urea (LY3009120) as a Pan-RAF Inhibitor with ... - ACS Publications

Drug Annotation pubs.acs.org/jmc

Discovery of 1‑(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl2-(methylamino)pyrido[2,3‑d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a Pan-RAF Inhibitor with Minimal Paradoxical Activation and Activity against BRAF or RAS Mutant Tumor Cells James R. Henry,*,† Michael D. Kaufman,‡ Sheng-Bin Peng,† Yu Mi Ahn,‡ Timothy M. Caldwell,‡ Lakshminarayana Vogeti,‡ Hanumaiah Telikepalli,‡ Wei-Ping Lu,‡ Molly M. Hood,‡ Thomas J. Rutkoski,‡ Bryan D. Smith,‡ Subha Vogeti,‡ David Miller,‡ Scott C. Wise,‡ Lawrence Chun,§ Xiaoyi Zhang,† Youyan Zhang,† Lisa Kays,† Philip A. Hipskind,† Aaron D. Wrobleski,† Karen L. Lobb,† Julia M. Clay,† Jeffrey D. Cohen,† Jennie L. Walgren,† Denis McCann,† Phenil Patel,† David K. Clawson,† Sherry Guo,† Danalyn Manglicmot,† Chris Groshong,† Cheyenne Logan,† James J. Starling,† and Daniel L. Flynn‡ †

Eli Lilly and Company, Indianapolis, Indiana 46285, United States Deciphera Pharmaceuticals, LLC, Waltham, Massachusetts 02451, United States § Emerald Biostructures, Bainbridge Island, Washington 98110, United States ‡

S Supporting Information *

ABSTRACT: The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

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INTRODUCTION The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. Mutations that lead to constitutive activation of the MAPK pathway are among the most common mutations in human cancers.1,2 The kinase inhibitors vemurafenib (1) and dabrafenib (2), which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation (Chart 1).3−5 However, both vemurafenib and dabrafenib have been shown to induce dimerization of RAF proteins and promote paradoxical pathway activation in BRAF wild type (WT) cells.6−8 Clinically, paradoxical activation promotes drug induced skin lesions including karatoacanthomas and squamous cell carcinoma.4,5 Because of paradoxical pathway activation, both vemurafenib and dabrafenib were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations.8,9 The precise mechanism of paradoxical activation is still unclear, but compound induced RAF dimerization appears to be an essential first step. In one © XXXX American Chemical Society

Chart 1

model, it is hypothesized that a BRAF selective inhibitor triggers a RAS dependent BRAF-CRAF heterodimer. Paradoxical activation then follows when the BRAF selective inhibitor is not able to effectively inhibit both partners of the dimer, leaving the unoccupied CRAF protomer activated for Received: January 14, 2015

A

DOI: 10.1021/acs.jmedchem.5b00067 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Drug Annotation

Table 1. Activities of RAF Inhibitorsa

compd

BRAF V600E IC50 (μM)

BRAF WT IC50 (μM)b

CRAF WT IC50 (μM)b

A375 pERK IC50 (μM)b

HCT116 pERK IC50 (μM)b

KDR IC50 (μM)

A375 proliferation IC50 (μM)

HCT116 proliferation IC50 (μM)

MEK IC50 (μM)b

1 2 3 4

0.0061 NA 0.0010 0.0012

0.034 0.0097 0.018 0.010

0.41 NA 0.0031 0.0029

0.15 0.011 0.0068 0.096

16.6 5.88 0.057 0.091

0.36 NA 0.00046 0.0019

0.17 NA 0.0061 0.056

>10 NA 0.032 0.16

1.5 >20 2.9 4.0

a

NA is not available. bAverage of triplicate determinations.

Figure 1. X-ray crystal structure of 3 bound to BRAF V600E.

binding format. Cellular profiling consisted of monitoring the change in phospho-ERK in the BRAF V600E driven melanoma cell line A375 as well as antiproliferative activity in the same line upon compound treatment for 72 h. Further cellular profiling was performed in the KRAS G13D mutant (BRAF WT) cell line HCT116, again measuring both impact on phospho-ERK and proliferation upon compound treatment. Our program began with the observation that 3 is a potent inhibitor of BRAF V600E (IC50 = 1 nM) and CRAF (IC50 = 3.1 nM) as determined in biochemical assays. Cellular potency was demonstrated in the BRAF V600E driven A375 cell line (IC50 = 6.8 nM) and also in the CRAF driven KRAS mutant HCT116 cell line (IC50 = 57 nM) as determined by inhibition of cellular phospho-ERK respectively (Table 1). Further, compound 3 showed an antiproliferative effect in A375 cells of 6 nM and in HCT116 cells of 32 nM. On the basis of the promising in vitro profile, a cocrystal Xray structure of 3 was obtained bound to the BRAF V600E protein (Figure 1). The structure reveals a type II binding mode within the protein wherein the methylamino naphthyridone heterocycle forms a bidentate hydrogen bond with the Cys-532 hinge residue. Phe-595 of the DFG motif (activation loop) is displaced by the tert-butylisoxazole and adopts the DFG-out or inactive conformation of the kinase. The urea then

signaling. In another model, paradoxical activation of RAF proteins can be explained by transactivation. A drug binding to one member of the RAF homodimer or heterodimer inhibits one partner but results in transactivation of the second drugfree partner likely due to a conformational change.7 Therefore, effective inhibition of the transactivated partner is required for minimalizing the paradoxical activation. According to either model, a pan-RAF inhibitor with true cellular activities against all RAF isoforms is hypothesized to be essential for minimizing paradoxical activation.6 Moreover, mutant RAS-driven cancers account for approximately one-third of all human cancers, and RAS-driven tumor cells have been shown to signal through RAF homodimers/heterodimers.10,11 In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF.

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RESULTS AND DISCUSSION In general, compounds were evaluated for their ability to inhibit recombinant BRAF V600E in a biochemical assay monitoring ADP formation via the coupled pyruvate kinase/lactate dehydrogenase system. Wild type BRAF, CRAF, and MEK biochemical assays were run with recombinant protein in a filter B



Drug Annotation

Figure 2. IVTI dose−response with compound 4 in mice.

Figure 3. Xenograft efficacy of 4 in HT-29 tumors implanted in mice: (A) xenograft growth curve; (B) individual animal phospho-ERK Western blot tumor analysis after dosing.

makes hydrogen bond interactions with α-C helix Glu-501 residue and the activation loop Asp-594 residue, while the tetrahydrofuran is oriented toward solvent. This binding mode is in contrast to that of vemurafenib which binds in a so-called type IIb binding mode with the DFG motif in the active conformation and the α-C helix out.12,13 We next turned our attention to evaluating 3 in vivo. Despite measured clearances less than hepatic blood flow in rats and dogs, 50 and 6 mL min−1 kg−1, respectively, when dosed as a suspension, the compound showed very poor bioavailability in both species of less than 10%, suggesting an issue with oral absorption. Further study revealed the compound to have very low solubility in simulated fed (FedSIF) and fasted intestinal fluid (FasSIF) of only 0.002 and 0.001 mg/mL, respectively.

Further optimization of 3 led to 4 which maintained potent BRAF V600E activity in our biochemical assay of 1.2 nM, phospho-ERK inhibition in A375 cells of 96 nM, and antiproliferative activity of 56 nM. Importantly, no compounds in this study showed potent inhibition of MEK in a biochemical assay, as MEK inhibition would impact phospho-ERK measurements (Table 1). In contrast to 3 however, 4 showed marked improvement in solubility measures with FedSIF of 0.31 mg/ mL and FasSIF of 0.041 mg/mL. When dosed orally as a suspension, the compound showed desirable clearance profiles of 13 mL min−1 kg−1 in rat and 14 mL min−1 kg−1 in dog while showing considerable improvement in oral bioavailabilities of 28% and 69% in rat and dog, respectively. We next moved to study the effects of 4 on in vivo target inhibition (IVTI) based on phospho-ERK activity. A375 cells C


CH3 H H CH3 CH3 CH3 CH3

R2

CH3 CH3 CH3 CH3 CH3 H CH3

R3

CH3 CH3 CH3 CH3 H CH3 CH3

R4

CH N CH CH N N N

X

BRAF WT IC50 (μM)b 0.011 0.039 0.027 0.018 0.0084 0.028 NA 0.0044 0.015

BRAF V600E IC50 (μM) 0.0042 0.0077 0.0043 0.0076 0.0030 0.018 0.0079 0.002 0.0058

NA is not available. bAverage of triplicate determinations.

H F F F F F F

5 6 7 8 9 10 11 12 13

a

R1

compd

Table 2. Structure−Activity Relationship of RAF Inhibitorsa

0.0052 0.0072 0.0067 0.0028 0.0016 0.0056 NA 0.0012 0.0043

CRAF WT IC50 (μM)b 0.040 0.022 0.14 0.11 0.082 0.047 NA 0.027 0.037

A375 pERK IC50 (μM)b 0.12 0.16 0.27 0.048 0.046 0.12 NA 0.016 0.15

HCT116 pERK IC50 (μM)b 0.025 0.75 NA 0.75 0.033 0.17 0.54 1.9 3.9

KDR IC50 (μM) 0.034 0.010 0.035 0.026 0.040 0.011 0.033 0.018 0.0092

A375 proliferation IC50 (μM)

0.13 0.174 0.51 0.28 0.16 0.32 0.19 0.11 0.22

HCT116 proliferation IC50 (μM)

2.5 15 >20 >20 >20 5.9 NA >20 >20

MEK IC50 (μM)b

Journal of Medicinal Chemistry Drug Annotation

D



Drug Annotation

Figure 4. (A) X-ray crystal structure of 6 bound to BRAF V600E. (B) X-ray crystal structure of 13 bound to BRAF V600E.

were implanted on the flank of nu/nu mice and allowed to grow to a size of 300 mm3. The test compound was dosed orally, and after 2 h, the mice were sacrificed and tumor samples were evaluated for phospho-ERK inhibition (Figure 2). Compound 4 required a dose of 10.4 mg/kg to inhibit phospho-ERK at 50%, while a dose of 20.5 mg/kg inhibited phospho-ERK at 80%. A corresponding increase of exposure from 0.35 μM (167 ng/mL) to 1.8 μM (843 ng/mL) was observed between the ED50 and ED80 doses. With IVTI data in hand, 4 was next evaluated in an HT-29 (BRAF V600E) xenograft efficacy model. At doses corresponding to the ED50 and ED80 for phospho-ERK in the IVTI model, 4 showed a dose responsive efficacy in HT-29 tumors when dosed for 28 days without significant weight loss. Analysis of the tumors via Western blot after dosing confirmed significant phospho-ERK inhibition (Figure 3). With a link between in vitro and in vivo target inhibition and in vivo efficacy in BRAF V600E tumors now established, we turned to the toxicological evaluation of 4 in rats and dogs. A number of dose-limiting toxicities were observed including profound inflammation, hyperbilirubinemia, skeletal muscle degeneration, and marked bone marrow hypocellularity. While some of the effects could be at least partially attributed to exaggerated pharmacology from RAF inhibition, other potential drivers for the undesirable toxicology profile of 4 were not clear, and the team embarked on further SAR study. The goal

was to maintain desired BRAF V600E, CRAF activity, and in vivo pharmacokinetic properties while aiming to improve or modify overall kinase selectivity. As a surrogate sentinel for overall selectivity, we chose to routinely monitor activity against the tyrosine kinase KDR. Work to optimize 4 led to compound 5 where the tertbutylisoxazole had been modified to a tert-butylethylurea. Surprisingly, this change maintained all of the desired activity against BRAF V600E (IC50 = 4 nM) and CRAF (IC50 = 5 nM) while providing a significant decrease in KDR activity (Table 2). While these new data were encouraging, compound 5 suffered from poor oral exposure in mice (data not shown) and required further optimization. Turning our attention to the hinge binding moiety of the inhibitor, it was discovered that the naphthyridone of 5 could be replaced with a naphthyridine to give 10. This change maintained the desirable in vitro BRAF and CRAF profile of 5 while unexpectedly providing an additional improvement in selectivity vs KDR (IC50 = 172 nM). Further, this compound showed a significant improvement in in vivo parameters in rat with bioavailability of 30% when dosed as a suspension and an iv clearance of 3.36 mL min−1 kg−1. Compound 6 was also found to have interesting properties, demonstrating improvement in KDR activity over 10, to 0.75 μM while maintaining very desirable BRAF and CRAF activities. Removal of the R2 methyl group of 10 gave compound 9, resulting in a 2-fold loss E



Drug Annotation

Figure 5. Paradoxical activation of RAS-RAF-MEK signaling by vemurafenib (1), compound 6, and 13 in BRAF WT HCT-116 cells.

but it is also possible that through reinforcement of the orthogonal conformation, selectivity is achieved. We next tested our hypothesis that pan-RAF inhibition would prevent paradoxical activation. As shown in Figure 5, evaluation of phospho-ERK in the BRAF WT cell line HCT116 shows that while 1 (vemurafenib) produces significant paradoxical activation as doses increase, pan-RAF inhibitors 6 and 13 give only very minor activation at very low doses, with near complete inhibition of phospho-ERK at concentrations above 100 nM. We believe this difference in behavior is driven by the pan-RAF activity of 6 and 13 and in particular the potent inhibition of CRAF. Indeed, the IC50 value for biochemical inhibition of CRAF is 7 and 15 nM for 6 and 13, respectively, compared to 414 nM for 1 in our assay conditions. Thus, the ability to inhibit all isoforms of RAF appears to be critical to avoid paradoxical activation and inhibit signaling through phospho-ERK in RAF WT cell lines. To confirm compound 13 as a pan-RAF inhibitor, it was evaluated in a whole cell-based KiNativ assay developed by ActivX Biosciences Inc. Compound 13 was incubated with A375 whole cell lysate for 15 min, and the binding affinities of over 170 kinases were determined by direct competitive binding with an ATP analog. Among the kinases measured, six proteins have binding affinities equal to or less than 100 nM, and eight targets have binding affinity between 290 and 1000 nM. The remaining kinases (over 150 examined) are inactive at 1 μM (Table 3). As summarized in Table 4, 13 bound ARAF, BRAF, and CRAF native proteins with IC50 values of 44, 31− 47, and 42 nM, respectively. Vemurafenib was able to bind to BRAF and ARAF with IC50 values of 260−360 and 950 nM, respectively; however, its binding affinity to CRAF was greater than 10,000 nM. Dabrafenib bound BRAF and ARAF potently with IC50 values of 6 and 26 nM, respectively, while binding to CRAF was mild with an IC50 of 150 nM, about 25-fold less than its binding affinity to BRAF. On the basis of its superior in vitro profile, 6 was moved into BRAFV600E A375 xenograft IVTI studies in mice. As illustrated in Figure 6, a single dose oral treatment of mice from 3 to 50 mg/kg showed a dose dependent inhibition of

in activity in the A375 phospho-ERK IC50 and also a drop in activity in the cell proliferation assay. Further, this change drove an undesirable 5-fold increase in KDR activity. Removal of the fluorine from the central phenyl ring of 10 afforded 7. Compound 7 exhibited a loss of desired BRAF cellular potency of nearly 3-fold compared to 10 (140 nM vs 47 nM) and 2 fold loss in CRAF cellular potency (267 nM vs 122 nM). Further modification of the hinge binder led to the pyridopyrimidine LY3009120 (13, Deciphera Pharmaceuticals ID DP-4978).14 While this structural change had little impact on BRAF and CRAF activity relative to 10, the KDR selectivity was improved by greater than 20-fold to 3.9 μM. As seen in the naphthyridine SAR, removal of the methyl group from the central phenyl ring to give 8 results in an increase in KDR activity of 5-fold to 0.75 μM. Removal of either the N-methyl group of 13 to give 11 or the 5-methyl group of the pyridopyrimidine to give 12 has little impact on the desired in vitro biology, although 11 does show more potent KDR activity. In each case, however, removal of the methyl group has a negative impact on in vivo performance, resulting in high iv clearance and poor oral bioavailability. On the basis of their promising in vitro profiles, cocrystal Xray structures of 6 and 13 were obtained bound to the BRAF V600E protein (Figure 4). Similar to 3, the structures reveal a type II binding mode wherein the methylamino napthyridine or methylamino pyridopyrimidine warheads form a bidentate hydrogen bond with Cys-532 hinge residue. Phe-595 of the DFG motif is displaced by the tert-butylethyl group and adopts the DFG-out or inactive conformation of the kinase. The urea then makes hydrogen bond interactions with Glu-501 and Asp594. In compound 6, the hydroxyl group seems to make an additional interaction with a binding site water. As shown with 8 vs 13, the methyl substituent at R2 plays an important role for KDR selectivity (Table 2). The central phenyl ring bearing R2 occupies the region near gatekeeper Thr-529 with a nearly orthogonal orientation to the heterocyclic warhead. The gatekeeper residue in KDR is valine compared to threonine in BRAF. The impact of the R2 methyl substituent on KDR selectivity may be driven by differences in gatekeeper residues, F



Drug Annotation

Table 3. General KiNativ Profiling of 13 in A375 Cellsa

a

kinase

IC50 (nM)

EphA2 ZAK p38 FYN MAP3K1 EphB4 CSK ABL IRAK1 GCN2 JNK MAP2K5 KHS1 SRC

20 39 61−97 91 98 100 290−400 380−520 390 450−480 470 800 970 310 to >1000

(Figure 7). As shown, 6 produced regressions in the A375 model when dosed at 20 and 30 mg/kg b.i.d. while also demonstrating significant efficacy in the KRAS mutant HCT116 xenograft at 40 mg/kg b.i.d. In both studies the compound was dosed for 15 days and was well tolerated with no significant weight loss in any group. We next evaluated 13 in xenograft efficacy models. Upon oral treatment of rats bearing BRAF V600E ST019VR PDX tumors with 15 or 30 mg/kg b.i.d. of 13, a dose-dependent tumor growth inhibition was observed (Figure 8). Additional efficacy for 13 in BRAF, NRAS, and KRAS mutant xenograft models has also been demonstrated.15 Altogether, these in vivo efficacy studies suggest that the pan-RAF inhibitors 6 and 13 are effective in inhibiting tumor growth with BRAF, KRAS, or NRAS mutations. Pharmacokinetic parameters for compound 13 have been determined in rat, dog, and monkey and are summarized in Table 5. In each species the compound was dosed as a 1 mg/kg solution in the iv arm and a 10 mg/kg formulation in the oral arm. In each species, iv clearance was moderate at 30−55% of hepatic blood flow and volumes of distribution between 0.84 and 1.78 L/kg. The oral bioavailability was dependent on the formulation used. In rat and dog, HEC suspension of API in capsule gave very low oral exposure. Compound 13 is a weak base with a pKa of 4.52, and the solubility is very low across the physiologically relevant pH range, as well as in simulated gastric and intestinal fluids. Solubility in water is 150) have IC50 values of >1μM.

Table 4. RAF KiNativ Data in A375 Cells IC50 (nM) compd

ARAF

BRAF

CRAF

13 1 2

44 950 26

31−47 260−360 6

42 >10,000 150

phospho-MEK and phospho-ERK, and the calculated dose for 50% inhibition of phospho-ERK (EC50) was 1.7 mg/kg (Figure 6A), with a plasma concentration to achieve 50% inhibition of phospho-ERK (EC50) of 1347 ng/mL or 3.1 μM (Figure 6B). Similarly, 13 was evaluated in nude rats bearing A375 xenograft tumors. Single dose oral treatment with 13 from 3 to 50 mg/kg showed a dose dependent inhibition of phospho-ERK, with a calculated dose for 50% inhibition of phospho-ERK (EC50) of 4.36 mg/kg, with plasma concentration to achieve 50% inhibition of phospho-ERK (EC50) of 68.9 ng/mL or 165 nM, significantly lower than the EC50 for compound 6.15 On the basis of the in vivo target inhibition data for 6, we initiated efficacy studies in A375 and HCT116 xenografts

Figure 6. Mouse IVTI of 6 in A375 xenograft: (A) phospho-ERK dose response; (B) phospho-ERK concentration response; (C) Western blot. G



Drug Annotation

Figure 7. Compound 6 in vivo efficacy in mice dosed b.i.d. for 15 days in (A) A375 BRAF V600E and (B) HCT116 xenograft tumors.

to the crystalline drug.18,19 Various polymers were evaluated with PVP-VA (Kollidon VA-64), resulting in a solid dispersion that was both chemically and physically stable under accelerated stability testing, as well as long-term storage under ambient conditions. Pharmacokinetic studies in dogs and pilot toxicology studies contained spray dried solid dispersion with 13/PVP-VA in ratio of 20:80, with added 2% sodium lauryl sulfate. Further evaluations of physical stability and in vivo pharmacokinetics in dogs were done, followed by GLP toxicology studies using higher drug/polymer ratio (40:60) and 1% added sodium lauryl sulfate. For human studies the solid dispersion containing 13/PVP-VA (40:60) was used. Dosing the molecule as an enabled formulation of 20% cyclodextrin in rat and monkey or solid dispersion in dog led to significantly improved oral exposure and bioavailability.

Figure 8. Compound 13 in vivo efficacy in rat BRAF V600E ST019VR PDX model. Compound was dosed at 15 and 30 mg/kg b.i.d., respectively, for 28 days.

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CHEMISTRY Compounds 3, 4, and 5 were prepared in a manor analogous to previous reports.20 Compounds 6, 7, 9, and 10 were prepared by the general methods illustrated in Scheme 1.21 In general, compound 18 is reacted with compound 22 (for 7, 9, and 10) or 23 (for 6) to provide 24 or 25, respectively. Further treatment of 24 or 25 with ispropenyl chloroformate under

because of an inability to achieve a solubility that would support high drug dose. Evaluation of solid dispersion technologies was next pursued where the drug is dispersed in an inert polymeric matrix and rendered in an amorphous form which results in faster dissolution rate and/or higher extent and duration of supersaturation leading to enhanced oral bioavailability relative

Table 5. Pharmacokinetic Parameters for Compound 13 across Species species SD rat: male, fasted, crossover (n = 3)

dog: male, fasted, crossover (n = 4)

dose (po) (mg/kg):

10 (1% HEC suspension)

10 (20% cyclodextrin, suspension)

AUC0−24 (ng·h/mL) Cmax (ng/mL) half-life (h) bioavailability (%)

169 ± 138 34.4 ± 37.1 1.4 3.6 ± 2.8

1667 ± 717 396 ± 142 2.7 31.7 ± 7.6

10 (API capsule) 20.5 ± 8.6 2.8 ± 0.3 7.6 ± 4.4 0.2 ± 0.1 species

monkey: male, fasted

10 (solid dispersion)

10 (20% cyclodextrin, suspension)

2093 ± 1229 511 ± 36 7.9 ± 8.5 16.7 ± 10.0

1260 ± 141 492 ± 68 3.5 ± 0.3 20 ± 0

SD rat: male, fasted, crossover (n = 3)

dog: male, fasted, crossover (n = 4)

monkey: male, fasted

dose (iv) (mg/kg):

1 (20% cyclodextrin solution)



half-life (h) clearance (mL min−1 kg−1) Qh (mL min−1 kg−1) vol. of dist. (L/kg)

1.1 ± 0.4 34.1 ± 8.5 55.2 1.08 ± 0.02

1.8 ± 0.1 16.1 ± 3.0 30.9 1.78 ± 0.33 H

2.1 ± 0.4 14.6 ± 1.3 1.53 ± 0.14

1.0 ± 0.8 25.9 ± 1.9 43.6 0.84 ± 0.42 DOI: 10.1021/acs.jmedchem.5b00067 J. Med. Chem. XXXX, XXX, XXX−XXX


Drug Annotation

Scheme 1a

a Reagents and conditions. (a) For 6, 9, 10: rt, 16 h, 52%; (b) EtOAc, Pd/C (10%), H2 1 atm, 16 h, 75%; (c) EtOH, KOH, 60 °C, 1 h, 60%. (d) For 7: dioxane, K2CO3, Pd(PPh3)4, 60 °C, 1 h, 84%. (e) 22: NMP, 185 °C, 64 h, 43%. 23: CH3NH2 30% EtOH, 120 °C microwave, 24 h, 98%; (f) EtOAc, H20, rt, 1 h, 95%. (g) 26: dioxane, RNH2, 60 °C, 4 h, carried on crude. 27: dioxane, NMP, 80 °C, 16 h, 89%; (h) 26, TFA, rt, 1 h, 53%.

Schotten−Baumann conditions provides 26 and 27, respectively. Further reaction of 26 or 27 with the appropriate amine in the presence of a base followed by removal of the PMB group as necessary gives the products. In turn, two routes to compound 18 were employed. Compound 14 reacts with acetic anhydride in the presence of 1-methylimidazole to provide 15. Nitro compound 15 is reduced to provide amine 16 which is condensed with aldehyde 17 in the presence of potassium hydroxide to yield naphthyridine 18. Alternatively, compound 18 can be synthesized via Pd(PPh3)4-mediated coupling of boronate 21 with triflate 19 or bromide 20. Compounds 8 and 13 are prepared as described in Scheme 2.22 Compound 28 is reacted with acetone and potassium hydroxide to provide compound 29, which is further reacted with methylamine to provide 30. Compound 30 is then converted to bromide 31 with N-bromosuccinimide. Compound 28 can also be reacted with 1-hydroxypropan-2-one and potassium hydroxide to provide compound 32, which can be converted to triflate 33 with trifluoromethanesulfonic anhydride. Compound 33 is then reacted with bleach followed by methylamine to provide compound 34. Compound 35 is reacted with bis(pinacolato)diboron and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)−dichloromethane complex to give compound 36 which is treated with isopropenyl chloroformate to give compound 37, which is further reacted with a suitable amine to produce compound 38.

Palladium coupling of boronate 38 with either bromide 31 or triflate 34 provided the desired final compounds. Compound 12 is prepared as described in Scheme 3. Aldehyde 39 and protected N-methylguanidine are reacted to give 40. Compounds 40 and 41 are reacted under palladium coupling conditions to provide compound 42, which is further reacted with isopropenyl chloroformate to give compound 43. Compound 43 was then converted to desired compound 12 as described above.

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DISCUSSION/CONCLUSION We have identified a series of compounds that are shown to be type II pan-RAF kinase inhibitors both biochemically and in cell based assays. Unlike more selective BRAF inhibitors, compounds 6 and 13 show minimal paradoxical pathway activation in RAF WT cell lines. Interestingly, as reported by others, we see an apparent increase in dimerization upon compound treatment.12,15,23 As proposed by others, our data suggest that even when compound induced dimerization of RAF proteins occurs, effective inhibition of dimer partners can still function to prevent significant paradoxical activation provided that both the BRAF and CRAF protomers are inhibitor-occupied.24 Compounds 1 and 2 have both been shown to bind with the DFG-in and αC-helix-out. This change in conformation of the αC-helix likely negatively impacts dimerization, explaining the increased dimer seen with type II I



Drug Annotation

Scheme 2a

Reagents and conditions: (a) acetone, KOH, rt, 10 min, carried on crude; (b) methylamine (33% in EtOH), 110 °C, 16 h, 65%, 2 steps; (c) MeCN, NBS, 0−5 °C 4 days, 31%; (d) NaOH, H2O, rt, 16 h, 56%; (e) pyridine, trifluoromethanesulfonic anhydride, 0 °C, 2 h, 70%; (f) DMF, glacial acetic acid, bleach, then methylamine (2.0 M in THF) 0 °C to rt, 2 h, 73%; (g) bis(pinacolato)diboron, KOAc, dioxane, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)−dichloromethane complex, 100 °C, 16 h, 85%; (h) EtOAC, aq NaHCO3, isopropenyl chloroformate, rt, 6 h, carried on crude; (i) 3,3-dimethylbutan-1-amine, NMP, dioxane, 75 °C, 16 h, 88% 2 steps. (j) From 34: dioxane, NaHCO3, 38, H2O, tetrakis(triphenylphosphine)palladium, 50 °C, 16 h, 77%. a

Scheme 3a

a Reagents and conditions: (a) MeCN, TEA, 180 °C microwave, 15 min, 24%; (b) 41, dioxane, K2CO3, H2O, Pd(PPh3)4, 60 °C, 3 h, 80%; (c) isopropenyl chloroformate, EtOAc, aq NaHCO3, rt, 2 h, 99%; (d) NMP, THF, 65 °C, 4 h, 75%; (e) TFA, 0 °C to rt, 16 h, 81%.

inhibitors. However, this same required αC-helix movement likely explains the inability of compounds 1 and 2 to inhibit signaling once dimerization has occurred.12 Importantly, panRAF inhibition has also been shown to provide in vivo efficacy against a variety of BRAF, NRAS, and KRAS mutant tumors. We drove our SAR predominantly using RAF dependent assays in BRAF and KRAS mutant cells and KDR as a surrogate marker to improve overall selectivity which was also followed by a significant improvement in the toxicity profile of compound 13 over the initial leading compound 5. On the

basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

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EXPERIMENTALS

General. All commercial chemicals and solvents are reagent grade and were used without further purification unless otherwise noted. All liquid chromatography (LC) analyses were performed using an Agilent 1100 LC system, consisting of a 1100 HiP degasser, 1100 binary pump, 1100 autosampler, 1100 thermostated column compartment, and a 1100 diode array detector (G4212B) coupled to an J



Drug Annotation

temperature at 20−30 °C. The reaction was monitored by TLC (EA/PE = 1:10). After the completion of the reaction, the pH value was adjusted to 2−3 using NaHCO3, and Celite (0.56 kg) was added. The mixture was stirred for 0.5−1 h and filtered. The solid was washed with toluene (2 × 2.6 L). The filtrates were combined and stirred for another 0.5−1 h before separation of the layers. The aqueous layer was extracted with toluene (2.6 L). The organic layers were combined, washed with brine (3 L), and filtered through a silica gel pad (2−3 cm). The filtrate was concentrated to 2−2.5 L and the residue mixed with n-heptane (3.3 L). After concentration to 2−2.5 L again, nheptane (1.65L) was added. The mixture was cooled to −10 to ∼0 °C and stirred for 1 h. The precipitate was collected by filtration, washed with n-heptane (0.5 L, precooled to −10 to 0 °C), and dried to afford the crude product of the title compound as a brown solid. Recrystallization of crude product with n-heptane (3.3L) gave offwhite to brown solid of the title compound (527 g, 52% yield, and 98% purity detected by UV absorption at 210 nm). 1H NMR (400 MHz, DMSO-d6): δ 6.97 (m, 2H), 5.18 (s, 2H), 2.16 (s, 3H). 13C NMR (500 MHz, CDCl3): δ 21.41, 116.60, 118.30, 119.66, 127.22, 132.88, (149.37,151.12). 19F NMR (400 MHz, CDCl3): δ 151.14 2-Fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline 36, R1 = CH3. Method A. Combine 5-bromo-2fluoro-4-methylaniline (3.1 g, 15.2 mmol), bis(pinacolato)diboron (4.24 g, 16.7 mmol), and potassium acetate (4.47 g, 45.6 mmol) in dioxane (40 mL) and sparge with argon. Add [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)−dichloromethane complex (0.620 g, 0.760 mmol). Sparge again with argon, and heat at 100 °C overnight. Filter the reaction mixture and concentrate in vacuo. Purify by silica gel chromatography (0−50% EtOAc/ hexanes) to give the title compound (3.24 g, 85% yield). MS (m/z): 252.1 (M + 1). Method B. To a four-necked round-bottom flask equipped with mechanical agitation, thermometer, and N2 inlet were added 5-bromo2-fluoro-4-methylaniline (200g, 0.98 mol), CH3COOK (192 g, 1.95 mol, 2.0 equiv), bis(pinacolato)diboron (248 g, 0.98 mol, 1.0 equiv), and IPAC (3 L). After degassing with N2 for 30 min, the mixture was warmed to 50 °C and Pd(dppf)Cl2 (8g, 4 wt %) was added. The reaction mixture was heated under reflux for at least 10 h until the content of starting material was ≤2% (GC). The mixture was cooled to 20−30 °C, filtered through a pad of Celite, and rinsed with IPAC (1 L). The filtrate was concentrated to 400−500 mL. The residue was mixed with n-heptane (700 mL), filtered through a SiO2 pad, and eluted with IPAC/n-heptane (1/5 first, ∼2 L, and then 2/5, ∼3 L). The filtrate was concentrated to 350−400 mL. n-Heptane (300 mL) was added and the mixture was again concentrated to 350−400 mL. The residue (suspension) was cooled to −10 to −20 °C and filtered after stirring for 2−5 h. The crude product was dissolved in MeOH (200 mL) at 30−40 °C. Then slowly add H2O (600 mL) dropwise in 0.5−1 h. The suspension was cooled to 20−30 °C and filtered after stirring for 1−2 h.The solid was dried under high vacuum to afford the title compound as off-white solid (183g, 74% yield, and 99% purity detected by UV absorption at 210 nm. 1H NMR (400 MHz, CDCl3): δ 7.42 (d, J = 10.2 Hz, 1H), 7.01 (d, J = 12.4 Hz, 1H), 3.76 (s, 2H), 2.64 (s, 3H), 1.55 (s, 12H). 13C NMR (500 MHz, CDCl3): δ 20.66, 20.67, 24.40, 82.93, 116.20, 124.37, 130.64, 135.02, (151.93,153.37). 19 F NMR (400 MHz, CDCl3): δ 145.72. Prop-1-en-2-yl-2-fluoro-4-methyl-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenylcarbamate 37, R1 = CH3. Add 2fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (5.0 g, 19.91 mmol) and isopropenyl chloroformate (2.40 mL, 21.90 mmol) in EtOAc (60 mL) and saturated aqueous NaHCO3 (60 mL) and stir at rt for 6 h. Separate the layers, extract the aqueous layer with EtOAc (2×), wash the combined organics with brine, dry over Na2SO4, and concentrate to obtain the title compound. Use for the next reaction without further purification (assuming 100% yield). MS (m/z): 336.2 (M + 1). 3,3-Dimethylbutan-1-amine Hydrochloride. To a four-necked round-bottom flask equipped with mechanical agitation, thermometer, and N2 inlet was added 3,3-dimethylbutanal (200g, 2.0 mol). Then 1phenylmethanamine (214 g, 2.0 mol, 1.0 equiv) was added dropwise.

Agilent 1956 single quadrupole mass spectrometry (MS) detector. The injection volume was set to 1 μL by default. The UV (DAD) acquisition was performed at 80 Hz, with a scan range of 200−400 nm (by 2 nm step). The MS instrument was operated with an electrospray ionization source (ESI) in both positive and negative ion modes. The nebulizer pressure was set to 50 psi, and the drying gas temperature and flow were set to 350 °C and 12.0 L/min, respectively. The capillary voltages used were 4000 V in positive mode and 3500 V in negative mode. The MS acquisition range was set to 120−500 m/z with a step size of 0.25 m/z in both polarity modes. Fragmentor voltage was set to 80 (ESI+) or 80 (ESI−), gain to 1.00 (ESI+) or 1.00 (ESI−), and the ion count threshold to 100 (ESI+) or 100 (ESI−). The overall MS scan cycle time was 0.83 s/cycle. Data acquisition was performed with Agilent Chemstation software. Analyses were carried out on a Phenomenex Gemini-NX C18 column of 50 mm length, 2.1 mm internal diameter, and 3 μm particle size. The mobile phase used was A2 = water with 10 mM ammonium bicarbonate, pH = 9, B2 = acetonitrile. The run was performed at a temperature of 50 °C and a flow rate of 1.0 mL/min, with a gradient elution from 5% to 95% (B1) over 3.0 min followed by a 0.4 min hold at 95% (B1) and a total run time of 3.75 min. The second mobile phase used was A1 = water with 0.1% formic acid, B1 = acetonitrile with 0.1% formic acid. The run was performed at a temperature of 50 °C and a flow rate of 1.0 mL/min, with a gradient elution from 5% to 95% (B1) over 3.0 min followed by a 0.4 min hold at 95% (B1) and a total run time of 3.75 min. All final compounds were analyzed using one of these analytical methods and were at least 95% pure. 5-Bromo-2-fluoro-4-methylaniline 35, R1 = CH3. Method A. Combine 1-bromo-4-fluoro-2-methylbenzene (30.0 g, 159 mmol) in concentrated sulfuric acid (100 mL), cool to about −5 °C, and treat dropwise with nitric acid (11.00 mL, 174 mmol) over 20 min. Allow reaction mixture to warm to rt and stir for 30 min. Pour onto crushed ice with stirring and partition with tert-butyl methyl ether (MTBE) (200 mL). Separate the aqueous layer and extract with MTBE (2 × 50 mL). Combine organic layers, dry, and concentrate under reduced pressure to provide 1-bromo-4-fluoro-2-methyl-5-nitrobenzene as an orange-colored viscous oil (39.0 g). Combine crude 1-bromo-4-fluoro-2-methyl-5-nitrobenzene (32.4 g, 138 mmol), ethanol (100 mL), and Raney nickel (1.00 g, 17.04 mmol) in a shaker flask. Charge the flask with hydrogen (275 kPa) and agitate until the absorption of hydrogen ceases. Depressurize the reaction vessel, remove the catalyst by filtration, and evaporate the filtrate to dryness. Add MTBE, then filter again and evaporate the filtrate. Stir residue in hexanes. Collect the solids by filtration, wash with cold hexanes, and dry in vacuo to provide the title compound (17.8 g, 63% yield) as a dark solid. MS (m/z): 204.0 (M + 1)/206.0 (M + 3). Method B. To a four-necked round-bottom flask equipped with mechanical agitation, thermometer, ice bath, and N2 inlet were added 1-bromo-4-fluoro-2-methylbenzene (1.4 kg, 7.4 mol), then concentrated H2SO4 (6.3 kg) at 0−10 °C. After stirring for 10−20 min, the mixture was cooled to −10 to 0 °C, and KNO3 (0.82 kg, 7.8 mol, 1.05 equiv) was added in portions in about 6 h while maintaining the temperature at −10 to 0 °C. After the addition, the reaction mixture was warmed to 10−20 °C and monitored by TLC (EA/PE = 1:20) and HPLC until the content of 1-bromo-4-fluoro-2-methylbenzene was

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