A PROTAC compound derived from triptolide, preparation method and application thereof

Abstract

The present invention provides a PROTAC compound derived from triptolide, a preparation method and an application, relating to the technical field of PROTAC compounds. The structure of the PROTAC compound derived from triptolide is shown in Formula I; in Formula I, X is -(CH2) m - or -CH2CH2(OCH2CH2) n -, m is any integer from 2 to 8, and n is any integer from 1 to 3. Through activity tests, the PROTAC compound derived from triptolide has significant anti-proliferation effects on various cancer cells such as colorectal cancer, lung cancer, breast cancer, etc., and its toxicity has decreased significantly. It is expected to prepare new anti-cancer drugs and has good development prospects. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of PROTAC compounds, and in particular to a PROTAC compound derived from triptolide, a preparation method and an application thereof. Background Art

[0002] Cancer is the second leading cause of death in humans and poses a serious threat to human health. In 2020, there were 19.3 million new cases of cancer and 10 million cancer deaths worldwide. The current situation of cancer in my country is equally worrying. According to the latest national cancer statistics released by the National Cancer Center in January 2019, there were approximately 3.929 million cancer cases and 2.338 million deaths in my country in 2015, and the annual medical expenses caused by cancer exceeded 220 billion. Cancer has become one of the major public health issues threatening human health. Therefore, finding new anti-cancer targets and developing new anti-cancer drugs has become a top priority.

[0003] Protein degradation targeting chimeras (PROTAC) are one of the most popular drug development technologies. PROTAC technology is different from the mechanism of action of traditional small molecule inhibitors that inhibit the function of target proteins by binding to target proteins. Instead, PROTAC directly degrades the target protein to achieve the effect of disease treatment. PROTAC is a bifunctional molecular compound composed of three parts. One end is a ligand that binds to the target protein, and the other end is a ligand that binds to the ubiquitin ligase E3. The two are connected by a chain linker to form a ternary complex. Compared with traditional small molecule inhibitors, PROTAC technology has many advantages: (1) It does not need to bind tightly to the target protein, and only needs to be "labeled" to induce the degradation of the target protein; (2) It can act on undruggable targets; (3) Only a catalytic amount of drug is required; (4) Overcoming drug resistance; (5) High selectivity, etc. It is of great value to develop triptolide derivatives based on the PROTAC strategy.

[0004] Triptolide is a natural epoxyditerpene lactone compound, one of the main active ingredients of the Celastraceae plant Tripterygium wilfordii, and is a hot natural product that has attracted widespread attention. Triptolide has multiple pharmacological activities, including anti-tumor, anti-inflammatory, antioxidant, and antiviral effects. As a broad-spectrum anticancer natural drug, triptolide has good anticancer effects on colorectal cancer, breast cancer, lung cancer, etc. However, triptolide has serious side effects, especially strong toxicity, and its clinical application is strictly restricted. Therefore, the development of PROTAC compounds derived from triptolide is of great research value. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a PROTAC compound derived from triptolide, a preparation method and an application thereof. The PROTAC compound derived from triptolide can effectively inhibit the proliferation of various cancer cells while effectively reducing the toxicity and side effects of triptolide, and can be used to prepare a new anticancer drug.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A PROTAC compound derived from triptolide, the structure of which is shown in Formula I:

[0008] (I).

[0009] Furthermore, in the formula I, X is -(CH 2 ) m -or-CH 2 CH 2 (OCH 2 CH 2 ) n -, m is any integer from 2 to 8, and n is any integer from 1 to 3.

[0010] A method for preparing a PROTAC compound derived from triptolide, comprising the following steps: preparing an intermediate compound 2, preparing an intermediate compound 3, and synthesizing.

[0011] The method for preparing the intermediate compound 2 is that in a solvent environment, 4-fluorothalidomide (compound 1) and an amino derivative undergo a nucleophilic substitution reaction under the action of DIPEA to obtain the intermediate compound 2.

[0012] The structure of the amino derivative is shown in Formula II:

[0013] (II);

[0014] In the formula II, X is -(CH 2 ) m -or-CH 2 CH 2 (OCH 2 CH 2 ) n -, m is any integer from 2 to 8, and n is any integer from 1 to 3.

[0015] The method for preparing the intermediate compound 3 is as follows: in a solvent environment, the intermediate compound 2 is treated with trifluoroacetic acid to remove the protecting group on the carboxyl group, thereby obtaining the intermediate compound 3.

[0016] The synthesis method is that in a solvent environment, triptolide and the intermediate compound 3 are reacted with condensing agents EDCI and DMAP to form an ester, thereby obtaining a PROTAC compound derived from triptolide.

[0017] The PROTAC compound derived from triptolide is one of the following:

[0018] .

[0019] The preparation method of the PROTAC compound derived from triptolide, the synthesis route is as follows:

[0020] ;

[0021] Wherein the group X is -(CH 2 ) m -or-CH 2 CH 2 (OCH 2 CH 2 ) n -, m is any integer from 2 to 8, and n is any integer from 1 to 3.

[0022] Preferably, in the preparation of the intermediate compound 2, the solvent used is N,N-dimethylformamide;

[0023] In the preparation of the intermediate compound 3, the solvent used is dichloromethane;

[0024] In the synthesis, the solvent used is dichloromethane.

[0025] Preferably, in the preparation of the intermediate compound 2, the molar amount of the amino derivative is at least 1.5 times that of 4-fluorothalidomide.

[0026] In the synthesis, the molar amount of the intermediate compound 3 is at least 1.4 times that of triptolide.

[0027] A pharmaceutical composition, using the aforementioned PROTAC compound derived from triptolide or a pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and a pharmaceutically acceptable carrier to prepare a pharmaceutically acceptable dosage form.

[0028] A use of the aforementioned triptolide-derived PROTAC compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumor diseases or inflammatory diseases.

[0029] The tumor diseases include: colorectal cancer, lung cancer, and breast cancer.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) The PROTAC compound derived from triptolide of the present invention can effectively reduce the toxicity and side effects of triptolide while effectively inhibiting the proliferation of various tumor cells. It has broad prospects for drug development and is expected to be prepared into a new anti-tumor drug.

[0032] 2) The inhibitory IC of the PROTAC compound 4e derived from triptolide on the human colorectal cancer cell line SW620 was 50 The value can reach 0.0006μM. Compared with 0.003μM that can be achieved by triptolide, the anti-tumor activity of PROTAC compound 4e derived from triptolide is enhanced by 5 times.

[0033] 3) After testing, the PROTAC compounds derived from triptolide of the present invention have significantly lower toxicity to the normal rat glomerular mesangial cell line HBZY-1 than triptolide, effectively avoiding the side effects and toxicity of triptolide.

[0034] 4) The preparation method of the PROTAC compound derived from triptolide of the present invention is simple and efficient, has mild reaction conditions, high product yield and purity, simple post-treatment, and a green and environmentally friendly preparation process, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound (compound 4a) derived from triptolide in Example 1.

[0036] Figure 2 This is the carbon NMR spectrum of the PROTAC compound (compound 4a) derived from triptolide in Example 1.

[0037] Figure 3 This is the nuclear magnetic resonance hydrogen spectrum of the PROTAC compound (compound 4b) derived from triptolide in Example 2.

[0038] Figure 4 This is the carbon NMR spectrum of the PROTAC compound (compound 4b) derived from triptolide in Example 2.

[0039] Figure 5 This is the nuclear magnetic resonance hydrogen spectrum of the PROTAC compound (compound 4c) derived from triptolide in Example 3.

[0040] Figure 6 This is the carbon NMR spectrum of the PROTAC compound (compound 4c) derived from triptolide in Example 3.

[0041] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound (compound 4d) derived from triptolide in Example 4.

[0042] Figure 8 This is the carbon NMR spectrum of the PROTAC compound (compound 4d) derived from triptolide in Example 4.

[0043] Fig. 9 This is the nuclear magnetic resonance hydrogen spectrum of the PROTAC compound (compound 4e) derived from triptolide in Example 5.

[0044] Fig.10 This is the carbon NMR spectrum of the PROTAC compound (compound 4e) derived from triptolide in Example 5.

[0045] Fig.11 This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound (compound 4f) derived from triptolide in Example 6.

[0046] Fig.12 This is the carbon NMR spectrum of the PROTAC compound (compound 4f) derived from triptolide in Example 6.

[0047] Fig.13 This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound derived from triptolide in Example 7 (compound 4g).

[0048] Fig.14 This is the carbon NMR spectrum of the PROTAC compound derived from triptolide (compound 4g) in Example 7.

[0049] Fig.15 This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound (compound 4h) derived from triptolide in Example 8.

[0050] Fig.16 This is the carbon NMR spectrum of the PROTAC compound (compound 4h) derived from triptolide in Example 8.

[0051] Fig.17 This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound derived from triptolide (compound 4i) in Example 9.

[0052] Fig.18 This is the carbon NMR spectrum of the PROTAC compound (compound 4i) derived from triptolide in Example 9.

[0053] Fig.19This is the hydrogen nuclear magnetic resonance spectrum of the PROTAC compound derived from triptolide (compound 4j) in Example 10.

[0054] Fig. 20 This is the carbon NMR spectrum of the PROTAC compound (compound 4j) derived from triptolide in Example 10. DETAILED DESCRIPTION

[0055] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0056] Example 1

[0057] This example provides a method for preparing a PROTAC compound (compound 4a) derived from triptolide, and its synthetic route is as follows:

[0058]

[0059] The specific preparation method is:

[0060] 1) Preparation of intermediate compound 2

[0061] 4-Fluorothalidamide (0.50 g, 1.81 mmol), tert-butyl 3-aminopropionate (0.41 mL, 2.73 mmol), N,N-diisopropylethylamine DIPEA (1.50 mL, 9.10 mmol) were added to a 100 mL round-bottom flask, and 40 mL of N,N-dimethylformamide was added. The mixture was heated to 100 °C and kept for 5 h. After TLC showed that the reaction was completed, ethyl acetate (20 mL × 3) was used for extraction to obtain an extract. The extract was washed with saturated brine (20 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, and the dried organic phase was freed from the solvent by a rotary evaporator to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 2:1) to obtain the intermediate compound 2-1, 0.35 g, with a yield of 48%.

[0062] 2) Preparation of intermediate compound 3

[0063] Compound 2-1 (0.3 g, 0.72 mmol) was added to a 100 mL round-bottom flask, and 4 mL of trifluoroacetic acid and 8 mL of dichloromethane were added. The reaction was carried out at room temperature for 0.5 h. After TLC showed that the reaction was completed, the organic phase was dried to obtain a crude product. The crude product was purified by column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain the intermediate compound 3-1, 0.18 g, with a yield of 70%.

[0064] 3) Synthesis

[0065] In a 100 mL round-bottom flask, compound triptolide (0.1 g, 0.28 mmol), compound 3-1 (0.13 g, 0.39 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI (0.22 g, 1.12 mmol), 4-dimethylaminopyridine DMAP (0.10 g, 0.82 mmol) were added, and 15 mL of dichloromethane was added. The mixture was reacted at room temperature for 24 h. After TLC showed that the reaction was completed, ethyl acetate (20 mL × 3) was used for extraction to obtain an extract; the extract was washed with saturated brine (20 mL × 3), and the organic phases were combined; the organic phase was dried with anhydrous sodium sulfate, and the dried organic phase was freed from the solvent by a rotary evaporator to obtain a crude product; the crude product was purified by column chromatography (eluent: dichloromethane: methanol = 60:1) to obtain compound 4a, a yellow powder, 0.15 g, with a yield of 82%.

[0066] NMR data of compound 4a:

[0067] 1 H-NMR(400MHz,DMSO)δ11.10(s,1H),7.61(t, J =7.6Hz,1H),7.19(d, J =8.4Hz,1H),7.06(d, J =6.8Hz,1H),6.71(t, J =4.8Hz,1H),5.04(dd, J =4.8Hz, J =12.4Hz,1H),5.00(s,1H),4.72-4.90(m,2H),3.95(s,1H),3.55-3.70(m,4H),2. 83-2.95(m,1H),2.70-2.79(m,2H),2.54-2.65(m,2H),2.17-2.28(m,1H),2.09(d, J =16.8Hz,1H),1.88-2.05(m,2H),1.66-1.85(m,2H),1.20-1.37(m,3H),0.88(s,3H),0.79(d, J =6.4Hz,3H),0.65(d, J =6.4Hz,3H);

[0068] 13C-NMR(101MHz,DMSO)δ173.59,173.29,171.27,170.48,169.15,167.74, 162.64,146.33,136.80,132.66,123.57,117.67,111.27,110.01,71.58, 70.71,63.77,63.04,61.55,59.77,55.50,55.39,48.99,38.45,35.48,31 .44,29.60,27.76,27.70,22.77,22.65,17.78,17.04,16.79(2C),14.20.

[0069] This example also provides a PROTAC compound (compound 4a) derived from triptolide prepared by the aforementioned method and its application.

[0070] Example 2

[0071] This example provides a method for preparing a PROTAC compound (compound 4b) derived from triptolide, and its synthetic route is as follows:

[0072]

[0073] The preparation method of compound 4b in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with tert-butyl 4-aminobutyrate (0.46 mL, 2.73 mmol) to obtain intermediate compound 2-2; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-2 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-2; in the synthesis step, intermediate compound 3-2 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4b, yellow powder, 0.16 g, yield 82%.

[0074] NMR data of compound 4b:

[0075] 1 H-NMR(400MHz,DMSO)δ11.11(s,1H),7.59(t, J =7.6Hz,1H),7.17(d, J =8.4Hz,1H),7.04(d, J =6.8Hz,1H),6.66(s,1H),5.05(dd, J =4.8Hz, J=12.4Hz,1H),5.02(s,1H),4.72-4.90(m,2H),3.97(s,1H),3.70(s,1H),3.61(d, J =4.8Hz,1H),3.34-3.45(m,2H),2.80-2.95(m,1H),2.52-2.68(m,3H),2.38-2.50(m,2H),2.18-2.28(m ,1H),2.06-2.16(m,1H),1.75-1.95(m,4H),1.48-1.60(m,1H),1.23-1.40(m,3H),0.89(s,3H),0.87(d, J =6.8Hz,1H),0.75(d, J =6.8Hz,1H);

[0076] 13 C-NMR(101MHz,DMSO)δ173.61,173.32,172.40,170.57,169.28,167.77,16 2.66,146.77,136.75,132.71,123.58,117.62,110.98,109.66,71.32,70. 71,63.97,63.79,63.08,61.57,55.48,54.91,48.99,41.43,40.23,35.49, 31.45,30.63,28.02,24.71,22.78,22.62,19.08,17.92,16.99(2C),14.02.

[0077] This example also provides a PROTAC compound (compound 4b) derived from triptolide prepared by the aforementioned method and its application.

[0078] Example 3

[0079] This example provides a method for preparing a PROTAC compound (compound 4c) derived from triptolide, and its synthetic route is as follows:

[0080]

[0081] The preparation method of compound 4c in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with tert-butyl 5-aminovalerate (0.50 mL, 2.73 mmol) to obtain intermediate compound 2-3; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-3 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-3; in the synthesis step, intermediate compound 3-3 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4c, a yellow powder, 0.17 g, with a yield of 84%.

[0082] NMR data of compound 4c:

[0083] 1 H-NMR (400MHz, DMSO) δ11.10(s,1H),7.58(t,J=7.6Hz,1H),7.12(d,J=8.4Hz,1H),7.03(d,J=6.8Hz,1H),6.57(s,1H ),5.04(dd,J=4.8Hz,J=12.8Hz,1H),4.99(s,1H),4.72-4.90(m,2H),3.94(s,1H),3.68(s,1H),3.58(d,J=4.8Hz,1H) ,3.27-3.38(m,2H),2.80-2.95(m,1H),2.55-2.66(m,2H),2.35-2.47(m,2H),2.17-2.27(m,1H),2.06-2.16(m,1H),1 .86-2.05(m,2H),1.74-1.80(m,2H),1.58-1.72(m,4H),1.20-1.37(m,3H),0.83-0.93(m,6H),0.75(d,J=6.4Hz,3H);

[0084] 13 C-NMR(101MHz,DMSO)δ173.59,173.29,172.57,170.55,169.36,167.77,162 .68,146.86,136.72,132.66,123.57,117.69,110.87,109.51,71.14,70.70 ,63.76,63.09,61.43,59.86,55.38(2C),48.98,41.95,40.18,35.49,33.75 ,31.44,29.56,28.39,28.03,22.80,22.63,17.92,17.03,16.98(2C),14.14.

[0085] This example also provides a PROTAC compound (compound 4c) derived from triptolide prepared by the aforementioned method and its application.

[0086] Example 4

[0087] This example provides a method for preparing a PROTAC compound (compound 4d) derived from triptolide, and its synthetic route is as follows:

[0088]

[0089] The preparation method of compound 4d in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with tert-butyl 6-aminohexanoate (0.55 mL, 2.73 mmol) to obtain intermediate compound 2-4; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-4 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-4; in the synthesis step, intermediate compound 3-4 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4d, a yellow powder, 0.17 g, with a yield of 84%.

[0090] NMR data of compound 4d:

[0091] 1 H-NMR(400MHz,DMSO)δ11.11(s,1H),7.58(t, J =7.6Hz,1H),7.09(d, J =8.4Hz,1H),7.03(d, J =6.8Hz,1H),6.54(s,1H),5.06(dd, J =4.8Hz, J =12.4Hz,1H),4.98(s,1H),4.70-4.90(m,2H),3.94(s,1H),3.68(s,1H),3.56(d, J =4.8Hz,1H),3.24-3.34(m,2H),2.80-2.95(m,1H),2.52-2.66(m,3H),2.30-2.45(m,2H),2.16-2.27(m,1H),1.8 7-2.16(m,3H),1.72-1.85(m,2H),1.52-1.70(m,4H),1.36-1.50(m,2H),1.21-1.35(m,2H),0.89(s,3H),0.87(d, J =6.8Hz,3H),0.75(d, J=6.8Hz,3H);

[0092] 13 C-NMR (101MHz, DMSO) δ173.61,173.32,172.60,170.58,169.37,167.78,162. 67,146.85,136.76,132.65,123.57,117.60,110.85,109.47,71.11,70.70,63 .76,63.10,61.39,59.85,55.37(2C),48.99,42.22,40.24,35.49,34.07,31.4 4,29.56,28.85,28.06,26.05,22.80,22.63,17.91,17.03,16.98(2C),14.18.

[0093] This example also provides a PROTAC compound (compound 4d) derived from triptolide prepared by the aforementioned method and its application.

[0094] Example 5

[0095] This example provides a method for preparing a PROTAC compound (compound 4e) derived from triptolide, and its synthetic route is as follows:

[0096]

[0097] The preparation method of compound 4e in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with tert-butyl 7-aminoheptanoate (0.59 mL, 2.73 mmol) to obtain intermediate compound 2-5; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-5 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-5; in the synthesis step, intermediate compound 3-5 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4e, a yellow powder, 0.18 g, with a yield of 85%.

[0098] NMR data of compound 4e:

[0099] 1 H-NMR(400MHz,DMSO)δ11.11(s,1H),7.57(t, J =7.6Hz,1H),7.09(d, J =8.8Hz,1H),7.02(d, J =6.8Hz,1H),6.52(s,1H),5.03(dd, J=4.8Hz, J =12.4Hz,1H),4.97(s,1H),4.70-4.90(m,2H),3.93(s,1H),3.68(s,1H),3.56(d, J =4.8Hz,1H),3.24-3.34(m,2H),2.80-2.98(m,1H),2.70(s,1H),2.55-2.65(m,2H),2.26-2.45(m,2H),2.15-2.25(m,2H),2.05-2.1 5(m,1H),1.98-2.05(m,2H),1.86-1.98(m,1H),1.72-1.85(m,2H),1.50-1.68(m,4H),1.20-1.45(m,6H),0.82-0.94(m,6H),0.76(d, J =6.8Hz,3H);

[0100] 13 C-NMR (101MHz, DMSO) δ173.62,173.32,172.64,170.57,169.41,167.77,162.6 7,146.88,136.75,132.64,123.58,117.62,110.84,109.44,71.10,70.70,63.7 6,63.09,61.34,59.87,55.36,55.33,48.99,42.25,40.24,35.49,34.17,31.44,29.01,28.40,28.09,26.50,25.00,22.81,22.62,17.91,17.02,16.98,14.14.

[0101] This example also provides a PROTAC compound (compound 4e) derived from triptolide prepared by the aforementioned method and its application.

[0102] Example 6

[0103] This example provides a method for preparing a PROTAC compound (compound 4f) derived from triptolide, and its synthetic route is as follows:

[0104]

[0105] The preparation method of compound 4f in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with tert-butyl 8-aminooctanoate (0.65 mL, 2.73 mmol) to obtain intermediate compound 2-6; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-6 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-6; in the synthesis step, intermediate compound 3-6 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4f, a yellow powder, 0.17 g, with a yield of 83%.

[0106] NMR data of compound 4f:

[0107] 1 H-NMR (400MHz, CDCl 3 )δ11.11(s,1H),7.56(t, J =7.6Hz,1H),7.08(d, J =8.4Hz,1H),7.01(d, J =6.8Hz,1H),6.50(s,1H),5.03(dd, J =4.8Hz, J =12.4Hz,1H),4.96(s,1H),4.68-4.87(m,2H),3.92(s,1H),3.67(s,1H),3.54(d, J =4.8Hz,1H),3.23-3.31(m,2H),2.80-2.95(m,1H),2.51-2.64(m,3H),2.25-2.45(m,2H),2.15-2.25(m,1H),1.9 9-2.15(m,2H),1.85-1.98(m,1H),1.70-1.85(m,2H),1.45-1.65(m,4H),1.20-1.42(m,8H),0.88(s,3H),0.86(d, J =6.8Hz,1H),0.75(d, J =6.8Hz,1H);

[0108] 13 C-NMR (101 MHz, CDCl 3)δ178.36,178.06,177.41,175.33,174.17,172.52,167.41,151.64,141.48, 137.38,128.33,122.39,115.58,114.18,75.82,68.51,67.85,66.07,64.60, 60.12,60.06,59.66,47.03,44.99,40.23,38.92,36.19,34.31,33.85,33.70 ,33.39,32.80,31.42,29.77,27.56,27.37,22.66,21.77,21.72(2C),18.88.

[0109] This example also provides a PROTAC compound (compound 4f) derived from triptolide prepared by the aforementioned method and its application.

[0110] Example 7

[0111] This example provides a method for preparing a PROTAC compound (compound 4g) derived from triptolide, and its synthetic route is as follows:

[0112]

[0113] The preparation method of compound 4g in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with tert-butyl 9-aminononanoate (0.63 g, 2.73 mmol) to obtain intermediate compound 2-7; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-7 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-7; in the synthesis step, intermediate compound 3-7 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4g, yellow powder, 0.18 g, yield 85%.

[0114] NMR data of compound 4g:

[0115] 1 H-NMR(400MHz,DMSO)δ11.12(s,1H),7.57(t, J =7.6Hz,1H),7.08(d, J =8.4Hz,1H),7.02(d, J =6.8Hz,1H),6.51(s,1H),5.05(dd, J =4.4Hz, J=12.4Hz,1H),4.99(s,1H),4.7-4.90(m,2H),3.93(s,1H),3.68(s,1H),3.56(d, J =4.8Hz,1H),3.23-3.32(m,2H),2.83-2.97(m,1H),2.55-2.68(m,2H),2.27-2.44(m,2H),2.17-2.27(m, 1H),1.88-2.15(m,3H),1.73-1.86(m,2H),1.48-1.68(m,4H),1.20-1.42(m,11H),0.91(s,3H),0.88(d, J =6.4Hz,3H),0.77(d, J =6.8Hz,3H);

[0116] 13 C-NMR (101MHz, DMSO) δ173.56,173.28,172.64,170.55,169.43,167.76,162.61,14 6.87,136.70,132.64,123.60,117.57,110.83,109.46,71.06,70.67,63.75,63.09, 61.33,59.85,55.37,55.32,49.01,42.32,40.25,35.50,34.17,31.47,29.57,29.17 ,29.13,28.65,28.07,26.77,25.04,22.83,22.63,17.90,17.03,16.98(2C),14.13.

[0117] This example also provides a PROTAC compound (compound 4g) derived from triptolide prepared by the aforementioned method and its application.

[0118] Example 8

[0119] This example provides a method for preparing a PROTAC compound (compound 4h) derived from triptolide, and its synthetic route is as follows:

[0120]

[0121] The preparation method of compound 4h in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with amino-polyethylene glycol-tert-butyl propionate (n=1, 0.52 mL, 2.73 mmol in amino derivative structural formula II) to obtain intermediate compound 2-8; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-8 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-8; in the synthesis step, intermediate compound 3-8 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4h, an orange-yellow powder, 0.17 g, with a yield of 84%.

[0122] NMR data of compound 4h:

[0123] 1 H-NMR(400MHz,DMSO)δ11.11(s,1H),7.58(t, J =6.0Hz,1H),7.14(d, J =8.0Hz,1H),7.05(d, J =6.4Hz,1H),6.60(s,1H),5.05(d, J =8.8Hz,1H),5.00(s,1H),4.70-4.91(m,2H),3.95(s,1H),3.65-3.80(m,3H),3.53-3.65(m,3H),3.42-3.53(m,3H ),2.89(t,1H),2.55-2.70(m,4H),1.90-2.27(m,4H),1.70-1.90(m,2H),1.20-1.40(m,2H),0.90(s,3H),0.85(d, J =5.6Hz,3H),0.72(d, J =5.6Hz,3H);

[0124] 13C-NMR(100MHz,DMSO)δ173.61,173.31,170.85,170.55,169.35,167.77,162 .66,146.85,136.72,132.54,123.58,117.86,111.18,109.73,71.32,70.71, 69.27,66.51,63.78,63.16,61.40,59.71,55.41,55.38,49.01,42.17,40.15 ,35.50,31.45,29.59,27.67,22.79,22.61,17.83,17.04,16.92(2C),14.23.

[0125] This example also provides a PROTAC compound (compound 4h) derived from triptolide prepared by the aforementioned method and its application.

[0126] Example 9

[0127] This example provides a method for preparing a PROTAC compound (compound 4i) derived from triptolide, and its synthetic route is as follows:

[0128]

[0129] The preparation method of compound 4i in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with amino-diethylene glycol-tert-butyl propionate (n=2 in amino derivative structural formula II, 0.63 mL, 2.73 mmol) to obtain intermediate compound 2-9; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-9 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-9; in the synthesis step, intermediate compound 3-9 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4i, an orange-yellow powder, 0.17 g, with a yield of 84%.

[0130] NMR data of compound 4i:

[0131] 1 H-NMR(400MHz,DMSO)δ11.11(s,1H),7.59(t, J =7.6Hz,1H),7.15(d, J =8.4Hz,1H),7.05(d, J =6.8Hz,1H),6.61(s,1H),5.05(dd, J =4.4Hz, J=12.4Hz,1H),4.98(s,1H),4.72-4.90(m,2H),3.94(s,1H),3.65-3.72(m,3H),3.59-3.65(m,2H),3.50-3.59(m,5H),3.43-3.50(m,2H), 2.82-2.95(m,1H),2.54-2.66(m,4H),2.17-2.29(m,1H),2.07-2.17(m,1H),1.74-2.06(m,4H),1.20-1.35(m,3H),0.90(s,3H),0.86(d, J =6.4Hz,3H),0.73(d, J =6.4Hz,3H);

[0132] 13 C-NMR (101MHz, DMSO) δ173.59,173.29,170.94,170.55,169.40,167.76,162.64 ,146.86,136.68,132.55,123.58,117.90,111.14,109.71,71.26,70.69,70.19 ,70.14,69.38,66.63,63.77,63.19,61.34,59.68,55.39(2C),49.02,42.15,40 .26,35.50,31.46,29.59,27.52,22.80,22.61,17.84,17.04,16.92(2C),14.22.

[0133] This example also provides a PROTAC compound (compound 4i) derived from triptolide prepared by the aforementioned method and its application.

[0134] Example 10

[0135] This example provides a method for preparing a PROTAC compound (compound 4j) derived from triptolide, and its synthetic route is as follows:

[0136]

[0137] The preparation method of compound 4j in this example is the same as that in Example 1, except that, in the step of preparing intermediate compound 2, tert-butyl 3-aminopropionate is replaced with amino-triethylene glycol-tert-butyl propionate (n=3 in amino derivative structural formula II, 0.74 mL, 2.73 mmol) to obtain intermediate compound 2-10; in the step of preparing intermediate compound 3, the protecting group on the carboxyl group of intermediate compound 2-10 is removed under the action of trifluoroacetic acid to obtain intermediate compound 3-10; in the synthesis step, intermediate compound 3-10 is reacted with triptolide, and other reaction conditions and operations remain unchanged to obtain compound 4j, an orange-yellow powder, 0.18 g, with a yield of 85%.

[0138] NMR data of compound 4j:

[0139] 1 H-NMR(400MHz,DMSO)δ11.12(s,1H),7.58(t, J =7.6Hz,1H),7.15(d, J =8.4Hz,1H),7.05(d, J =7.2Hz,1H),6.61(s,1H),5.08(dd, J =4.4Hz, J =12.4Hz,1H),4.99(s,1H),4.72-4.90(m,2H),3.94(s,1H),3.60-3.71(m,5H),3.52-3.60(m,5H),3.45-3.52(m,5H),2.83-2.96(m,1H), 2.52-2.67(m,5H),2.18-2.30(m,1H),2.08-2.17(m,1H),1.90-2.07(m,2H),1.75-1.90(m,2H),1.21-1.37(m,3H),0.91(s,3H),0.87(d, J =6.4Hz,3H),0.74(d, J =6.4Hz,3H);

[0140] 13C-NMR (101MHz, DMSO) δ173.58,173.28,170.93,170.54,169.40,167.76,162.61,14 6.86,136.67,132.55,123.60,117.88,111.13,109.70,71.25,70.69,70.30,70.24 (2C),70.18,69.35,66.56,63.76,63.19,61.36,59.68,55.40,55.37,49.03,42.16 ,40.26,35.50,31.47,29.59,27.50,22.81,22.62,17.83,17.05,16.91(2C),14.21.

[0141] This example also provides a PROTAC compound (compound 4j) derived from triptolide prepared by the aforementioned method and its application.

[0142] Test Example 1

[0143] The CCK-8 method was used to detect the cellular antitumor activity and toxicity of compounds 4a-4j, and triptolide and pomalidomide were used as positive controls.

[0144] Cell lines: human colorectal cancer cell line SW620, human non-small cell lung cancer cell line NCI-H358, human breast cancer cell line MDA-MB-231, and rat normal glomerular mesangial cell line HBZY-1.

[0145] Experimental steps: The above cell lines were inoculated in 96-well plates, 5,000 cells per well, and cultured overnight. The compound to be tested was added to the 96-well plate with gradient concentrations and incubated for 48 hours (HBZY-1 cells) or 96 hours (SW620 cells, NCI-H358 cells, MDA-MB-231 cells), 10 μL CCK8 solution was added to every 100 μL culture medium, mixed and incubated for 1 hour to 2 hours, and the absorbance value of OD (wavelength 450nm) was read.

[0146] Cell inhibition rate (%) = [(OD value of blank control group - OD value of drug-treated group) / OD value of blank control group] × 100%.

[0147] The experiments showed that the compounds 4a-4j of the present invention had inhibitory effects on human colorectal cancer cell line SW620, human non-small cell lung cancer cell line NCI-H358, and human breast cancer cell line MDA-MB-231, as shown in the following table:

[0148]

[0149] It can be seen that the PROTAC compounds 4a-4j derived from triptolide of the present invention showed high anti-proliferative activity against human colorectal cancer cell line SW620, human non-small cell lung cancer NCI-H358 and human breast cancer cell MDA-MB-231 cells; the anti-proliferative activity of some PROTAC compounds derived from triptolide was higher than that of the positive control triptolide.

[0150] Among them, compound 4e has the strongest anti-cancer cell proliferation activity, and its anti-proliferation activity against the aforementioned cancer cells is significantly higher than that of the positive control triptolide. For human colorectal cancer cells, the anti-proliferation activity of compound 4e is enhanced by 5 times compared with the positive control triptolide.

[0151] The inhibitory effects of compounds 4a-4j of the present invention on rat normal glomerular mesangial cell line HBZY-1 are shown in the following table:

[0152]

[0153] It can be seen that the toxicity of the PROTAC compounds 4a-4j derived from triptolide to normal rat kidney cells is significantly lower than that of the positive control triptolide, which effectively solves the problem of the strong toxicity of triptolide. Among them, the toxicity of compound 4e is reduced by more than 5.2 times compared with the positive control triptolide.

[0154] In summary, the PROTAC compounds 4a-4j developed based on triptolide of the present invention have high anti-tumor activity and low toxicity, and are expected to be prepared into new anti-tumor drugs, with great prospects for drug development.

[0155] Unless otherwise specified, all percentages used in the present invention are by mass.

[0156] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A PROTAC compound derived from triptolide, characterized in that: The structure of the PROTAC compound derived from triptolide is shown in Formula I: （I）； In the formula I, X is -(CH2) m -, m is any integer from 4 to 6.

2. A method for preparing a PROTAC compound derived from triptolide, characterized in that: The method comprises the following steps: preparing intermediate compound 2, preparing intermediate compound 3, and synthesizing; The method for preparing intermediate 2 is that 4-fluorothalidomide and an amino derivative undergo a nucleophilic substitution reaction under the action of DIPEA to obtain intermediate compound 2; The structure of the amino derivative is shown in Formula II: (II); In the formula II, X is -(CH2) m -, m is any integer from 4 to 6; The method for preparing the intermediate compound 3 is as follows: the intermediate compound 2 is treated with trifluoroacetic acid to remove the protective group on the carboxyl group to obtain the intermediate compound 3; The synthesis method is that triptolide and the intermediate compound 3 are reacted with condensing agents EDCI and DMAP to form an ester, thereby obtaining a PROTAC compound derived from triptolide; The synthetic route of the preparation method is as follows: 。 3. The method for preparing a PROTAC compound derived from triptolide according to claim 2, characterized in that: In the preparation of the intermediate compound 2, the molar amount of the amino derivative is at least 1.5 times that of 4-fluorothalidomide.

4. The method for preparing a PROTAC compound derived from triptolide according to claim 2, characterized in that: In the synthesis, the molar amount of the intermediate compound 3 is at least 1.4 times that of triptolide.

5. A pharmaceutical composition, characterized in that A pharmaceutically acceptable dosage form is prepared by using the PROTAC compound derived from triptolide or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an effective ingredient or a main effective ingredient and a pharmaceutically acceptable carrier.

6. A use of the triptolide-derived PROTAC compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating tumor diseases; The tumor disease is selected from: colorectal cancer, non-small cell lung cancer, and breast cancer.

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