RAF; p38 MAPK

Kinome profiling of Exarafenib/KIN-2787 revealed exquisite selectivity with only 2 of 669 non-RAF family kinases inhibited > 75% at 1 M Exarafenib/KIN-2787 and retained ̃10x and 70x selectivity window against those two kinases, DDR1 and p38b, respectively, relative to RAF kinases. We previously reported KIN-2787 activity across BRAF, NRAS, and KRAS mutant tumor cell lines with greatest sensitivity in Class II and III dimer-driven BRAF models [1]. KIN-2787 is a highly selective, potent, next-generation, pan-RAF inhibitor with activity across BRAF and RAS mutant human tumor cell models [1].

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In Vitro Characterization of Exarafenib/15 [2]
The pan-RAF inhibitor 15/Exarafenib is potent in biochemical assays, showing low nanomolar activity across the isoforms (ARAF IC50 = 2.4 nM, BRAF IC50 = 3.5 nM, and CRAF IC50 = 1.4 nM; Table 3). In a full kinase panel of >600 kinases (Reaction Biology HotSpot Kinome Selectivity assay; see Supporting Information) including both wild-type and mutants, compound 15/Exarafenib at 1 μM concentration possesses a class-leading selectivity profile (Figure 4a), with only one off-target wild-type kinase observed at >90% inhibition (DDR1 IC50 107 nM). Similarly, in the mutant panel, beyond RAF family mutants, only a DDR mutant is inhibited (Supporting Information). In expanded RAF-mutant and RAF-dependent cellular studies (Table 3), compound 15 inhibits pERK signaling in monomeric class I altered cell lines (A375 EC50 = 62 nM and Colo800 EC50 = 103 nM), dimer-driven class II altered cell lines (NCI-H2405 EC50 = 10 nM, BxPC-3 EC50 = 51 nM, and OV-90 EC50 = 26 nM) and heterodimer class III altered cell lines (WM3629 EC50 = 9 nM, and CAL-12T EC50 = 18 nM) at unbound plasma concentrations readily achieved in the clinic (vide infra). In contrast, MAPK-dysregulated cell lines with BRAFWT background are less sensitive to 15, which has only modestly potent pERK inhibition (SK-MEL-2 EC50 = 144 nM, NCI-H358 EC50 = 351 nM, and CHL-1 EC50 = 580 nM). To test for drug-induced paradoxical activation, an NRASQ61L mutant cell line with BRAFWT background, IPC-298, was treated with 15 and known paradoxical activators 1 and 2. Consistent with the anticipated ability of 15 to inhibit both protomers of BRAFWT dimer signaling in a cellular context, a dose dependent inhibition of pERK is observed (EC50 = 265 nM), whereas 1 and 2 clearly induce paradoxical activation of pERK (Figure 4b).

Here, we evaluated NRAS mutant, BRAF WT melanoma for combination potential with binimetinib. Melanoma tumor cell lines bearing NRAS hotspot mutations demonstrated synergistic benefit with Exarafenib/KIN-2787 combined with binimetinib. Daily KIN-2787 plus binimetinib treatment in NRAS mutant melanoma xenograft models resulted in significant tumor growth inhibition benefit relative to either agent alone and was associated with added MAPK pathway biomarker suppression.
Preclinical in vivo studies using Exarafenib/KIN-2787 in combination with binimetinib demonstrated significant combination benefit in NRAS mutant melanoma models. Taken together with its unique selectively, these data support use of KIN-2787 in combination therapy in this patient segment. A Phase 1/1b dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285) [1].

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Pharmacology of Exarafenib/15 [2]
Efficacy was assessed in BALB/c nude mice implanted with BxPC-3 cells, a human pancreatic ductal adenocarcinoma (PDAC) model with a class II BRAFindel(NVTAP) alteration. Dosing groups treated with compound 15/Exarafenib at 1.5, 3, 5, and 10 mg/kg twice daily (BID) for 14 days exhibited dose-dependent tumor growth inhibition (TGI) of 68%, 79%, 88%, and 118% respectively, compared to the vehicle-treated group (Figure 5a). All dose levels were well-tolerated, as determined by no changes in body weight during the study period (Supporting Information). End-of-study free plasma concentrations confirmed a near-linear dose-proportional exposure (Figure 5b). In vivo biomarker modulation was measured in a separate cohort of BxPC-3 tumor-bearing animals treated for 3 days. The measured ratio of pERK relative to total ERK was suppressed in a dose-dependent manner at 1 h post-dose and returned to baseline by 12 h (Figure 5c). These data demonstrate robust in vivo efficacy associated with the degree and duration of pharmacodynamic mechanism modulation by compound 15/Exarafenib in a class II BRAF-mutant cancer model.

To evaluate compound 15/Exarafenib in a class III BRAF-mutant setting, mice bearing WM3629, a model of human melanoma with a class III BRAFD594G alteration and an upstream NRASG12D activating mutation, were treated for 21 days. Doses of compound 15 at 3, 5, 10, and 20 mg/kg of BID were well-tolerated (see Supporting Information) and resulted in robust and dose-dependent TGI of 81%, 91%, 100%, and 105%, respectively (Figure 6a).

In addition to the class II and class III BRAF-altered tumors, which signal through BRAF dimers, compound 15/Exarafenib was likewise tested in the monomeric class I BRAFV600E human melanoma xenograft model, A375. Consistent with in vitro cellular data showing weaker potency in class I BRAF-mutant tumor cell lines versus class II or class III, A375 xenografts required higher exposures of compound 15 to achieve efficacy in vivo. The 10 mg/kg BID treatment group resulted in modest efficacy of 21% TGI (Figure 6b), while the 30 mg/kg BID group led to 130% TGI and tumor regression (Figure 6b). Importantly, compound 15 remained well-tolerated even at the higher dose level (see Supporting Information).

Based on in vitro MAPK pathway inhibition data indicating that NRAS-mutant melanoma is also dependent on RAF dimer signaling, efficacy was evaluated in a human melanoma model with NRASQ61R mutation and BRAFWT background (SK-MEL-2). Treatment of NOD SCID mice bearing SK-MEL-2 tumors with compound 15/Exarafenib resulted in a modest 13% TGI at 10 mg/kg BID, while 30 mg/kg BID resulted in 135% TGI and tumor regressions (Figure 6c). This data validate the potential for compound 15 to be effective in RAF-dependent cancers that are otherwise BRAFWT, such as NRAS-mutant melanoma.

Altogether, the in vivo studies establish tolerability and anti-tumor activity of compound 15/Exarafenib across tumor models with different classes of BRAF alterations or RAF-dependency.

Kinome profiling was evaluated by radiometric enzyme assay at Reaction Biology across 688 kinases (including wild type, atypical, and mutant) [1].

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BRAF Kinase biochemical assay: [2]
Small molecule inhibition of the BRAF kinase was measured using ADP-Glo assay. In the assay, ADP is converted to ATP in the presence of test kinase and substrate, resulting in luciferase reaction and luminescent readout with light generated proportional to the relative kinase activity. Compounds diluted in DMSO were used in 10-point, 3-fold dose curve for both assays. Final concentrations of 6 nM BRAF and 30 nM MEK1 substrate were incubated with 3 μM ATP, 10 mM MgCl2, 0.003% Brij-35, 2 mM DTT, 0.05% BSA, 1 mM EGTA, and 50 mM HEPES for 90 minutes at room temp prior to addition of ADP-Glo reagent for 40 minutes, and detection reagent for 45 minutes. Luminescence was read on an Envision plate reader and percent remaining activity was used to calculate IC50 using a four-parameter fit model using Dotmatics Knowledge Solutions Studies curve fitting.

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Kinome selectivity panel: [2]
The kinase specificity profile for exarafenib was determined as previously described.1 Exarafenib was assessed in the Reaction Biology HotSpot Kinome Selectivity assay at 10uM ATP with activity presented as % Inhibition (=100 – Enzyme activity which is presented relative to DMSO controls). Data for the 688 mutant and wildtype kinases in Table S3.

Cellular activity was assessed by suppression of downstream MAPK pathway signaling and cell growth inhibition in human tumor cell lines. Combination cell growth inhibition studies were performed in 9x5 dose matrices with KIN-2787 and binimetinib, respectively. Extended cell growth inhibition effects were assessed by Incucyte imaging [1].

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Cellular assay: [2]
Cells were seeded at 8000 cells/well in 24 µl growth media in a 384-well plate and allowed to adhere at 37°C with 5% CO2 overnight. The following day, compounds were serially diluted into 10-point, 3-fold dilution curves in 384 well plates. Compound was transferred to cell plates using Echo550 such that the final concentration range was 0.508 nM to 10 mM in 0.1% DMSO with 0.1% DMSO being used as negative control. Cells were incubated with compounds for 1 hour at 37°C with 5% CO2. Cells were lysed by addition of 8 µl 4X lysis buffer provided with HTRF kit plus 1X protease/phosphatase inhibitor cocktail. 20 µl lysate was transferred to HTRF plate followed by 2.5 µl each of anti-ERK1/2-Europium/Terbium Cryptate and antiphospho-ERK1/2 antibody solutions per manufacturer’s instructions and incubated. Specific signal was measured at 665nm (donor) and 620nm (acceptor) on a Perkin Elmer Envision 2105 and the ratios used to calculate IC50 values within the Dotmatics Knowledge Solutions Studies curve fitting environment and are presented in Table 1-3.

In vivo KIN-2787 and combination efficacy was evaluated in NRAS mutant xenograft models. [1]

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In vivo pharmacology: [2]
iPC-3, WM3629, or A375 cells were inoculated into BALB/c nude mice, while SKMEL-2 cells were inoculated into NOD SCID mice. Animals were injected subcutaneously on the flank with 5 x 106 or 1 x 107 tumor cells in 0.1 ml of a 1:1 mixture of media and Matrigel. Bodyweights were monitored every other day, and tumor volumes (width2 × length × 0.5) were monitored 2-3 times per week. Efficacy studies were initiated when average tumor volumes reached ∼200-300 mm3 . Animals were randomized into treatment groups (n= 9/group for all models except n= 6/group for A375) and dosed twice daily for 2-4 weeks with vehicle (0.5% methylcellulose/0.1% Tween80 in water) or compound 15/Exarafenib from 1.5 mg/kg up to 30 mg/kg. Tumor growth inhibition (TGI) was calculated as [1 − (TVf − TV0)treated/(TVf − TV0)control] × 100, where “TVf” and “TV0” are the mean tumor volumes on the final day and the initial day of treatment, respectively. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc comparisons. Significance was set at 5% or p<0.05 for all tests. End-of-study plasma samples were collected at multiple timepoints following the last dose and analyzed by LC/MS/MS. [2]

In Vitro ADME and Physical-Chemical Properties [1]
Compound 15/Exarafenib was tested for metabolic stability in hepatocytes across species, demonstrating >60% remaining after 60 min incubation (h = 85%, d = 72%, r = 69%, m = 62%, Table 3). Compound 15/Exarafenib has a molecular weight of 521.58, a melting point of 193 °C, a measured pKa of 5.3, and a measured log D of 3.82. The increase in basicity, compared to ethylene glycol 14 (pKa = 3.8), enables conventional formulation of 15 as an HCl salt, which affords favorable solubility across physiologically relevant pHs (7.4–2.0) and thermodynamic solubility in biorelevant media (FaSSGF, FaSSIF). The properties allow for a reliably measured fraction of unbound in human plasma of 6.7%. With the criteria met for a potent pan-RAF inhibitor coupled with minimal paradoxical activation and excellent ADME and pharmaceutical properties, 15 was further profiled in vivo.

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Pharmacokinetics of 15/Exarafenib [1]
The pharmacokinetic properties of compound 15/Exarafenib were determined across different species as summarized in Table 4. Compound 15 exhibits low to moderate i.v. clearance in preclinical species (mouse = 7.5, rat = 17.4, dog = 11.7 mL/min/kg), low to moderate Cmax (mouse = 7.0, rat = 1.4, dog = 4.8 μM), and moderate volume of distribution (mouse = 1.6, rat = 2.5, dog = 1.6 L/kg). Compound 15 was readily absorbed after oral administration, resulting in excellent exposures (mouse = 16.7, rat = 15, dog = 31 μM·h) with corresponding oral bioavailability >40%.

[1]. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in combination with MEK inhibition in preclinical models of human NRAS mutant melanoma.2022 Jun 2;40(16): e15099.

[2]. The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition. J Med Chem. 2024 Feb 8;67(3):1747-1757.

Exarafenib is an orally available inhibitor of class I, II, and III B-Raf (BRAF) protein kinases, with potential antineoplastic activity. Upon administration, Exarafenib binds to and inhibits Class I, Class II, or Class III B-Raf mutations. This prevents B-Raf-mediated signal transduction pathways, which may lead to an inhibition of tumor growth in B-Raf mutant cells. B-Raf protein kinases play a key role in the RAF/MEK/ERK signaling pathway, which is often deregulated and mutated in human cancers, and plays a key role in tumor cell proliferation and survival.

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NRAS mutations which activate MAPK signaling represent oncogenic driver alterations in approximately 20% melanoma cases in the US. NRAS mutant melanomas are uniquely dependent upon CRAF rather than BRAF for activation of downstream MEK/ERK signaling. In BRAF mutant melanoma, approved RAF-targeted therapies are commonly used in combination with a MEK inhibitor which provides clinical benefit by inhibition of two targets within the oncogenic MAPK signaling pathway. Emerging data with pan-RAF inhibitors in early clinical development suggests benefit with and without combined MEK inhibition, yet no approved targeted therapy exists for NRAS mutant melanoma patients. KIN-2787 is a novel, orally available pan-RAF inhibitor designed to be effective in RAF-dependent cancers, regardless of isoform. [1]

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RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of Exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.[2]

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FDA-approved RAF inhibitors have demonstrated clinical efficacy in treating BRAFV600E mutant cancers. However, they prove to be ineffective against BRAF class II and III and NRAS mutant tumors, which derive their signaling activity through dimers. Moreover, these inhibitors have the unintended consequence of inducing an active dimer conformation, leading to the paradoxical activation of the MAPK pathway where dimer signaling predominates, such as in normal tissues bearing BRAFWT. The heterogeneity of RAF alterations underscores the challenges in developing targeted therapies while avoiding undesirable side effects. We have discovered Exarafenib (15), a pan-RAF inhibitor that intercepts the αC-helix-IN conformation of the kinase domain and is capable of inhibiting both mono- and dimeric signaling. Exarafenib displays an exquisitely clean kinome selectivity profile and remarkable cellular potency against a broad range of RAF alteration types without inducing paradoxical activation. With the goal of achieving broad target coverage in vivo, multi-parameter optimization was employed to integrate drug-like properties into the molecular designs. As a result, exarafenib exhibits outstanding ADME/DMPK properties and shows favorable solubility at physiologically relevant pH levels. Accordingly, in vivo treatment of exarafenib in class I, II, and III and NRAS tumor models is well tolerated and results in a dose-dependent efficacy as a single agent. These findings culminated in the selection of exarafenib as a clinical candidate. The safety and efficacy of exarafenib in cancer patients harboring RAF and NRAS alterations will be reported in due course.[2]

C26H34F3N5O3

521.575076580048

521.26

C, 59.87; H, 6.57; F, 10.93; N, 13.43; O, 9.20

2639957-39-2

2639957-39-2

156297592

White to yellow solid

4

3

9

7

37

739

2

CC1=C(C=C(C=C1)NC(=O)N2CC[C@H](C2)CC(F)(F)F)C3=CC(=NC(=C3)N4CCOCC4)N[C@H](C)CO

GZMYLSJUNSCMTD-MOPGFXCFSA-N

InChI=1S/C26H34F3N5O3/c1-17-3-4-21(31-25(36)34-6-5-19(15-34)14-26(27,28)29)13-22(17)20-11-23(30-18(2)16-35)32-24(12-20)33-7-9-37-10-8-33/h3-4,11-13,18-19,35H,5-10,14-16H2,1-2H3,(H,30,32)(H,31,36)/t18-,19+/m1/s1

(3S)-N-[3-[2-[[(2R)-1-hydroxypropan-2-yl]amino]-6-morpholin-4-ylpyridin-4-yl]-4-methylphenyl]-3-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide

Exarafenib; RAF/KIN_2787; 2639957-39-2; KIN-2787 free base; 2VPX8HL8AB; compound 15 [Chen et al., 2024]; example 14 [WO2021081375A1]; KIN 2787; KIN2787; KIN-2787

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~191.7 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.79 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.79 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)