Bioorganic & Medicinal Chemistry Letters 22 (2012) 6237–6241

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Pyrazolopyridine inhibitors of B-RafV600E. Part 4: Rational designand kinase selectivity profile of cell potent type II inhibitors

Steve Wenglowsky a, David Moreno a,⇑, Ellen R. Laird a, Susan L. Gloor a, Li Ren a, Tyler Risom a,Joachim Rudolph b, Hillary L. Sturgis a, Walter C. Voegtli a

a Array BioPharma, 3200 Walnut Street, Boulder, CO 80301, United Statesb Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 May 2012Revised 24 July 2012Accepted 1 August 2012Available online 10 August 2012

Keywords:B-RafV600E

Inactive conformationDFG-outStructure-based designKinase selectivity

0960-894X/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.bmcl.2012.08.007

⇑ Corresponding author.E-mail address: david.moreno@arraybiopharma.co

Cell potent inhibitors of B-RafV600E that bind to the kinase in the DFG-out conformation are reported.These compounds utilize the hinge-binding group and lipophilic linker from a previously disclosed seriesof B-RafV600E inhibitors that bind to the kinase in an atypical DFG-in, aC-helix-out conformation. This newseries demonstrates that DFG-out kinase inhibitors can be rationally designed from related inhibitorswhich utilize an unconventional binding mode. Kinase selectivity profiles are compared. The pattern ofkinase selectivity was found to be determined by the feature of the inhibitor which extends into the backpocket of the kinase and leads to the kinase conformation, rather than by the hinge-binding group orother minor modifications.

� 2012 Elsevier Ltd. All rights reserved.

HN

O

X

FNH

SO O

N

HNN

OMe

1: X = FB-Raf IC50 = 4.8 nMpERK IC50 = 19 nM

2: X = ClB-Raf IC50 = 1.7 nMpERK IC50 = 20 nM

Figure 1. B-RafV600E inhibitors 1 and 2.

The Ras/Raf/MEK/ERK (MAPK) signaling pathway transducessignals from cell surface receptors to the nucleus leading to cellularproliferation, differentiation and survival.1 Mutations in the BRAFgene may lead to the expression of constitutively active B-Raf ki-nase which results in amplification of the MAPK pathway. MutatedB-Raf is present in approximately 7% of all cancers and is most fre-quently associated with melanoma.2 Over 90% of the detectedmutations in B-Raf are a glutamic acid for valine substitution atresidue 600 (V600E).2 This mutation leads to constitutive kinaseactivity 500-fold greater than for B-RafWT and correlates with in-creased malignancy and decreased response to chemotherapy.3

The approval of vemurafenib in 2011 demonstrated that small-molecule inhibitors of B-RafV600E are an effective strategy for can-cer therapy.4

Using structure-based design, our laboratory discovered a seriesof potent and selective ATP-competitive B-RafV600E inhibitorswhich utilized 3-alkoxy pyrazolopyridine as a novel hinge-bindinggroup.5 Optimization led to compounds 1 and 2 (Fig. 1) which werehighly active against a broad panel of melanoma and colon cancercell lines driven by this activating mutation. The X-ray crystalstructure of 1 in complex with B-Raf revealed that the kinaseadopts the DFG-in conformation, in which the sulfonamide formshydrogen bonds with the backbone nitrogens of Asp594, Phe595and Gly596, and the propyl group occupies the small lipophilic

ll rights reserved.

m (D. Moreno).

pocket formed by an outward shift of the aC-helix.5 Few kinasesare known to achieve this peculiar variant of the DFG-in conforma-tion,6,7 and this likely contributes to the excellent selectivity ofinhibitors 1 and 2 towards the Raf kinase family.5

An additional feature of the DFG-in, aC-helix-out conformationis the disruption of the salt bridge between the catalytic lysine andthe conserved glutamic acid of the aC-helix (Lys483 and Glu501 inB-Raf). This renders the kinase in an inactive state despite the DFGtriad residing in the ‘in’ conformation. In the typical inactive kinasestate the conserved DFG triad at the beginning of the activationloop shifts to an ‘out’ conformation. As a result, the phenylalanineside chain vacates its position deep inside the protein and creates alipophilic pocket available to inhibitor binding. Compounds thatbind to the kinase in the DFG-out conformation are classified astype II inhibitors. Since only a subset of kinases can achieve thisconformation, these inhibitors tend to demonstrate distinct kinaseselectivity profiles8 and generally improved overall kinase selectiv-

HN NH

N

N

N N

N

HNHN

ON N

N

OCF3

N

N

NH

NH

OCl

CF3

O

N

O NH

Imatinib Nilotinib

Sorafenib

Figure 2. Clinically approved DFG-out kinase inhibitors.

Table 1B-RafV600E activity of 3-alkoxy pyrazolopyridines 3 and 4

HN

O

F

FNH

N

HNN

OMe

OR

Compd R B-Raf IC50a (nM) pERK IC50

a (nM)

3 H 39 >120004 3-C(CH3)2CN 2.3 180

a Values are means of at least two experiments.

6238 S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6237–6241

ity compared to typical DFG-in inhibitors.9 In addition, some of themost clinically and commercially successful kinase inhibitors re-side in the DFG-out conformation, including imatinib,10 nilotinib11

and sorafenib12 (Fig. 2). Thus, the conversion of the 3-alkoxy pyraz-olopyridine hinge-binding group and lipophilic linker of 1 and 2into a series of B-RafV600E type II inhibitors was investigated and ki-nase selectivity profiles between the two series were compared.

The established type II pharmacophore consists of two mainparts.13 The first is a hinge-binding scaffold which forms up tothree hydrogen bonds to the hinge region of the kinase and makeslipophilic interactions to the adenine region of the ATP bindingpocket. This feature is shared in common with type I inhibitors.The second is a moiety that is comprised of a hydrogen bonddonating and accepting pair (usually a urea, amide or amide bioiso-stere) attached to a lipophilic group that occupies the pocketformed from the displacement of the phenylalanine side chain.The hinge binder and donor–acceptor pair can be connectedthrough a linker that resides in a pocket adjacent to the gatekeeperresidue (Thr529 in B-Raf). As a result, type II inhibitors are more of-ten observed for kinases with smaller gatekeeper residues such asthreonine or valine.

In an extension of the hybrid-design approach described by Liuand Gray, in which a type II tail group is grafted onto a type I hinge-binding scaffold,8,13 the propyl sulfonamide of compound 1 wasmodified to satisfy the type II pharmacophore. Replacement ofthe sulfonamide with an amide, and propyl with phenyl, led tobenzamide 3 (Fig. 3). The amide of 3 was predicted to form the re-quired hydrogen bonds to the conserved Glu501 of the aC-helixand the backbone amide of Asp594 from the DFG triad, while thephenyl ring would occupy the hydrophobic back pocket. The goodbiochemical activity of 3 against B-RafV600E supported this hypoth-esis (Table 1).14 However, improvements in cellular activity werenecessary and substitution of the benzamide was examined next.The 3-(2-cyanopropan-2-yl) group has been reported to be an opti-

HN

O

F

FNH

SO O

N

HNN

OMe 1

Figure 3. Proposal of DFG-out B-R

mized substituent in related type II inhibitors of B-Raf.15 Incorpo-ration of this group led to compound 4 with a significantimprovement in cellular activity over 3, and a new lead compoundthat was only 10-fold less active than sulfonamides 1 and 2.

Recently, we reported a study which demonstrated that 3-alk-oxy pyrazolopyridine hinge-binders provided more potent inhibi-tors of B-RafV600E than pyrrolopyridine.16 This study wasperformed across a broad range of inhibitors which bound to thekinase in the DFG-in, aC-helix-out conformation. It was of interestto know whether similar SAR existed for the current series of DFG-out inhibitors in light of the reorganization of residues near theATP-binding pocket. Three additional hinge-binding groups wereexamined, and their potencies are compared in Table 2. Pyrrolo-pyridine 5 was less active than 3-methoxy pyrazolopyridine 4 inboth the enzymatic and cellular assays, a result which indicateda parallel SAR to the earlier study. Pyrazolopyridine 6, with no sub-stituent at the 3-position of the hinge-binding scaffold, was alsoless potent than 4. It had been previously demonstrated that 3-cyclopyropyl was nearly equipotent to 3-methoxy for B-RafV600E

HN

O

F

FNN

HNN

OMe

O

3

H

O-

Glu501

O

NAsp594

H

afV600E inhibitor 3 based on 1.

Table 2B-RafV600E activity of pyrrolo-pyrazolopyridines 4–7

HN

O

F

FNH

N

HNA

R

O

CN

Compd A R B-Raf IC50a (nM) pERK IC50

a (nM)

4 N OMe 2.3 1805 CH H 6.8 4406 N H 3.5 430

7 N 3.0 520

a Values are means of at least two experiments.

S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6237–6241 6239

inhibitors that utilize the DFG-in, aC-helix-out conformation.17 Inthe present series however, compound 7 was threefold less activethan 4 in the cellular assay, despite similar activity in the enzy-matic assay. Thus, 3-alkoxy pyrazolopyridine proved to be thesuperior hinge-binding group for potency against B-RafV600E acrosstwo series of compounds that inhibit this kinase by two distinctbinding modes.

An X-ray co-crystal structure of compound 4 with B-Raf was ob-tained (Fig. 4).18 The expected interactions with the hinge regionand Thr529 were seen, and the DFG-out binding mode was ob-served including the two critical hydrogen bonds to Glu501 andAsp594. In addition, the benzamide carbonyl was observed to beonly 2.6 Å away from the fluoro at the 2-position of the centralphenyl ring. Although a fluoro or chloro substituent at this positionwas shown to be important for the potency of the sulfonamide ser-ies of B-RafV600E inhibitors,16 this close interatomic distance none-theless suggested that removal of this substituent might improvethe potency of the DFG-out series. SAR on this central ring was nextcarried out to test this hypothesis.

As hypothesized, removal of the fluoro substituent adjacent tothe amide carbonyl improved enzymatic activity and, significantly,led to a ninefold improvement in cellular activity (8 vs 4, Table 3).

Figure 4. X-ray crystal structure of 4 in complex with B-RafWT. The cleft surface is reHydrogen-bonding interactions are illustrated with yellow dashed lines. The inter-atom

Removal of both fluoro substituents led to compound 9 and re-sulted in a sevenfold loss in cellular activity compared to 8,although enzyme activity remained unchanged. The single 2-fluorosubstitution on the central ring of compound 10 resulted in the ex-pected loss of both enzymatic and cellular activity. The 3-(2-cyano-propan-2-yl) group on the benzamide ring of 8 was replaced withthe 3-CF3,4-Cl substitution known to be active in DFG-out inhibi-tors. While compound 11 showed similar enzymatic activity, anearly fivefold loss in cellular activity versus 8 was observed, inline with previously reported results.15 The 6-F substitution ofthe central phenyl ring of 8 was next replaced with both 6-Cland 6-Me to provide compounds 12 and 13, respectively. Thesecompounds were equipotent to 8 and all three achieved similarcellular activity (pERK = 18–22 nM) to the lead sulfonamides 1and 2. Preliminary ADME studies were carried out and in vitro pre-dicted clearance in mouse, rat and human microsomes was deter-mined for this series. Each compound demonstrated medium tohigh stability suggesting the potential for a favorable pharmacoki-netic profile. Although slightly less active in the cellular assay,compound 11, which possesses the 3-CF3,4-Cl substitution, dem-onstrated improved predicted human clearance and a possiblepharmacokinetic advantage over the 3-(2-cyanopropan-2-yl)group. Compounds 1, 2, 8, 12 and 13 reveal that similar potencycan be achieved by B-Raf inhibitors that utilize the identicalhinge-binding group but inhibit the kinase via two different bind-ing modes.

The key donor–acceptor pair of the DFG-out pharmacophorecan also be satisfied by an amide in its inverted configuration. Anotable example of this strategy is nilotinib which utilizes theidentical hinge-binding and aromatic linker groups as imatinib,but inverts the donor–acceptor amide that extends toward thehydrophobic back pocket ( Fig. 2). This strategy was applied tothe current series of inhibitors and examples are presented in Table4. Compound 14 lacks key substituents on both the central phenylring and the aromatic tail but still demonstrates moderate enzy-matic activity. Incorporation of established substituents on bothof these groups provided compound 15 with a >10-fold improve-ment in enzymatic activity and a significant gain in cellular activ-ity. Furthermore, the cellular activity of this alternative DFG-outinhibitor was within threefold of compounds 8, 12 and 13.19,20

ndered in violet, select residues are depicted in white, and the inhibitor is green.distance is indicated in orange. The kinase adopts the DFG-out conformation.

Table 3B-RafV600E activity and predicted clearance of DFG-out inhibitors 4, 8–13

HN

O

R1

R2NH

N

HNN

OMe

O

R32

6

Compd R1 R2 R3 B-Raf IC50a (nM) pERK IC50

a (nM) Microsomal CLb

Rat Mouse Human

4 F F 3-C(CH3)2CN 2.3 180 17 38 118 F H 3-C(CH3)2CN 1.0 20 11 42 139 H H 3-C(CH3)2CN 0.9 140 16 37 1310 H F 3-C(CH3)2CN 5.1 1800 18 46 1111 F H 3-CF3,4-Cl 1.3 96 12 32 3.112 Cl H 3-C(CH3)2CN 1.2 22 13 44 1313 Me H 3-C(CH3)2CN 1.9 18 19 35 16

a Values are means of at least two experiments.b mL/min/kg

Table 4B-RafV600E activity of inverted amides 14 and 15

HN

O

R1HN

N

HNN

OMe

R2

O

Compd R1 R2 B-Raf IC50a (nM) pERK IC50

a (nM)

14 H H 80 >1000015 Me CF3 6.6 52

a Values are means of at least two experiments.

6240 S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6237–6241

Although DFG-out inhibitors were prepared with similar po-tency to sulfonamide leads 1 and 2, it was critical to assess howthis modification affected kinase selectivity. Compounds 4 and 15were screened against a panel of 64 kinases at 1 lM and theirselectivity profiles were compared to compound 1. In addition,B-RafV600E inhibitor AZ-628,21 which bears the identical 3-(2-cyanopropan-2-yl) substituted benzamide tail as 4 and binds in

1

HN

O

F

NH

SN

HNN

OMe

F

O O

15

HN

O

MeHN

N

HNN

OMe

CF3

O

4

HN

O

F

NH

N

HNN

OMe

FCN

O

AZ-628

N

N

ONH

Me

NH

O

CN

Figure 5. Kinase selectivity of B-RafV600E inhibitors 1, 4,15 and AZ-628. Compounds werethe X-axis. Red indicates 100% of Kinase inhibition, blue indicates 0% of kinase inhibitio

the type II binding mode,18 was included in the selectivity screenin order to help elucidate how inhibitor structure relates to kinaseselectivity. The results are displayed as the heat map in Figure 5.

Compounds 1, 4, 15 and AZ-628 were potent inhibitors of B-RafV600E and demonstrated activity against B-RafWT and C-Raf(Raf1). The three DFG-out inhibitors 4, 15 and AZ-628 all had sim-ilar selectivity profiles with potent activity against Epha2, FGR,LCK, PDGFRa, and RET, while two of these three compounds dem-onstrated activity against Abl, KDR and Src. Every kinase targetedby these inhibitors possesses small gatekeeper residues. This sim-ilarity between the selectivity profiles is striking given the struc-tural differences between 4, 15 and AZ-628 with respect to thehinge-binder, the central phenyl ring substitution, the amide con-figuration and the terminal phenyl substitution. Despite these dif-ferences, compliance with the DFG-out pharmacophore via thedonor–acceptor pair attached to a lipophilic group was constantand seems to drive the pattern of selectivity.22,23 In contrast, inhib-itor 1 possesses a distinct kinase selectivity profile and superioroverall selectivity for the Raf kinases. Since compounds 1 and 4possess identical hinge-binding and linker groups, and only differby a sulfonamide versus a benzamide group, the altered, and supe-rior, selectivity profile of 1 must arise from its atypical binding

screened at 1 lM and are an average of two experiments. Kinases are plotted alongn and gray indiccates 70% of kinase inhibition.

S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6237–6241 6241

mode and the resulting distinct interactions the propyl sulfon-amide makes in the kinase back pocket.

In conclusion, a series of DFG-in, aC-helix-out B-RafV600E inhib-itors was converted to an alternative cell potent series by replacingthe propyl sulfonamide with a benzamide group to satisfy the DFG-out pharmacophore. 3-Methoxy pyrazolopyridine proved to be themost potent hinge-binder in this new series just as in the progen-itor sulfonamide series. Despite maintaining excellent potencyagainst the target, the new inhibitor binding mode led to an ero-sion of kinase selectivity, particularly via inhibition of kinases withsmall gatekeepers. This loss of selectivity extended to structurallydiverse inhibitors that nonetheless fulfilled the DFG-out pharma-cophore requirements. Thus, the pattern of kinase selectivity dis-played by the inhibitors presented in this work is determined bythe feature of the compound which extends into the back pocketof the kinase and leads to the kinase conformation, rather thanby the hinge-binding group or other minor modifications.

References and notes

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Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.;Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.;Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.;Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.

3. (a) Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V.M.; Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004,116, 855; (b) Samowitz, W. S.; Sweeney, C.; Herrick, J.; Albertsen, H.; Levin, T.R.; Murtaugh, M. A.; Wolff, R. K.; Slattery, M. L. Cancer Res. 2005, 65, 6063; (c)Riesco-Eizaguirre, G.; Gutierrez-Martinez, P.; Garcia-Cabezas, M. A.; Mistal, M.;Santisteban, P. Endocr.-Relat. Cancer 2006, 3, 257; (d) Houben, R.; Becker, J. C.;Kappel, A.; Terheyden, P.; Bröcker, E. B.; Goetz, R.; Rapp, U. R. J. Carcinog. 2004,3, 6.

4. (a) Flaherty, K. T.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G. A.; Sosman, J.A.; O’Dwyer, P. J.; Lee, R. J.; Grippo, J. F.; Nolop, K.; Chapman, P. B. N. Engl. J. Med.2010, 363, 809; (b) Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. N.; Cho, H.;Spevak, W.; Zhang, C.; Zhang, Y.; Habets, G.; Burton, E. A.; Wong, B.; Tsang, G.;West, B. L.; Powell, B.; Shellooe, R.; Marimuthu, A.; Nguyen, H.; Zhang, K. Y. J.;Artis, D. R.; Schlessinger, J.; Su, F.; Higgins, B.; Iyer, R.; D’Andrea, K.; Koehler, A.;Stumm, M.; Lin, P. S.; Lee, R. J.; Grippo, J.; Puzanov, I.; Kim, K. B.; Ribas, A.;McArthur, G. A.; Sosman, J. A.; Chapman, P. B.; Flaherty, K. T.; Xu, X.;Nathanson, K. L.; Nolop, K. Nature 2010, 467, 596.

5. Wenglowsky, S.; Ren, L.; Ahrendt, K. A.; Laird, E. R.; Aliagas, I.; Alicke, B.;Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Feng, B.; Gloor, S. L.; Gould, S. E.; Gross,S.; Gunzner-Toste, J.; Hansen, J. D.; Hatzivassiliou, G.; Liu, B.; Malesky, K.;Mathieu, S.; Newhouse, B.; Raddatz, N. J.; Ran, Y.; Rana, S.; Randolph, N.; Risom,T.; Rudolph, J.; Savage, S.; Selby, L. T.; Shrag, M.; Song, K.; Sturgis, H. L.; Voegtli,W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.-I.; Young, W. B.; Grina, J. A.C. S. Med. Chem. Lett. 2011, 2, 342.

6. Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.;Kong, J.; Haass, N. K.; Sproesser, K.; Li, L.; Smalley, K. S. M.; Fong, D.; Zhu, Y.-L.;Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu,C.; Settachatgul, C.; Shellooe, R.; Cantwell, J.; Kim, S.-H.; Schlessinger, J.; Zhang,K. Y. J.; West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth, P.;Artis, D. R.; Herlyn, M.; Bollag, G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3041.

7. Via a substituted benzyl ether lapatinib also traps the DFG-in, aC-helix-outconformation in EGFR kinase which similarly leads to high kinase selectivity:Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan, D.; Hassell, A.; Dickerson, S.H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.; Alligood, K. J.; Rusnak, D. W.;Gilmer, T. M.; Shewchuk, L. Cancer Res. 2004, 64, 6652.

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13. Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358.14. Inhibitor enzymatic activity was determined utilizing full-length B-RafV600E.

Inhibition of basal ERK phosphorylation in Malme-3 M cells was used as themechanistic cellular assay and to drive the structure-activity relationships. Forcomplete experimental details regarding the enzymatic and mechanisticcellular screening assays, please refer to Ref. 1.

15. Lyne, P. D.; Aquila, B.; Cook, D. J.; Dakin, L. A.; Ezhuthachan, J.; Ioannidis, S.;Pontz, T.; Su, M.; Ye, Q.; Zheng, X.; Block, M. H.; Cowen, S.; Deegan, T.; Lee, J.W.; Scott, D. A.; Custeau, D.; Drew, L.; Poondru, S.; Shen, M.; Wu, A. Bioorg. Med.Chem. Lett. 2009, 19, 1026.

16. Wenglowsky, S.; Ahrendt, K.; Buckmelter, A. J.; Feng, B.; Gloor, S. L.; Gradl, S.;Grina, J.; Hansen, J. D.; Laird, E. R.; Lunghofer, P.; Mathieu, S.; Moreno, D.;Newhouse, B.; Ren, L.; Risom, T.; Rudolph, J.; Seo, J.; Sturgis, H. L.; Voegtli, W.C.; Wen, Z. Biorg. Med. Chem. Lett. 2011, 21, 5533.

17. Wenglowsky, S.; Moreno, D.; Rudolph, J.; Ran, Y.; Ahrendt, K. A.; Arrigo, A.;Colson, B.; Gloor, S.; Hastings, G. Bioorg. Med. Chem. Lett. 2012, 22, 912.

18. Coordinates for the B-Raf crystal structures have been deposited in the ProteinData Bank for compounds 4 (accession code 4G9C) and AZ-628 (accession code4G9R).

19. A third class of presumed DFG-out inhibitor was prepared which utilized a ureafor the donor–acceptor pair in place of an amide. This single example, whilevery active in the enzymatic assay, did not have cellular activity.

HN

ONH

NH

N

HNN

O

B-Raf IC50 = 0.7 nMpERK IC50 > 10 µM

20. Preparation of the compounds described in this manuscript are adapted fromgeneral synthetic methods described in Refs. 5,15–17.

21. Aquila, B.; Dakin, L.; Ezhuthachan, J.; Lee, S.; Lyne, P.; Ponntz,T.; Zheng, X. WO2006/024834.

22. Although compounds 4, 15 and AZ-628 do not possess identical selectivityprofiles (e.g., only AZ-628 is active against p38a and only 15 is active againstSRMS), it can nonetheless be stated that this group B-RafV600E inhibitorspossesses a very similar pattern of selectivity against these 64 kinases: Posy, S.L.; Hermsmeier, M. A.; Vaccaro, W.; Ott, K.-H.; Todderud, G.; Lippy, J. S.;Trainor, G. L.; Loughney, D. A.; Johnson, S. R. J. Med. Chem. 2011, 54, 54.

23. Recently, two groups have described B-RafV600E inhibitors which, despite evengreater structural diversity to the inhibitors in this report, comply with theDFG-out pharmacophore and demonstrate a similar pattern of kinaseselectivity to compounds 4, 15 and AZ-628 with activity against Abl, EPHA2,KDR, LCK, PDGFRa and b, and RET kinases:

NH

CF3

O

NMe2

O

N

HN

O ML-721

Gould, A. E.; et. al. J. Med. Chem. 2011, 54, 1836.

O NH

O

NH

ON CF3NN

MeOOMe CEP-32496

Rowbottom, M. W.; Faraoni, R.; Chao, Q.; Campbell, B. T.; Lai, A. G.; Setti, E.;Ezaqa, M.; Sprankle, K. G.; Abraham, S.; Tran, L.; Struss, B.; Gibney, M.;Armstrong, R. C.; Gunawardane, R. N.; Nepomuceno, R. R.; Valenta, I.; Hua, H.;Gardner, M. F.; Cramer, M. D.; Gitnick, D.; Insko, D. E.; Apuy, J. L.; Jones-Bolin,S.; Ghose, A. K.; Herbertz, T.; Ator, M. A.; Dorsey, B. D.; Ruggeri, B.; Willimas,M.; Bhagwat, S.; James, J.; Holladay, M. W. J. Med. Chem. 2012, 55, 1082.