Tovorafenib (MLN2480; TAK-580; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) is an orally bioactive, potent and selective pan-Raf kinase inhibitor with potential anticancer activity. It is being tested clinically on people with advanced solid tumors or melanoma. At concentrations that are tolerated in vivo, MLN2480 inhibits MAPK pathway signaling in some RAS mutant and BRAF mutant preclinical cancer models. At very low concentrations, it is found to activate phosphorylated MEK, but at higher concentrations, it inhibits this same activity. Different models and genetic contexts are found to have different MLN-2480 inhibitory effects. The Raf kinases (A-Raf, B-Raf, and C-Raf) are important mediators of the mitogen-activated protein kinase (MAPK) pathway, which controls cell growth and survival. In many cases, Ras or Raf activating mutations lead to the MAPK pathway becoming dysregulated in human cancers.

Tovorafenib (OJEMDA™) is a once-weekly oral, selective, brain-penetrant, type II RAF kinase inhibitor being developed by Day One Biopharmaceuticals, Inc., under a license from Takeda Oncology, for the treatment of paediatric low-grade glioma (pLGG) and solid tumours. Most pLGGs harbour alterations in the MAPK pathway, such as a BRAF mutation or BRAF fusion, which result in aberrant intracellular signalling. Tovorafenib is an inhibitor of mutant BRAF V600E, wild-type BRAF and wild-type CRAF kinases and BRAF fusions. In April 2024, tovorafenib received its first approval in the USA for the treatment of patients aged ≥ 6 months with relapsed or refractory pLGGs harbouring a BRAF fusion or rearrangement, or BRAF V600 mutation. It received accelerated approval for this indication based on the response rate and duration of response achieved in this population in the ongoing, pivotal, phase 2 FIREFLY-1 study. Clinical development of tovorafenib is underway in numerous countries worldwide. This article summarizes the milestones in the development of tovorafenib leading to this first approval for relapsed or refractory pLGG with an activating BRAF alteration [1].

Physicochemical Properties

Molecular Formula	C17H12CL2F3N7O2S
Molecular Weight	506.29
Exact Mass	505.01
Elemental Analysis	C, 40.33; H, 2.39; Cl, 14.01; F, 11.26; N, 19.37; O, 6.32; S, 6.33
CAS #	1096708-71-2
Related CAS #	1096708-71-2
PubChem CID	25161177
Appearance	White to off-white solid powder
Density	1.64
LogP	5.024
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	5
Heavy Atom Count	32
Complexity	695
Defined Atom Stereocenter Count	1
SMILES	ClC1C(N([H])[H])=NC([H])=NC=1C(N([H])[C@]([H])(C([H])([H])[H])C1=NC([H])=C(C(N([H])C2C([H])=C(C(F)(F)F)C(=C([H])N=2)Cl)=O)S1)=O
InChi Key	VWMJHAFYPMOMGF-ZCFIWIBFSA-N
InChi Code	InChI=1S/C17H12Cl2F3N7O2S/c1-6(28-15(31)12-11(19)13(23)27-5-26-12)16-25-4-9(32-16)14(30)29-10-2-7(17(20,21)22)8(18)3-24-10/h2-6H,1H3,(H,28,31)(H2,23,26,27)(H,24,29,30)/t6-/m1/s1
Chemical Name	2-[(1R)-1-[(6-amino-5-chloropyrimidine-4-carbonyl)amino]ethyl]-N-[5-chloro-4-(trifluoromethyl)pyridin-2-yl]-1,3-thiazole-5-carboxamide
Synonyms	BIIB-024; TAK-580; AMG2112819; TAK 580; MLN2480; TAK580; AMG 2112819; BIIB024; AMG-2112819; MLN 2480; MLN-2480; BIIB 024; BIIB-024
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Solubility Data

Targets	Raf kinase
ln Vitro	MLN2480 inhibits MAPK pathway signaling at concentrations that are tolerated in vivo in BRAF mutant and some RAS mutant preclinical cancer models[1]. At very low concentrations, it is found to activate phosphorylated MEK, whereas at higher concentrations, it inhibits this same activity. It has been discovered that MLN-2480's inhibitory effects differ between models and genetic contexts[2]. In vitro testing of the drug combination of MLN2480 and TAK-733 (an investigational allosteric MEK kinase inhibitor) in cell proliferation assays shows synergistic activity. Additionally, western blot analysis shows how MLN2480 reverses the feedback activation of MEK in response to TAK-733, resulting in more concerted MAPK pathway inhibition. PRAK is only weakly inhibited by MLN-2480 [1][2]. Potency of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and naporafenib across RAF isoforms [7] To better understand the RAF selectivity of tovorafenib and naporafenib, we measured their inhibition of the RAF complexes described above using our adapted TR-FRET assay. BRAFWT, BRAFV600E, and CRAFSSDD were assayed at a concentration of 1 nM, while CRAFWT and ARAFSSDD were assayed at 4 nM and 10 nM, respectively, due to their lower enzymatic activities. An ATP concentration of 200 μM was used for all assays, and the WT MEK1 substrate concentration was 250 nM. Measured IC50 values and calculated Ki values are provided in Table 1, and representative concentration-response curves from which they were derived are shown in Figure 2. [7] Both Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and naporafenib were most potent as inhibitors of CRAF, with IC50 values of 94.2 nM and 3.7 nM, respectively, against the WT CRAF kinase. Potency against the CRAFSSDD mutant was essentially the same as for CRAFWT (Table 1). Both agents exhibited intermediate potency against BRAFWT and BRAFV600E (633 nM for tovorafenib and 13.4 nM for naporafenib on BRAFWT) and were much weaker inhibitors of ARAFSSDD. Tovorafenib did not completely inhibit ARAFSSDD even at 10 μM, the highest inhibitor concentration we could achieve in this assay. While tovorafenib and naporafenib share potency trends across the RAF isoforms, naporafenib is consistently more potent than tovorafenib against each enzyme by at least an order of magnitude. A prior study of naporafenib activity against purified ARAF, BRAF, and CRAF reported relative potencies similar to those we observe but with markedly lower IC50 values (0.07 nM for CRAF) (42). Reaction conditions were not provided for this study, precluding meaningful comparison with our results. [7] The very steep concentration-response curves for Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and naporafenib against CRAF and WT BRAF suggest positive cooperativity of inhibition of these RAF dimers (Fig. 2). Fitting of these curves with a four-parameter model to allow for a variable Hill slope resulted in Hill slopes ranging from −2.6 to −3.2 (Table 1). These values indicate that tovorafenib and naporafenib inhibit BRAF and CRAF dimers with marked positive cooperativity; that is, that binding of inhibitor to the active site of one protomer increases the affinity for inhibitor binding to the second protomer in the RAF dimer. We did not observe this effect with either ARAFSSDD or with BRAFV600E, which is monomeric in this assay (Table 1).
ln Vivo	MLN2480 exhibits antitumor activity in vivo in xenograft models for pancreatic, lung, colon, and melanoma cancer[3]. MLN-2480 (37.5 mg/kg) in a tumor xenograft model is tolerable. An SK-MEL-30 xenograft model benefits from the combination of MLN-2480 (12.5 mg) and TAK-733 (1 mg/kg), but neither drug by itself has much of an impact[2]. The dose expansion phase provided a preliminary indication of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) efficacy. Partial responses were seen in 8 (50%) of 16 patients in the BRAF mutation-positive, RAF and MEK inhibitor-naïve cohort who received the Q2D RP2D. This level of monotherapy activity is in line with that seen in phase 1 studies of first-generation agents in a similar setting. The PK analyses showed that Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) has a moderately fast absorption rate, with an overall median Tmax of 2–4 h post-dose. Overall mean accumulation following 21 days of Q2D dosing was 2.5-fold. By contrast, QW dose administration was associated with minimal to no apparent accumulation of tovorafenib in systemic circulation in the dose range of 400 mg to 800 mg. Steady-state AUC increased in an approximately dose-proportional manner for both Q2D and QW dose ranges tested. The plasma terminal half-life (t1/2) of tovorafenib was approximately 70 h.[6] The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitro analysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. [2]
Enzyme Assay	Kinase inhibition assays [7] Inhibition assays were performed using a modified HTRF KinEASE tyrosine kinase assay kit. Rather than the provided kit substrate, we purified MEK135-393 and biotinylated it (MEK-B) in-house using birA enzyme. Inhibitors were dispensed into black 384-well plates using an HP300e dispenser and normalized to 1% final DMSO concentration per well. Kit assay buffer was supplemented with purified RAF at a final concentration of 1 nM for MEK1SASA:BRAFKD:14-3-3 and MEK1SASA:CRAFSSDD:14-3-3, 4 nM for MEK1:CRAFKD:14-3-3, and 10 nM for MEK1SASA:ARAFSSDD-14-3-3	Solubility (In Vitro) DMSO: ~100 mg/mL (~197.5 mM) Water: <1 mg/mL Ethanol: ~100 mg/mL (~197.5 mM) Solubility (In Vivo) Solubility in Formulation 1: ≥ 0.67 mg/mL (1.32 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 6.7 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. Solubility in Formulation 2: ≥ 0.67 mg/mL (1.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 6.7 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. Solubility in Formulation 3: ≥ 0.67 mg/mL (1.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 6.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.) Preparing Stock Solutions 1 mg 5 mg 10 mg 1 mM 1.9752 mL 9.8758 mL 19.7515 mL 5 mM 0.3950 mL 1.9752 mL 3.9503 mL 10 mM 0.1975 mL 0.9876 mL 1.9752 mL *Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.