Methods of making sting agonists and intermediates thereof

Abstract

The present application relates to a kind of preparation method of STING agonist and its intermediate, overcome the low yield in prior art, two-step yield is only 7.2%, the shortcoming of poor atomic economy, the target product can be prepared only by ring closure, deprotection reaction, and reaction yield is high, can reach 82%, compared with prior art route, overall yield is increased by more than 70%, with unexpected technical effect.Moreover, the synthesis method provided by the present application has mild reaction conditions, simple and convenient operation method, not only suitable for small amount preparation in laboratory, but also suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention relates to a method for preparing a STING agonist and its intermediates, and belongs to the field of organic synthesis technology. Background Technology

[0002] In recent years, immunotherapy has emerged as a rising star in the field of cancer treatment. Immunotherapy-based drugs have rapidly entered clinical trials, marking a breakthrough in cancer treatment and ushering in the era of immunotherapy, bringing good news to more cancer patients. Small molecule immune agonists have the unique advantage of achieving higher tumor distribution, potentially activating the immune response within tumors while also reducing the systemic toxicity of protein and peptide immune agonists.

[0003] STING (stimulator of interferon genes, also known as MITA or ERIS), is an endoplasmic reticulum transmembrane protein that primarily participates in the innate immune response triggered by DNA viruses and pathogenic bacteria. After viral infection, the cytoplasmic nucleotide transferase cGAS in the host cell senses viral-derived DNA and catalyzes the synthesis of the second messenger 2',3'-cGAMP from adenosine triphosphate (ATP) and guanosine monophosphate (GTP). Prokaryotic bacteria can also secrete cyclic diadenosine monophosphate (cGAMP) during infection. STING is activated upon binding to cyclic dinucleotides, regulating the host's innate immune response against viral / bacterial infections by inducing type I interferon and initiating autophagy. STING agonist-20 (CAS: 2591300-72-8) is an effective STING agonist that can be used as a vaccine adjuvant in research on cancer and other inflammatory and immune diseases.

[0004] Examples 44 and 45 of patent CN114585623A disclose the synthesis of STING agonist-20. The synthetic route is shown below. According to the disclosed technical solution, the target product is synthesized in two steps using 2591301-47-0 as the starting material. The reaction is at the milligram level, and the total yield of the two-step reaction is only 7.2%, which is extremely low and has poor atom economy, making it unsuitable for industrial scale-up.

[0005]

[0006] The inventors repeated the method of patent CN114585623A and found that the raw material (CAS: 2591301-47-0) could not be completely consumed. Through LCMS analysis, they deduced that they obtained unilateral cyclization and byproducts of unilateral cyclization with simultaneous removal of the TBS protecting group. They could not reproduce the preparation of the intermediate compound (CAS: 2591301-48-1) by the existing technology. The inventors tried adding cyanogen bromide, raising the temperature to 40-50℃, or extending the reaction time, but still failed to obtain the target product.

[0007]

[0008] Therefore, designing and implementing a synthesis method that is suitable for industrial production, easy to operate, and has a high yield has become a key focus of research and development for those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing STING agonists and their intermediates to solve the problems mentioned in the background art.

[0010] To address the aforementioned technical problems, a first aspect of the present invention provides a method for preparing compound 3, comprising the following steps: a first stage in which compound 1 reacts with compound 2 in an organic solvent; and a second stage in which a condensing agent is added to close the ring, thereby obtaining compound 3.

[0011]

[0012] The R is a hydroxyl protecting group; preferably, the R is selected from one of TBS, TMS, and TBDPS; more preferably, the R is TBS.

[0013] As a further improvement of the present invention, the preparation method of compound 3 includes the following steps: in the first stage, compound 1 and organic solvent are added to the reaction flask, the temperature is lowered, and a mixed solution of compound 2 and organic solvent is added dropwise to react, and the reaction of the raw materials is detected by LCMS to be complete; in the second stage, the reaction solution is heated, a condensing agent is added to the reaction solution, and after the reaction is detected by LCMS to be complete, the product is obtained by post-processing; preferably, the post-processing step is: adding water to the reaction solution, filtering, washing and drying, dissolving the filter cake and mixing the sample before column chromatography.

[0014] As a further improvement of the present invention, the molar ratio of compound 1 to compound 2 is 1:(2-3); preferably, the molar ratio of compound 1 to compound 2 is 1:2.5.

[0015] As a further improvement of the present invention, the organic solvent in the first stage is selected from one or more of acetonitrile, methanol, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide; preferably, the organic solvent is N,N-dimethylformamide.

[0016] As a further improvement of the present invention, the reaction temperature in the first stage is 0 to 5°C.

[0017] As a further improvement of the present invention, the reaction time in the first stage is 1 to 5 hours; preferably, the reaction time is 2 to 4 hours; more preferably, the reaction time is 3 hours.

[0018] As a further improvement of the present invention, the condensing agent in the second stage is DCC.

[0019] As a further improvement of the present invention, the molar ratio of compound 1 to condensing agent is 1:(2-6); preferably, the molar ratio of compound 1 to condensing agent is 1:3.

[0020] As a further improvement of the present invention, the reaction temperature in the second stage is 40-50°C; preferably, the reaction temperature is 50°C.

[0021] As a further improvement of the present invention, the reaction time in the second stage is 8 to 16 hours; preferably, the reaction time is 12 to 16 hours; more preferably, the reaction time is 12 hours.

[0022] A second aspect of the present invention provides a method for synthesizing compound 4, comprising the following steps: Step 1: In a first stage, compound 1 reacts with compound 2 in an organic solvent; in a second stage, a condensing agent is added to close the ring, yielding compound 3; Step 2: Compound 3 is deprotected to yield compound 4.

[0023]

[0024] The R is a hydroxyl protecting group; preferably, the R is selected from one of TBS, TMS, and TBDPS; more preferably, the R is TBS.

[0025] As a further improvement of the present invention, the preparation method of step 2 includes the following steps: dissolving compound 3 in an organic solvent, adding a deprotection agent, and after the reaction is completed, performing post-treatment to obtain the product.

[0026] As a further improvement of the present invention, the deprotection method described in step 2 is a conventional method well known to those skilled in the art, wherein the deprotection reagent described in step 2 includes, but is not limited to, acids and TBAF.

[0027] As a further improvement of the present invention, the preparation method of step 2 includes the following steps: dissolving compound 3 in an organic solvent, adding acid, concentrating under reduced pressure after the reaction is completed, adjusting to alkalinity with sodium bicarbonate solution, filtering, and drying to obtain the product.

[0028] As a further improvement of the present invention, the acid mentioned in step 2 is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, hydrochloric acid, sulfuric acid, sulfurous acid, carbonic acid, phosphoric acid, nitric acid, and trifluoroacetic acid; preferably, the acid is hydrochloric acid; more preferably, the acid is a 1,4-dioxane solution of hydrochloric acid; preferably, the concentration of hydrochloric acid in the 1,4-dioxane solution of hydrochloric acid is 3-5 mol / L.

[0029] As a further improvement of the present invention, the organic solvent in step 2 is selected from one or more of acetonitrile, methanol, N,N-dimethylformamide, and N,N-dimethylacetamide; preferably, the organic solvent is methanol.

[0030] As a further improvement of the present invention, the reaction time in step 2 is 3 to 5 hours; preferably, the reaction time is 4 hours.

[0031] As a further improvement of the present invention, the reaction temperature in step 2 is 15-30°C.

[0032] A third aspect of the present invention provides an intermediate compound for the preparation of a STING agonist, having the following structure:

[0033] R is selected from one of TBS, TMS, and TBDPS.

[0034] Beneficial effects

[0035] Compared with the prior art, the beneficial effects of the present invention include:

[0036] 1) This invention provides a method for preparing STING agonists and their intermediates, which overcomes the shortcomings of low yield, two-step yield of only 7.2% and poor atom economy in the prior art. This invention can obtain the target product through only cyclization and deprotection reactions, and the reaction yield is high, with a two-step yield of up to 82%. Compared with the prior art, the overall yield is improved by more than 70%, which has unexpected technical effects.

[0037] 2) This invention targets special substrate structures, especially complex molecules containing both pyridine and benzene rings. It preferably uses specific condensing agents and specific reaction conditions to effectively reduce the formation of by-products and achieve simultaneous ring closure, thereby further improving the reaction yield.

[0038] 3) By changing the reaction route and optimizing the reaction conditions, this invention can effectively reduce the difficulty of post-reaction processing. In particular, the deprotection step can be directly washed and filtered to obtain the target product with high purity, which greatly improves the experimental efficiency.

[0039] 4) The synthesis method has mild reaction conditions and is simple and convenient to operate, making it suitable not only for small-scale preparation in the laboratory but also for large-scale industrial production. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0041] Unless otherwise specified, all raw materials or reagents used in the examples are commercially available.

[0042] In the examples, room temperature refers to 10–20°C. Unless otherwise specified, the reagents are used directly without purification. All solvents were purchased from commercial suppliers, such as Aldrich, and are ready for use without treatment.

[0043] The reaction was analyzed by TLC and / or LCMS, with termination determined by the consumption of starting materials. Analytical thin-layer chromatography (TLC) was performed on glass plates (EMD Chemicals) pre-coated with silica gel 60F254 0.25 mm plates, using UV light (254 nm) and / or iodine development on the silica gel, and / or heating with TLC staining agents such as alcohol-modified phosphomolybdic acid, ninhydrin hydrate solution, potassium permanganate solution, or cerium persulfate solution.

[0044] The abbreviations used in this invention have their conventional meanings in the art, such as: DMF for N,N-dimethylformamide; MeOH for methanol; 1,4-dioxane for 1,4-dioxane; TBS for tert-butyldimethylsilyl protecting group; TMS for trimethylsilyl protecting group; TBDPS for tert-butyldiphenylsilyl protecting group; DCC for N,N'-dicyclohexylcarbodiimide; DIC for N,N'-diisopropylcarbodiimide; EDCI for 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride; HATU for 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIPEA for N,N-diisopropylethylamine; and BP (By Product) refers to the byproducts generated in this reaction.

[0045]

[0046] Example 1

[0047] First stage: Compound 1-1 (1.0 g, 1.84 mmol) and DMF (10 mL) were added to a 100 mL dry and clean three-necked flask. The temperature was lowered to 0–5 °C, and a DMF solution (4.5 mL) of compound 2 (0.90 g, 4.60 mmol) was added dropwise. After the addition was complete, the reaction was kept at this temperature for 3 h. LCMS analysis showed that the starting material was completely converted. Second stage: The reaction solution was heated to 50 °C, and N,N-dicyclohexylcarbodiimide DCC (1.14 g, 5.52 mmol) was added to the reaction solution. After the addition was complete, the reaction was kept at 50 °C for 12 h. LCMS analysis showed that the intermediate reaction was complete and no BP was detected. Water (30 mL) was added to the reaction solution, and the mixture was stirred for 2 h. The mixture was then filtered, the filter cake was washed with water and dried, and the filter cake was dissolved in dichloromethane and mixed with the solution. After purification by column chromatography (DCM:MeOH = 100:1 to 75:25), pure compound 3-1 (1.42 g, 89%) was obtained.

[0048] 1 H NMR(400MHz,DMSO-d6)δ8.69(d,J=1.9Hz,1H),8.17–8.09(m,2H),7.95(s,1H),7.62 (s,1H),7.52(s,1H),7.32(s,2H),5.90–5.77(m,2H),4.89(d,J=5.1Hz,2H),4.75(d, J=5.0Hz,2H),4.53(t,J=6.4Hz,2H),2.79(p,J=7.5Hz,4H),2.40(d,J=3.7Hz,6H),1. 75(t,J=6.2Hz,2H),0.98(td,J=7.6,3.0Hz,6H), 0.85(d,J=3.7Hz,9H),0.01(s,6H).

[0049] Example 2

[0050]

[0051] Compound 3-1 (1 g, 1.15 mmol) was dissolved in methanol (2 mL), and a 1,4-dioxane solution of HCl (4 mol / L, 0.5 mL) was added. The mixture was stirred at 25 °C for 4 h. After the reaction was completed, the mixture was concentrated under reduced pressure, adjusted to alkaline with 5% sodium bicarbonate solution, stirred until homogeneous, filtered, and dried to obtain compound 4 (0.80 g, 92%, purity 99%).

[0052] 1H NMR(400MHz,DMSO-d6)δ8.69(d,J=1.9Hz,1H),8.17–8.09(m,2H),7.95(s,1 H),7.62(s,1H),7.52(s,1H),7.32(s,2H),5.90–5.77(m,2H),4.89(d,J=5. 1Hz,2H),4.75(d,J=5.0Hz,2H),4.12(t,J=6.4Hz,2H),2.79(p,J=7.5Hz,4H ), 2.40 (d, J = 3.7Hz, 6H), 1.75 (t, J = 6.2Hz, 2H), 0.98 (td, J = 7.6, 3.0Hz, 6H).

[0053] Example 3

[0054] The operation methods of Examples 3.1 to 3.9 are the same as those of Example 1, except that the reaction temperature, solvent and condensing agent of the second stage are changed to prepare compound 3-3. The specific reaction conditions are shown in Table 1 below.

[0055] Table 1

[0056]

[0057]

[0058] The above comparison shows that the reaction temperature has a significant impact on the reaction. If the temperature is too low, the conversion rate of the target product is low and more by-products are obtained, while if the temperature is too high, other unknown impurities will be generated. The reaction effect is best when the reaction temperature is 50℃. In addition, the solvent and condensing agent also have a significant impact on the reaction. The reaction effect is best when the solvent is DMF and the condensing agent is DCC.

[0059] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A process for the preparation of compound 3, characterized in that, The method comprises the following steps: in a first stage, compound 1 is reacted with compound 2 in an organic solvent; and in a second stage, a condensing agent is added to close the ring to obtain compound 3; ； The R is selected from one of TBS, TMS and TBDPS; The condensing agent is DCC; The organic solvent is N,N-dimethylformamide or N,N-dimethylacetamide; The reaction temperature in the second stage is 40-50 DEG C.

2. A process for the preparation of compound 4 characterized in that, The method comprises the following steps: in a first stage, compound 1 is reacted with compound 2 in an organic solvent; and in a second stage, a condensing agent is added to close the ring to obtain compound 3; and in a second stage, a deprotecting agent is added to obtain compound 4. ； The R is selected from one of TBS, TMS and TBDPS; The condensing agent is DCC; The organic solvent is N,N-dimethylformamide or N,N-dimethylacetamide; The reaction temperature in the second stage is 40-50 DEG C.

3. The production method according to claim 1 or 2, characterized by, The molar feeding ratio of compound 1 to compound 2 is 1: (2-3).

4. The production method according to claim 1 or 2, wherein The molar feeding ratio of compound 1 to the condensing agent is 1: (2-6).

5. The production method according to claim 4, wherein The molar feeding ratio of compound 1 to the condensing agent is 1:

3.

6. The production method according to claim 1 or 2, wherein The reaction temperature is 50 DEG C.

7. The production method according to claim 1 or 2, wherein The preparation method of compound 3 comprises the following steps: in a first stage, compound 1 and an organic solvent are added into a reaction bottle, and then compound 2 and an organic solvent are added dropwise to react, and LCMS is used to detect the complete reaction of raw materials; in a second stage, the reaction liquid is heated, a condensing agent is added into the reaction liquid, and after LCMS is used to detect the complete reaction of an intermediate state, post-treatment is performed to obtain the compound 3.

8. The production method according to claim 2, wherein The preparation method of step 2 comprises the following steps: compound 3 is dissolved in an organic solvent, a deprotecting agent is added, and after the reaction is completed, post-treatment is performed to obtain the compound 4.

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