrazol-3-one (26, 5.12 g, 23.7 mmol) and
`urea), 9.12 (s, 1H, urea). MS (NH3-CI): m/e369 (MH+). Anal.
`2,6-di-tert-butyl-4-methyl pyridine (6.12 g, 29.8 mmol) in
`(C20H21ClN4O‚CH3OH) C, H, N.
`CH2Cl2 (50 mL) was added dropwise trifluoromethanesulfonic
`anhydride (4.4 mL, 7.4 g, 26 mmol) at -78 °C. The resulting
`1-[5-(1-Methylcyclohexyl)-2-phenyl-2H-pyrazol-3-yl]-3-
`solution was warmed to 0 °C, saturated NaHCO3 solution (100
`phenyl-urea (20). A solution of cyclohexane-1-methyl-1-
`mL) was added, and the mixture was stirred vigorously for 10
`carboxylic acid (1.31 g, 9.21 mmol), oxalyl chloride (5.5 mL of
`min. The layers were separated, and the aqueous layer was
`a 2.0 M solution in CH2Cl2, 11.05 mmol), and a drop of
`extracted with CH2Cl2 (3×). The combined organic layers were
`anhydrous dimethylformamide (DMF) in CH2Cl2 (5 mL) was
`washed with brine and dried (Na2SO4). Removal of the volatiles
`heated to reflux for 3 h and cooled to ambient temperature to
`in vacuo provided a residue, which was purified by flash
`give 17. In a separate flask to a solution of cyanoacetic acid
`chromatography eluting unpolar impurities with hexanes and
`(1.57 g, 18.4 mmol, freshly dried with MgSO4) and a catalytic
`subsequently eluting the product with hexanes:ethyl acetate
`amount of 2,2′-bipyridine in anhydrous tetrahydrofuran (THF)
`(20:1). Concentration in vacuo of the product-rich fractions
`(68 mL) at -70 °C under an inert atmosphere was added
`gave 8.19 g (99%) of yellow oil 27. 1H NMR (400 MHz,
`dropwise n-butyllithium (15 mL of a 2.5 M solution in hexanes,
`CDCl3): δ 1.34 (s, 9H, tert-butyl), 6.19 (s, 1H, pyrazole), 7.35
`37.2 mmol). The mixture was slowly warmed to 0 °C until a
`(dd, 3J1 ) 3J2 ) 7.4 Hz, 1H, phenyl), 7.4 (dd, 3J1 ) 3J2 ) 7.8
`persistent red-colored slurry was obtained. The mixture was
`Hz, 2H, phenyl), 7.54 (d, 7.96 Hz, 2H, phenyl).
`cooled to -70 °C, and 17 in CH2Cl2 was slowly added. The
`3-tert-Butyl-1-phenyl-5-vinyl-1H-pyrazole (28). A solu-
`mixture was slowly warmed to room temperature, stirred for
`1 h, and quenched with 2 N aqueous HCl. The aqueous layer
`tion of 27 (3.74 g, 10.7 mmol) in dioxane (90 mL) in a sealable
`was extracted twice with CHCl3. The combined organic layers
`tube was degassed under vacuum and charged with nitrogen.
`LiCl (2.91 g, 68.8 mmol) was added, and the mixture was
`were washed with saturated aqueous NaHCO3 and brine and
`degassed and charged with nitrogen again. Pd(PPh3)4 (0.491
`dried (MgSO4). Removal of the volatiles in vacuo provided a
`g, 0.425 mmol) was added, and the mixture was degassed and
`residue, which was purified by silica gel chromatography using
`ethyl acetate in hexanes as the eluent. Concentration in vacuo
`charged with nitrogen again. Tributyl(vinyl) tin (4.0 mL, 4.3
`of the product-rich fractions provided 1.26 g of 18. A mixture
`g, 14 mmol) was added, and the mixture was degassed and
`of 18 (0.80 g, 4.8 mmol) and phenylhydrazine (0.48 mL, 4.8
`charged with nitrogen again. The tube was sealed, and the
`mmol) in dry toluene (6 mL) was heated to reflux overnight.
`mixture was heated to 100 °C overnight. After it was cooled
`Removal of the volatiles in vacuo provided a residue, which
`to room temperature, the volatiles were removed in vacuo and
`was purified by silica gel chromatography. Concentration in
`the residue was purified by flash chromatography eluting
`vacuo of the product-rich fractions provided 1.10 g (90%) of
`unpolar im