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 physicochemical properties [1]
Compound 15/Exarafenib was tested for metabolic stability in hepatocytes of different species. The results showed that >60% of the residue remained after 60 minutes of incubation (h = 85%, d = 72%, r = 69%, m = 62%, Table 3). The molecular weight of compound 15/Exarafenib was 521.58, the melting point was 193 °C, the pKa value was 5.3, and the log D value was 3.82. Compared with ethylene glycol 14 (pKa = 3.8), its basicity was enhanced, which allowed compound 15 to be routinely prepared in the form of hydrochloride, thus exhibiting good solubility in the physiologically relevant pH range (7.4–2.0) and good thermodynamic solubility in biologically relevant media (FaSSGF, FaSSIF). The properties of this compound allowed its free fraction in human plasma to be reliably measured as 6.7%. Compound 15 was further analyzed in vivo due to its meeting the criteria for highly effective pan-RAF inhibitors and its minimal paradoxical activation, excellent ADME and pharmaceutical properties.

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Pharmacokinetics of Compound 15/Exarafenib [1]
The pharmacokinetic properties of Compound 15/Exarafenib in different species are summarized in Table 4. Compound 15 showed low to moderate intravenous clearance (mice = 7.5, rats = 17.4, dogs = 11.7 mL/min/kg), low to moderate Cmax (mice = 7.0, rats = 1.4, dogs = 4.8 μM) and moderate volume of distribution (mice = 1.6, rats = 2.5, dogs = 1.6 L/kg) in preclinical animal models. Compound 15 was readily absorbed after oral administration, resulting in excellent exposure (mice = 16.7, rats = 15, dogs = 31 μM·h) and 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 administered class I, II, and III B-Raf (BRAF) protein kinase inhibitor with potential antitumor activity. After administration, Exarafenib binds to and inhibits the activity of class I, II, or III B-Raf mutations. This blocks the B-Raf-mediated signal transduction pathway, thereby inhibiting tumor growth in B-Raf-mutant cells. B-Raf protein kinases play a crucial role in the RAF/MEK/ERK signaling pathway, which is frequently dysregulated and mutated in human cancers and plays a vital role in tumor cell proliferation and survival. NRAS mutations activate the MAPK signaling pathway; in the United States, oncogene alterations are present in approximately 20% of melanoma cases. NRAS-mutant melanomas specifically rely on CRAF, rather than BRAF, to activate the downstream MEK/ERK signaling pathway. In BRAF-mutant melanomas, approved RAF-targeted therapies are often used in combination with MEK inhibitors to achieve clinical efficacy by inhibiting two targets in the oncogenic MAPK signaling pathway. Emerging data from pan-RAF inhibitors in early clinical development suggest efficacy regardless of combination with MEK inhibitors, but there are currently no approved targeted therapies for patients with NRAS-mutant melanoma. KIN-2787 is a novel oral pan-RAF inhibitor designed to be effective against RAF-dependent cancers, regardless of RAF subtype. [1] RAF is a core signaling component of the MAPK kinase cascade and is frequently mutated in a variety of cancers, including melanoma, lung cancer, and colorectal cancer. Approved inhibitors primarily target BRAFV600E mutations, which lead to constitutive activation of monomeric protein (class I) kinase signaling. However, these inhibitors also present a paradox: they activate the kinase signaling pathway of RAF dimers, resulting in enhanced MAPK signaling in normal tissues. In recent years, attention has been paid to targeting RAF alterations that activate dimer signaling (class II and III BRAF and NRAS). However, finding an effective, selective, and biopharmaceutical inhibitor that maintains stable target inhibition in a clinical setting remains a challenge. This article reports the discovery of Exarafenib (15), a potent and selective inhibitor that blocks RAF proteins in a dimer-compatible αC-helix-IN conformation and has shown antitumor efficacy in preclinical models of BRAF class I, II, and III and NRAS alterations. [2] FDA-approved RAF inhibitors have shown clinical efficacy in treating BRAFV600E mutant cancers. However, these inhibitors are ineffective against BRAF class II and III and NRAS-mutant tumors, whose signaling activity is mediated through dimers. Furthermore, these inhibitors have the unintended consequence of inducing an active dimer conformation, leading to paradoxical activation of the MAPK pathway, which is dominant in dimer signaling, for example, in normal tissues carrying BRAF wild-type (BRAFWT). The heterogeneity of RAF alterations highlights the challenge of developing targeted therapies while avoiding adverse side effects. We discovered Exarafenib (15), a pan-RAF inhibitor that blocks the αC-helix-IN conformation of the kinase domain and inhibits monomeric and dimeric signaling. Exarafenib exhibits high kinase selectivity and significant cellular activity against a variety of RAF alterations without inducing paradoxical activation. To achieve broad target coverage in vivo, we used a multi-parameter optimization approach to integrate drug-like properties into the molecular design. The results showed that esarafenib has excellent ADME/DMPK properties and good solubility within a physiologically relevant pH range. Therefore, esarafenib was well tolerated in vivo in class I, II, and III tumor models, as well as in NRAS-mutant tumor models, and showed dose-dependent efficacy as monotherapy. Based on these findings, we ultimately selected esarafenib as a clinical candidate. We will report on the safety and efficacy of esarafenib in cancer patients with RAF and NRAS mutations 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.)