A chiral isomer of remdesivir, preparation method and application thereof

Abstract

The present invention discloses a chiral isomer of Remdesivir, a preparation method thereof and an application thereof. The present invention provides a compound as shown in Formula I and a compound as shown in Formula II. In addition, the preparation methods of the compounds as shown in Formula I and the compounds as shown in Formula II in the present invention are simple in reaction, mild in conditions, high in yield, without extreme reaction conditions of high and low temperatures, without the use of highly dangerous and highly toxic substances, simple in operation, high in chiral purity, without special requirements, stable in process, suitable for laboratory synthesis and industrial production, and have broad industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the field of drug synthesis, and in particular to a chiral isomer of remdesivir, a preparation method thereof and applications thereof. Background Art

[0002] Remdesivir was developed by Gilead in the United States to treat Ebola virus. After the outbreak of the new coronavirus, it was used as a clinical drug for the new coronavirus. In 2020, the US FDA has approved Remdesivir for the treatment of the new coronavirus. Nature, 2016, Doi: 10.1038 / nature17180 (Nature, 2016, 531 (7594): 381-385.) provides a preparation method for Remdesivir, which is the second-generation synthesis method of Gilead Remdesivir. The synthesis route is as follows:

[0003]

[0004] Gilead's Chinese patents CN110636884A and CN103052631A report the preparation method and crystal form patent of Remdesivir. In order to study the preparation process of Remdesivir, it is necessary to study the chiral impurities generated by the process.

[0005] Journal of Medicinal Chemistry 2017, 60, 5, 1648-1661 and US20160122374 reported the synthesis of a chiral isomer of remdesivir, RDV-ISO-2. The intermediate C-3' debenzylate was reacted with phosphoryl chloride F to obtain product G (containing R and S configurations, S type is remdesivir, R type is RDV-ISO-2), and then chiral preparative HPLC was used to separate the product S type, which is remdesivir, and R type, which is RDV-ISO-2. The route is as follows:

[0006]

[0007] The above method for preparing the chiral isomer RDV-ISO-2 of Remdesivir uses expensive chiral HPLC separation, and the final yield of the reaction is only about 20%, which still needs further improvement. There is no literature reporting the synthesis of other important chiral isomers, nor the use of chiral isomers of Remdesivir. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the defects of high cost and low yield of the existing chiral isomers of Remdesivir. The present invention provides a chiral isomer of Remdesivir process different from the prior art, a preparation method thereof and its application, and specifically provides a compound as shown in Formula I and a compound as shown in Formula II. The present invention unexpectedly found through research that the compound as shown in Formula I and the compound as shown in Formula II both have the effect of inhibiting Hela cells, COLO 205 cells and U-87 MG cells, and the present invention also found for the first time that the compound as shown in Formula III also has the effect of inhibiting Hela cells, COLO 205 cells and U-87 MG cells. In addition, the preparation method of the compound as shown in Formula I and the compound as shown in Formula II in the present invention has simple reaction, mild conditions, high yield, no high and low temperature extreme reaction conditions, no use of high-risk and highly toxic substances, simple operation, high chiral purity, no special requirements, stable process, suitable for laboratory synthesis and industrial production, and has broad industrial application prospects.

[0009] The present invention provides a compound as shown in Formula I or Formula II, or a pharmaceutically acceptable salt thereof:

[0010]

[0011] The present invention also provides a method for preparing a compound as shown in Formula I, comprising the following steps: in a solvent, in the presence of an acid, compound E is subjected to a deprotection reaction as shown below to obtain a compound as shown in Formula I;

[0012]

[0013] The conditions and steps of the deprotection reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0014] In the deprotection reaction, the solvent may be a conventional solvent in the art. Preferably, the solvent is one or more of an ether solvent, a nitrile solvent and a ketone solvent; more preferably, the solvent is one or more of tetrahydrofuran, acetonitrile, 1,4-dioxane and acetone.

[0015] In the deprotection reaction, the acid of the deprotection reaction can be an acid conventionally used in this type of deprotection reaction in the art. Preferably, the acid is one or more of hydrochloric acid, sulfuric acid and phosphoric acid.

[0016] In the deprotection reaction, the reaction temperature may be a conventional temperature for such deprotection reactions in the art. Preferably, the reaction temperature is 0°C to 30°C; more preferably, the reaction temperature is 0°C to 5°C or 20°C to 25°C.

[0017] In the deprotection reaction, the disappearance of the compound E is regarded as the end point of the deprotection reaction. Preferably, the reaction time is 2 hours to 5 hours, and more preferably, the reaction time is 3 hours.

[0018] The preparation method of the compound as shown in Formula I may further comprise the following steps: in a solvent, in the presence of a catalyst and a base, compound C and compound D undergo a substitution reaction as shown below to obtain compound E;

[0019]

[0020] The conditions and steps of the substitution reaction may be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0021] In the substitution reaction, the solvent may be a conventional solvent for such substitution reactions in the art, preferably an anhydrous solvent, preferably an ether solvent and / or a nitrile solvent, more preferably, the solvent is one or more of acetonitrile, 1,4-dioxane and tetrahydrofuran.

[0022] In the substitution reaction, the catalyst may be a conventional catalyst for such substitution reactions in the art. Preferably, the catalyst is magnesium chloride, and more preferably, the catalyst is anhydrous magnesium chloride.

[0023] In the substitution reaction, the base can be a base conventionally used in this type of substitution reaction in the art. Preferably, the base is N,N-diisopropylethylamine.

[0024] In the substitution reaction, the molar ratio of the compound C to the compound D can be a conventional ratio for such substitution reactions in the art, preferably 1:1 to 1:1.5, more preferably 1:1.1 or 1:1.2.

[0025] In the substitution reaction, the reaction temperature may be a conventional reaction temperature for such substitution reactions in the art. Preferably, the reaction temperature is 10°C to 50°C, and more preferably, the reaction temperature is 35°C to 45°C.

[0026] In the substitution reaction, the disappearance of the compound C is regarded as the end point of the substitution reaction. Preferably, the reaction time is 1 to 5 hours, and more preferably, the reaction time is 3 hours.

[0027] The preparation method of the compound as shown in Formula I may further comprise the following steps: in a solvent, in the presence of an acid, compound C-3 and 2,2-dimethoxypropane undergo a condensation reaction as shown below to obtain compound C;

[0028]

[0029] The conditions and steps of the condensation reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0030] In the condensation reaction, the solvent may be a conventional solvent for this type of condensation reaction in the art. Preferably, the solvent is a ketone solvent, and more preferably, the solvent is acetone.

[0031] In the condensation reaction, the acid may be an acid conventionally used in this type of condensation reaction in the art. Preferably, the acid is sulfuric acid.

[0032] In the condensation reaction, the reaction temperature can be a conventional temperature for such condensation reactions in the art. Preferably, the reaction temperature is 35-60°C, more preferably, the reaction temperature is 40-50°C, and most preferably, the reaction temperature is 45°C.

[0033] In the condensation reaction, the disappearance of the compound C-3 is regarded as the end point of the condensation reaction. Preferably, the reaction time is 1 to 2 hours.

[0034] The preparation method of the compound as shown in Formula I may further comprise the following steps: in a solvent, in the presence of a debenzylation agent, compound C-2 undergoes a debenzylation reaction as shown below to obtain compound C-3;

[0035]

[0036] The conditions and steps of the debenzylation reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0037] In the debenzylation reaction, the solvent may be a solvent conventionally used in this type of debenzylation reaction in the art. Preferably, the solvent is one or more of a halogenated alkane solvent, a nitrile solvent and an ether solvent. More preferably, the solvent is one or more of dichloromethane, acetonitrile, 1,4-dioxane and tetrahydrofuran. Most preferably, the solvent is dichloromethane.

[0038] In the debenzylation reaction, the debenzylation reagent may be a reagent conventionally used in this type of debenzylation reaction in the art. Preferably, the debenzylation reagent is BCl 3 and / or BBr 3 More preferably, the debenzylation agent is BCl 3 .

[0039] In the debenzylation reaction, the molar ratio of the compound C-2 to the debenzylation agent can be a conventional ratio for such debenzylation reactions in the art, preferably 1:4 to 1:5.

[0040] In the debenzylation reaction, the reaction temperature may be a conventional reaction temperature for such debenzylation reactions in the art. Preferably, the reaction temperature is -70°C to -40°C. More preferably, the reaction temperature is -65°C, -60°C or -50°C.

[0041] In the debenzylation reaction, the debenzylation reaction takes the compound C-2 hours as the reaction end point, and preferably, the reaction time is 2 hours to 5 hours.

[0042] After the debenzylation reaction is completed, it is preferred to include post-treatment, and the post-treatment preferably includes the following steps: adding methanol, NaHCO 3 and water, adjust the pH to 8, and separate by preparative HPLC to obtain the compound C-3.

[0043] The preparation method of the compound shown in Formula I may further comprise the following steps: adding TMSOTf and TMSCN in a solvent at 0°C, and subjecting compound C-1 to a cyanation reaction as shown below at 15-20°C to obtain compound C-2;

[0044]

[0045] In the cyanation reaction, the solvent can be a conventional solvent for this type of cyanation reaction in the art. Preferably, the solvent is one or more of a halogenated alkane solvent, a nitrile solvent and an ether solvent. More preferably, the solvent is one or more of dichloromethane, acetonitrile, 1,4-dioxane and tetrahydrofuran. Most preferably, the solvent is dichloromethane.

[0046] In the cyanation reaction, the cyanation reaction may not include trifluoromethanesulfonic acid.

[0047] In the cyanation reaction, the cyanation reaction may only include the solvent, the TMSOTf, the TMSCN, and the compound C-1.

[0048] In the cyanation reaction, the molar ratio of the compound C-1 to the TMSCN can be a conventional ratio for such cyanation reactions in the art, preferably 1:4 to 1:5.

[0049] In the cyanation reaction, the molar ratio of the compound C-1 to the TMSOTf can be a conventional ratio for such cyanation reactions in the art, preferably 1:4 to 1:4.2.

[0050] In one embodiment, the cyanation reaction preferably comprises the following steps: in the presence of compound C-1 and a solvent, after adding TMSCN, TMSOTf is added to carry out the cyanation reaction to obtain the compound C-2.

[0051] In a certain embodiment, the cyanation reaction preferably comprises the following steps: in the presence of compound C-1 and a solvent, adding 4 equivalents of TMSCN relative to the amount of compound C-1, then adding 2 to 2.1 equivalents of TMSOTf relative to the amount of compound C-1, and then adding 2 to 2.1 equivalents of TMSOTf relative to the amount of compound C-1 to carry out the cyanation reaction to obtain the compound C-2.

[0052] In the cyanation reaction, the cyanation reaction takes compound C-1 hour as the reaction endpoint, preferably, the reaction time is 2 hours to 5 hours, more preferably, the reaction time is 2.5 hours or 4.5 hours.

[0053] The preparation method of the compound shown in Formula I may further comprise the following steps: in a solvent, in the presence of compound B, TMSCl and PhMgCl, adding compound A to carry out the following Grignard reaction to obtain compound C-1;

[0054]

[0055] The conditions and steps of the format reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0056] In the Grignard reaction, the solvent can be a solvent conventionally used in this type of Grignard reaction in the art. Preferably, the solvent is an ether solvent. More preferably, the solvent is tetrahydrofuran.

[0057] In the format reaction, the reaction temperature can be a conventional temperature for this type of format reaction in the art. Preferably, the reaction temperature is -40°C to 0°C, preferably -5°C, 5°C or -20°C.

[0058] In the Grignard reaction, the raw materials of the Grignard reaction preferably further include iPrMgCl; the molar ratio of the iPrMgCl to the compound B is preferably 1:1.

[0059] In the Grignard reaction, the disappearance of the compound A is regarded as the reaction endpoint. Preferably, the reaction time is 0.5 hours to 2 hours, and more preferably, the reaction time is 1 hour.

[0060] The present invention also provides a method for preparing a compound as shown in Formula II, which comprises the following steps: in a solvent, in the presence of an acid, compound E' is subjected to the following deprotection reaction to obtain a compound as shown in Formula II;

[0061]

[0062] The conditions and steps of the deprotection reaction can be as described above.

[0063] The preparation method of the compound shown in Formula II may further comprise the following steps: in a solvent, in the presence of a catalyst and a base, compound C and compound D' undergo a substitution reaction as shown below to obtain compound E';

[0064]

[0065] The conditions and steps of the substitution reaction may be as described above;

[0066] The preparation method of compound C can be as described above.

[0067] The present invention also provides a method for preparing compound C-2, which comprises the following steps: in a solvent, in the presence of TMSOTf and TMSCN, compound C-1 is subjected to a cyanation reaction as shown below to obtain compound C-2;

[0068]

[0069] The conditions and operation of the cyanation reaction can be as described above.

[0070] The present invention also provides a compound C:

[0071]

[0072] The present invention also provides a compound E:

[0073]

[0074] The present invention also provides a compound E':

[0075]

[0076] The present invention also provides a pharmaceutical composition, which includes one or more of substance X, substance Y and substance Z, and pharmaceutical excipients; the substance X is the compound shown in formula I or a pharmaceutically acceptable salt thereof, the substance Y is the compound shown in formula II or a pharmaceutically acceptable salt thereof, and the substance Z is the compound shown in formula III or a pharmaceutically acceptable salt thereof. The amount of the substance X, substance Y or substance Z may be a therapeutically effective amount;

[0077]

[0078] The present invention also provides a use of the substance X or the pharmaceutical composition in preparing a cell proliferation inhibitor; the cells are one or more of Hela cells, COLO 205 cells and U-87 MG cells.

[0079] The present invention also provides a use of the substance X or the pharmaceutical composition in preparing a drug for treating and / or preventing cancer, wherein the cancer is one or more of cervical cancer, colon cancer and glioma.

[0080] The present invention also provides an application of the substance Y or the pharmaceutical composition in the preparation of a cell proliferation inhibitor; the cells are one or more of Hela cells, COLO 205 cells and U-87 MG cells.

[0081] The present invention also provides a use of the substance Y or the pharmaceutical composition in preparing a drug for treating and / or preventing cancer, wherein the cancer is one or more of cervical cancer, colon cancer and glioma.

[0082] The present invention also provides an application of the substance Z or the pharmaceutical composition in the preparation of a cell proliferation inhibitor; the cells are one or more of Hela cells, COLO 205 cells and U-87 MG cells.

[0083] The present invention also provides a use of the substance Z or the pharmaceutical composition in preparing a drug for treating and / or preventing cancer, wherein the cancer is one or more of cervical cancer, colon cancer and glioma.

[0084] In the above applications, the cell proliferation inhibitor can be used in mammals; it can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the art to provide rapid detection of cell inhibition effects. Unless otherwise specified, the following terms appearing in the present invention have the following meanings:

[0085] The pharmaceutical excipients can be excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide methods to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipients can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: buffers, chelating agents, preservatives, cosolvents, stabilizers, excipients and surfactants colorants, flavoring agents and sweeteners.

[0086] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a relatively nontoxic, pharmaceutically acceptable acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes an inorganic acid, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acid includes organic acids, including but not limited to acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0087] The term "treatment" or its equivalent, when used, for example, for cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient or to alleviate the symptoms of cancer. "Treatment" of cancer or another proliferative disorder does not necessarily mean that cancer cells or other disorders will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the symptoms of cancer or other disorders will actually be alleviated. In general, a method of treating cancer will be performed even if it has only a low probability of success, but it is still believed to induce an overall beneficial course of action, taking into account the patient's medical history and estimated survival expectancy.

[0088] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0089] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0090] The reagents and raw materials used in the present invention are commercially available.

[0091] The positive and progressive effects of the present invention are that the compound shown in Formula I, the compound shown in Formula II and the compound shown in Formula III of the present invention can not only be used as a standard or reference substance to control the quality of remdesivir drugs, but also have the effect of inhibiting Hela cells, COLO 205 cells and U-87 MG cells. In addition, the preparation method of the present invention has simple reaction, mild conditions, high yield, no high and low temperature extreme reaction conditions, no use of high-risk and highly toxic substances, simple operation, high chiral purity, no special requirements, stable process, suitable for laboratory synthesis and industrial production, and has broad industrial application prospects. DETAILED DESCRIPTION

[0092] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the embodiments. The present invention is further described in detail below in conjunction with the examples. These specific embodiments only show the implementation technical solutions selected to achieve the purpose of the present application, and the improvements to the technical solutions of the present application in combination with the prior art all belong to the scope of protection of the present application.

[0093] The implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments. Among them, the chemical reagents used in the examples are all commercially available chemical reagents.

[0094] Example 1 Preparation of C-1 Hydroxyl Compound

[0095]

[0096] Add compound B iodide (50 g, 192.3 mmol, 1.0 eq) to a 5 L reaction bottle, add tetrahydrofuran (1.25 L) and stir to dissolve, replace argon three times, add TMSCl (48.8 mL, 384.6 mmol, 2.0 eq) dropwise, stir at room temperature for 40 min, cool to 0 ° C, add PhMgCl (192.5 mL, 384.6 mmol, 2.0 eq, 2 M in THF) dropwise, and continue stirring for 1 hour after addition. Under the condition of controlling the internal temperature at 0-5°C, iPrMgCl··LiCl (200 mL, 192.3 mmol, 1 eq) was slowly added dropwise. After the addition, the mixture was stirred for about 30 minutes. The temperature was lowered to -20°C, and compound A (88 g, 211.53 mmol, 1.1 eq, in 250 mL of THF) was added dropwise. The reaction was continued to stir for 1 hour. Samples were taken for TLC detection (developing solvent was isoacetic acid ester). After the reaction was completed, the temperature was controlled to about 0°C, and methanol (200 mL), acetic acid (200 mL) and water (200 mL) were slowly added to quench the reaction. The mixture was concentrated under reduced pressure, and ethyl acetate (2 L) and 1N HCl (2 L) were added. The organic phase was washed with saturated NaHCO 3 The mixture was washed twice with saturated sodium sulfate (1 L × 2) and saturated brine (1 L × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The filtrate obtained was a light oily liquid and passed through a silica gel column to obtain a white solid C-1 hydroxy compound (50.6 g, yield: 47.7%).

[0097] Compound C-1:

[0098] 1 H NMR (400 MHz, DMSO-d 6 )δ8.049(brs,1H),7.985(s,1H),7.362-7.226(m,11H),7.157-7.006(m,3H),7.1 57-7.126(m,2H),6.941(d,J=4.8Hz,1H),5.385(d,J=5.6Hz,1H),5.033(d,J=5.2H z,1H),4.595-4.554(m,2H),4.495-4.447(m,4H),4.0142-4.001(m,1H),3.927(dd ,J=5.9,4.4Hz,1H),3.688(dd,J=10.1,3.4Hz,1H),3.472(dd,J=10.0,6.4Hz,1H); 13 C-NMR (100 MHz, DMSO-d 6)δ187.893,155.786,148.900,138.605,138.105,128.570,128.194,128.172,12 8.100,128.090,127.776,127.706,127.668,127.553,127.492,127.424,127.39 7,127.339,127.222,127.147,127.043,126.963,118.499,117.425,102.162,81 .870,80.790,72.421,72.243,71.643,71.340,69.341; MS(ESI):m / z553.1[M+H] + .

[0099] Example 2 Preparation of Compound C-2 Nitrile

[0100]

[0101] Compound C-1 hydroxy (40 g, 0.0724 mmol, 1.0 eq) was added to a 5 L reaction bottle, and dichloromethane (1.2 L) was added and stirred to dissolve. Argon was replaced three times, and the temperature was lowered to 0 ° C, TMSCN (28.74 g, 0.2897 mol, 4.0 eq) was added, and stirred for 30 minutes. TMSOTf (33.8 g, 0.1521 mol, 2.1 eq) was added dropwise, and the reaction was carried out at 0 ° C for 2 hours. TMSOTf (33.8 g, 0.1521 mol, 2.1 eq) was added and reacted for 1 hour. The temperature was raised to 15 ° C and reacted for 2 hours. The temperature was lowered to 0 ° C, 2.4 L was added to dilute, and the reaction solution was poured into 3.6 L of saturated NaHCO 3 The solution was used to quench the reaction, and the aqueous phase was extracted once with 2 L of dichloromethane. The organic phases were combined, washed once with saturated brine, and concentrated to obtain a crude product, which was crystallized with isopropanol and n-heptane to obtain a white solid C-2 nitrile compound (35.1 g, yield: 86.3%).

[0102] Compound C-2:

[0103] 1 H NMR (400 MHz, DMSO-d 6)δ7.931-7.881(m,3H),7.367-7.252(m,15H),6.947(d,J=4.5Hz,1H),6.874(d,J=4.5Hz,1H),6.758(dd,J=4.5,5.9Hz,1H), 6.660 (dd,J=4.5,5.8Hz,1H),4.921-4.710(m,2H),4.646-4.503(m,4H),4.472-4.386(m,1H),4.140-4.028(m,1H),3.808-3.580(m,2H); 13 C-NMR (100MHz, DMSO-d6): δ155.530,155.453,148.029,147.832,138.167,138.079,137.938,137.689,137.3 64,137.311,128.250,128.159,128.127,128.095,127.911,127.843,127.732,127.703,127.504,127.390,12 6.963,122.632,121.959,116.758,116.720,115.608,110.480,100.780,81.884,80.408,80.369,79.236,78 .288,77.611,76.940,76.260,73.108,72.324,72.293,72.265,71.891,71.448,68.779,68.695; MS(ESI):m / z 562.1[M+H] + .

[0104] Comparative Example 2 Preparation of Compound C-2 Nitrile

[0105]

[0106] Compound C-1 hydroxy (1.1 g, 2 mmol, 1.0 eq) was added to a 500 mL reaction bottle, and dichloromethane (50 mL) was added to stir and dissolve. Argon was replaced three times, and the temperature was lowered to 0 ° C. TMSCN (0.794 g, 8 mmol, 4.0 eq) was added, and stirred for 30 minutes. TMSOTf (2 g, 9 mmol, 4.5 eq) was slowly added dropwise, and the reaction was carried out at 30 ° C for 1 hour. TLC (petroleum ether: ethyl acetate = 1:2) showed that the reaction was completed, but some impurities were produced. The temperature was lowered to 0 ° C, 100 mL of dichloromethane was added to dilute, and the reaction solution was poured into 100 mL of saturated NaHCO 3The solution was used to quench the reaction, and the aqueous phase was extracted once with 100 mL of dichloromethane. The organic phases were combined, washed once with saturated brine, and concentrated to obtain a crude product, which was subjected to silica gel chromatography with a gradient elution of 0-75% ethyl acetate and n-heptane to obtain a white solid C-2 nitrile compound (0.62 g, yield: 55%).

[0107] Example 3 Preparation of Compound C-3

[0108]

[0109] C-2 (10 g, 0.0178 mol, 1.0 eq) was added to a 1 L reaction bottle and dissolved in dichloromethane (320 mL). The temperature was cooled to -65 °C under argon protection. 1 M boron trichloride hexane solution (89 mL, 0.089 mol, 5.0 eq) was added dropwise. The internal temperature was controlled at about -50 °C. The addition was completed in about 25 minutes. The reaction was completed at about -65 °C for about 2 hours. Methanol (100 mL) was added. NaHCO 3 (12 g) and water (20 mL), stirred and naturally heated to 0 ° C, measured pH = 8, stirred for 20 minutes, filtered, the filtrate was concentrated and the crude product was separated by C18 preparative HPLC, and then separated by H 2 O (0.1% TFA), 10-60% MeOH gradient elution to obtain white solid compound C-3' (debenzylated product, remdesivir intermediate) (2.3 g, yield: 44.4%), white solid compound C-3 (2.4 g, yield: 46.3%).

[0110] Compound C-3':

[0111] 1 H NMR (400MHz, water-d2): δ8.13(s,1H),7.42(d,J=4.9Hz,1H),7.16(d,J=4.9Hz,1H), 4.94(d,J=5.4Hz,1H), 4.43(td,J=4.8,3.2Hz,1H),4.38-4.31(m,1H),3.88(dd,J=12.8,3.2Hz,1H),3.80(dd,J=12.8,4.8Hz,1H); 13 C-NMR (100 MHz, DMSO-d 6 ): δ155.634,147.872,123.887,117.344,116.540,110.788,100.816,85.444,78.574,74.245,70.087,60.962; MS(ESI):m / z 292.1[M+H] + .

[0112] Compound C-3:

[0113] 1 H NMR (400MHz, water-d2): δ8.11(s,1H),7.43(d,J=4.8Hz,1H),7.10(d,J=4.8Hz,1H),5.14(d,J=4.4Hz,1H), 4.75(dd,J=9.0,4.5Hz,1H),4.33-4.26(m,1H),4.07(dd,J=12.9,2.1Hz,1H),3.88(dd,J=12.9,4.5Hz,1H); 13 C-NMR (100MHz, water-d2): δ148.833,136.031,128.111,116.941,113.587,112.978,109.950,83.305,77.589,76.376,70.608,60.261; MS (ESI): m / z 292.1[M+H] + .

[0114] The preparation method here comes from CN103052631A, and the structural data of compound C-3' remdesivir intermediate is consistent with the data reported in Nature, 2016, Doi: 10.1038 / nature17180 (Nature, 2016, 531(7594): 381-385.).

[0115] Example 4 Preparation of Compound C

[0116]

[0117] Compound C-3 (6.8 g, 0.02336 mmol, 1.0 eq) and acetone (200 mL) were added to a 1000 mL reaction bottle, and 2,2-dimethoxypropane (12.16 g, 0.1168 mol, 5.0 eq) and concentrated sulfuric acid (2 ml) were added. After stirring at room temperature for 0.5 hours, the temperature was raised to 45 ° C. After reacting for 1 hour, the temperature was lowered to room temperature, and then solid sodium bicarbonate (7 g) and water (7 mL) were added. The mixture was stirred for 15 minutes and concentrated under reduced pressure. The crude product obtained by concentration was added with water (50 mL) and ethyl acetate (150 mL). Stir, stand, separate the lower aqueous phase, and extract the aqueous phase with ethyl acetate (2×50 mL). Combine the organic phases, dry with sodium sulfate, concentrate under reduced pressure, and dry under vacuum to obtain compound C (7.52 g, yield: 97.2%).

[0118] Compound C:

[0119] 1 H NMR (400 MHz, DMSO-d 6)δ7.92(s,1H),7.81(s,2H),6.89(d,J=4.5Hz,1H),6.68(d,J=4.5Hz,1H),5.50(d,J=5.8Hz,1H),5.21(t,J=5. 0Hz, 1H), 5.04 (dd, J = 5.8, 0.7Hz, 1H), 4.43 (t, J = 6.1Hz, 1H), 3.71 (t, J = 5.3Hz, 2H), 1.17 (s, 3H), 0.89 (s, 3H); 13 C-NMR (100 MHz, DMSO-d 6 ): δ155.364,147.687,122.727,118.573,115.442,112.515,108.992,100. 726,86.651,85.772,82.688,79.520,60.455,25.464,24.973; MS(ESI):m / z 332.2[M+H] + .

[0120] Example 5 Preparation and Synthesis of Compound E (Ester)

[0121]

[0122] Compound C (1.1 g, 0.00332 mol, 1.0 eq) and compound D (1.8 g, 0.004 mol, 1.2 eq) were placed in a 100 mL reaction bottle, anhydrous acetonitrile (16 mL) was added to dissolve, anhydrous magnesium chloride was added at room temperature, the temperature was raised to 50°C with stirring for 20 minutes, DIPEA (1.074 g, 0.0093 mol, 2.5 eq) was added dropwise, the temperature was kept for reaction for 1 hour, cooled to room temperature, ethyl acetate (50 mL) was added to dilute, and the mixture was washed with 5% citric acid (20 mL × 2), saturated ammonium chloride solution (20 mL), 5% potassium carbonate aqueous solution (20 mL × 2), and saturated sodium chloride (20 mL) once, dried over anhydrous magnesium sulfate, filtered, and the organic phase was concentrated under reduced pressure to an oily substance, which was passed through a column to obtain a white solid compound E (1.6 g, yield: 75.1%).

[0123] Compound E:

[0124] 1 H NMR (400MHz,methanol-d 4)δ7.83(s,1H),7.38(t,J=7.8Hz,2H),7.30(dd,J=8.8,1.1Hz,2H),7.21(t,J=7.3Hz ,1H),6.88(d,J=4.5Hz,1H),6.77(d,J=4.5Hz,1H),5.59(d,J=5.8Hz,1H),5.08(dd, J=5.8,1.2Hz,1H),4.70(t,J=5.2Hz,1H),4.40(ddd,J=19.5,11.1,5.7Hz,2H),4.26 –4.03(m,3H),1.43–1.24(m,11H),1.22(s,3H),0.96(s,3H),0.90(t,J=7.5Hz,6H); 13 C-NMR (100MHz,methanol-d 4 ): δ175.079,175.027,156.998,152.219,152.150,147.879,130.831,126.235, 124.900,121.534,121.485,119.359,116.868,115.019,110.914,102.399,87.8 62,86.294,84.041,81.730,68.286,67.141,67.086,51.642,41.777,33.034,30.142,26.037,25.339,24.342,24.306,23.730,20.697,20.629,14.411,11.368; 31 P-NMR(162MHz, methanol-d4): δ3.480(s); MS(ESI): m / z 642.9[M+H] + .

[0125] Example 6 Preparation and Synthesis of RDV-ISO-1 (i.e., the compound shown in Formula I)

[0126]

[0127] In a 250ml reaction bottle, add ester compound E (1.8g, 0.0028mol) and tetrahydrofuran (20mL), stir until dissolved, cool to 0-5℃, add concentrated hydrochloric acid (4mL), add until complete, heat to 20-25℃ and keep warm for 5 hours. Add 20mL of water, adjust pH to 7-8 with saturated sodium bicarbonate solution. Add ethyl acetate (50mL), stir, separate the organic phase, wash with sodium chloride aqueous solution (20mL×2), dry with anhydrous sodium sulfate, filter, concentrate to dryness under reduced pressure, and pass the crude product through a column to obtain RDV-ISO-1 (1.26g, yield: 75%).

[0128] Compound RDV-ISO-1:

[0129] 1 H NMR (400MHz,methanol-d 4 )δ7.84(s,1H),7.42–7.32(m,2H),7.32–7.27(m,2H),7.18(td,J=7.3,0.8Hz,1H), 6.90(d,J=4.5Hz,1H), 6.80(d,J=4.5Hz,1H), 5.00(d,J=4.3Hz,1H), 4.60(dd,J=8. 6,4.3Hz,2H),4.48(dd,J=9.2,6.0Hz,1H),4.38–4.28(m,2H),4.06(tdd,J=16.5,1 0.3, 5.9Hz, 3H), 1.54–1.43 (m, 1H), 1.43–1.26 (m, 8H), 0.86 (td, J = 7.5, 2.4Hz, 6H). 13 C-NMR (100MHz,methanol-d 4 ): δ175.13,175.06,156.95,152.23,152.16,148.07,130.73,126.08,124.62,121.54,121.49,118.77,116.87,111.91,1 02.65,82.56,82.48,79.70,77.21,72.66,68.25,67.41,67.35,51.56,41.64,24.23,24.18,20.78,20.72,11.35,11.29; 31 P-NMR(162MHz, methanol-d4): δ3.641(s); MS(ESI): m / z 602.9[M+H] + .

[0130] Example 7 Preparation and Synthesis of E-ISO-2 (Ester R 70%, S 30%)

[0131]

[0132] In a 100 mL reaction bottle, C' (1 g, 0.003 mol, 1.0 eq) and D' (1.62 g, 0.0036 mol, 1.2 eq, R 70%, S 30%) were added, anhydrous acetonitrile (16 mL) was added, dissolved, anhydrous magnesium chloride was added at room temperature, stirred and heated to 50 ° C for 20 minutes, DIPEA (0.969 g, 0.0075 mol, 2.5 eq) was added dropwise, and the reaction was kept warm for 1 hour. The mixture was cooled to room temperature, and ethyl acetate (50 mL) was added for dilution. The mixture was washed with 5% citric acid (20 mL × 2), saturated ammonium chloride solution (20 mL), 5% potassium carbonate aqueous solution (20 mL × 2), and saturated sodium chloride (20 mL) once, dried over anhydrous magnesium sulfate, filtered, and the organic phase was concentrated under reduced pressure to an oily substance. The white solid E-ISO-2 (1.5 g, yield: 77.3%) was obtained by column chromatography.

[0133] Compound E-ISO-2:

[0134] 1 H NMR (400MHz,methanol-d 4 )δ7.86(s,1H),7.26(dd,J=15.6,7.8Hz,2H),7.18–7.02(m,3H),6.89(dt,J=4.6,3.9Hz,2H),5.26(dd,J=63.7,6.6Hz,1H),4.97(ddd,J=15.6 ,6.6,3.3Hz,1H),4.67–4.50(m,1H),4.40–4.25(m,2H),4.19–3.78(m,3H),1.70(d,J=2.2Hz,3H),1.54–1.20(m,12H),0.87(t,J=7.5Hz,6H); 13 C-NMR (100MHz,methanol-d 4): δ175.14,175.09,174.96,174.90,157.26,152.15,152.08,148.52,148.49,130.68,126.20,126.10,124. 72,124.61,121.40,121.37,121.36,121.32,118.34,118.31,117.80,117.62,117.06,117.02,112.35,112.2 8,102.46,85.70,84.97,84.94,84.89,84.85,83.09,83.03,82.73,82.60,68.12,68.07,67.03,66.97,66.95,66.90,51.61,51.48,41.75,41.73,26.52,26.43,25.54,25.45,24.24,24.22,20.38,20.31,11.33,11.30; 31 P-NMR(162MHz, methanol-d4): δ3.533(s),3.219(s); MS(ESI): m / z 665.0[M+Na] + .

[0135] Example 8 Preparation and Synthesis of RDV-ISO-2

[0136]

[0137] In a 100mL reaction bottle, add ester E-ISO-2 (1g, 0.00156mol) and tetrahydrofuran (16mL), stir until dissolved, cool to 0-5℃, add concentrated hydrochloric acid (4mL), add until complete, heat to 20-25℃ and react for 5 hours. Add 20mL of water, adjust pH to 7-8 with saturated sodium bicarbonate solution. Add ethyl acetate (50mL), stir, separate the organic phase, wash with sodium chloride aqueous solution (20mL×2), dry with anhydrous sodium sulfate, filter, concentrate to dryness under reduced pressure, and separate the crude product by preparative HPLC to obtain RDV-ISO-2 (492mg, yield: 52.6%).

[0138] Compound RDV-ISO-2:

[0139] 1 H NMR (400MHz,methanol-d 4)δ7.85(s,1H),7.28(dd,J=10.0,5.7Hz,2H),7.21–7.08(m,3H),6.91(dd,J=10.7,4 .6Hz,2H),4.79(d,J=5.5Hz,1H),4.53–4.36(m,2H),4.36–4.26(m,1H),4.21(t,J=5. 4Hz, 1H), 4.00 (qd, J=10.9, 5.7Hz, 2H), 3.86 (dq, J=14.3, 7.1Hz, 1H), 1.46 (dq, J=12. 1,6.0Hz,1H),1.39–1.27(m,4H),1.25(dd,J=7.1,1.0Hz,3H),0.86(t,J=7.5Hz,6H); 13 C-NMR (100MHz,methanol-d 4 ): δ175.15,175.11,157.26,152.17,152.10,148.34,130.68,126.11,125.54,121.43,121.39,118.00,117.64,112.37,1 02.63,84.61,84.53,81.09,75.68,71.55,68.09,66.83,66.77,51.61,41.73,24.23,24.21,20.45,20.38,11.33,11.30; 31 P-NMR (162MHz,methanol-d 4 ): δ3.545(s); MS(ESI): m / z602.9[M+H] + .

[0140] Example 9 Preparation and Synthesis of Compound E' (Ester)

[0141]

[0142] Compound C (1.1 g, 0.00332 mol, 1.0 eq) and compound D' (R, about 70%; S, about 30%) (1.8 g, 0.004 mol, 1.2 eq) were placed in a 100 mL reaction bottle, anhydrous acetonitrile (16 mL) was added to dissolve, anhydrous magnesium chloride was added at room temperature, the temperature was raised to 50°C with stirring for 20 minutes, DIPEA (1.074 g, 0.0093 mol, 2.5 eq) was added dropwise, the temperature was kept for 1 hour, cooled to room temperature, ethyl acetate (50 mL) was added to dilute, and the mixture was washed with 5% citric acid (20 mL × 2), saturated ammonium chloride solution (20 mL), 5% potassium carbonate aqueous solution (20 mL × 2), and saturated sodium chloride (20 mL) once, dried over anhydrous magnesium sulfate, filtered, and the organic phase was concentrated under reduced pressure to an oily substance, which was passed through a column to obtain a white solid compound E' (1.5 g, yield: 70%).

[0143] Compound E':

[0144] 1 H NMR (400MHz,methanol-d 4 )δ7.85(s,1H),7.45–7.35(m,2H),7.35–7.27(m,2H),7.23(t,J=7.8Hz,1H),6.88 (t,J=4.4Hz,1H),6.76(t,J=4.8Hz,1H),5.28(d,J=5.7Hz,1H),4.96(dd,J=5.7,1 .0Hz,1H),4.78–4.66(m,1H),4.42(t,J=5.1Hz,2H),4.17–4.00(m,3H),1.52(dd, J=12.3,6.2Hz,1H),1.45–1.15(m,11H),0.96(s,1H),0.90(td,J=7.2,2.5Hz,7H); 13 C-NMR (100MHz,methanol-d 4): δ175.33,175.28,156.99,152.31,152.24,147.87,147.82,130.90,130.83,126.37,126.24,125.1 9,124.91,121.64,121.59,121.53,121.49,119.44,119.36,116.86,115.02,114.84,110.92,110.82 ,102.35,87.87,86.38,86.29,86.19,86.09,84.07,81.96,81.73,68.29,68.18,67.38,67.32,67.14,61.53,51.69,51.65,41.82,41.78,26.04,25.88,25.34,25.18,24.32,20.63,20.57,14.46,11.36; 31 P-NMR(162MHz, methanol-d4): δ3.705(s),3.482(s); MS(ESI): m / z665.0[M+Na] + .

[0145] Example 10 Preparation and Synthesis of RDV-ISO-3 (i.e., the compound shown in Formula II)

[0146]

[0147] In a 100mL reaction bottle, add ester compound E' (1g, 0.00156mol) and tetrahydrofuran (16mL), stir until dissolved, cool to 0-5℃, add concentrated hydrochloric acid (4mL), add until complete, heat to 20-25℃ and react for 5 hours. Add 20mL of water, adjust pH to 7-8 with saturated sodium bicarbonate solution. Add ethyl acetate (50mL), stir, separate the organic phase, wash with sodium chloride aqueous solution (20mL×2), dry with anhydrous sodium sulfate, filter, concentrate to dryness under reduced pressure, and separate the crude product by preparative HPLC to obtain RDV-ISO-3 (528mg, yield: 56.3%).

[0148] Compound RDV-ISO-3:

[0149] 1 H NMR (400MHz,methanol-d 4)δ7.86(s,1H),7.40–7.33(m,2H),7.32–7.25(m,2H),7.20(td,J=7.5,0.9Hz,1H),6.9 0(d,J=4.5Hz,1H),6.80(d,J=4.5Hz,1H),4.97(d,J=4.2Hz,1H),4.60(dd,J=8.7,4.3Hz ,1H),4.57–4.50(m,1H),4.35–4.24(m,2H),4.11(tt,J=14.2,7.1Hz,1H),4.01(d,J=5. 7Hz,2H),1.46(dq,J=12.1,6.0Hz,1H),1.42–1.25(m,8H),0.85(td,J=7.4,1.8Hz,6H);

[0150] 13 C-NMR (100MHz,methanol-d 4 ): δ175.34,175.29,157.01,152.23,152.16,148.06,130.72,126.16,124.76,121.72,121.67,118.75,116.92,111.85,1 02.67,82.50,82.41,79.81,77.32,72.15,68.07,66.13,66.08,51.57,41.73,24.22,24.21,20.71,20.64,11.32,11.30; 31 P-NMR(162MHz, methanol-d4): δ3.654(s); MS(ESI): m / z 603.3[M+H] + .

[0151] Example 11 Effect Test

[0152] The three process impurities of Remdesivir prepared by the present invention, RDV-ISO-1 (Formula I), RDV-ISO-2 (Formula III) and RDV-ISO-3 (Formula II), have obvious inhibitory activity against Hela (human cervical cancer cells), COLO 205 (human colon cancer cells) and U-87 MG (human brain astroglioma cells). The results are as follows:

[0153] Cytotoxicity Assay:

[0154] Drug treatment concentration

[0155] The compounds were prepared in DMSO at 10 -2The stock solution of 100 μM was stored at -80℃ for future use after small aliquoting. During the experiment, the initial concentration of the compound detection was set to 100 μM, 10 gradient dilution concentrations were selected, the gradient dilution multiple was 3 times, and 2 replicates were tested for each concentration. The control group without drug addition was the corresponding culture medium solution containing 0.2% DMSO without drug addition.

[0156] CTG detection method

[0157] Hela (human cervical cancer cells), COLO 205 (human colon cancer cells) and U-87MG (human astrocytoblastoma cells, i.e., glioma cells) in the logarithmic growth phase were seeded in 384-well plates at a certain cell density. The cell culture plates were placed at 37 degrees Celsius and 5% CO 2 The compounds were diluted in series and cultured for 24 h at 37 °C and 5% CO. 2 The cell culture plate was taken out of the incubator and equilibrated at room temperature for 30 minutes. 50 μl of the equilibrated medium was added to each well. The Luminescent Cell Viability Assay reaction solution was shaken at 500 rpm for 2 minutes at room temperature and centrifuged at 1000 rpm for 1 minute. After equilibration at room temperature for 10 minutes, the fluorescence signal value was measured. The in vitro inhibitory activity of the compound was calculated according to the following formula:

[0158] Inhibition rate (%) = (signal value control - signal value administration) / signal value control × 100%. According to the inhibition rate of each concentration, the 50% inhibitory concentration (50% inhibitory concentration, IC50) was calculated using the LOGIT method.

[0159]

[0160] Note: Rf IC50 and Abs IC50 represent relative half inhibition rate and absolute half inhibition rate, respectively.

[0161] From the data in the table above, the IC50 values ​​of RDV-ISO-2 against Hela (human cervical cancer cells), COLO 205 (human colon cancer cells) and U-87 MG (human brain glioblastoma cells) are significantly lower than those of RDV-ISO-1 and RDV-ISO-3, and RDV-ISO-2 has better inhibitory activity. All three compounds have potential anticancer activity and are worth further study.

Claims

1. A compound as shown in formula I, or a pharmaceutically acceptable salt thereof:

2. A method for preparing a compound as shown in formula I, characterized in that: The preparation method of the compound shown in Formula I comprises the following steps: in a solvent, in the presence of an acid, compound E is subjected to a deprotection reaction as shown below to obtain the compound shown in Formula I; 3. The preparation method according to claim 2, characterized in that: The preparation method of the compound as shown in Formula I further comprises the following steps: in a solvent, in the presence of a catalyst and a base, compound C and compound D undergo a substitution reaction as shown below to obtain the compound E; 4. The preparation method according to claim 3, characterized in that: In the substitution reaction, the catalyst is magnesium chloride; In the substitution reaction, the base is N,N-diisopropylethylamine.

5. The preparation method according to claim 3, characterized in that: The preparation method of the compound as shown in Formula I further comprises the following steps: in a solvent, in the presence of an acid, compound C-3 and 2,2-dimethoxypropane undergo a condensation reaction as shown below to obtain compound C; 6. The preparation method according to claim 5, characterized in that: The preparation method of the compound as shown in Formula I further comprises the following steps: in a solvent, in the presence of a debenzylation agent, compound C-2 undergoes a debenzylation reaction as shown below to obtain compound C-3; 7. The preparation method according to claim 6, characterized in that: In the debenzylation reaction, the debenzylation reagent is BCl3 and / or BBr3.

8. The preparation method according to claim 6, characterized in that: The preparation method of the compound shown in formula I further comprises the following steps: TMSOTf and TMSCN are added to a solvent at 0°C, and compound C-1 is subjected to a cyanation reaction as shown below at 15-20°C to obtain compound C-2.

9. The preparation method according to claim 8, characterized in that: The preparation method of the compound shown in Formula I further comprises the following steps: in a solvent, in the presence of compound B, TMSCl and PhMgCl, adding compound A to carry out the following Grignard reaction to obtain compound C-1; 10. The preparation method according to claim 9, characterized in that: In the cyanation reaction, the solvent is one or more of a halogenated alkane solvent, a nitrile solvent and an ether solvent; and / or, in the cyanation reaction, the molar ratio of the compound C-1 to the TMSCN is 1:4 to 1:5; and / or, the cyanation reaction does not include trifluoromethanesulfonic acid; And / or, the cyanation reaction only comprises the solvent, the TMSOTf, the TMSCN, and the compound C-1; and / or, in the cyanation reaction, the molar ratio of the compound C-1 to the TMSOTf is 1:4 to 1:4.2; And / or, the cyanation reaction comprises the following steps: in the presence of compound C-1 and a solvent, after adding TMSCN, adding TMSOTf to carry out the cyanation reaction to obtain the compound C-2; And / or, in the cyanation reaction, the reaction time is 2 hours to 5 hours.

11. The preparation method according to claim 10, characterized in that: In the cyanation reaction, the solvent is one or more of dichloromethane, acetonitrile, 1,4-dioxane and tetrahydrofuran.

12. The preparation method according to claim 10, characterized in that: The cyanation reaction comprises the following steps: in the presence of compound C-1 and a solvent, adding 4 to 5 equivalents of TMSCN relative to the amount of compound C-1, then adding 2 to 2.1 equivalents of TMSOTf relative to the amount of compound C-1, and then adding 2 to 2.1 equivalents of TMSOTf relative to the amount of compound C-1 to carry out the cyanation reaction to obtain the compound C-2.

13. A compound E:

14. A pharmaceutical composition comprising one or more of a substance X and a substance Y, and a pharmaceutical excipient; the substance X is a compound as shown in formula I or a pharmaceutically acceptable salt thereof; the substance Y is a compound as shown in formula II or a pharmaceutically acceptable salt thereof; and the amount of the substance X and the substance Y is a therapeutically effective amount; 15. Use of a substance X, a substance Y, a substance Z or the pharmaceutical composition as claimed in claim 14 in the preparation of a cell proliferation inhibitor; the definitions of the substance X and the substance Y are as described in claim 14, the substance Z is a compound as shown in formula III or a pharmaceutically acceptable salt thereof, and the cell is one or more of Hela cells, COLO 205 cells and U-87MG cells; 16. Use of a substance X, a substance Y, a substance Z or the pharmaceutical composition as claimed in claim 14 in the preparation of a drug for treating and / or preventing cancer, wherein the cancer is one or more of cervical cancer, colon cancer and glioma, and the definitions of the substance X, substance Y and substance Z are as described in claim 15.

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