A carnosic acid derivative and its preparation method and application

Abstract

The present invention discloses a carnosic acid derivative and a preparation method and application thereof, which is selected from one of the following compounds:

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor drugs, and specifically relates to a carnosic acid derivative and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Malignant tumors, also known as cancer, are chronic non-communicable diseases that seriously threaten human health. With the improvement of medical and health conditions, most diseases among residents, such as nutritional diseases, have been significantly controlled, making cancer the main cause of death, accounting for about 20% of total deaths, and has become a major disease that seriously endangers human life and health and restricts economic and social development.

[0004] The transcriptional activation target genes of β-catenin and its nuclear partner protein-protein interaction (PPI), mainly including β-catenin / TCF, β-catenin / BCL9, β-catenin / CBP and β-catenin / p300PPI, are the last step of the wnt / β-catenin signaling pathway, indicating that the disruption of these nuclear complexes is the most promising strategy to prevent the abnormal activation of Wnt / β-catenin. TCF, BCL9 and CBP / p300 are highly expressed in a variety of tumors, especially the disruption of β-catenin / BCL9 interaction may not cause damage to normal cells.

[0005] BCL9 enhances tumor cell proliferation, survival, migration, invasion, and metastatic potential. In addition, BCL9 deficiency can effectively improve the survival of xenograft mice by inhibiting tumor growth, including colorectal cancer, breast cancer, multiple myeloma, and hepatocellular carcinoma. In addition, inhibition of BCL9 can reduce tumor growth, promote CD8+ T cell tumor invasion, and enhance the response to anti-PD-1 therapy.

[0006] The β-catenin / BCL9 complex is dominant in tumor tissues but not in normal cells. The β-catenin / BCL9 PPI interface buries approximately The moderate Kd value is about 0.50 μM. The β-catenin / BCL9PPI is mediated by a helical segment of approximately 25 residues of BCL9, with "two hot zones". In hot zone 1, β-catenin residues D162, E163 and D164 form an acidic knob and form salt bridge interactions with BCL9 residues H358 and R359, but no obvious drug design binding pocket was observed in this hot zone. Hot zone 2 is the β-catenin hydrophobic pocket, including residues A149, A152, L156, L159, L160, V167, A171, M174 and L178, which form hydrophobic interactions with residues L366, I369 and L373 of BCL9. Since BCL9 residues L366, I369, L373, and F374 bind in a wider and slightly shallower pocket of β-catenin, hot zone 2 has been applied to the design and optimization of β-catenin / BCL9 PPI inhibitors.

[0007] Carnosic acid (CA) is a natural active ingredient derived from rosemary, sage, salvia miltiorrhiza and other plants of the Lamiaceae family. It is a phenolic diterpene and a secondary metabolite of terpenes. Studies have found that carnosic acid has a variety of biological activities, including anti-cancer, anti-inflammatory, antioxidant and anti-obesity. Among them, the study of anti-cancer activity is favored by a wide range of researchers. It has been reported that CA can directly target β-catenin to exert anti-cancer effects, inhibit the binding of BCL9 to β-catenin in a dose-dependent manner in vitro, and has specificity. However, β-catenin / BCL9PPI inhibitors based on the structure of carnosic acid derivatives have not been reported so far. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention aims to provide a carnosic acid derivative and a preparation method and application thereof.

[0009] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0010] In a first aspect, the present invention provides a carnosic acid derivative selected from one of the following compounds:

[0011]

[0012]

[0013] In some embodiments, the synthetic route of CA-1 is:

[0014] Preferably, the synthetic route of CA-2 is:

[0015]

[0016] Preferably, the synthetic route of CA-3 is:

[0017]

[0018]

[0019] Preferably, the synthetic route of CA-4 is:

[0020] Preferably, the synthetic route of CA-5 is:

[0021]

[0022]

[0023] Preferably, the synthetic route of CA-6 is:

[0024]

[0025] Preferably, the synthetic route of CA-7 is:

[0026]

[0027] Preferably, the synthetic route of CA-8 is:

[0028]

[0029] In some embodiments, the synthetic route of CA-9 is:

[0030]

[0031] In a third aspect, the present invention provides the use of the carnosic acid derivative in the preparation of β-catenin / BCL9PPI inhibitors and cell anti-proliferation drugs.

[0032] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0033] An amide linker is introduced into carnosic acid to extend its backbone length and connect some derivative groups with benzene rings so that it can be tightly bound to the target protein interface. By extending the backbone of carnosic acid and modifying the groups, the affinity of its derivative compounds to the target protein can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 It is a schematic diagram of the docking mode of carnosic acid targeting β-catenin / BCL9 and the design of its derivatives;

[0036] Figure 2 This is the possible binding mode of CA-8 and β-catenin. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] Design of carnosic acid derivatives targeting β-catenin / BCL9

[0039] Based on the exploration of the docking mode of carnosic acid and β-catenin / BCL9 target molecules, it can bind tightly to the lower end of the target PPI interface, and the binding sites are ASP145 and LYS181. However, there are extensive protein hydrophobic channels and a large number of binding sites at the upper end of the PPI where carnosic acid and β-catenin / BCL9 target molecules dock. We consulted the literature to evaluate the possibility of its synthesis, creatively introduced an amide bond linker on carnosic acid, extended its backbone length, and connected some derivative groups with benzene rings, so that it can bind tightly to the target protein interface. It is hoped that the extension of the carnosic acid backbone and the modification of the groups can improve the affinity of its derivative compounds with the target protein.

[0040]

[0041] Using carnosic acid (CA) as the starting material, two phenolic hydroxyl groups were esterified by acetic anhydride to synthesize compound CA-1; the carboxyl group was esterified by iodomethane to obtain intermediate C1, the benzyl methylene group of intermediate C1 was oxidized to carbonyl group by chromium trioxide to obtain intermediate C2, and the ester group of intermediate C2 was hydrolyzed under sodium hydroxide to obtain compound CA-2. The two phenolic hydroxyl groups and ester groups of CA were protected by benzyl bromide to obtain intermediate C3, the benzyl methylene group of intermediate C3 was oxidized to carbonyl group to obtain intermediate C4, and C4 and methylamine hydrochloride were reductively aminized to obtain key intermediate C5. 3-bromophenol and 4-isopropyl-phenylboronic acid pinacol ester were reacted by Suzuki coupling reaction to obtain intermediate C8; intermediate C8 was etherified with methyl bromoacetate to obtain intermediate C9, and then hydrolyzed to obtain intermediate 10. C5 was alkylated and amide condensed to obtain intermediates C6, C7 and 11a-d; intermediates C6, C7 and 11a-d were hydrogenated under 10% palladium hydroxide carbon to remove the benzyl group to give compounds CA-3-CA-8. Using 3-bromo-4-hydroxy-benzoic acid methyl ester as the starting material, intermediate C12 was prepared by Mitsunobu etherification reaction, and intermediate C12 was obtained by Suzuki coupling reaction to obtain intermediate C13; intermediate C13 was hydrolyzed, condensed, debenzylated and Boc protected to obtain compound CA-9. For the modification of CA, CA-1 and CA-2 were synthesized according to the method described in the literature, while CA-3, CA-4, CA-5, CA-6, CA-7, CA-8 and CA-9 were new compounds synthesized through completely new modification sites, and there is no literature report at present.

[0042]

[0043] According to the listed synthetic routes, a total of 9 compounds were synthesized. The structures of all target compounds were 1 H NMR, 13 C NMR and ESI-MS confirmed the structure of the target compound.

[0044] Table 1 Structures of carnosic acid derivatives

[0045]

[0046] Table 2 Experimental instruments

[0047]

[0048]

[0049] Table 3 Experimental reagents

[0050]

[0051]

[0052] Synthesis and structure confirmation of compounds CA-1 to CA-9

[0053] Synthesis of CA-1

[0054]

[0055] Add CA (1.00 g, 3.01 mmol) to a 50 ml dry reaction bottle, dissolve it with 20 ml DCM, then add DMAP (1.54 g, 15.05 mmol), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 3:1). After the reaction is completed, add 30 ml of water to the reaction solution and extract it with DCM (30 ml × 3). Collect the organic phase, wash it with 50 ml of saturated brine, dry it with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 3:1 as eluent] to obtain CA-1 (white solid 1.21 g, yield 96.8%). 1 H NMR(400MHz,Chloroform-d)δ7.05(q,J=1.0Hz,1H),3.23(d,J=1.0Hz,1H),2.88(d d,J=18.0,1.0Hz,1H),2.72(dd,J=18.0,1.0Hz,1H),2.37(d,J=0.6Hz,7H),2.25(d, J=12.9Hz,1H),2.17(s,1H),2.00(s,1H),1.81(dd,J=13.0,8.2Hz,2H),1.65–1.54( m,2H),1.36(d,J=13.0Hz,1H),1.24(s,3H),1.19(s,3H),0.98(s,3H),0.93(s,3H). 13 C NMR(100MHz,Chloroform-d)δ176.08,168.97,168.86,141.61,139.80,139.57,131.50,128.58,122 .65,50.72,50.15,40.40,34.21,32.36,30.89,26.97,26.78,23.20,21.48,20.77,19.67.ESI-Mass for C 24 H 32 O6: m / z(MH) + 415.51.

[0056] Synthesis of compound C1

[0057]

[0058] Add CA-1 (0.23 g, 0.55 mmol) to a 50 ml dry reaction bottle, dissolve it with 20 ml DMF, then add cesium carbonate (0.36 g, 1.10 mmol), iodomethane (0.094 g, 0.66 mmol), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 10:1). After the reaction is completed, add 30 ml of water to the reaction solution and extract it with ethyl acetate (30 ml × 3). Collect the organic phase, wash it with 40 ml of saturated brine and 40 ml of saturated ammonium chloride aqueous solution, dry it with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 30:1 as eluent] to obtain compound C1 (white solid, 0.22 g, yield 90.2%).

[0059] Synthesis of compound C2

[0060]

[0061] Add compound C1 (0.2 g, 0.46 mmol) to a 50 ml dry reaction bottle, dissolve it with 20 ml glacial acetic acid, then add chromium trioxide (0.18 g, 0.92 mmol), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction is completed, concentrate under reduced pressure to remove the solvent, add 30 ml of water and 20 ml of saturated sodium carbonate aqueous solution to the residual liquid, and extract with ethyl acetate (30 ml×3). Collect the organic phase, wash with 40 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 20:1 as eluent] to obtain C2 (white solid, 0.15 g, yield 72.8%).

[0062] Synthesis of compound CA-2

[0063]

[0064] Compound C2 (0.15 g, 0.35 mmol) was added to a 50 ml dry reaction bottle, dissolved with 10 ml THF, and then lithium hydroxide (0.087 g, 0.21 mmol) dissolved in 2.5 ml water was added, and stirred at room temperature overnight. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction was completed, the solvent was removed by concentration under reduced pressure, 30 ml of water was added to the residual liquid, the pH was adjusted to about 4 with 1M HCl, and then extracted with ethyl acetate (30 ml×3). The organic phase was collected, washed with 40 ml of saturated brine, dried with anhydrous Na2SO4, and the organic phase was concentrated under reduced pressure. The residual liquid was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain CA-2 (white solid, 0.084 g, yield 70.2%). 1 HNMR(400MHz,Chloroform-d)δ8.00(s,1H),7.66(s,1H),7.59(d,J=1.0Hz,1 H),3.23(d,J=1.0Hz,1H),2.83(s,1H),2.61(s,1H),2.39(d,J=13.0Hz,1H),1 .99(d,J=13.0Hz,1H),1.78(d,J=13.0Hz,1H),1.57(dd,J=13.0,11.7Hz,2H) ,1.35(d,J=13.0Hz,1H),1.25(s,3H),1.20(s,3H),0.99(s,3H),0.94(s,3H). 13 C NMR(100MHz,Chloroform-d)δ199.41,175.46,145.69,143.05,135.89,128.30,124.80, 122.62,48.25,46.50,40.11,39.92,34.71,33.06,27.23,26.08,22.82,19.73.ESI-Mass for C 20 H 26 O5: m / z(MH) + 34-.42.

[0065] Synthesis of compound C3

[0066]

[0067] Add CA (2.00 g, 6.02 mmol) to a 100 ml dry reaction bottle, dissolve it with 60 ml DMF, then add cesium carbonate (11.78 g, 36.14 mmol) and benzyl bromide (4.12 g, 30.12 mmol), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 10:1). After the reaction is completed, add 50 ml of water to the reaction solution and extract it with ethyl acetate (30 ml × 3). Collect the organic phase, wash it with 40 ml of saturated brine and 40 ml of saturated ammonium chloride aqueous solution, dry it with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 60:1 as eluent] to obtain compound C3 (light yellow oily liquid, 3.50 g, yield 96.52%).

[0068] Synthesis of compound C4

[0069]

[0070] Compound 3 (3.50 g, 5.81 mmol) was added to a 50 ml dry reaction bottle, dissolved with 30 ml glacial acetic acid, and then chromium trioxide (2.34 g, 11.63 mmol) was added, and stirred at room temperature overnight. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and 40 ml of water and 20 ml of saturated sodium carbonate aqueous solution were added to the residual liquid, and extracted with ethyl acetate (30 ml × 3). The organic phase was collected, washed with 40 ml of saturated brine, dried with anhydrous Na2SO4, and the organic phase was concentrated under reduced pressure. The residual liquid was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 40:1 as eluent] to obtain C4 (light yellow oily liquid, 2.15 g, yield 60.0%).

[0071] Synthesis of compound C5

[0072]

[0073] Compound C4 (1.0 g, 1.60 mmol) was added to a 100 ml dry glass tube, dissolved with 5 ml dioxane, then methylamine hydrochloride (0.53 g, 7.99 mmol), zinc chloride (0.43 g, 3.19 mmol), sodium cyanoborohydride (4.79 g, 0.30 mmol) were added, and stirred at 80-100 ° C for 24 h. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 5: 1). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and 40 ml of water and 10 ml of saturated sodium carbonate aqueous solution were added to the residual liquid, and extracted with DCM (30 ml × 3). The organic phase was collected, washed with 40 ml of saturated brine, dried with anhydrous Na2SO4, and the organic phase was concentrated under reduced pressure. The residual liquid was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 10: 1 as eluent] to obtain C5 (light yellow oily liquid, 0.54 g, yield 53.60%).

[0074] Synthesis of compound C6

[0075]

[0076] Add intermediate C5 (0.13 g, 0.21 mmol) to a 50 ml dry reaction bottle, dissolve it with 20 ml acetonitrile, then add cesium carbonate (0.08 g, 0.25 mmol), iodomethane (0.035 g, 0.25 mmol), and stir at room temperature for 1 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction is completed, concentrate under reduced pressure to remove the solvent, add 20 ml of water to the residual liquid, and extract it with ethyl acetate (10 ml × 3). Collect the organic phase, wash it with 20 ml of saturated brine, dry it with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 20:1 as eluent] to obtain compound C6 (colorless solid, 0.08 g, yield 60.58%).

[0077] Synthesis of compound CA-3

[0078]

[0079] Add intermediate C6 (0.08 g, 0.12 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.16 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration through diatomaceous earth. The filtrate is concentrated under reduced pressure, and the residual liquid is purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain compound CA-3 (white solid, 0.03 g, yield 65.22%). 1HNMR(400MHz,Chloroform-d)δ7.97(s,1H),7.76(s,1H),6.95(t,J=1.0Hz,1H),3. 73(d,J=1.0Hz,1H),3.22(d,J=1.0Hz,1H),2.37(d,J=13.0Hz,1H),2.24(s,6H),2.1 5(s,1H),2.04(s,1H),2.00(d,J=13.0Hz,1H),1.80(d,J=13.0Hz,1H),1.67–1.54( m,2H),1.35(d,J=13.0Hz,1H),1.24(s,3H),1.19(s,3H),0.97(s,3H),0.92(s,3H). 13 C NMR(100MHz,Chloroform-d)δ176.27,145.35,142.82,134.85,129.52,127.29,120.16,62. 51,46.70,46.44,41.62,40.08,34.35,32.55,28.06,26.97,26.70,22.82,19.68.ESI-Mass forC 22 H 33 NO4: m / z(MH) + 374.52.

[0080] Synthesis of compound C7

[0081]

[0082] Add intermediate C5 (0.13 g, 0.21 mmol) to a 50 ml dry reaction bottle, dissolve it with 10 ml DCM, then add DIPEA (0.08 g, 0.63 mmol), stir at 0 ° C for 30 min, then add methyl chlorothiocyanate (0.028 g, 0.24 mmol), and after the addition is complete, warm to room temperature and stir for 1 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5: 1). After the reaction is completed, add 20 ml of water to the reaction solution and extract with DCM (10 ml × 3). Collect the organic phase, wash with 20 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 20: 1 as eluent] to obtain compound C7 (colorless solid, 0.11 g, yield 78.6%).

[0083] Synthesis of compound CA-4

[0084]

[0085] Add intermediate C7 (0.11 g, 0.15 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.022 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration through diatomaceous earth. The filtrate is concentrated under reduced pressure, and the residual liquid is purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain compound CA-4 (white solid, 0.05 g, yield 73.58%). 1 HNMR(400MHz,Chloroform-d)δ7.97(s,1H),7.76(s,1H),7.03(t,J=1.0Hz,1H),4.46( d,J=1.0Hz,1H),3.22(d,J=1.0Hz,1H),2.90(s,3H),2.63(s,3H),2.37(d,J=13.0Hz,1 H),2.25(s,1H),2.15(s,1H),2.00(d,J=13.0Hz,1H),1.80(d,J=13.0Hz,1H),1.67–1. 54(m,2H),1.35(d,J=13.0Hz,1H),1.24(s,3H),1.19(s,3H),0.97(s,3H),0.92(s,3H). 13 C NMR(100MHz,Chloroform-d)δ176.27,145.35,142.82,135.26,131.03,126.16,122.21,59.81, 46.62,46.11,40.08,38.49,34.30,32.93,32.55,27.93,26.97,26.70,22.82,19.68.ESI-Mass for C 22 H 33 NO6S:m / z(MH) + 438.57.

[0086] Synthesis of compound C8

[0087]

[0088] 3-Bromophenol (0.5 g, 2.87 mmol) was added to a 50 ml dry reaction bottle and dissolved with dioxane / EtOH / H2O (5:2:1, 32 ml), followed by potassium acetate (1.12 g, 11.49 mmol), 4-isopropyl-phenylboronic acid pinacol ester (0.85 g, 3.45 mmol), and Pd(dppf)Cl2 (0.21 g, 0.28 mmol). The mixture was stirred at 80°C for 24 h under N2 protection and monitored by TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction was completed, the reaction solution was cooled to room temperature, the insoluble matter was removed by filtration, the filtrate was concentrated, and the residual solution was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 20:1 as eluent] to obtain light yellow liquid C8 (0.48 g, yield 78.56%).

[0089] Synthesis of compound C9

[0090]

[0091] Compound C8 (0.2 g, 0.94 mmol) was added to a 50 ml dry reaction bottle, dissolved with 20 ml acetonitrile, then methyl bromoacetate (0.28 g, 1.88 mmol) and potassium carbonate (0.39 g, 2.83 mmol) were added, and stirred under reflux at 85 ° C overnight. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 10: 1), and then the reaction solution was cooled to room temperature, filtered to remove part of the potassium carbonate, and the filtrate was concentrated. 40 ml of water was added to the residue, and extracted with ethyl acetate (20 ml × 3). The organic phase was collected, washed with 30 ml of saturated brine, dried with anhydrous Na2SO4, and the organic phase was concentrated under reduced pressure. The residual liquid was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 30: 1 as eluent] to obtain a colorless transparent liquid product compound C9 (0.25 g, yield 95.56%).

[0092] Synthesis of compound C10

[0093]

[0094] Add compound C9 (0.25 g, 0.88 mmol) to a 50 ml dry reaction bottle, dissolve with 20 ml THF, dissolve lithium hydroxide (0.15 g, 3.54 mmol) in 5 ml water, add to the reaction solution, and stir at room temperature for 4 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 10:1). After the reaction is completed, concentrate the reaction solution, add 20 ml water to the residual solution, adjust the pH to about 4-5 with 1 M HCl, and extract with ethyl acetate (30 ml × 3). Combine the organic phases, wash with 40 ml saturated brine, and dry three times with anhydrous Na2SO4. Concentrate the organic phase to obtain compound C10 (light yellow solid, 0.23 g, yield 99%).

[0095] Synthesis of compound 11a

[0096]

[0097] Add intermediate C5 (0.13 g, 0.21 mmol) to a 50 ml dry reaction bottle, dissolve it with 10 ml DCM, then add DIPEA (0.08 g, 0.63 mmol), HATU (0.08 g, 0.21 mmol), glacial acetic acid 0.013 g, 0.21 mmol), and stir at room temperature for 6 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction is completed, add 20 ml of water to the reaction solution and extract with DCM (10 ml × 3). Collect the organic phase, wash with 20 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 8:1 as eluent] to obtain compound 11a (colorless solid, 0.1 g, yield 72.46%).

[0098] Synthesis of compound 11b

[0099]

[0100] Add intermediate C5 (0.13 g, 0.21 mmol) to a 50 ml dry reaction bottle, dissolve it with 10 ml DCM, then add DIPEA (0.08 g, 0.63 mmol), HATU (0.08 g, 0.21 mmol) and benzoic acid 0.027 g, 0.21 mmol) and stir at room temperature for 6 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction is completed, add 20 ml of water to the reaction solution and extract with DCM (10 ml × 3). Collect the organic phase, wash with 20 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 8:1 as eluent] to obtain compound 11d (colorless solid, 0.14 g, yield 77.78%).

[0101] Synthesis of compound 11c

[0102]

[0103] Add intermediate C5 (0.13 g, 0.21 mmol) to a 50 ml dry reaction bottle, dissolve it with 10 ml DCM, then add DIPEA (0.08 g, 0.63 mmol), HATU (0.08 g, 0.21 mmol) and 2-(4-(ethylsulfonyl)phenyl)acetic acid (0.045 g, 0.21 mmol) and stir at room temperature for 6 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction is completed, add 20 ml of water to the reaction solution and extract with DCM (10 ml × 3). Collect the organic phase, wash with 20 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 8:1 as eluent] to obtain compound C11c (colorless solid, 0.11 g, yield 64%).

[0104] Synthesis of compound 11d

[0105]

[0106] Add intermediate C5 (0.13 g, 0.21 mmol) to a 50 ml dry reaction bottle, dissolve it with 10 ml DCM, then add DIPEA (0.08 g, 0.63 mmol), HATU (0.08 g, 0.21 mmol), intermediate 10 (0.055 g, 0.21 mmol) and stir at room temperature for 6 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 5:1). After the reaction is completed, add 20 ml of water to the reaction solution and extract with DCM (10 ml × 3). Collect the organic phase, wash with 20 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 8:1 as eluent] to obtain compound C11d (colorless solid, 0.14 g, yield 78%).

[0107] Synthesis of compound CA-5

[0108]

[0109] Add intermediate 11a (0.1 g, 0.15 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.02 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration through diatomaceous earth. The filtrate is concentrated under reduced pressure, and the residual liquid is purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain compound CA-5 (white solid, 0.03 g, yield 50%). 1 HNMR(400MHz,Chloroform-d)δ7.92(s,1H),7.79(s,1H),7.01(t,J=1.0Hz,1H),4.72( d,J=1.0Hz,1H),3.22(d,J=1.0Hz,1H),2.94(s,3H),2.37(d,J=13.0Hz,1H),2.25(s,1 H),2.12(s,1H),2.00(d,J=13.0Hz,1H),1.97(s,3H),1.80(d,J=13.0Hz,1H),1.67–1. 54(m,2H),1.35(d,J=13.0Hz,1H),1.24(s,3H),1.19(s,3H),0.97(s,3H),0.92(s,3H). 13C NMR(100MHz,Chloroform-d)δ176.27,172.10,145.35,142.82,134.85,130.58,126.54,120.28,55 .00,46.71,46.41,40.08,34.35,33.36,32.55,28.71,26.97,26.52,22.82,22.11,19.68.ESI-Mass for C 23 H 33 NO5: m / z(M+H) + 404.52.

[0110] Synthesis of compound CA-6

[0111]

[0112] Add intermediate 11b (0.12 g, 0.16 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.024 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration on diatomaceous earth. The filtrate is concentrated under reduced pressure, and the residual liquid is purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain compound CA-6 (white solid, 0.05 g, yield 71.43%). 1 HNMR(400MHz,Chloroform-d)δ7.92(s,1H),7.79(s,1H),7.77–7.70(m,2H),7.59–7.49(m,1H),7 .48–7.38(m,2H),6.99(t,J=1.0Hz,1H),4.85(d,J=1.0Hz,1H),3.22(d,J=1.0Hz,1H),2.96(s,3H) ,2.37(d,J=13.0Hz,1H),2.28(s,1H),2.12(s,1H),2.00(d,J=13.0Hz,1H),1.80(d,J=13.0Hz,1H ),1.67–1.54(m,2H),1.35(d,J=13.0Hz,1H),1.24(s,3H),1.19(s,3H),0.97(s,3H),0.92(s,3H). 13C NMR(100MHz,Chloroform-d)δ176.27,169.49,145.35,142.82,135.87,134.85,131.29,129.39,128.94,128.48,1 25.83,120.59,59.27,46.71,46.46,40.08,34.35,34.32,32.55,28.23,26.97,26.52,22.82,19.68.ESI-Massfor C 28 H 35 NO5: m / z(MH) + 444.59.

[0113] Synthesis of compound CA-7

[0114]

[0115] Add intermediate 11c (0.11 g, 0.13 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.022 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration through diatomaceous earth. The filtrate is concentrated under reduced pressure, and the residual liquid is purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain compound CA-7 (white solid, 0.04 g, yield 54.1%). 1 HNMR(400MHz,Chloroform-d)δ7.92(s,1H),7.79(s,1H),7.77–7.70(m,2H),7.40–7.32(m,2H),7.0 1(t,J=1.0Hz,1H),4.78(d,J=1.0Hz,1H),3.56(d,J=1.0Hz,2H),3.30–3.19(m,3H),2.96(s,3H),2.3 7(d,J=13.0Hz,1H),2.26(s,1H),2.12(s,1H),2.00(d,J=13.0Hz,1H),1.80(d,J=13.0Hz,1H),1.67– 1.54(m,2H),1.35(d,J=13.0Hz,1H),1.26(d,J=15.1Hz,6H),1.19(s,3H),0.97(s,3H),0.92(s,3H). 13C NMR(100MHz,Chloroform-d)δ176.27,171.81,145.35,142.82,139.27,136.29,134.85,130.50,129.24,128.60,126.54,1 20.28,56.71,51.79,46.71,46.41,41.62,40.08,34.35,33.38,32.55,28.43,26.97,26.52,22.82,19.68,8.71.ESI-Mass for C 31 H 41 NO7S:m / z(MH) + 570.73.

[0116] Synthesis of compound CA-8

[0117]

[0118] Add intermediate 11d (0.14 g, 0.16 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.028 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration through diatomaceous earth. The filtrate is concentrated under reduced pressure, and the residual liquid is purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:1 as eluent] to obtain compound CA-8 (white solid, 0.07 g, yield 72.16%). 1 HNMR(400MHz,Chloroform-d)δ7.53(d,J=7.9Hz,2H),7.33(dd,J=8.0,5.2Hz,2H),7.25–7.19(m,2H),6.94(td,J=9.7 ,8.8,6.1Hz,2H),6.42(d,J=9.8Hz,1H),5.53(d,J=6.4Hz,1H),4.92–4.78(m,2H),3.48(t,J=13.7Hz,1H),3.20–3.13( m,1H),2.97(q,J=6.1,5.2Hz,1H),2.91(d,J=5.7Hz,3H),2.08(d,J=6.2Hz,1H),1.92(d,J=14.0Hz,1H),1.78(d,J=14. 5Hz,1H),1.68–1.60(m,2H),1.53–1.47(m,1H),1.31(dd,J=7.0,4.0Hz,6H),1.12(dt,J=6.9,4.4Hz,6H),0.83(s,3H). 13C NMR(101MHz,Chloroform-d)δ180.22,169.07,158.06,148.30,143.49,143.00,142 .46,138.27,134.53,129.92,127.50,127.11,126.88,124.72,123.88,120.64,120 .41,113.57,112.77,67.98,53.53,48.91,48.60,42.16,34.64,34.11,33.82,33.0 8,32.39,31.37,27.14,26.24,24.17,24.02,22.44,22.01,21.47,20.34.ESI-Mass for C 38 H 47 NO6: m / z(MH) + 612.80.

[0119] Synthesis of compound C12

[0120]

[0121] 3-Bromo-4-hydroxy-benzoic acid methyl ester (0.3 g, 1.3 mmol) was added to a 50 ml dry reaction bottle, dissolved in 15 ml of anhydrous THF, and then triphenylphosphine (0.41 g, 1.56 mmol) and 1-BOC-pyrrole-(S)-3-ol (0.26 g, 1.56 mmol) were added. The mixture was cooled to 0°C and stirred for 30 min, and then DIAD (0.41 g, 1.56 mmol) dissolved in 5 ml of anhydrous THF was added dropwise and stirred at room temperature overnight. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 3:1). After the reaction was completed, the solvent was concentrated and removed. The residual liquid was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 10:1 as eluent] to obtain compound C12 (light yellow oily liquid, 0.5 g, yield 98%).

[0122] Synthesis of compound C13

[0123]

[0124] To a 50 ml dry reaction bottle, add intermediate C12 (0.5 g, 1.25 mmol), dissolve with dioxane / EtOH / H2O (5:2:1, 32 ml), then add potassium acetate (0.49 g, 5.0 mmol), 4-isopropyl-phenylboronic acid pinacol ester (0.37 g, 1.5 mmol), Pd(dppf)Cl2 (0.09 g, 0.125 mmol), and stir the reaction at 80 ° C for 48 h under N2 protection. The reaction was monitored by TLC plate (petroleum ether: ethyl acetate = 3:1). After the reaction, the reaction solution was cooled to room temperature, the insoluble matter was filtered out, and the filtrate was concentrated. The residual liquid was purified by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 10:1 as eluent] to obtain C13 (light yellow oily liquid, 0.32 g, yield 59.3%).

[0125] Synthesis of compound C14

[0126]

[0127] Add compound C13 (0.32 g, 0.73 mmol) to a 50 ml dry reaction bottle, dissolve it with 20 ml THF, dissolve lithium hydroxide (0.12 g, 2.91 mmol) in 5 ml water, add it to the reaction solution, and stir at room temperature for 4 hours. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 3:1). After the reaction is completed, concentrate the reaction solution, add 20 ml water to the residual solution, adjust the pH to about 4-5 with 1M HCl, and extract with ethyl acetate (30 ml × 3). Combine the organic phases, wash with 40 ml saturated brine, dry three times with anhydrous Na2SO4, and concentrate the organic phase to obtain compound C14 (white solid, 0.3 g, yield 97%).

[0128] Synthesis of compound C15

[0129]

[0130] Add intermediate C5 (0.15 g, 0.24 mmol) to a 50 ml dry reaction bottle, dissolve with 10 ml DCM, then add DIPEA (0.12 g, 0.96 mmol), HATU (0.09 g, 0.24 mmol), intermediate C14 (0.10 g, 0.24 mmol) and stir at room temperature for 6 h. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, add 20 ml of water to the reaction solution and extract with DCM (10 ml × 3). Collect the organic phase, wash with 20 ml of saturated brine, dry with anhydrous Na2SO4, concentrate the organic phase under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1 as eluent] to obtain compound C15 (light yellow solid, 0.18 g, yield 75.0%).

[0131] Synthesis of compound C16

[0132]

[0133] Add intermediate C15 (0.17 g, 0.23 mmol) to a 50 ml dry reaction bottle, dissolve with 6 ml THF, then add 10% palladium hydroxide on carbon (0.034 g, 20% wt), and stir at room temperature overnight. Monitor the reaction with a TLC plate (petroleum ether: ethyl acetate = 1:1). After the reaction is completed, remove the insoluble matter by filtration through diatomaceous earth, concentrate the filtrate under reduced pressure, and purify the residual liquid by silica gel column chromatography [V (petroleum ether): V (ethyl acetate) = 1:2 as eluent] to obtain compound C16 (white solid, 0.10 g, yield 76.92%).

[0134] Synthesis of compound CA-9

[0135]

[0136] Add intermediate C16 (0.1 g, 0.13 mmol) to a 50 ml dry reaction bottle, dissolve it with 10 ml DCM, then add 1 ml TFA and stir at room temperature for 3 h. Monitor the reaction with a TLC plate (dichloromethane: methanol = 20: 1). After the reaction is completed, concentrate and remove the solvent, add 20 ml DCM again to dissolve the residual liquid, remove the residual trifluoroacetic acid, and repeat the operation 3 times. Then dissolve the product with acetonitrile / water (1: 1, 2 ml), freeze it at -80 ° C for 4 h, and freeze-dry it in a freeze dryer for 18 h. Compound CA-9 (white powder, 0.07 g, yield 81.39%) was obtained. 1H NMR(400MHz,Chloroform-d)δ8.03(d,J=2.0Hz,1H),7.92(s,1H),7.79(s,1H),7.70(dd,J=7.5,2.0Hz,1H),7.41–7.34(m,2H),7.24 –7.16(m,2H),7.02–6.93(m,2H),4.85(d,J=1.0Hz,1H),4.62(s,1H),3.22(d,J=1.0Hz,1H),3.15(dd,J=1.6,0.6Hz,2H),3.06–2.95 (m,2H),2.96(s,3H),2.94–2.87(m,2H),2.37(d,J=13.0Hz,1H),2.27(s,1H),2.11(d,J=4.4Hz,2H),2.00(d,J=13.0Hz,1H),1.87(s ,1H),1.80(d,J=13.0Hz,1H),1.67–1.54(m,2H),1.35(d,J=13.0Hz,1H),1.24(dd,J=20.0,16.6Hz,12H),0.97(s,3H),0.92(s,3H). 13 C NMR(100MHz,Chloroform-d)δ176.27,169.06,154.67,146.41,145.35,142.82 ,138.17,134.85,132.51,129.66,129.39,127.95,127.29,127.05,126.22,125 .83,120.59,115.32,78.37,59.27,49.15,46.71,46.46,43.73,40.08,34.35,3 4.32,33.56,32.55,30.76,28.06,26.97,26.52,24.63,22.82,19.68.ESI-Mass forC 41 H 52 N2O6: m / z(MH)+667.88.

[0137] Biological Activity Test Methods

[0138] The biological activity test in this part is the inhibitory activity of the target product on β-catenin / BCL9 in vitro, and carnosic acid CA is used as the positive control compound. The inhibitory activity of carnosic acid derivatives on β-catenin / BCL9PPI is evaluated by competitive fluorescence polarization (FP) analysis, and the IC50 value is calculated. This method can directly reflect the potency of the inhibitor. The CTG cell proliferation assay is used to evaluate the antiproliferative activity of carnosic acid derivatives on SW480 cells in vitro, and the IC50 value is calculated.

[0139] Experimental Methods A competitive fluorescence polarization (FP) assay was established using full-length β-catenin and BCL9HD2 to evaluate

[0140] Inhibitory activity of the target compound on β-catenin / BCL9 PPI. 200μL FP buffer (25mM HEPES, 100Mm NaCl, 0.01% TritonX-100, 0.1% BSA) and 1μM β-catenin were added to a black 96-well plate, and different concentrations of the target compound (DMSO dissolved) were added to the wells, with a concentration gradient of 0.625, 1.250, 2.500, 5.000, and 10.000μmol / L, respectively. Three replicate wells were made for each concentration condition. Incubate on a horizontal shaker for 2h, and then add 2nM FAP-BCL9 (residues 350-375) tracer to each well. After incubation on a horizontal shaker for 2h, the absorbance was measured. The positive control representing 100% inhibition contains only the tracer. The negative control representing 0% inhibition contains the tracer and β-catenin. The microplate was set at excitation and emission wavelengths of 485 and 535 nm, respectively. The polarization values ​​were then read using Synergy H1. All experiments were repeated three times and the average values ​​were taken. The inhibition rate was calculated according to the formula: percent inhibition = 100 [1-(MP-MP positive) / (MP negative-MP positive)].

[0141] Cell Counting Kit-8 (CCK-8) kit was used to detect cell proliferation. HCT116 cells were plated in a 96-well plate (flat bottom transparent), with 10,000 cells / 100 μl DMEM (10% FBS) per well. The cell status was observed the next day, and the drug was added after the cells were well attached to the wall; the target product was diluted with DMEM (2% FBS) in a gradient dilution to a concentration of 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, 0.15625μM, and 0μM (equal volume of DMSO); 100μL of the target product of different concentrations was added to each well, and 2 replicates were made for each concentration gradient; 3 blank wells were reserved (only DMEM 2% FBS culture medium was added, without cells); the cells were cultured at 37°C for 24 hours; 10μL of CCK-8 enhanced solution was added to each well: since the amount of CCK-8 added to each well was relatively small, errors may be caused by the reagent sticking to the well wall, and the culture plate was gently tapped after the reagent was added to help mix; the cells were incubated in an incubator for 0.5-4 hours: the amount of Formazan formed was different for different cell types, and in most cases, incubation for 1 hour was sufficient. If the color development is not enough, you can continue to culture to confirm the optimal conditions. Measure the absorbance at 450nm and 600nm (excluding the interference of the reagent background color and the absorbance value of the well plate itself); the final absorbance value uses OD450nm-OD600nm, and all experiments are repeated 3 times to calculate the inhibition rate by taking the average value; Inhibition rate = [(Ac-As) / (Ac-Ab)]×100% As: absorbance of the experimental well (culture medium containing cells, CCK-8, and the drug to be tested); Ac: absorbance of the control well (culture medium containing cells, CCK-8, and no drug to be tested); Ab: absorbance of the blank well (culture medium without cells and the drug to be tested, CCK-8).

[0142] Cell proliferation was detected using the CellTiter Glo (Promega, G7572) kit. SW480 cells in the logarithmic growth phase were cultured in a cell culture incubator. Since SW480 is an adherent cell, 4 ml of trypsin was added to the dish after discarding the supernatant of the 10 cm cell culture dish. Digestion was performed in a 37 °C cell culture incubator for 2 minutes. After removal, 4 ml of DMEM medium containing 10% FBS was added to the dish to stop digestion, and the bottom of the culture dish was gently blown with a pipette to form a cell suspension of adherent cells. The cell suspension was transferred to a 15 ml centrifuge tube with a pipette and centrifuged at 1000 rpm and 20 °C in a centrifuge for 5 minutes. After discarding the supernatant in the centrifuge tube, it was resuspended with DMEM medium containing opti-MEM + 1% FBS + 1% PS, and the cell suspension (5×104 cells per well) was added to a 96-well plate with a volume of 100 μL per well. The solvent control group, control group and treatment group were treated with DMSO, culture medium and compounds with different concentration gradients, respectively. The concentration gradient of the compound was 100, 50, 25, 12.5 and 6.25 μM. Three parallel wells were prepared for each concentration. After adding the compound, the plate was gently shaken by hand to mix the compound in the well and incubated for 72 hours. After the incubation, 100 μL of CellTiterGlo (Promega, G7572) was added to each well and placed in a 37°C cell culture incubator for 0.5 hours. After the end, the absorbance at 450 nm was detected using a BioTek SynergyH1 microplate reader.

[0143] 4.3.1 Analysis of activity results of carnosic acid derivatives

[0144] The inhibitory effects of carnosic acid and its derivatives on β-catenin / BCL9 and the antiproliferative activities on SW480 cells are shown in Table 4.

[0145] Table 4 Results of carnosic acid and its derivatives on β-catenin / BCL9 inhibition and cell anti-proliferation

[0146]

[0147] Activity results of carnosic acid derivatives

[0148] According to the analysis of the test results, among the 9 carnosic acid derivatives synthesized from carnosic acid, some compounds maintained the same inhibitory activity as the parent nucleus carnosic acid, and the effects of CA-3 and CA-8 were improved compared with the anti-proliferative effect of carnosic acid.

[0149] In order to verify whether the designed molecule is reasonable, molecular simulation docking was performed on CA-8 to predict the binding mode of CA-8 with β-catenin. Since CA-8 is a pair of racemates, the binding modes of (S)-CA-8 and (R)-CA-8 with β-catenin were predicted during molecular simulation docking. Figure 2 As shown. Obviously, both conformations can occupy the hydrophobic pocket of β-catenin well. Compared with (R)-CA-8, the skeleton of (S)-CA-8 can better mimic the α-helical structure of BCL9 residues L366, I369, L373 and F374. In addition, the phenolic hydroxyl groups of both conformations are predicted to form hydrogen bond interactions with β-catenin residue D145; the carboxyl group forms hydrogen bonds and salt bridge interactions with residue K181, which is similar to the parent core.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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 carnosic acid derivative, characterized in that: One of the following compounds:

2. The carnosic acid derivative of claim 1, selected from: CA-3:

3. Use of the carnosic acid derivative according to claim 1 in the preparation of β-catenin / BCL9 PPI inhibitors.

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