Small-molecule coupling medicine as well as preparation method and application thereof

Abstract

The invention relates to the field of medicinal chemistry, in particular to a small-molecule coupling medicine as well as a preparation method and application thereof. The structural formula of the micromolecular coupling drug is shown as a formula 1: # imgabs0, wherein A in the formula 1 is a cytotoxic drug. According to the small-molecule coupled drug provided by the invention, two parts of structures are connected to a cytotoxic drug, one part is a tetrafluoro azidophenyl connexon which can be activated by X-rays to release the cytotoxic drug, and the other part is a small-molecule inhibitor aprepitant with targeting NK1R, so that drug targeting can be better realized; meanwhile, medicine activation can be realized in a time-space controllable manner, the compound can be well applied to tumor treatment, and the compound shows higher cytotoxicity to tumor cells after being irradiated by X rays.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical 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, do not preferentially target tumor sites, and often lead to toxicity to normal cells. In the past three decades, the discovery and production of anticancer drugs have undergone tremendous changes, from traditional chemotherapy drugs to targeted drugs, which have greatly improved the accuracy 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, target units, and imaging agents. This method can accurately target chemotherapy drugs to cancer cells and avoid cytotoxicity to normal cells. The various parts of small molecule drug conjugates are generally connected 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 release active drugs after reaching the vicinity of the tumor 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 accounts for 70-80% of tissue weight), resulting in water radiolysis, a process that produces two main reactive species: hydrated electrons (e - aq ) and hydroxyl radical (·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 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 can be used as a means of prodrug activation and have been extensively studied. Moreover, compared with other external stimuli, ionizing radiation has high tissue penetration, spatiotemporal resolution, and clinical relevance, and the X-ray radiotherapy-induced prodrug activation strategy shows 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 realize 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 a preparation method and application thereof. 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, thereby further improving the targeting of cytotoxic drugs, and showing good application prospects in the treatment of tumors.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect of the present invention, a small molecule drug conjugate is provided, wherein the structural formula of the small molecule drug conjugate is as 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 conjugated drug, wherein the preparation method comprises the steps of: subjecting compound 1 and compound 2 to an amidation reaction to obtain a small molecule conjugated drug as shown in 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 conjugated drug of Formula 1 specifically comprises:

[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 as shown in formula 1.

[0019] Optionally, the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, the activator is one of 1-hydroxybenzotriazole, N-hydroxy-7-azabenzotriazole, 4-dimethylaminopyridine, the base is one of triethylamine, N,N-diisopropylethylamine, 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 conjugate drug described in the present invention in the preparation of anti-tumor drugs.

[0024] Beneficial effects: The present invention has developed a small molecule conjugated drug, which connects two structures to the cytotoxic drug, one is a tetrafluoroazidophenyl linker that can be activated by X-rays to release the cytotoxic drug, and the other is a small molecule inhibitor aprepitant that targets NK1R, wherein 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 cytotoxic drugs. The small molecule conjugated drug provided by the present invention can better achieve drug targeting, and can achieve drug activation in a controllable manner in time and space, can be well used in 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 conjugated drug 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 SMDC+60Gy group, SMDC group and MMAE group.

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

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

[0033] Fig. 9 This is a graph showing 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 conjugated drug and a preparation method and application thereof. To make the purpose, technical scheme 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 explain the present invention and are not intended to limit the present invention.

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

[0036]

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

[0038] The embodiment of the present invention provides a small molecule conjugated drug, by connecting two structures on the cytotoxic drug (A), one is a tetrafluoroazidophenyl linker that can be activated by X-rays to release the cytotoxic drug, and the other is a small molecule inhibitor aprepitant targeting NK1R, wherein the tetrafluoroazidophenyl linker can be activated by X-rays to release the cytotoxic drug, and the small molecule inhibitor aprepitant targeting NK1R can actively target tumor cells, further improving the targeting of the cytotoxic drug. The small molecule conjugated drug provided by the embodiment of the present invention can better achieve drug targeting, and at the same time, achieve drug activation in a controllable manner in time and space, 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 conjugate provided by the embodiment of the present invention are at least that, first, the X-ray activated prodrug strategy can produce a certain spatiotemporally controllable selective drug release capability by irradiating X-rays at a fixed point; second, a small molecule inhibitor aprepitant that actively targets NK1R is added, thereby further improving the targeting of the drug.

[0040] The embodiment of the present invention selects aprepitant, a small molecule inhibitor targeting NK1R (Neurokinin 1 receptor, neurokinin receptor 1) as the targeting molecule. The reason for selecting NK1R as the target is that it is a receptor membrane protein overexpressed on a variety of tumor cells. At the same time, many studies have proved that NK1R inhibitors produce anti-cancer effects by affecting downstream pathways.

[0041] The cytotoxic drug in the embodiment 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] The embodiment of the present invention provides a method for preparing the small molecule conjugate drug described in the above 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 shown in 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 of 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 as shown in 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 1-3:1; 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 activating agent and the base is 1:1-3:1-3:1-3; preferably, the molar ratio of the compound 2, the condensing agent, the activating agent 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 obtained 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 comprises: dissolving compound B1 in DMF, and then dropping trimethylsilyl azide (TMS 3 ), and then tetrabutylammonium fluoride (nBu 4 NF), and react at room temperature for 30 min to obtain compound B2.

[0060] In this embodiment, the preparation method of compound B3 obtained 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 obtained 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, which comprises the small molecule conjugate drug described in any of the above 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 conjugate drug described in any of the above 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 thereof 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 to react for 48 h. After the reaction, the solvent was rotary evaporated to obtain a mixture, which was directly carried out to the next step. 1 mL of methanol was added to the mixture as a solvent, and oxalyl chloride (3 eq, 3 mmol, 380 mg) was slowly added dropwise, and reacted at room temperature for 5 h. After the reaction, semi-preparative liquid chromatography was used for purification to obtain compound 2 (279 mg, yield 38%), which appeared 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 (400 MHz, Methanol-d 4 )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,1 H), 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 O 6 N 5 F 7 ,[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 trimethylsilane (1.05 eq., 29.4 mmol, 3.3 g) was added dropwise, and then tetrabutylammonium fluoride (1 mol / L in THF, 10 mol%, 2.8 mmol, 2.6 mL) was added dropwise. The mixture was reacted 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 / ether = 10:1. The organic phase was washed with a saturated sodium chloride solution and then dried over anhydrous sodium sulfate to obtain compound B2 (5.5 g, yield 95%), which appeared 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 mixture was reacted 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. The reaction was quenched after 5 min of reaction. After the reaction, the solvent was rotary evaporated, and compound B3 (1.85 g, yield 58%) was obtained by column chromatography separation and purification 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), dicyclohexylcarbocyanine (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 respectively. The mixture was reacted at room temperature overnight. After the reaction, the solvent was rotary evaporated. 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 O 4 N 4 F 4 Na,[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 evaporated by rotary evaporation to obtain a mixture, which was directly carried out 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] 13 C NMR (101 MHz, CDCl 3)δ174.53,173.84,172.72,170.50,169.69,169.47,169.34,169.16,168.72,167.93,167.62,162. 55,155.76,154.58,146.28,143.89,141.70,141.23,139.19,127.99,127.21,126.29,119.95,115 .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 N 9 F 4 Na,[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 reacted for 2 h. After the reaction, saturated NaHCO 3 The solution was used to quench the reaction, and then extracted with dichloromethane, dried over anhydrous sodium sulfate, and then pumped with an oil pump for 2 hours to obtain compound 2-1, which can be directly used in the next step.

[0106] (7) Synthesis of small molecule drug conjugate 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, and 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 conjugated drug SMDC (207 mg, yield 72%) was separated and purified by column chromatography (dichloromethane / methanol = 20: 1), which appeared as 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 (376 MHz, CDCl 3 )δ-62.96,-113.59,-142.25,-151.82.

[0112] 13 C NMR (101 MHz, CDCl 3)δ174.57,170.50,169.90,169.77,169.56,169.42,169.08,168.65,164.05,161.59,155.81,155.70,155.51,145.41, 141.34,128.01,127.23,124.35,121.64,121.47,115.24,115.02,95.13,81.98,75.71,72.22,70.47,70.08,68.58,65. 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 drug conjugate SMDC.

[0116] Weigh 2.1 mg of the small molecule conjugate drug SMDC prepared in Example 1 and dissolve it in 12 mL of DMSO, slowly pour 108 mL of deionized water, and prepare a SMDC aqueous solution with a concentration of 10 uM and 10% DMSO. Ultrasonicate it for 5 min, then take 1 mL of sample (retain the sample as 0 Gy, as a control group), and evenly divide the remaining solution into 6 glass bottles for X-ray irradiation, first bubble with argon for 20 min to remove oxygen, seal it with a sealing film after deoxygenation, and then use RS2000-Pro-225 small animal X-ray irradiator to irradiate 2 Gy, 4 Gy, 6 Gy, 12 Gy, 24 Gy, 48 Gy, 60 Gy of X-rays respectively. After irradiation, HPLC is used to detect product generation, wherein the injection volume is set to 50 μL, the flow rate is 1 mL / min, and the mobile phase is water / acetonitrile.

[0117] Figure 5It is a relationship diagram between X-ray dose and release rate of cytotoxic drug MMAE. According to the figure, as the X-ray dose increases, the MMAE released by the small molecule conjugate drug SMDC gradually increases, and there is obvious release when a trace 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 release rate of the cytotoxic drug MMAE reaches about 70%.

[0118] Example 3

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

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

[0121] Cell recovery: Take out the cryopreservation tubes of frozen cells from the -80℃ freezer, which are the three cell lines of Hela, HCT116, and A549, and immediately put them in a 37℃ water bath to thaw, and gently shake them to accelerate the thawing process. When only a small piece of ice core remains in the cryopreservation tube, stop shaking and place it in the clean bench, which takes about 1-2 minutes. After thawing, transfer the cells to a 15mL centrifuge tube containing 2mL DMEM high-glucose medium, put it into a centrifuge for centrifugation (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 on a clean bench, remove the culture medium, and add 4 mL PBS to wash once. Then add 2 mL of 0.25% trypsin (containing EDTA) to digest for 1-2 minutes. After digestion, add culture medium containing 10% fetal bovine serum to terminate digestion, collect cells into a 15 mL centrifuge tube for centrifugation, remove the supernatant, add DMEM culture medium to resuspend the cells, and finally transfer Hela, HCT116, and A549 cells to new culture medium respectively.

[0123] CCK-8 test cytotoxicity experiment:

[0124] Take out 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 2mL, 0.25% trypsin (containing EDTA), and incubate at 37°C for 2min. After observing that the cell layer becomes loose, add 2mL of DMEM culture medium containing 10% serum to terminate digestion. Then transfer the cells to a 15mL centrifuge tube, centrifuge (1000rpm, 3min) 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] The cell suspension was inoculated into 96-well plates at 100 μL per well (4000 cells / well) and incubated in a cell culture incubator (37°C, 5% CO 2 , >90% humidity) and the 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 added 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 added with 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 of small molecule conjugate drug SMDC. Experimental group 3 (labeled as MMAE group) was added with 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 of small molecule conjugate drug SMDC. The cytotoxic drug MMAE was added at 100000 nmol / L, 250 nmol / L, 62.5 nmol / L, 15.63 nmol / L, 3.91 nmol / L, 0.98 nmol / L, 0.24 nmol / L, 0.06 nmol / L, and 0.02 nmol / L, and the drug aprepitant (Aprepitant) was added at a concentration of 100000 nmol / L, 25000 nmol / L, 6250 nmol / L, 1562.5 nmol / L, 390.63 nmol / L, 97.66 nmol / L, 24.41 nmol / L, 6.10 nmol / L, and 1.53 nmol / L, and the blank control group was 10% fetal bovine serum culture medium. The obtained 96-well plate was placed in a cell culture incubator and cultured for 72 hours, and 3 replicates were set for each group.

[0127] After 72 hours of culture, the 96-well plate was taken out, the cell morphology was observed under a microscope, and then the culture medium was removed. 100 μL of CCK-8 (concentration of 1 mg / mL) was added to each well, and the plates were placed in a carbon dioxide incubator for 1 hour. The absorbance value (OD) at a wavelength of 450 nm was measured on an ELISA 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 result 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 8This is the CCK-8 cell experiment results of A549 cells in the SMDC+60Gy group, SMDC group and MMAE group. 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. However, when the cells were incubated with 60Gy of X-ray irradiation, the small molecule conjugate drug SMDC showed stronger cytotoxicity in the three cell lines of Hela, HCT116, and A549.

[0129] Fig. 9 This is the result of CCK-8 cell experiment on Hela, HCT116 and A549 cells in aprepitant group. The results show that the drug aprepitant has only weak cytotoxicity to the three groups of cells at a concentration of 100000nM.

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

[0131] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, 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 conjugate drug 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 conjugate drug 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 steps of: 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 a 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 conjugated drug as shown in 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 the compound 1 to the 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 composition comprises the small molecule conjugated drug as described in any one of claims 1 to 3 and a pharmaceutically acceptable excipient, carrier, adjuvant, stabilizer and / or diluent.

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

Images