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. 2011 Mar 8;2(5):342–347. doi: 10.1021/ml200025q

Pyrazolopyridine Inhibitors of B-RafV600E. Part 1: The Development of Selective, Orally Bioavailable, and Efficacious Inhibitors

Steve Wenglowsky †,*, Li Ren †, Kateri A Ahrendt †, Ellen R Laird †, Ignacio Aliagas ‡, Bruno Alicke ‡, Alex J Buckmelter †, Edna F Choo ‡, Victoria Dinkel †, Bainian Feng ‡, Susan L Gloor †, Stephen E Gould ‡, Stefan Gross †, Janet Gunzner-Toste ‡, Joshua D Hansen †, Georgia Hatzivassiliou ‡, Bonnie Liu ‡, Kim Malesky ‡, Simon Mathieu ‡, Brad Newhouse †, Nicholas J Raddatz †, Yingqing Ran ‡, Sumeet Rana †, Nikole Randolph †, Tyler Risom †, Joachim Rudolph ‡, Scott Savage ‡, LeAnn T Selby †, Michael Shrag †, Kyung Song ‡, Hillary L Sturgis †, Walter C Voegtli †, Zhaoyang Wen ‡, Brandon S Willis †, Richard D Woessner †, Wen-I Wu †, Wendy B Young ‡, Jonas Grina †
PMCID: PMC4017973  PMID: 24900315

Abstract

graphic file with name ml-2011-00025q_0008.jpg

The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-RafV600E was developed. Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent, selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft model driven by B-RafV600E with no effect on body weight. On the basis of their in vivo efficacy and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.

Keywords: B-RafV600E, MAPK pathway, targeted therapy, pyrazolopyridine, amorphous spray-dried dispersion

The Ras/Raf/MEK/ERK (MAPK) signaling pathway transduces signals from cell surface receptors to the nucleus leading to cellular proliferation, differentiation, and survival.1 The Raf family consists of serine/threonine kinases A-Raf, B-Raf, and C-Raf (Raf-1), which phosphorylate and activate MEK, although with different biochemical potencies (B-Raf > C-Raf ≫ A-Raf).1 Mutations in the BRAF gene may lead to MAPK pathway amplification via constitutive activation of B-Raf kinase and are present in approximately 7% of all cancers.2 Mutated B-Raf is most frequently associated with melanoma and has been detected in up to 82% of cutaneous melanocyte nevi,3 66% of primary melanomas,2 and 40−68% of metastatic melanomas.4,5 Additional cancers with aberrant activation of B-Raf due to oncogenic mutations include papillary thryroid cancer (29−83%),2,6,7 colorectal cancer (5−22%),2 cholangiocarcinoma (22%),8 and ovarian cancer (30%).2 Over 90% of the detected mutations in B-Raf are a glutamic acid for valine substitution at residue 600 (V600E),2 which leads to constitutive kinase activity 500-fold greater than B-RafWT and correlates with increased malignancy and decreased response to chemotherapy.9−12 Thus, cancers induced via constitutive activation of the MAPK signaling pathway arising from B-RafV600E should be treatable by a small molecule inhibitor targeted to disable this aberrant enzyme.13 Herein is described the discovery of selective, orally bioavailable, and efficacious inhibitors of B-RafV600E that utilize a novel 3-methoxy pyrazolopyridine hinge-binding template.

The targeted approach to cancer therapy development has been validated by the growing number of marketed small-molecule protein kinase inhibitors.14 Although there are no approved kinase inhibitors that are selective for B-RafV600E, four are currently in clinical trials including RAF265 (Chiron/Novartis),15 XL281/BMS-908662 (Exelixis/BMS),16 GSK2118436 (GlaxoSmithKline),17 and PLX4032 (1, Plexxikon/Roche),18 with others in preclinical development.19−22 A detailed account of the discovery and clinical development of 1, a selective B-RafV600E inhibitor that binds to the active conformation of the kinase (DFG-in), has recently been published.23 The authors report that the propyl group of the sulfonamide effects B-RafV600E selectivity by trapping a small lipophilic pocket enlarged by an outward shift of the αC-helix.24 It was further reported that the difluorophenyl ring of 1 occupies a hydrophobic pocket adjacent to the gatekeeper residue, Thr529.

graphic file with name ml-2011-00025q_0002.jpg

From structural evaluation and molecular modeling, it was hypothesized that an amide linker could couple an aryl group in this hydrophobic pocket to a hinge-binding heterocycle, while forming a hydrogen bond to the hydroxyl of Thr529. Linkage via an amide bond would also allow for rapid and efficient screening of several potential hinge-binding groups. The initial series of amide-linked B-RafV600E inhibitors utilized pyridine as a hydrogen bond acceptor, which was postulated to bind to the −NH of hinge residue Cys532. Scheme 1 illustrates the synthesis of pyridine-amides 3−5 and the structures of the two benzoic acid coupling partners used in this report (2a and 2b).25

Scheme 1. Preparation of Pyridine Amides 3−5.

Scheme 1

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Reagents and conditions: (a) Compound 2a, EDCI, HOBt, DMF, 22 °C, 5−36%.

Inhibitor enzymatic activity was determined utilizing full-length B-RafV600E. Inhibition of basal ERK phosphorylation in Malme-3M cells was used as the mechanistic cellular assay and to drive the structure−activity relationships for the amide series.26 Inhibition of ERK phosphorylation was also determined in the A375 cell line and, while up to an order of magnitude less sensitive than the Malme-3M cell line, generated an identical SAR.

The micromolar enzymatic and cellular activity of pyridine amide 3 revealed that additional interactions would be necessary to achieve useful potency (Table 1). Molecular modeling indicated that the addition of a 6-amino group to the pyridine of 3 could yield a hydrogen bond to the carbonyl of Cys532. The 4-fold improvement in enzymatic activity and nearly 3-fold improvement in cellular activity of aminopyridine 4 substantiated this approach. Addition of bromine at the 5-position of 4 led to compound 5 and a further 8-fold increase in enzymatic and cellular activity. The presumed binding model, with hydrogen bonds to Cys532 and Thr529, was confirmed by an X-ray crystal structure of amide 5 with B-Raf (Figure 1). The crystal structure also revealed key lipophilic contacts made by the 5-bromo group with the side chains of Ile463, Val471, Trp531, and Phe583, which accounted for the potency increase over 4. However, despite the >20-fold improvement in potency from 3 to 5, the cellular activity of the pyridine amides remained insufficient for in vivo efficacy.

Table 1. Enzymatic and Cellular Activities of B-RafV600E Amide Series Inhibitors.

  IC50 (nM)
compd enzymea Malme-3Mb A375b
3 3900 6700 >3000
4 920 2400 >3000
5 110 310 1200
8 61 2400 >3000
9 38 94 290
10 3.4 39 190
13 17 62 430
14 4.0 44 260
15 12 86 830
16 8.2 40 650
17 4.8 19 150
18 6.3 56 560
19 1.7 20 240
1 31c 61 190
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a

Biochemical assay (B-RafV600E).

b

Cellular phosphorylation assay (pERK) using B-RafV600E mutant Malme-3M or A375 cell line.

c

Ref (23).

Figure 1.

Figure 1

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X-ray crystal structure of 5 in complex with B-RafWT. The cleft surface is rendered in violet, select residues are depicted in white, and the inhibitor is green. Hydrogen-bonding interactions are illustrated with yellow dashed lines. Several residues that are involved in hydrophobic interactions with 5 are omitted for clarity and described in the text. The propyl group resides in a pocket that is enlarged by an outward shift of the αC-helix. The DFG sequence (D594-G596) resides in its active (DFG-in) conformation.

To further improve the potency of the amide series, bicyclic heterocycles were prepared as replacements for the pyridine hinge-binding template. Cyclization of the hinge donor amine of 4 to the 5-position of the pyridine via a five-membered ring suggests three potential bicyclic cores: imidazopyridine, pyrrolopyridine, and pyrazolopyridine. These bicycles were designed to form a bidentate hydrogen-bonding interaction at the hinge, enhance the interaction with the indole of Trp531, and provide a point of diversity for analogue synthesis. Scheme 2 illustrates the synthesis of imidazopyridine and pyrrolopyridine amide series analogues 8−10. Although imidazopyridine 8 had encouraging enzymatic activity, cellular activity was poor (Table 1). This result was surprising given that a related imidazopyrimidine hinge binder has demonstrated excellent cellular activity in an alternative series of B-Raf inhibitors.22 While pyrrolopyridine 9 had similar enzymatic activity to imidazopyridine 8, it was 20-fold more active in the cellular assay. In addition, pyrrolopyridine 9 was significantly more potent than aminopyridines 4 and 5, demonstrating the advantage of a bicyclic hinge binder. Bromination at the 3-position of pyrrolopyridine 9 afforded compound 10 and a 2-fold improvement in cellular activity. This result indicated that potency could be increased by an appropriate substitution at the 3-position of the bicyclic hinge-binding template.

Scheme 2. Preparation of Imidazopyridine and Pyrrolopyridine Amides 8−10.

Scheme 2

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Reagents and conditions: (a) Compound 2a, EDCI, HOBt, DMF, 22 °C, 9% for 8, 53% for 9. (b) Compound 9, n-bromosuccinimide, CHCl3, 82%.

The pyrazolopyridine hinge-binding template was prepared concurrently and ultimately provided the most potent B-RafV600E inhibitors from the amide series. A key advantage of this template included the facility with which substituents of varying electronics could be synthetically incorporated at the 3-position of the core (Scheme 3). Nitromalonaldehyde condensation27 with 3-substituted-5-aminopyrazoles 11a−c provided pyrazolopyridines 12a−c. Bromination of 12a introduced a synthetic precursor (12d) from which a 3-amino pyrazolopyridine (12e) was obtained. Nitro reduction of 12a−e followed by coupling to benzoic acids 2a or 2b provided amides 13−19.

Scheme 3. Preparation of Pyrazolopyridines 13−19.

Scheme 3

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Reagents and conditions: (a) Sodium nitromalonaldehyde monohydrate, AcOH or water, 90 °C, 38−94%. (b) Br2, NaOH, dioxane, 0−22 °C, 69%. (c) Forty percent HNMe2/water, microwave, 160 °C, 54%. (d) H2, 10% Pd/C, EtOAc/MeOH, 22 °C, or SnCl2, EtOAc, reflux, 82−99%. (e) Benzoic acid 2a or 2b, EDCI, HOBt, DMF, 22 °C, 35−82%.

Varying the substituent at the 3-position of the pyrazolopyridine resulted in improvements in the enzymatic and cellular activity of the inhibitors while significantly modulating their pharmacokinetic and physicochemical properties. Unsubstituted pyrazolopyridine 13 was similarly active to the corresponding pyrrolopyridine 9 (Table 1). Most substitutions at the 3-position [Br- (14), Me- (15), and Me2N- (16)] produced inhibitors within a narrow range of activity and with no significant improvement in potency over 13. One exception to the flat SAR observed at the 3-position of the pyrazolopyridine template was obtained by the introduction of a methoxy group.28 This led to 3-methoxy pyrazolopyridine 17, the most cell potent inhibitor in the amide series. Compounds with halogen substitutions on the benzamide moiety were also examined. While substitution of chloro for fluoro at the 6-position of the benzamide resulted in a 3-fold improvement in the enzymatic activity within two pairs of compounds, there was no change in cellular activity (18 vs 13 and 19 vs 17). Compounds 17 and 19 both had similar potency to PLX4032 in the A375 cell line but were 3-fold more potent in Malme-3M cells. The X-ray crystal structure of 17 in complex with B-Raf further confirmed the amide series binding model (Figure 2). Importantly, the proposed interaction of the bicyclic hinge-binding core with Trp531 was validated: A π-stacking interaction between the pyrazole portion of the bicycle and the side chain indole of Trp531 was revealed and likely contributes to the improvement in potency over the pyridine amides 3−5.22

Figure 2.

Figure 2

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X-ray crystal structure of 17 in complex with B-RafWT. Notation is as described for Figure 1. In addition to the hydrogen-bonding interactions described in Figure 1, the sulfonamide NH of 17 is in close contact to the main chain NH of Asp594, indicating that the sulfonamide is deprotonated.23

Both the nature of the bicyclic template and its substituents were found to have significant effects on the pharmacokinetic and physicochemical properties of the amide series inhibitors (Table 2). Pyrrolo- and pyrazolopyridine amides were dosed orally in mice at 30 mg/kg as a solution in 40% PEG400/10% ethanol/50% water, while select compounds were also dosed intravenously. These compounds typically exhibited high oral bioavailability (% F) and oral exposure (AUC). The total clearance (CL) and volume of distribution (Vd) were low and, with the exception of 10, correlated with plasma protein binding (PPB). Aqueous solubility varied across the series and correlated with melting point (mp), which broadly ranged from 173 to 261 °C; these data suggest that subtle crystal packing effects exist within this series, which could be modulated by varying the substituents around the molecule. Unfortunately, the compounds with the highest aqueous solubilities also exhibited the lowest oral exposures (13, 15, and 16). The substitution of chloro for fluoro at the 6-position of the benzamide ring had pronounced effects on pharmacokinetic and physicochemical properties: attenuated aqueous solubility, increased PPB, lower IV clearance, and significantly higher oral AUC (18 vs 13 and 19 vs 17). Overall, compounds 17 and 19 possessed the best combined potency and exposure from this series, which illustrates the advantage of the 3-methoxy pyrazolopyridine hinge-binding template. However, when these two compounds were dosed orally in mice at 100 mg/kg using the same solution formulation, compound 17 exhibited a dose proportional increase in exposure, while the oral bioavailability of 19 dropped to 1%. The low aqueous solubility and high melting point of this compound likely led to crystallization in the gut preventing absorption.

Table 2. Mouse Pharmacokinetics and Physicochemical Properties of Pyrrolo- and Pyrazolopyridine Amides.

compd 30 mg/kg po AUCa (% F) 100 mg/kg po AUCa (% F) CLb Vdc mouse PPBd mp (°C) sol. at pH 6.5, 7.4e
9 200 (76)   1.9 0.20 97.9 243 1, 4
10 49 (71)   7.2 0.24 99.4 227 <1, <1
13 40 (100)   15 0.68 87.1 173 80, 150
14 52.5       98.4 220 1, 3
15 18.3       89.9 181 380, 740
16 1.09       90.8 181 290, 450
17 190 (48) 664 (51) 1.3 0.11 97.4 229 4, 9
18 147 (67)   2.3 0.17 97.1 236 15, 48
19 1080 (53) 50.7 (1.2) 1 0.11 99.1 261 <1, 2
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a

μg h/mL.

b

mL/min/kg.

c

L/kg.

d

Plasma protein binding (%).

e

μg/mL.

In an effort to rescue the poor pharmacokinetics of compound 19 at higher doses and because of concerns with daily dosing of high concentrations of PEG400 in potential preclinical toxicology and xenograft studies, 17 and 19 were reformulated as a 25% amorphous spray-dried dispersion (SDD) on hydroxypropyl methylcellulose acetate succinate (HPMCAS) polymer.29 Bridging pharmacokinetic studies in mice with 17 dosed orally at 2.5, 10, 40, and 80 mg/kg revealed a linear dose response, albeit with oral bioavailabilities lower than with the soluble PEG400-based solution formulation (F = 19−25 vs 48−51%). Nonetheless, exposure exceeded the levels projected to achieve efficacy in mouse models of cancer driven by B-RafV600E. Compound 19 was also dosed as the SDD formulation in mice at 10, 30, 60, and 100 mg/kg and demonstrated decreasing oral bioavailability with increasing dose (F = 53, 28, 19, and 13%). Although a linear dose response was not achieved, this formulation provided a significant improvement in exposure at 100 mg/kg for 19 (13 vs 1%), higher bioavailability than 17 at 10 mg/kg (53 vs 25%), and likewise exceeded the levels projected to achieve efficacy.

To further characterize in vitro potency and in preparation for mouse xenograft studies, inhibition of cellular viability by compound 17 was measured against a panel of 13 melanoma and 10 colon cancer cell lines driven by the B-RafV600E mutation, and potencies were compared to PLX4032 (1). In melanoma, 17 was 2−3-fold more potent than 1 in 8 of the 13 cell lines tested and equivalent in the remaining 5, while in colon cancer, 17 was 2−14-fold more potent than 1 in 9 of 10 cell lines examined. Compound 19 was screened against four of the colon cancer cell lines and was similarly more potent than 1. For Colo205, in which mouse xenograft studies were run, the EC50 values for 17 and 19 were 80 and 70 nM as compared to 240 nM for 1.

The activity of 17 and 19 was also determined against a diverse panel of kinases and demonstrated similar selectivity profiles with >100-fold selectivity for 223/228 kinases examined. Compounds 17 and 19 displayed no inhibition of cytochrome P450 enzymes at 25 μM, no hERG channel inhibition at 100 μM, and were negative in the Ames test for genotoxicity. On the basis of their in vitro potency and favorable pharmacokinetic properties, 3-methoxy pyrazolopyridines 17 and 19 were selected for dose-ranging tumor growth inhibition studies in the Colo205 mouse xenograft model.

The SDD of 17 and 19 were administered at ascending doses once daily for 17 or 20 days in mice with established Colo205 xenografts (Table 3). Tumor growth inhibition was observed for both compounds with a maximum tumor regression of 76% at 125 mg/kg for 17 and 95% at 100 mg/kg for 19. Analysis of tumor samples indicated that in vivo efficacy requires sustained inhibition of pERK levels of at least 50% for 6 h.30 Given that compound 19 was similarly active to 17 in the biochemical assay and in several cellular assays, the superior efficacy of 19 across the entire dose range of the xenograft study was presumably due to its higher exposure. The low volumes of distribution for both compounds (Table 2) did not abate the efficacy of the compounds. Significantly, over the course of the dosing period, no toxicity and no significant alterations in body weight were observed at any dose for either 17 or 19.

Table 3. Dose-Ranging Tumor Growth Inhibition Studies in Colo205 Mouse Xenografts.

compd no. of days dosed ED50a ED90a max TGI (%)b
17 20 20 80 76
19 17 11 50 95
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a

mg/kg.

b

Tumor growth inhibition as a percent of vehicle control.

In summary, selective, orally bioavailable, and efficacious inhibitors of B-RafV600E were developed. These inhibitors were based on a novel 3-methoxy pyrazolopyridine hinge-binding template, which evolved through structure-based design from a simple pyridine lead (3). While the activity of 3 was micromolar in both enzymatic and cellular assays, the introduction of a hydrogen bond donor, utilization of a bicyclic hinge-binding template, and addition of lipophilic contacts provided potent pyrrolo- and pyrazolopyridine inhibitors of B-RafV600E. Optimization at the 3-position ultimately led to 3-methoxy pyrazolopyridines 17 and 19, which demonstrated a 300-fold improvement in potency over the lead pyridine 3. Compounds 17 and 19 were highly potent against a broad panel of cancer cell lines driven by B-RafV600E, particularly those derived from colon cancer, and displayed significant antitumor activity in an in vivo colon cancer model driven by B-RafV600E. Although 17 and 19 were well tolerated in mice during the xenograft studies, RAF inhibitors have been reported to activate the MAPK pathway in B-RafWT cells.19,20,22,31−33 This paradoxical activation has been linked to enhanced proliferation and stimulated tumor growth in vivo, and hyperplasia of normal epithelial cells was observed in mice.34 Pyrazolopyridine Raf inhibitors likewise activate the MAPK pathway, and the results from these studies will be presented elsewhere.

Acknowledgments

We thank Josh Ballard for HRMS data and Ben Colson for solubility determinations. We also thank Jim Blake for helpful discussions.

Supporting Information Available

Experimental procedures for the synthesis of 3−5, 8−10, 13−19, kinase selectivity, and cell viability data for 17 and 19 and details of in vitro and in vivo assays. This material is available free of charge via the Internet at http://pubs.acs.org.

Supplementary Material

ml200025q_si_001.pdf (212.2KB, pdf)

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