Small-molecule conjugated drug, preparation method and application thereof

Abstract

The present application relates to the field of pharmaceutical chemistry, and particularly relates to a small molecule conjugated drug and a preparation method and application thereof.The structural formula of the small molecule conjugated drug is shown as formula 1: wherein A in formula 1 is a cytotoxic drug.The small molecule conjugated drug provided in the present application connects two structures on the cytotoxic drug, one is a tetrafluoroazidophenyl linker capable of being activated by X-rays to release the cytotoxic drug, and the other is a small molecule inhibitor aripiprazole having targeting NK1R, which can better achieve drug targeting, and can achieve drug activation in space-time control, can be well used in tumor treatment, and exhibits stronger cytotoxicity on tumor cells after X-ray irradiation.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a small molecule coupled drug and a preparation method and application thereof. Background Art

[0002] Traditional anticancer chemotherapy drugs are not selective for tumors and do not preferentially target tumor sites, often leading to toxicity to normal cells. Over the past three decades, the discovery and production of anticancer drugs has undergone tremendous changes, from traditional chemotherapy drugs to targeted drugs, which have greatly improved the precision of tumor treatment and reduced side effects.

[0003] Small molecule-drug conjugates (SMDCs) are drugs synthesized by covalently coupling molecules of multiple different components, including cytotoxic drugs, activation units, targeting units, and imaging agents. This approach allows for the precise delivery of chemotherapy drugs to cancer cells while avoiding cytotoxicity to normal cells. The various parts of small molecule drug conjugates are generally linked together by unstable chemical bonds, which are easily affected by factors such as overexpressed reactive oxygen species (ROS), glutathione (GSH), pH, and various enzymes in the tumor microenvironment. Through the targeting effect of specific ligands, SMDCs reach the vicinity of the tumor and release active drugs under the influence of the microenvironment, achieving the purpose of precision treatment. In this field, a variety of small molecule compounds have been studied as targeting ligands and have attracted considerable attention.

[0004] On the other hand, in radiotherapy, ionizing radiation interacts primarily with water (which constitutes 70-80% of tissue weight), leading to water radiolysis, a process that produces two main reactive species: hydrated electrons (e - aq ) and hydroxyl radicals (·OH), where ·OH is an oxidative free radical with a standard potential of 2.7 V, which can react with DNA to induce cell apoptosis; e - aq X-ray and hydrogen radicals are highly active reducing agents with standard potentials of -2.9 V and -2.3 V, respectively. These species exhibit high chemical reactivity and have been extensively studied as a means of prodrug activation. Furthermore, compared to other external stimuli, ionizing radiation has high tissue penetration, spatiotemporal resolution, and clinical relevance. Therefore, X-ray radiotherapy-induced prodrug activation strategies have shown great promise in clinical development.

[0005] Therefore, there is an urgent need in this field to develop a small molecule drug conjugate based on X-ray activation to better achieve the targeting of anti-tumor drugs and to achieve drug activation in a spatiotemporally controllable manner. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a small molecule drug conjugate and its preparation method and application. The small molecule drug conjugate provided by the present invention can be activated by X-rays to release cytotoxic drugs, and at the same time actively targets tumor cells through small molecule inhibitors, further improving the targeting of cytotoxic drugs, and showing good application prospects in the treatment of tumors.

[0007] The technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides a small molecule drug conjugate, wherein the structural formula of the small molecule drug conjugate is shown in Formula 1:

[0009]

[0010] Wherein, A in Formula 1 is a cytotoxic drug.

[0011] Optionally, A is one of monomethyl auristatin E, doxorubicin, camptothecin, mitomycin C, methotrexate, paclitaxel, and vinblastine.

[0012] Optionally, the structural formula of the small molecule drug conjugate is:

[0013]

[0014] The second aspect of the present invention provides a method for preparing a small molecule conjugate drug, wherein the preparation method comprises the steps of: subjecting Compound 1 and Compound 2 to an amidation reaction to obtain a small molecule conjugate drug represented by Formula 1;

[0015] The synthetic route of the small molecule conjugated drug is:

[0016]

[0017] Optionally, the step of subjecting Compound 1 and Compound 2 to an amidation reaction to obtain the small molecule conjugate drug represented by Formula 1 specifically includes:

[0018] Compound 1 and compound 2 are dissolved in an organic solvent, and then a condensing agent, an activating agent and a base are added, and the mixture is reacted at 25° C. for 5 to 10 hours to obtain a small molecule conjugated drug represented by formula 1.

[0019] Optionally, the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the activator is one of 1-hydroxybenzotriazole, N-hydroxy-7-azabenzotriazole, and 4-dimethylaminopyridine; the base is one of triethylamine, N,N-diisopropylethylamine, and pyridine; and the organic solvent is one of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, toluene, and ethyl acetate.

[0020] Optionally, the molar ratio of compound 1 to compound 2 is 1 to 3:1.

[0021] Optionally, the molar ratio of the compound 2, the condensing agent, the activating agent and the base is 1:1-3:1-3:1-3.

[0022] The third aspect of the present invention provides a pharmaceutical composition, which comprises the small molecule conjugate drug described in the present invention and a pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer and / or diluent.

[0023] The fourth aspect of the present invention provides a use of the small molecule drug conjugate of the present invention in the preparation of anti-tumor drugs.

[0024] Beneficial Effects: The present invention has developed a small molecule drug conjugate that connects two structures to a cytotoxic drug: a tetrafluoroazidophenyl linker that can be activated by X-rays to release the cytotoxic drug, and a small molecule inhibitor aprepitant that targets NK1R. The tetrafluoroazidophenyl linker can be activated by X-rays to release the cytotoxic drug, while the small molecule inhibitor aprepitant that targets NK1R can actively target tumor cells, further improving the targeting of the cytotoxic drug. The small molecule drug conjugate provided by the present invention can better achieve drug targeting and can achieve drug activation in a spatiotemporally controllable manner. It is well suited for tumor treatment and exhibits stronger cytotoxicity to tumor cells after X-ray irradiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the hydrogen spectrum of compound 1.

[0026] Figure 2 is the hydrogen spectrum of compound B4.

[0027] Figure 3 is the hydrogen spectrum of compound B5-1.

[0028] Figure 4 This is the hydrogen spectrum of the small molecule drug conjugate SMDC.

[0029] Figure 5 This is a graph showing the relationship between X-ray dose and the release rate of the cytotoxic drug MMAE.

[0030] Figure 6 This is the CCK-8 cell experiment results of Hela cells in the SMDC+60Gy group, SMDC group and MMAE group.

[0031] Figure 7 This is the CCK-8 cell experiment results of HCT116 cells in the SMDC+60Gy group, SMDC group and MMAE group.

[0032] Figure 8 This is the CCK-8 cell experiment results of A549 cells in the SMDC+60Gy group, SMDC group and MMAE group.

[0033] Figure 9 The graph shows the results of CCK-8 cell experiments on Hela, HCT116, and A549 cells in the aprepitant group. DETAILED DESCRIPTION

[0034] The present invention provides a small molecule drug conjugate and its preparation method and application. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0035] An embodiment of the present invention provides a small molecule drug conjugate, wherein the structural formula of the small molecule drug conjugate is shown in Formula 1:

[0036]

[0037] Wherein, A in Formula 1 is a cytotoxic drug.

[0038] The present invention provides a small molecule drug conjugate, which connects two structures to a cytotoxic drug (A): a tetrafluoroazidophenyl linker that can be activated by X-rays to release the cytotoxic drug, and a small molecule inhibitor aprepitant that targets NK1R. The tetrafluoroazidophenyl linker can be activated by X-rays to release the cytotoxic drug, and the small molecule inhibitor aprepitant that targets NK1R can actively target tumor cells, further improving the targeting of the cytotoxic drug. The small molecule drug conjugate provided by the embodiment of the present invention can better achieve drug targeting and achieve drug activation in a spatiotemporally controllable manner. It can be well used in tumor treatment and exhibits stronger cytotoxicity to tumor cells after X-ray irradiation.

[0039] The advantages of the small molecule drug conjugates provided by the embodiments of the present invention are at least that, first, the X-ray activated prodrug strategy is capable of producing a certain spatiotemporally controllable selective drug release by targeted X-ray irradiation; second, the addition of a small molecule inhibitor aprepitant that actively targets NK1R further improves the targeting of the drug.

[0040] In the present invention, aprepitant, a small molecule inhibitor targeting NK1R (neurokinin 1 receptor), is selected as the targeting molecule. NK1R is selected as the target because it is a receptor membrane protein overexpressed on a variety of tumor cells. At the same time, multiple studies have demonstrated that NK1R inhibitors produce anti-cancer effects by affecting downstream pathways.

[0041] The cytotoxic drug in the embodiments of the present invention refers to a class of drugs that can effectively kill cells and inhibit their proliferation, and is commonly used as an anti-tumor drug in clinical practice. Preferably, the cytotoxic drug is one of monomethyl auristatin E, doxorubicin, camptothecin, mitomycin C, methotrexate, paclitaxel, and vinblastine.

[0042] In this embodiment, the structural formula of monomethyl auristatin E (MMAE) is

[0043]

[0044] In some embodiments, the structural formula of the small molecule drug conjugate is:

[0045]

[0046] An embodiment of the present invention provides a method for preparing the small molecule conjugate drug described in the aforementioned embodiment, wherein the preparation method comprises the steps of: subjecting Compound 1 and Compound 2 to an amidation reaction to obtain the small molecule conjugate drug represented by Formula 1;

[0047] The synthetic route of the small molecule conjugated drug is:

[0048]

[0049] In some embodiments, the step of subjecting Compound 1 and Compound 2 to an amidation reaction to obtain the small molecule conjugate drug represented by Formula 1 specifically comprises:

[0050] Compound 1 and compound 2 are dissolved in an organic solvent, and then a condensing agent, an activating agent and a base are added, and the mixture is reacted at 25° C. (room temperature) for 5 to 10 hours to obtain a small molecule conjugated drug represented by formula 1.

[0051] In some embodiments, the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N,N'-dicyclohexylcarbodiimide (DCC), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); the activating agent is one of 1-hydroxybenzotriazole (HOBt), N-hydroxy-7-azabenzotriazole (HOAt), and 4-dimethylaminopyridine (DMAP); the base is one of triethylamine (TEA), N,N-diisopropylethylamine, and pyridine; and the organic solvent is one of N,N-dimethylformamide (DMF), dichloromethane, dimethyl sulfoxide, toluene, and ethyl acetate.

[0052] In some embodiments, the molar ratio of Compound 1 to Compound 2 is 1 to 3:1; preferably, the molar ratio of Compound 1 to Compound 2 is 1.2:1.

[0053] In some embodiments, the molar ratio of the compound 2, the condensing agent, the activator and the base is 1:1-3:1-3:1-3; preferably, the molar ratio of the compound 2, the condensing agent, the activator and the base is 1:1.5:1.5:1.5.

[0054] In some embodiments, the synthetic route of compound 1 is:

[0055]

[0056] In this embodiment, the preparation method of compound 1 by reacting compound A1 and compound A2 includes: dissolving compound A1 (aprepitant) in DMF, adding compound A2 (polyethylene glycol chain, PEG) and potassium carbonate, and reacting at room temperature for 48 hours. After the reaction, the solvent is rotary evaporated to obtain a mixture, and the next step is directly performed; methanol is added to the mixture, and oxalyl chloride is slowly added dropwise, and the mixture is reacted at room temperature for 5 hours to obtain compound 1.

[0057] In some embodiments, the synthetic route of compound 2 is:

[0058]

[0059] In this embodiment, the preparation method of compound B2 by reacting compound B1 includes: dissolving compound B1 in DMF, then dropwise adding trimethylsilyl azide (TMSN3), and then dropwise adding tetrabutylammonium fluoride (nBu4NF), and reacting at room temperature for 30 minutes to obtain compound B2.

[0060] In this embodiment, the preparation method of compound B3 by reacting compound B2 includes: dissolving compound B2 in dichloromethane (DCM), adding diisopropylethylamine (DIPEA) and acetic acid, and then slowly adding trimethylsilyl trifluoromethylsulfonate (TMSOTf) dropwise, reacting at room temperature for 2 hours, and then adding ethanol and trifluoroacetic acid to the system for 5 minutes to obtain compound B3.

[0061] In this embodiment, the preparation method of compound B4 by reacting compound B3 includes: dissolving compound B3 in dichloromethane, adding tert-butyl sarcosine hydrochloride, dicyclohexylcarboximide (DCC), 1-hydroxybenzotriazole (HOBt) and triethylamine (TEA), respectively, and reacting at room temperature overnight to obtain compound B4.

[0062] In this embodiment, the preparation method of compound B5 by reacting compound B4 includes: dissolving compound B4 in dichloromethane, adding p-nitrophenyl chloroformate, TEA and 4-dimethylaminopyridine (DMAP) respectively, reacting at room temperature overnight, and after the reaction, rotary evaporating the solvent to obtain a mixture, and directly proceeding to the next step; adding DMF, TEA, AH (i.e., a cytotoxic drug) and HOBt to the mixture, reacting at room temperature for 12 hours to obtain compound B5.

[0063] In this embodiment, the preparation method of compound 2 by reacting compound B5 comprises: dissolving compound B5 in dichloromethane, cooling to 0° C., adding TMSOTf dropwise and reacting for 2 h to obtain compound 2.

[0064] An embodiment of the present invention provides a pharmaceutical composition, comprising the small molecule conjugate drug described in any of the aforementioned embodiments and a pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer and / or diluent.

[0065] An embodiment of the present invention provides a use of the small molecule drug conjugate described in any of the aforementioned embodiments in the preparation of an anti-tumor drug.

[0066] The present invention will be further described below by means of specific examples.

[0067] Example 1

[0068] This embodiment provides a small molecule drug conjugate SMDC and a preparation method thereof, wherein the structural formula of SMDC is:

[0069] The preparation method comprises the following steps:

[0070] (1) Synthesis of Compound 1

[0071]

[0072] Compound A1 (aprepitant, 1.0 eq., 1 mmol, 534 mg) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and compound A2 (PEG, 1.1 eq., 1.1 mmol, 392 mg) and potassium carbonate (2 eq., 2 mmol, 276 mg) were added and reacted for 48 h. After completion of the reaction, the solvent was rotary evaporated to obtain a mixture, which was directly carried out to the next step. To the mixture was added 1 mL of methanol as solvent, and oxalyl chloride (3 eq., 3 mmol, 380 mg) was slowly added dropwise. The mixture was reacted at room temperature for 5 h. After completion of the reaction, it was purified by semi-preparative liquid chromatography to obtain compound 2 (279 mg, 38% yield) as a white solid.

[0073] The hydrogen spectrum, fluorine spectrum, carbon spectrum and high-resolution mass spectrum data of the compound 1 are as follows ( Figure 1 is the hydrogen spectrum of compound 1):

[0074] 1 H NMR (400MHz, Methanol-d4) δ7.91(s,3H),7.68(s,2H),7.44(t,J=8.4Hz,2H),5.29-5.24(m,1H),4.75(t,J=12.4Hz,1H),4.63(d,J=15.5Hz,1H),4.38(d ,J=15.5Hz,1H),4.20(d,J=10.3Hz,1H),4.02(d,J=3.7Hz,8H),3.88(t,J=5 .0Hz, 4H), 3.73 (d, J = 26.0Hz, 5H), 3.67-3.44 (m, 6H), 1.80 (d, J = 6.3Hz, 3H).

[0075] 19 F NMR (376MHz, MeOD) δ-64.18,-111.84.

[0076] 13C NMR(101MHz,MeOD)δ166.14,163.66,161.43,160.65,160.02,159.19,159.17,156.01,150. 31,145.97,140.37,133.31,133.05,132.72,132.38,132.05,128.28,127.85,127.81,127. 00,125.57,122.87,122.49,122.45,122.41,120.17,117.22,117.01,94.96,74.45,71.06,71.03,70.89,70.86,69.14,68.41,67.58,57.17,53.72,53.62,53.51,42.73,40.44,24.13.

[0077] HRMS(ESI):m / z Calcd for C 31 H 39 O6N5F7,[M+H] + :710.27831,found:710.27881.

[0078] (2) Synthesis of Compound B2

[0079]

[0080] Compound B1 (1.0 eq., 28 mmol, 5.5 g) was dissolved in 50 mL of DMF, and then trimethylsilyl azide (1.05 eq., 29.4 mmol, 3.3 g) was added dropwise, followed by tetrabutylammonium fluoride (1 mol / L in THF, 10 mol%, 2.8 mmol, 2.6 mL). The reaction was allowed to proceed at room temperature for 30 min. After the reaction, the reaction solution was poured into 500 mL of water and extracted three times with petroleum ether / diethyl ether = 10:1. The organic phase was washed with saturated sodium chloride solution and then dried over anhydrous sodium sulfate to obtain compound B2 (5.5 g, yield 95%) as a light yellow solid.

[0081] (3) Synthesis of Compound B3

[0082]

[0083] Compound B2 (1.0 eq., 11.4 mmol, 2.5 g) was dissolved in 60 mL of dichloromethane, and diisopropylethylamine (DIPEA, 3.0 eq., 34 mmol, 4.42 g) and acetic acid (1.2 eq., 13.7 mmol, 0.82 g) were added, and then trimethylsilyl trifluoromethanesulfonate (TMSOTf, 2.6 eq., 30 mmol, 6.67 g) was slowly added dropwise. The reaction was carried out at room temperature for 2 h. After the reaction, 50 mL of ethanol and 2.5 mL of trifluoroacetic acid were added to the system, and the reaction was quenched after 5 min. After the reaction, the solvent was rotary evaporated and separated and purified by column chromatography to obtain compound B3 (1.85 g, yield 58%), which appeared as a light yellow solid.

[0084] The hydrogen spectrum and fluorine spectrum data of the compound B3 are as follows:

[0085] 1 H NMR (400MHz, Chloroform-d) δ 5.52 (dd, J = 9.5, 4.1 Hz, 1H), 3.21 (dd, J = 16.9, 9.4 Hz, 1H), 2.82 (dd, J = 16.9, 4.0 Hz, 1H).

[0086] 19 F NMR (376MHz, Chloroform-d) δ-142.63--143.22(m),-150.98--151.66(m).

[0087] (4) Synthesis of Compound B4

[0088]

[0089] Compound B3 (1.0 eq., 6.6 mmol, 1.85 g) was dissolved in 30 mL of dichloromethane, and tert-butyl sarcosine hydrochloride (1.5 eq., 10 mmol, 1.8 g), dicyclohexylcarboximide (DCC, 1.5 eq., 10 mmol, 2.06 g), 1-hydroxybenzotriazole (HOBt, 1.5 eq., 10 mmol, 1.35 g) and triethylamine (TEA, 1.5 eq., 10 mmol, 1.01 g) were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction, the solvent was rotary evaporated, and the compound B4 was separated and purified by column chromatography (petroleum ether / ethyl acetate = 2:1) to obtain compound B4 (yield 78%) as a white solid.

[0090] The hydrogen spectrum, fluorine spectrum, carbon spectrum and high-resolution mass spectrum data of the compound B4 are as follows ( Figure 2 is the hydrogen spectrum of compound B4):

[0091] 1 H NMR (400MHz, Chloroform-d) δ5.51 (dt, J=9.6, 3.1Hz, 1H), 4.14-3.79 (m, 2H), 2.99 (d, J=26.5Hz, 4H), 2.64 (ddd, J=53.7, 16.4, 3.3Hz, 1H), 1.44 (s, 9H).

[0092] 19 F NMR (376MHz, Chloroform-d) δ -142.50 (ddd, J=52.7, 20.7, 9.7Hz), -152.03 (ddd, J=21.4, 10.0, 4.3Hz).

[0093] 13 C NMR (101MHz, Chloroform-d) δ171.78 (d, J = 10.0Hz), 167.86 (d, J = 50.9Hz), 146.40 (ddd, J = 12.2, 8. 2,4.2Hz),146.39,143.92(ddd,J=12.3,8.2,4.1Hz),143.92,141.90,142.40-141.36(m),140.16-1 38.12(m),139.41,119.52,120.00-119.08(m),115.84(td,J=15.3,6.8Hz),115.83,82.76(d,J=87 .8Hz), 62.41, 65.65-57.75 (m), 51.22 (d, J = 224.2Hz), 38.90-37.90 (m), 34.92, 28.04 (d, J = 9.3Hz).

[0094] HRMS(ESI):m / z Calcd for C 16 H 18 O4N4F4Na,[M+Na] + :429.11564,found:429.11600.

[0095] (5) Synthesis of Compound B5-1

[0096]

[0097] Compound B4 (1.0 eq., 1 mmol, 406 mg) was dissolved in 5 mL of dichloromethane, and p-nitrophenyl chloroformate (1.2 eq., 1.2 mmol, 242 mg), TEA (1.3 eq., 1.3 mmol, 131 mg) and 4-dimethylaminopyridine (DMAP, 10 mol%, 0.1 mmol, 12 mg) were added respectively. The mixture was reacted at room temperature overnight. After the reaction, the solvent was rotary evaporated to obtain a mixture, which was directly subjected to the next step. 6 mL of DMF was added to the mixture as a solvent, followed by TEA (1.5 eq., 1.5 mmol, 150 mg), monomethyl auristatin E (MMAE, 1.5 eq., 1.5 mmol, 1.08 g) and HOBt (1.5 eq., 1.5 mmol, 202 mg), and the mixture was reacted at room temperature for 12 h. After the reaction, the solvent was rotary evaporated, and the compound B5-1 (542 mg, 47%) was separated and purified by column chromatography (ethyl acetate / methanol = 100:1) to obtain a white solid.

[0098] The hydrogen spectrum, fluorine spectrum, carbon spectrum and high-resolution mass spectrum data of the compound B5-1 are as follows ( Figure 3 is the hydrogen spectrum of compound B5-1):

[0099] 1 H NMR(400MHz,Chloroform-d)δ7.31(s,5H),6.60(s,1H),6.32(s,1H),4.79(d,J=104.2Hz,3H),4.04(dt,J=82.1,42.5Hz,6H),3.50(s,1H),3.3 9(s,3H),3.28(s,5H),3.07(s,4H),2.96(d,J=18.4Hz,3H),2.81(d,J=3 5.1Hz,5H),2.53-1.56(m,9H),1.53-1.10(m,13H),1.12-0.41(m,22H).

[0100] 19 F NMR(376MHz,Chloroform-d)δ-141.91(m),-142.38(m),-151.77(m),-152.13(m).

[0101] 13C NMR (101MHz, CDCl3) δ174.53,173.84,172.72,170.50,169.69,169.47,169.34,169.16,168.72,167.93 ,167.62,162.55,155.76,154.58,146.28,143.89,141.70,141.23,139.19,127.99,127.21,126.29,11 9.95,115.69,114.00,82.93,81.94,78.43,75.74,65.46,60.91,60.00,57.95,53.75,51.51,49.79,47.79,44.89,37.59,36.49,33.35,31.42,30.73,29.28,28.00,25.70,24.97,19.24,18.50,15.90,10.87.

[0102] HRMS(ESI):m / z Calcd for C 56 H 83 O 12 N9F4Na,[M+Na] + :1172.59895,found:1172.59937.

[0103] (6) Synthesis of Compound 2-1

[0104]

[0105] Compound B5-1 (0.235 mmol, 270 mg) was dissolved in 2 mL of dichloromethane, cooled to 0°C, TMSOTf (8 eq., 2.8 mmol, 500 μL) was added dropwise, and the mixture was allowed to react for 2 h. After the reaction, saturated NaHCO3 solution was added to quench the reaction, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and pumped with an oil pump for 2 h to obtain compound 2-1. The product can be directly used in the next step.

[0106] (7) Synthesis of small molecule drug conjugates SMDC

[0107]

[0108] Compound 1 (1.2 eq., 0.19 mmol, 134 mg) prepared in step (1) and compound 2-1 (1.0 eq., 0.16 mmol, 183 mg) prepared in step (6) were dissolved in 2 mL of DMF, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.5 eq., 0.24 mmol, 46 mg), HOBt (1.5 eq., 0.24 mmol, 32 mg) and TEA (1.5 eq., 0.24 mmol, 24 mg) were added. The mixture was reacted at room temperature for 5 h, and the reaction progress was monitored by TLC (dichloromethane / methanol = 10:1). After the reaction, the small molecule conjugate drug SMDC (207 mg, 72% yield) was isolated and purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a white solid.

[0109] The hydrogen spectrum, fluorine spectrum, carbon spectrum and high-resolution mass spectrum data of the small molecule drug conjugate SMDC are as follows ( Figure 4 This is the hydrogen spectrum of the small molecule drug conjugate SMDC):

[0110] 1 H NMR(400MHz,Chloroform-d)δ7.62(s,1H),7.49-7.27(m,6H),7.21(m,3H),7.03(m, 2H),6.73(d,J=131.5Hz,1H),6.22(d,J=85.9Hz,1H),4.99-4.45(m,4H),4.17(m,8H ),3.95-3.71(m,3H),3.63(m,4H),3.36(m,21H),3.15(m,4H),3.02(m,2H),2.79(m, 7H),2.41(m,7H),2.27-1.58(m,8H),1.47(m,3H),1.23(m,5H),1.10-0.56(m,22H).

[0111] 19 F NMR (376MHz, CDCl3) δ-62.96, -113.59, -142.25, -151.82.

[0112] 13C NMR (101MHz, CDCl3) δ174.57,170.50,169.90,169.77,169.56,169.42,169.08,168.65,164.05,161.59,155.81,155.70,15 5.51,145.41,141.34,128.01,127.23,124.35,121.64,121.47,115.24,115.02,95.13,81.98,75.71,72.22,70.47,70.08,6 8.58,65.31,64.93,60.93,60.02,59.32,57.96,52.27,51.42,47.83,44.98,41.03,39.33,37.49,36.98,36.51,33.25,29.75,29.17,25.74,25.73,24.94,24.92,24.50,19.26,18.98,18.42,18.08,15.86,14.36,14.27,13.90,13.57,10.85,10.69.

[0113] HRMS(ESI):m / z Calcd for C 83 H 111 O 17 N 14 F 11 Na,[M+Na] + :1807.79682,found:1807.79797.

[0114] Example 2

[0115] This example explores the in vitro release experiment of the small molecule conjugated drug SMDC.

[0116] 2.1 mg of the small molecule conjugate drug SMDC prepared in Example 1 was weighed and dissolved in 12 mL of DMSO. 108 mL of deionized water was slowly poured into the solution to prepare a 10 μM SMDC aqueous solution containing 10% DMSO. The solution was ultrasonicated for 5 min, and then 1 mL of the sample was taken (retained as 0 Gy for the control group). The remaining solution was evenly divided into 6 glass bottles for X-ray irradiation. Argon was first bubbled for 20 min to remove oxygen. After deoxygenation, the bottles were sealed with a sealing film and then irradiated with 2 Gy, 4 Gy, 6 Gy, 12 Gy, 24 Gy, 48 Gy, and 60 Gy of X-rays using an RS2000-Pro-225 small animal X-ray irradiator. After irradiation, product formation was detected by HPLC with a sample volume of 50 μL, a flow rate of 1 mL / min, and a mobile phase of water / acetonitrile.

[0117] Figure 5 This is a relationship diagram between X-ray dose and the release rate of the cytotoxic drug MMAE. According to the figure, as the X-ray dose increases, the small molecule conjugate drug SMDC releases gradually more MMAE, and there is obvious release when a small amount of X-ray irradiation is applied. This shows that the small molecule conjugate drug SMDC of the present invention can successfully release MMAE under X-ray irradiation, and the activation of the small molecule conjugate drug SMDC does not require a high X-ray dose. When the X-ray dose reaches 60Gy, the cytotoxic drug MMAE reaches a release rate of about 70%.

[0118] Example 3

[0119] This example explores the cytotoxicity experiment of the small molecule conjugate drug SMDC.

[0120] The entire experimental operation in this embodiment was performed in a clean bench to ensure the aseptic operation process.

[0121] Cell recovery: Take out the cryovials of frozen cells from the -80℃ freezer. For the three cell lines, Hela, HCT116, and A549, immediately thaw them in a 37℃ water bath while gently shaking to accelerate the thawing process. When only a small piece of ice core remains in the cryovial, stop shaking and place it in the clean bench. This takes about 1-2 minutes. After thawing, transfer the cells to a 15mL centrifuge tube containing 2mL of DMEM high-glucose medium and centrifuge (1000rpm, 3min). Discard the supernatant, add DMEM high-glucose medium to resuspend the cells, and transfer them to a 10cm culture medium.

[0122] Cell passaging: Place the cell culture medium in a clean bench, aspirate the medium, and wash once with 4 mL of PBS. Then, add 2 mL of 0.25% trypsin (containing EDTA) and digest for 1-2 minutes. After digestion, add medium containing 10% fetal bovine serum to terminate digestion. Collect the cells into a 15 mL centrifuge tube and centrifuge. Remove the supernatant and resuspend the cells in DMEM medium. Finally, transfer Hela, HCT116, and A549 cells to fresh culture medium.

[0123] CCK-8 test cytotoxicity experiment:

[0124] Remove Hela, HCT116, and A549 cells that have reached the logarithmic growth phase from the constant temperature incubator, discard the original culture medium, and gently wash them once with PBS solution. Add 2 mL of 0.25% trypsin (containing EDTA) and incubate at 37 ° C for 2 minutes. After observing that the cell layer becomes loose, add 2 mL of DMEM culture medium containing 10% serum to terminate the digestion. Then transfer the cells to a 15 mL centrifuge tube, centrifuge (1000 rpm, 3 minutes) and discard the supernatant. Add an appropriate amount of DMEM complete culture medium and gently blow with a pipette about 30 times to evenly disperse the cells into a single cell suspension. Take an appropriate amount of cell suspension and count it using a cell counting plate to ensure that the final concentration is 1×10 6 pieces / mL.

[0125] 100 μL of the cell suspension per well (4000 cells / well) was inoculated into a 96-well plate and cultured in a cell culture incubator (37° C., 5% CO 2 , >90% humidity). Cell morphology was observed under a microscope.

[0126] After 24 hours of incubation, the cells grew to about 70% adherent to the wall. The original culture medium in the 96-well plate was discarded, and 100 μL of experimental samples were added to the corresponding wells of the 96-well plate. Experimental group 1 (labeled as SMDC+60Gy group) was treated with 60Gy The concentrations of X-ray irradiation were 1000nmol / L, 250nmol / L, 62.5nmol / L, 15.63nmol / L, 3.91nmol / L, 0.98nmol / L, 0.24nmol / L, 0.06nmol / L, 0.02nmol / L of small molecule conjugate drug SMDC. Experimental group 2 (labeled as SMDC group) was treated with small molecule conjugate drug SMDC at concentrations of 1000nmol / L, 250nmol / L, 62.5nmol / L, 15.63nmol / L, 3.91nmol / L, 0.98nmol / L, 0.24nmol / L, 0.06nmol / L, 0.02nmol / L. Experimental group 3 (labeled as MMAE group) was treated with small molecule conjugate drug SMDC at concentrations of 1000nmol / L, 250nmol / L, 62.5nmol / L, 15.63nmol / L, 3.91nmol / L, 0.98nmol / L, 0.24nmol / L, 0.06nmol / L, 0.02nmol / L. The cells were cultured in a 96-well plate in a cell culture incubator for 72 hours, with three replicates per group.

[0127] After 72 hours of culture, the 96-well plate was removed and the cell morphology was observed under a microscope. The culture medium was then removed and 100 μL of CCK-8 (1 mg / mL) was added to each well. The plates were placed in a carbon dioxide incubator and cultured for 1 hour. The absorbance (OD) at a wavelength of 450 nm was measured on a microplate reader. When the reading of the blank control group was between 0.8 and 1.2, the proliferation inhibition rate of Hela, HCT116, and A549 tumor cell growth was calculated.

[0128] Figure 6 This is the CCK-8 cell experiment results of Hela cells in SMDC+60Gy group, SMDC group and MMAE group. Figure 7 This is the CCK-8 cell experiment results of HCT116 cells in the SMDC+60Gy group, SMDC group and MMAE group. Figure 8The results of CCK-8 cell experiments on A549 cells in SMDC+60Gy group, SMDC group and MMAE group are shown. Figure 6-Figure 8 It can be seen that the cytotoxicity of the small molecule conjugate drug SMDC is significantly weaker than that of the cytotoxic drug MMAE. When the cells are incubated with 60Gy of X-ray irradiation, the small molecule conjugate drug SMDC shows stronger cytotoxicity in the three cell lines of Hela, HCT116 and A549.

[0129] Figure 9 This is the result of the CCK-8 cell experiment on Hela, HCT116, and A549 cells in the aprepitant group. The results show that the drug aprepitant has only weak cytotoxicity to the three groups of cells at a concentration of 100,000 nM.

[0130] In summary, the small molecule conjugate drug provided by the present invention can release a cytotoxic drug (such as MMAE) when irradiated by X-rays in an aqueous solution; and, cytotoxicity experiments have shown that the small molecule conjugate drug, as a prodrug of a cytotoxic drug, has lower cytotoxicity, and after irradiation with X-rays, the cytotoxicity is enhanced. Therefore, the small molecule conjugate drug provided by the present invention can better achieve drug targeting, and can achieve drug activation in a spatiotemporally controllable manner, can be well used in tumor treatment, and exhibits stronger cytotoxicity to tumor cells after irradiation with X-rays.

[0131] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A small molecule conjugated drug, characterized in that: The structural formula of the small molecule drug conjugate is shown in Formula 1: ， Wherein, A in Formula 1 is a cytotoxic drug.

2. The small molecule drug conjugate according to claim 1, characterized in that: The A is one of monomethyl auristatin E, doxorubicin, camptothecin, mitomycin C, methotrexate, paclitaxel, and vinblastine.

3. The small molecule drug conjugate according to claim 1, characterized in that: The structural formula of the small molecule drug conjugate is: 。 4. A method for preparing a small molecule drug conjugate according to claim 1, characterized in that: The preparation method comprises the following steps: subjecting compound 1 and compound 2 to an amidation reaction to obtain a small molecule conjugated drug as shown in formula 1; The synthetic route of the small molecule conjugated drug is: 。 5. The method for preparing a small molecule drug conjugate according to claim 4, characterized in that: The step of subjecting Compound 1 and Compound 2 to an amidation reaction to obtain the small molecule conjugate drug represented by Formula 1 specifically comprises: Compound 1 and compound 2 are dissolved in an organic solvent, and then a condensing agent, an activating agent and a base are added, and the mixture is reacted at 25° C. for 5 to 10 hours to obtain a small molecule conjugate drug represented by formula 1.

6. The method for preparing a small molecule drug conjugate according to claim 5, characterized in that: The condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the activating agent is one of 1-hydroxybenzotriazole, N-hydroxy-7-azabenzotriazole, and 4-dimethylaminopyridine; the base is one of triethylamine, N,N-diisopropylethylamine, and pyridine; and the organic solvent is one of N,N-dimethylformamide, dichloromethane, dimethyl sulfoxide, toluene, and ethyl acetate.

7. The method for preparing a small molecule drug conjugate according to claim 5, characterized in that: The molar ratio of compound 1 to compound 2 is 1 to 3:

1.

8. The method for preparing a small molecule drug conjugate according to claim 5, characterized in that: The molar ratio of the compound 2, the condensing agent, the activating agent and the base is 1:1-3:1-3:1-3.

9. A pharmaceutical composition, characterized in that The invention comprises the small molecule conjugate drug according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, adjuvant, stabilizer and / or diluent.

10. Use of the small molecule drug conjugate according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

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