A process for the synthesis of tovorafenib

Abstract

The present application relates to a kind of drug synthesis process of Tovorafenib.The synthesis process described in the present application, the preparation method of chiral alpha-heteroamine involved, using biological catalyst amine dehydrogenase, directly under mild conditions, one-step reduction amination of latent chiral ketone is into optical pure chiral alpha-heteroamine, it can overcome the deficiency of existing catalytic technology, process route is simple, reaction condition is mild, yield is high, has the advantages, such as step economy, atom economy and other green synthesis, and the advantages, such as good stereoselectivity (ee value is as high as 99%).The condensation method of amino and carboxyl in the synthesis route described in the present application is mostly under HATU and alkaline environment, simple operation, high reaction yield, suitable for batch production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular, it relates to a drug synthesis process of Tovorafenib. BACKGROUND

[0002] Tovorafenib is an oral, brain-penetrating pan-RAF kinase inhibitor developed by Day One Biopharmaceuticals, which can inhibit wild-type and some mutant forms of BRAF, CRAF and ARAF protein kinases. On October 30, 2023, Day One announced that the FDA accepted the new drug application for Tovorafenib for the treatment of recurrent or progressive pediatric low-grade glioma (pLGG) and granted priority review. On April 23, 2024, Tovorafenib tablets (100MG) and Tovorafenib oral suspension (25MG / ML) developed by Day One Biopharmaceuticals were approved for marketing in the United States.

[0003] The NDA of Tovorafenib is based on the results of an open-label key Phase 2 trial evaluating Tovorafenib as a monotherapy once a week for patients aged 6 months to 25 years with recurrent or progressive pLGG. In November 2023, the results of this clinical Phase 2 (FIREFLY-1) trial of Tovorafenib were published in Nature Medicine, and the study results showed that Tovorafenib may be an effective treatment for recurrent / refractory pediatric low-grade glioma carrying BRAF mutations, bringing new hope for the treatment of pediatric low-grade glioma. The study results showed that: according to independent review assessment, in neuro-oncology high-grade glioma (RANO-HGG) patients, the overall response rate (ORR) was 67%, reaching the pre-set primary endpoint of Group 1, the median duration of response (DOR) was 16.6 months, and the median time to response (TTR) was 3.0 months (secondary endpoint). In pediatric neuro-oncology low-grade glioma (RAPNO) patients, the ORR was 51%, the median DOR was 13.8 months, and the median TTR was 5.3 months.

[0004] The chemical structural formula of Tovorafenib is shown as formula I:

[0005]

[0006] However, the current drug synthesis process for Tovorafenib still needs to be improved. SUMMARY

[0007] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to propose a pharmaceutical synthesis process of a compound Tovorafenib shown in Formula I. Compared with the prior art, the method described in the present application uses a potential chiral ketone compound (a compound shown in Formula 1) as a raw material, isopropylamine as an amine donor, water as a solvent, DMSO as a cosolvent, transaminase (ATASK-000250) as a biological catalyst, and PLA (phosphorylated pyridoxal) as a coenzyme regeneration system. The ketone is reduced and aminated in one step to form an optically pure compound shown in Formula 2 (a chiral α-heteroalkyl amine compound), then the compound shown in Formula 2 is subjected to a condensation reaction with a compound shown in Formula 3 to obtain a compound shown in Formula 4, and the compound shown in Formula 4 is subjected to ester hydrolysis in an alkaline environment to obtain a corresponding carboxylic acid compound (a compound shown in Formula 5), then the compound shown in Formula 5 is subjected to a condensation reaction with a compound shown in Formula 6 to obtain the product Tovorafenib shown in Formula I.

[0008] In one aspect of the present application, the present application provides a pharmaceutical synthesis process of a compound Tovorafenib shown in Formula I. According to an embodiment of the present application, the synthesis process comprises:

[0009] (1) contacting a compound shown in Formula 1 with transaminase to obtain a compound shown in Formula 2;

[0010] (2) contacting the compound shown in Formula 2 with a compound shown in Formula 3, HATU, and DIPEA to obtain a compound shown in Formula 4;

[0011] (3) contacting the compound shown in Formula 4 with LiOH to obtain a compound shown in Formula 5;

[0012] (4) contacting the compound shown in Formula 5 with a compound shown in Formula 6, HATU, and DIPEA to obtain the compound Tovorafenib shown in Formula I,

[0013]

[0014] The inventors have found that, by using the pharmaceutical synthesis process described in the present application, the starting material is a compound shown in Formula 1, and the target product Tovorafenib can be successfully synthesized by a total of 4 reaction steps.

[0015] The term "contacting" used herein should be interpreted broadly, which can be any way that can enable at least two reactants to chemically react, for example, the two reactants can be mixed under appropriate conditions. If necessary, the reactants to be contacted can be mixed under stirring, and thus the type of stirring is not particularly limited, for example, it can be mechanical stirring, i.e. stirring under the action of mechanical force.

[0016] In the present text, the terms "first", "second", etc. are used only for the purpose of description and cannot be understood as indicating or implying relative importance or implying a number of the technical features indicated. Thus, the features defined with "first", "second", etc. can include one or more of such features, either explicitly or implicitly. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly specified otherwise.

[0017] According to the embodiments of the present application, the above-mentioned method for preparing the compound of formula 2, the compound of formula 4, the compound of formula 5, the compound of formula I can further have at least one of the following additional technical features:

[0018] According to the embodiments of the present application, the chemical reactions described in the present application can be carried out according to any method known in the art. The source of the raw materials of the compound of formula 2, the compound of formula 4, the compound of formula 5, the compound of formula I is not particularly limited, which can be prepared by using any known method, or obtained commercially. For example, the cas of the compound of formula 1 is: 1261080-59-4.

[0019] According to the embodiments of the present application, in step (1), the way of contacting the compound of formula 1 with transaminase is not particularly limited. Thus, the efficiency of the contacting reaction of the compound of formula 1 with transaminase can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound of formula 2 by using the method can be further improved.

[0020] According to the embodiments of the present application, in step (1), the following steps are included: dissolving the compound of formula 1 in DMSO, placing it in a reaction bottle, adding a phosphate buffer with a pH of 7.5 and a concentration of 0.1 M and transaminase, oscillating the reaction at 30°C and 250 rpm for 48 hours, stopping the reaction by adding a 10 M NaOH solution to the reaction solution under ice water bath and stirring, extracting the reaction solution with ethyl acetate, separating the organic layer after filtering the mixture, washing with saturated brine, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to evaporate the solvent, purifying the concentrate with a silica gel column chromatography using a mixed solvent of petroleum ether / ethyl acetate with a volume ratio of 10:1, and obtaining the compound of formula 2. Thus, the efficiency of the contacting reaction of the compound of formula 1 with transaminase can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound of formula 2 by using the method can be further improved.

[0021] According to the embodiments of the present application, in step (1), the transaminase is purchased from Codex amine transaminase screening kit ATASK-000250.

[0022] According to the embodiments of the present application, in step (1), the phosphate buffer is composed of 0.25 mM pyridoxal phosphate and 1 M isopropylamine.

[0023] According to an embodiment of the present application, in step (1), the weight ratio (g / g) of the compound of formula 1 to the transaminase is 1:0.15. Thus, the efficiency of preparing the compound of formula 2 using the method can be further improved.

[0024] According to a specific embodiment of the present application, in step (1), the following steps are included: dissolving the compound of formula 1 (10.0 g, 54.0 mmol) in DMSO (100 mL), placing it in a 5 L reaction bottle, adding a phosphate buffer solution (2106 mL, 0.25 mM pyridoxal phosphate, 1 M isopropylamine) with a pH of 7.5 and a concentration of 0.1 M and transaminase (1.5 g, purchased from Codex amine transaminase screening kit ATASK-000250), oscillating the reaction at 30°C and 250 rpm for 48 hours, adding a 10 M NaOH solution (1736 mL) to the reaction solution under ice water bath, stirring to stop the reaction, extracting the reaction solution with ethyl acetate (2 x 2 L), separating the organic layer after filtering the mixture, washing with saturated brine, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to evaporate the solvent, purifying the concentrate with a silica gel column using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 10:1 to obtain the compound of formula 2, with a yield of 7.4 g, a yield of 73.6%, a purity of 99.5% (HPLC), and an optical purity of 99.3% (chiral HPLC).

[0025] According to an embodiment of the present application, in step (2), the contacting mode of the compound of formula 2 with the compound of formula 3, HATU, and DIPEA is not particularly limited. Thus, the efficiency of the contacting reaction of the compound of formula 2 with the compound of formula 3, HATU, and DIPEA can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound of formula 4 using the method can be further improved.

[0026] According to an embodiment of the present application, in step (2), the following steps are included: adding the compound of formula 2 and the compound of formula 3 to DMF at 0°C, stirring, and then adding 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA), stirring the reaction solution after naturally increasing the temperature to room temperature for 6-8 hours, adding water to the reaction solution after the reaction is completed, extracting with ethyl acetate, washing with saturated brine after combining the organic phases, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to evaporate the solvent, purifying the concentrate with a silica gel column using a petroleum ether / ethyl acetate mixed solvent to obtain the compound of formula 4. Thus, the efficiency of the contacting reaction of the compound of formula 2 with the compound of formula 3, HATU, and DIPEA can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound of formula 4 using the method can be further improved.

[0027] According to an embodiment of the present application, in step (2), the molar ratio of the compound of formula 2 to the compound of formula 3, HATU, and DIPEA is 1 : (1.0-1.2) : (1.3-2.2) : (1.5-3.5), preferably the molar ratio of the compound of formula 2 to the compound of formula 3, HATU, and DIPEA is 1 : 1.02 : 1.5 : 2.0. In this way, the efficiency of preparing the compound of formula 4 using the method can be further improved.

[0028] According to an embodiment of the present application, in step (2), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether / ethyl acetate mixed solvent is (3-6) : 1, preferably the volume ratio of the petroleum ether / ethyl acetate mixed solvent is 4 : 1.

[0029] According to an embodiment of the present application, in step (2), the reaction solution is preferably stirred for 6.5 hours after being naturally raised to room temperature.

[0030] According to an embodiment of the present application, in step (2), the following steps are included: at 0°C, the compound of formula 2 (6.5 g, 34.90 mmol) and the compound of formula 3 (6.18 g, 35.60 mmol) are added to DMF (65 mL) and stirred, then HATU (19.90 g, 52.35 mmol) and DIPEA (9.03 g, 69.80 mmol) are added, the reaction solution is naturally raised to room temperature and stirred for 6.5 hours, after the reaction is completed, 65 mL of water is added to the reaction solution and washed, then ethyl acetate (2 x 65 mL) is added to extract, 65 mL of saturated brine is added to the combined organic phase and washed, anhydrous sodium sulfate is added for drying, the filtrate is concentrated under reduced pressure to evaporate the solvent, and the concentrate is purified by silica gel column chromatography using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 4 : 1 to obtain the compound of formula 4, with a yield of 9.14 g and a yield of 76.6%.

[0031] According to an embodiment of the present application, in step (3), the contacting method of the compound of formula 4 and LiOH is not particularly limited. In this way, the efficiency of the contacting reaction of the compound of formula 4 and LiOH can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound of formula 5 using the method can be further improved.

[0032] According to an embodiment of the present application, in step (3), the following steps are included: mixing THF and H2O in a volume ratio of 1:1 in a reaction vessel, adding the compound shown as formula 4, stirring and cooling to 0-5°C, adding LiOH, allowing the reaction solution to naturally rise to room temperature, stirring and reacting for 3-4 hours, showing complete reaction of the compound shown as formula 1 by TLC, concentrating the reaction solution under reduced pressure, removing THF, adding diethyl ether to extract the aqueous layer, cooling the aqueous layer to 0°C, adding a HC1 solution to adjust the pH to 3, adding ethyl acetate to extract the aqueous phase after acidification, washing the combined organic phase with saturated brine, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to dryness, and obtaining the compound shown as formula 5.

[0033] According to an embodiment of the present application, in step (3), the molar ratio of the compound shown as formula 4 to LiOH is 1:(1.5-3.5), and preferably the molar ratio of the compound shown as formula 4 to LiOH is 1:2.2. Thus, the efficiency of preparing the compound shown as formula 5 using the method can be further improved.

[0034] According to an embodiment of the present application, in step (3), the stirring reaction time is preferably 3.5 hours.

[0035] According to an embodiment of the present application, in step (3), the following steps are included: mixing THF (400 mL) and H2O (400 mL) in a reaction vessel, adding the compound shown as formula 4 (80.0 g, 234.1 mmol), stirring and cooling to 0-5°C, adding LiOH (12.33 g, 515.0 mmol), allowing the reaction solution to naturally rise to room temperature, stirring and reacting for 3.5 hours, showing complete reaction of the compound shown as formula 1 by TLC, concentrating the reaction solution under reduced pressure, removing THF, adding diethyl ether (2 x 400 mL) to extract the aqueous layer, cooling the aqueous layer to 0°C, adding a 3M HC1 solution to adjust the pH to 3, adding ethyl acetate (2 x 400 mL) to extract the aqueous phase after acidification, washing the combined organic phase with 400 mL of saturated brine, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to dryness, and obtaining the compound shown as formula 5, with a yield of 64.52 g and a yield rate of 84.1%.

[0036] According to an embodiment of the present application, in step (4), the contacting mode of the compound shown as formula 5, the compound shown as formula 6, HATU and DIPEA is not particularly limited. Thus, the efficiency of the contacting reaction of the compound shown as formula 5, the compound shown as formula 6, HATU and DIPEA can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound shown as formula I using the method can be further improved.

[0037] According to an embodiment of the present application, in step (4), the following steps are included: the compound of formula 5 and the compound of formula 6 are added into DMF at 0°C, stirred, then HATU and DIPEA are added, the reaction solution is naturally raised to room temperature, then stirred for 6-7.5 hours, water is added to the reaction solution for washing, then ethyl acetate is added for extraction, the combined organic phase is washed with saturated brine, dried with anhydrous sodium sulfate, the filtrate is concentrated under reduced pressure to distill off the solvent, the concentrate is purified by silica gel column chromatography using petroleum ether / ethyl acetate mixed solvent to obtain the compound of formula I. In this way, the efficiency of the contact reaction of the compound of formula 5 with the compound of formula 6, HATU and DIPEA can be improved, the reaction speed can be accelerated, and the efficiency of preparing the compound of formula I by using the method can be further improved.

[0038] According to an embodiment of the present application, in step (4), the molar ratio of the compound of formula 5 to the compound of formula 6, HATU and DIPEA is 1:(1.0-1.2):(1.3-2.5):(1.6-3.0), preferably the molar ratio of the compound of formula 5 to the compound of formula 6, HATU and DIPEA is 1:1.05:1.6:2.2. In this way, the efficiency of preparing the compound of formula I by using the method can be further improved.

[0039] According to an embodiment of the present application, in step (4), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether / ethyl acetate mixed solvent is (3-6):1, preferably the volume ratio of petroleum ether to ethyl acetate in the petroleum ether / ethyl acetate mixed solvent is 4:1.

[0040] According to an embodiment of the present application, in step (4), preferably the reaction solution is stirred for 6.5 hours after being naturally raised to room temperature.

[0041] According to an embodiment of the present application, in step (4), the following steps are included: the compound of formula 5 (5.50 g, 16.78 mmol) and the compound of formula 6 (3.46 g, 17.62 mmol) are added into DMF (60 mL) at 0°C, stirred, then HATU (10.21 g, 26.85 mmol) and DIPEA (4.77 g, 36.92 mmol) are added, the reaction solution is naturally raised to room temperature, then stirred for 6.5 hours, after the reaction is completed, 60 mL of water is added to the reaction solution for washing, then ethyl acetate (2x60 mL) is added for extraction, the combined organic phase is washed with 60 mL of saturated brine, dried with anhydrous sodium sulfate, the filtrate is concentrated under reduced pressure to distill off the solvent, the concentrate is purified by silica gel column chromatography using petroleum ether / ethyl acetate mixed solvent with a volume ratio of 4:1 to obtain the compound of formula 7, the yield is 7.14 g, the yield is 84.0%, the purity is 99.6% (HPLC), and the optical purity is 99.4% (chiral HPLC).

[0042] According to a specific embodiment of the present application, the synthesis route of the compound Tovorafenib shown in formula I can be as follows:

[0043]

[0044] Compared with the prior art, the pharmaceutical synthesis process of Tovorafenib described in the present application has at least the following beneficial effects:

[0045] 1. Compared with the prior art, the method described in the present application uses a prochiral ketone compound (compound shown in formula 1) as a raw material, isopropylamine as an amine donor, water as a solvent, DMSO as a cosolvent, transaminase (ATASK-000250) as a biological catalyst, and PLA (phosphorylated pyridoxal) as a coenzyme regeneration system. The ketone is reduced and aminated in one step to form an optically pure compound shown in formula 2 (a chiral α-heteroalkyl amine compound), then the compound shown in formula 2 is subjected to a condensation reaction with a compound shown in formula 3 to obtain a compound shown in formula 4, and the compound shown in formula 4 is subjected to ester hydrolysis in an alkaline environment to obtain a corresponding carboxylic acid compound (compound shown in formula 5), then the compound shown in formula 5 is subjected to a condensation reaction with a compound shown in formula 6 to obtain the product Tovorafenib shown in formula I.

[0046] 2. Compared with the prior art method, the present application has the following significant advantages: (1) The most difficult step in the synthesis of Tovorafenib is the construction of chiral α-heteroalkyl amine. The commonly used method is to synthesize such chiral (hetero) aryl amines through asymmetric hydrogenation of olefins and asymmetric hydrogenation of N-containing intermediates (such as amides, amides or imines), as described in CN111032047A. The chemical catalysts for catalyzing such reactions are mostly transition metals, which are expensive, seriously polluting, sensitive to water and air. In addition, the coordination effect of heteroatoms and the easy degradation of heteroaromatic rings make the synthesis process complicated, the reaction conditions harsh, the product purification difficult, and the yield low. The synthesis process described in the present application involves a method for preparing chiral α-heteroalkyl amine, which uses amine dehydrogenase as a biological catalyst to directly reduce and amine prochiral ketones into optically pure chiral α-heteroalkyl amines under mild conditions. This method can overcome the shortcomings of existing catalytic technologies, has a simple process route, mild reaction conditions, high yield, and the advantages of green synthesis such as step economy and atom economy, as well as good stereoselectivity (ee value as high as 99%). (2) In the synthesis route, the method of condensation of amino and carboxyl groups in HATU and alkaline environment is simple to operate, has high reaction yield, and is suitable for batch production.

[0047] 3. In summary, the present route has the advantages of low raw material cost, high yield, simple purification, etc., and is easy to realize industrialization, and is very suitable for industrial large-scale production. DETAILED DESCRIPTION

[0048] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and are not to be understood as limiting the present application. In the embodiments, specific techniques or conditions are not mentioned unless the techniques or conditions described in the literature in the art or according to the product manual are used. The reagents or instruments used are not mentioned unless they are conventional products that can be commercially available.

[0049] Synthesis of the compound shown in Formula 2

[0050] The compound shown in Formula 1 (10.0 g, 54.0 mmol) was dissolved in DMSO (100 mL) in a 5 L reaction bottle, and a phosphate buffer (2106 mL, 0.25 mM pyridoxal phosphate, 1 M isopropylamine) having a pH of 7.5 and a concentration of 0.1 M and transaminase (1.5 g, purchased from Codex aminotransferase screening kit ATASK-000250) were added, and the reaction was stirred at 30°C and 250 rpm for 48 hours. The reaction solution was added with 10 M NaOH solution (1736 mL) in an ice water bath, and the reaction was stopped by stirring. The reaction solution was extracted with ethyl acetate (2 x 2 L), and the mixture was filtered to separate the organic layer. Saturated brine was added, and the mixture was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to distill off the solvent, and the concentrate was purified by column chromatography using a mixture of petroleum ether / ethyl acetate (10:1 by volume) as a solvent to obtain the compound shown in Formula 2 in an amount of 7.40 g, a yield of 73.6%, a purity of 99.5% (HPLC), and an optical purity of 99.3% (chiral HPLC).

[0051] LC-MS (APCI): m / z = 187.1 (M+1) + .

[0052] 1 H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 2.3 Hz, 1H), 4.44 (td, J = 6.7, 1.9 Hz, 1H), 3.92 (s, 3H), 1.92 (s, 2H), 1.57 (dd, J = 6.8, 1.9 Hz, 3H).

[0053] Synthesis of the compound shown in Formula 4

[0054] To a stirred solution of compound of formula 2 (6.5 g, 34.90 mmol) and compound of formula 3 (6.18 g, 35.60 mmol) in DMF (65 mL) was added HATU (19.90 g, 52.35 mmol) and DIPEA (9.03 g, 69.80 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 6.5 h. The reaction mixture was diluted with water (65 mL) and extracted with ethyl acetate (2 x 65 mL). The organic layers were combined, washed with saturated brine (65 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 4:1 petroleum ether / ethyl acetate as the eluent to afford compound of formula 4 (9.14 g, 76.6 % yield).

[0055] LC-MS (APCI): m / z = 342.0 (M+1) + .

[0056] Example 3: Synthesis of compound of formula 4

[0057] To a stirred solution of compound of formula 2 (6.5 g, 34.90 mmol) and compound of formula 3 (6.06 g, 34.90 mmol) in DMF (65 mL) was added HATU (17.25 g, 45.37 mmol) and DIPEA (6.77 g, 52.38 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 6 h. The reaction mixture was diluted with water (65 mL) and extracted with ethyl acetate (2 x 65 mL). The organic layers were combined, washed with saturated brine (65 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 3:1 petroleum ether / ethyl acetate as the eluent to afford compound of formula 4 (8.90 g, 74.6 % yield).

[0058] Example 4: Synthesis of compound of formula 4

[0059] To a stirred solution of compound of formula 2 (6.5 g, 34.90 mmol) and compound of formula 3 (7.27 g, 41.88 mmol) in DMF (80 mL) at 0 °C, HATU (29.19 g, 76.78 mmol) and DIPEA (15.79 g, 122.15 mmol) were added. The reaction mixture was allowed to warm to room temperature and stirred for 8 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (2 x 80 mL). The organic layers were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 6:1 petroleum ether / ethyl acetate as an eluent to afford compound of formula 4 (9.08 g, 76.1 % yield).

[0060] Synthesis of compound of formula 4 of comparative example 1

[0061] To a stirred solution of compound of formula 2 (6.5 g, 34.90 mmol) and compound of formula 3 (7.87 g, 45.37 mmol) in DMF (65 mL) at 0 °C, HATU (17.25 g, 45.37 mmol) and DIPEA (5.86 g, 45.37 mmol) were added. The reaction mixture was allowed to warm to room temperature and stirred for 9 h. The reaction mixture was diluted with water (65 mL) and extracted with ethyl acetate (2 x 65 mL). The organic layers were combined, washed with saturated brine (65 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 4:1 petroleum ether / ethyl acetate as an eluent to afford compound of formula 4 (8.85 g, 74.2 % yield).

[0062] It can be seen that changing the molar ratio of compound of formula 2, compound of formula 3, HATU and DIPEA to 1:1.3:1.3:1.3 and adjusting the stirring time to 9 h did not further improve the efficiency (yield) of preparing compound of formula 4.

[0063] Synthesis of compound of formula 4 of comparative example 2

[0064] To a reaction vessel, THF (400 mL) and H2O (400 mL) were mixed, then the compound of Formula 4 (80.0 g, 234.1 mmol) was added, stirred and cooled to 0-5 °C, then LiOH (12.33 g, 515.0 mmol) was added, the reaction was allowed to warm to room temperature, then stirred for 3.5 hours, TLC showed that the compound of Formula 1 was completely reacted, the reaction was concentrated under reduced pressure to remove THF, then the residue was extracted with diethyl ether (2 x 400 mL) to obtain the aqueous layer, the aqueous layer was cooled to 0 °C, then 3M HCl solution was added to adjust the pH value to 3, then the acidified aqueous phase was extracted with ethyl acetate (2 x 400 mL), then the combined organic phase was washed with 400 mL of saturated brine, dried over anhydrous sodium sulfate, then the filtrate was concentrated under reduced pressure to dryness to obtain the compound of Formula 5, the yield was 64.52 g, the yield was 84.1%.

[0065] As can be seen, the molar ratio of the compound of Formula 2 to the compound of Formula 3, HATU and DIPEA was changed to 1:0.96:2.5:4.0, and the stirring reaction was adjusted to 5.5 hours, which did not further improve the efficiency (yield) of preparing the compound of Formula 4.

[0066] Example 5 Synthesis of the compound of Formula 5

[0067] To a reaction vessel, THF (400 mL) and H2O (400 mL) were mixed, then the compound of Formula 4 (80.0 g, 234.1 mmol) was added, stirred and cooled to 0-5 °C, then LiOH (12.33 g, 515.0 mmol) was added, the reaction was allowed to warm to room temperature, then stirred for 3.5 hours, TLC showed that the compound of Formula 1 was completely reacted, the reaction was concentrated under reduced pressure to remove THF, then the residue was extracted with diethyl ether (2 x 400 mL) to obtain the aqueous layer, the aqueous layer was cooled to 0 °C, then 3M HCl solution was added to adjust the pH value to 3, then the acidified aqueous phase was extracted with ethyl acetate (2 x 400 mL), then the combined organic phase was washed with 400 mL of saturated brine, dried over anhydrous sodium sulfate, then the filtrate was concentrated under reduced pressure to dryness to obtain the compound of Formula 5, the yield was 64.52 g, the yield was 84.1%.

[0068] LC-MS (APCI): m / z = 328.0 (M+1) + .

[0069] Example 6 Synthesis of the compound of Formula 5

[0070] In a reaction vessel, THF (400 mL) and H2O (400 mL) were mixed, then the compound shown in formula 4 (80.0 g, 234.1 mmol) was added, and the mixture was stirred to be cooled to 0-5°C, LiOH (8.41 g, 351.15 mmol) was added, the reaction solution was naturally warmed to room temperature, and then stirred for 3 hours. TLC showed that the compound shown in formula 1 was completely reacted. The reaction solution was concentrated under reduced pressure, THF was removed, and the residue was extracted with diethyl ether (2x400 mL) to obtain an aqueous layer. The aqueous layer was cooled to 0°C, and 3M HCl solution was added to adjust the pH value to 3. After acidification, ethyl acetate (2x400 mL) was added to the aqueous phase to extract, and then the organic phase was combined, washed with 400 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound shown in formula 5. The yield was 62.91 g, and the yield was 82.0%.

[0071] Example 7 Synthesis of the compound shown in formula 5

[0072] In a reaction vessel, THF (400 mL) and H2O (400 mL) were mixed, then the compound shown in formula 4 (80.0 g, 234.1 mmol) was added, and the mixture was stirred to be cooled to 0-5°C, LiOH (8.41 g, 351.15 mmol) was added, the reaction solution was naturally warmed to room temperature, and then stirred for 3 hours. TLC showed that the compound shown in formula 1 was completely reacted. The reaction solution was concentrated under reduced pressure, THF was removed, and the residue was extracted with diethyl ether (2x400 mL) to obtain an aqueous layer. The aqueous layer was cooled to 0°C, and 3M HCl solution was added to adjust the pH value to 3. After acidification, ethyl acetate (2x400 mL) was added to the aqueous phase to extract, and then the organic phase was combined, washed with 400 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound shown in formula 5. The yield was 62.91 g, and the yield was 82.0%.

[0073] Example 8 Synthesis of the compound Tovorafenib shown in formula I

[0074] To a stirred solution of compound of formula 5 (5.50 g, 16.78 mmol) and compound of formula 6 (3.46 g, 17.62 mmol) in DMF (60 mL) was added HATU (10.21 g, 26.85 mmol) and DIPEA (4.77 g, 36.92 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 6.5 h. The reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (2 x 60 mL). The combined organic layer was washed with saturated brine (60 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 4:1 petroleum ether / ethyl acetate as an eluent to afford compound of formula I, Tovorafenib, in 7.14 g, 84.0% yield, 99.6% purity (HPLC), 99.4% optical purity (chiral HPLC).

[0075] LC-MS (APCI): m / z = 505.6 (M+1) + .

[0076] 1 H NMR (400 MHz, DMSO-d6) d ppm 11.88 (s, 1H) 9.63 (d, J = 7.82 Hz, 1H) 8.79 (s, 1H) 8.68 (s, 1H) 8.59 (s, 1H) 8.38 (s, 1H) 7.86 (br. s., 1H) 7.48 (br. s., 1H) 5.37 (tdd, J = 7.27, 7.27, 7.21, 7.09 Hz, 1H) 1.61 (d, J = 6.85 Hz, 3H)

[0077] Example 9: Synthesis of compound of formula I, Tovorafenib

[0078] To a stirred solution of compound of formula 5 (5.50 g, 16.78 mmol) and compound of formula 6 (3.46 g, 17.62 mmol) in DMF (60 mL) was added HATU (10.21 g, 26.85 mmol) and DIPEA (4.77 g, 36.92 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 6.5 h. The reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (2 x 60 mL). The combined organic layer was washed with saturated brine (60 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 4:1 petroleum ether / ethyl acetate as an eluent to afford compound of formula I, Tovorafenib, in 7.14 g, 84.0% yield, 99.6% purity (HPLC), 99.4% optical purity (chiral HPLC).

[0079] Synthesis of compound Tovorafenib of Formula I

[0080] To a stirred solution of compound of Formula 5 (5.5 g, 16.78 mmol) and compound of Formula 6 (3.96 g, 20.136 mmol) in DMF (80 mL) was added HATU (15.95 g, 41.95 mmol) and DIPEA (6.51 g, 50.37 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 7.5 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (2 x 80 mL). The organic layers were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 6:1 petroleum ether / ethyl acetate as the eluent to give compound Tovorafenib of Formula I in 7.03 g, 82.8% yield, 99.4% purity (HPLC), and 99.5% optical purity (chiral HPLC).

[0081] Synthesis of compound Tovorafenib of Formula I

[0082] To a stirred solution of compound of Formula 5 (5.5 g, 16.78 mmol) and compound of Formula 6 (3.96 g, 20.136 mmol) in DMF (80 mL) was added HATU (15.95 g, 41.95 mmol) and DIPEA (6.51 g, 50.37 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 7.5 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (2 x 80 mL). The organic layers were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 6:1 petroleum ether / ethyl acetate as the eluent to give compound Tovorafenib of Formula I in 7.03 g, 82.8% yield, 99.4% purity (HPLC), and 99.5% optical purity (chiral HPLC).

[0083] It can be seen that the yield of compound of Formula I was not further improved when the molar ratio of compound of Formula 5, compound of Formula 6, HATU, and DIPEA was changed to 1:0.98:1.2:1.3, but the purity of compound of Formula I was reduced.

[0084] Synthesis of compound Tovorafenib of Formula I

[0085] To a solution of compound of formula 5 (5.5 g, 16.78 mmol) and compound of formula 6 (4.62 g, 23.49 mmol) in DMF (100 mL) was added HATU (19.14 g, 50.34 mmol) and DIPEA (7.59 g, 58.73 mmol) at 0 °C and stirred. The reaction mixture was allowed to warm to room temperature and stirred for 8 h. The reaction mixture was diluted with water (80 mL) and extracted with ethyl acetate (2 x 80 mL). The combined organic layer was washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 4:1 petroleum ether / ethyl acetate mixture to obtain compound of formula I, Tovorafenib, in 6.89 g, 81.1% yield, 99.1% purity (HPLC), 98.9% optical purity (chiral HPLC).

[0086] It can be seen that the molar ratio of compound of formula 5, compound of formula 6, HATU and DIPEA was changed to 1:1.4:3.0:3.5 and the reaction was stirred for 8 h, which did not further improve the yield of compound of formula I, but the purity of compound of formula I was reduced.

[0087] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0088] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, substitutions and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method for the pharmaceutical synthesis of Tovorafenib, characterized in that, Comprise: (1) contacting a compound shown as formula 1 with transaminase to obtain a compound shown as formula 2; (2) contacting the compound shown as formula 2 with a compound shown as formula 3, HATU, DIPEA to obtain a compound shown as formula 4; (3) contacting the compound shown as formula 4 with LiOH to obtain a compound shown as formula 5; (4) contacting the compound shown as formula 5 with a compound shown as formula 6, HATU, DIPEA to obtain a compound shown as formula I Tovorafenib, ， In step (1), the following steps are included: dissolving the compound shown as formula 1 in DMSO, placing it in a reaction bottle, adding a phosphate buffer with a pH of 7.5 and a concentration of 0.1 M and transaminase, oscillating the reaction at 30°C and 250 rpm for 48 hours, adding 10 M NaOH solution to the reaction liquid in an ice water bath to stop the reaction, extracting the reaction solution with ethyl acetate, separating the organic layer after filtering the mixture, adding saturated brine for washing, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to evaporate the solvent, purifying the concentrate with a silica gel column chromatography using a mixed solvent of petroleum ether / ethyl acetate with a volume ratio of 10:1 to obtain the compound shown as formula 2; In step (1), the transaminase is purchased from Codex amine transaminase screening kit ATASK-000250; In step (1), the phosphate buffer is composed of 0.25 mM pyridoxal phosphate and 1 M isopropylamine; In step (1), the weight ratio of the compound shown as formula 1 to transaminase is 1:0.15; In step (2), the following steps are included: adding the compound shown as formula 2 and the compound shown as formula 3 into DMF at 0°C, stirring, then adding HATU and DIPEA, stirring the reaction liquid after it naturally rises to room temperature for 6-8 hours, adding water to the reaction liquid after the reaction is completed for washing, then adding ethyl acetate for extraction, adding saturated brine for washing after combining the organic phases, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to evaporate the solvent, purifying the concentrate with a silica gel column chromatography using a mixed solvent of petroleum ether / ethyl acetate to obtain the compound shown as formula 4; In step (2), the molar ratio of the compound shown as formula 2 to the compound shown as formula 3, HATU, DIPEA is 1:(1.0-1.2):(1.3-2.2):(1.5-3.5); In step (3), the following steps are included: mixing THF and H2O in a volume ratio of 1:1, then adding the compound shown as formula 4, stirring to cool to 0-5°C, adding LiOH, stirring the reaction liquid after it naturally rises to room temperature for 3-4 hours, adding ethyl ether to extract the water layer from the remaining liquid after TLC shows that the compound shown as formula 1 has completely reacted, cooling the water layer to 0°C, adding HCl solution to adjust the pH value to 3, adding ethyl acetate to extract the water phase after acidification, adding saturated brine for washing after combining the organic phases, drying with anhydrous sodium sulfate, concentrating the filtrate under reduced pressure to dryness to obtain the compound shown as formula 5; In step (3), the molar ratio of the compound shown as formula 4 to LiOH is 1:(1.5-3.5); In step (4), the following steps are included: the compound shown as formula 5 and the compound shown as formula 6 are added into DMF at 0°C, stirred, then HATU and DIPEA are added, the reaction solution is naturally raised to room temperature, then stirred for 6-7.5 hours, water is added to the reaction solution for washing, then ethyl acetate is added for extraction, the combined organic phase is washed with saturated brine, dried with anhydrous sodium sulfate, the filtrate is concentrated under reduced pressure to distill off the solvent, the concentrate is purified by silica gel column chromatography with petroleum ether / ethyl acetate mixed solvent to obtain the compound shown as formula I; In step (4), the molar ratio of the compound shown as formula 5, the compound shown as formula 6, HATU and DIPEA is 1: (1.0-1.2): (1.3-2.5): (1.6-3.0).

2. The method of claim 1, wherein, In step (2), the molar ratio of the compound shown as formula 2, the compound shown as formula 3, HATU and DIPEA is 1: 1.02: 1.5: 2.

0.

3. The method of claim 1, wherein, In step (2), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether / ethyl acetate mixed solvent is (3-6):

1.

4. The method of claim 3, wherein, In step (2), the volume ratio of the petroleum ether / ethyl acetate mixed solvent is 4:

1.

5. The method of claim 1, wherein, In step (2), the reaction solution is naturally raised to room temperature, then stirred for 6.5 hours.

6. The method of claim 1, wherein, In step (3), the molar ratio of the compound shown as formula 4 and LiOH is 1: 2.

2.

7. The method of claim 1, wherein, In step (3), the stirring reaction time is 3.5 hours.

8. The method of claim 1, wherein, In step (4), the molar ratio of the compound shown as formula 5, the compound shown as formula 6, HATU and DIPEA is 1: 1.05: 1.6: 2.

2.

9. The method of claim 1, wherein, In step (4), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether / ethyl acetate mixed solvent is (3-6):

1.

10. The method of claim 9, wherein, In step (4), the volume ratio of the petroleum ether / ethyl acetate mixed solvent is 4:

1.

11. The method of claim 1, wherein, In step (4), the reaction solution is naturally raised to room temperature, then stirred for 6.5 hours.

12. The method of claim 1, wherein, In step (1), the following steps are included: 10.0 g of the compound shown as formula 1 is dissolved in 100 mL of DMSO, placed in a 5 L reaction bottle, 2106 mL of phosphate buffer with pH of 7.5 and concentration of 0.1 M is added, wherein the phosphate buffer contains 0.25 mM pyridoxal phosphate and 1 M isopropylamine, and 1.5 g of transaminase purchased from Codex amine transaminase screening kit ATASK-000250, the reaction is oscillated at 30°C and 250 rpm for 48 hours, 1736 mL of 10 M NaOH solution is added to the reaction solution under ice water bath to stop the reaction, the reaction solution is extracted with 2 × 2 L of ethyl acetate, the mixture is filtered to separate the organic layer, saturated brine is added for washing, dried with anhydrous sodium sulfate, the filtrate is concentrated under reduced pressure to distill off the solvent, the concentrate is purified by silica gel column chromatography with petroleum ether / ethyl acetate mixed solvent with a volume ratio of 10:1 to obtain the compound shown as formula 2, the yield is 7.4 g, the yield is 73.6%, the HPLC purity is 99.5%, and the chiral HPLC optical purity is 99.3%; In step (2), the following steps are included: 6.5 g of the compound shown in formula 2 and 6.18 g of the compound shown in formula 3 are added to 65 mL of DMF at 0°C, stirred, and then 19.90 g of HATU and 9.03 g of DIPEA are added. After the reaction solution is naturally raised to room temperature, stirring is performed for 6.5 hours. After the reaction is completed, 65 mL of water is added to the reaction solution for washing, and then 2*65 mL of ethyl acetate is added for extraction. After the organic phases are combined, 65 mL of saturated brine is added for washing, anhydrous sodium sulfate is added for drying, the filtrate is concentrated under reduced pressure to remove the solvent, and the concentrate is purified by silica gel column chromatography using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 4:1 to obtain the compound shown in formula 4, with a yield of 9.14 g and a recovery rate of 76.6%; In step (3), the following steps are included: 400 mL of THF and 400 mL of H2O are mixed in a reaction container, and then 80.0 g of the compound shown in formula 4 is added. After stirring and cooling to 0°C-5°C, 12.33 g of LiOH is added. After the reaction solution is naturally raised to room temperature, stirring is performed for 3.5 hours. TLC shows that the compound shown in formula 1 is completely reacted. The reaction solution is concentrated under reduced pressure to remove THF. The residue is extracted with 2*400 mL of ether to obtain the aqueous layer. The aqueous layer is cooled to 0°C, and 3M HCl solution is added to adjust the pH value to 3. After acidification, the aqueous phase is extracted with 2*400 mL of ethyl acetate. After the organic phases are combined, 400 mL of saturated brine is added for washing, anhydrous sodium sulfate is added for drying, and the filtrate is concentrated under reduced pressure to dryness to obtain the compound shown in formula 5, with a yield of 64.52 g and a recovery rate of 84.1%; In step (4), the following steps are included: 5.50 g of the compound shown in formula 5 and 3.46 g of the compound shown in formula 6 are added to 60 mL of DMF at 0°C, stirred, and then 10.21 g of HATU and 4.77 g of DIPEA are added. After the reaction solution is naturally raised to room temperature, stirring is performed for 6.5 hours. After the reaction is completed, 60 mL of water is added to the reaction solution for washing, and then 2*60 mL of ethyl acetate is added for extraction. After the organic phases are combined, 60 mL of saturated brine is added for washing, anhydrous sodium sulfate is added for drying, and the filtrate is concentrated under reduced pressure to remove the solvent. The concentrate is purified by silica gel column chromatography using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 4:1 to obtain the compound shown in formula 7, with a yield of 7.14 g and a recovery rate of 84.0%, an HPLC purity of 99.6%, and a chiral HPLC optical purity of 99.4%.

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