Salicylaldehyde carbazole compounds and applications thereof

Abstract

The application discloses a kind of carbazole compounds containing salicylaldehyde, it is characterized in that: its structural formula is as follows: Wherein R2For halogen, methyl, methoxy or carbazole isopropyl alcohol group;R1For hydrogen or bromine.The application is based on carbazole;With isopropanol introduction different substituent salicylaldehyde, design and synthesis series salicylaldehyde carbazole compounds.Series compounds show good bacteriostatic activity by anti tea tree wheel spot and tea tree anthracnose experiment.It is found by anti colon cancer (MC38), melanoma (B16) and lung cancer cell (A549 and PC9) experiment and mechanism research, compound 1o has anticancer potential to MC38 and B16, can effectively inhibit cancer cell growth and proliferation.Therefore, carbazole salicylaldehyde derivative synthesized in the present application shows potential anticancer activity and antibacterial activity, provides important scientific basis for the research and creation of new antibacterial and anticancer drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to a preparation method of a salicylaldehyde carbazole compound containing different substituents and the application thereof in resisting plant fungi and resisting cancer. BACKGROUND

[0002] The chemical formula of carbazole is C 12 H9N, also known as 9-azafloren, iminodiphenylene, has the characteristics of low toxicity, simple structure and wide biological activity, so its skeleton structure is widely concerned in the fields of medicine and pesticide. Related studies have shown that natural products carbazole have biological activities such as antibacterial, antioxidant, anti-inflammatory, anticancer, antitumor, antihistamine, antiviral, anti-Alzheimer's disease and treatment of neurodegenerative diseases.

[0003]

[0004] The chemical structure of salicylaldehyde is C7H6O2, also known as 2-hydroxybenzaldehyde or o-hydroxybenzaldehyde. Salicylaldehyde, as a common natural product, has biological activities such as antibacterial, anti-leukemia, antiviral, anti-tuberculosis, anti-tumor and treatment of diabetes (for example: aspirin, also known as acetylsalicylic acid, has a high structural similarity with salicylaldehyde).

[0005]

[0006] Although there are few commercialized drugs of carbazole in the fields of medicine and pesticide, the literature in the aspects of antibacterial, anticancer, antitumor and treatment of HIV shows that carbazole has the potential to be deeply developed into medicine and pesticide. Salicylaldehyde is also a green and low-toxic natural product, which is widely used in the fields of medicine and pesticide in recent years. Therefore, in this study, propanolamine, which is often used to connect different active units in the fields of anticancer and antibacterial, is selected as a bridge to explore the influence of salicylaldehyde containing different substituents on the biological activity of carbazole skeleton. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a salicylaldehyde carbazole derivative with anti-plant fungal and anticancer properties, which is used for treating tea anthracnose, tea cercospora leaf spot, treating colon cancer, melanoma and lung cancer cells.

[0008] The technical scheme of the present application is a kind of carbazole derivative containing salicylaldehyde, and its structural formula is as follows: In the formula, R1 is hydrogen or bromine; R2 is ortho, meta or para halogen, methyl, methoxy or carbazole isopropyl alcohol group. The halogen, methyl or methoxy is ortho, meta or para substitution.

[0009] The reaction formula is as follows:

[0010]

[0011] The application of the salicylaldehyde carbazole compound to the preparation of a fungicide for preventing and treating plant fungi and an anticancer agent. The cancer includes colon cancer and lung cancer.

[0012] Advantages of the present application: There are many literatures on the application of carbazole and salicylaldehyde to antibacterial and anticancer, respectively, but there are few literatures on the combination of the two for antibacterial and anticancer. By connecting the two through propanolamine structure, different substituted salicylaldehydes such as fluorine, chlorine, bromine, methyl and methoxy are used, and the 3-position of carbazole is designed as "hydrogen" or "bromine". The two are combined to design and synthesize a series of salicylaldehyde carbazole derivatives containing different substituents. Through the biological activity test of the compounds on tea tree anthracnose, tea tree wheel spot, colon cancer, melanoma and lung cancer cells, it is found that the compounds exhibit good biological activity on tea tree anthracnose, tea tree wheel spot, colon cancer, melanoma and lung cancer cells. Finally, the carbazole double substitution of salicylaldehyde shows good inhibition activity on tea tree anthracnose, tea tree wheel spot, colon cancer, melanoma and lung cancer cells, which provides an important theoretical basis for the research and development of anticancer drugs and pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 To inhibit the growth and proliferation of cancer cells by compound 1o; (A-C) MC38 cells or (D-F) B16 cells were cultured in DMEM containing 5% FBS and treated with compound 1o at 5, 10 or 20 μM, DMSO as negative control or 5 μM 5-fluorouracil as positive control; cell viability was measured at 0, 24, 48 and 72 h using CCK-8; 2 replicates of experiments, n = 6;

[0014] Figure 2 To induce apoptosis and necrosis by compound 1o; (A-B) MC38 or (C-D) B16 cells were incubated with 5, 10 or 20 μM compound 1o or 5 μM 5-fluorouracil for 24 h; DMSO was used as control. Apoptosis was detected using Annexin V and propidium iodide (PI) staining; cell status was identified using FACS: Annexin V–PI– live cells (Q4), Annexin V+PI– early apoptotic cells (Q3), Annexin V+PI+ late apoptotic cells (Q2), Annexin V–PI+ necrotic cells (Q1); 2 replicates of experiments, n = 5-6, representative experimental results are shown;

[0015] Figure 3To reduce cancer cell ATP production by inhibiting glycolysis for compound 1o; (A) MC38 cells were cultured in DMEM containing 5% FBS and treated with DMSO (control), 5, 10 or 20 mM compound 1o for 24 h; ECAR was detected using seahorse technology; glycolysis and glycolytic capacity were calculated after addition of 10 mM glucose or 1 mM oligomycin, respectively;

[0016] Figure 4 To inhibit gene expression of Akt / mTOR and glycolytic enzymes for compound 1o; (A-G) MC38 cells were cultured in DMEM medium containing 5% FBS; then treated with 5 mM compound 1o for 12 h, DMSO as control; mRNAs were measured using qPCR; 2 experimental replicates, n = 6. DETAILED DESCRIPTION

[0017] Synthetic route of salicylaldehyde-containing carbazole derivatives

[0018]

[0019] Carbazole was used as the starting material to synthesize target compounds 1a-1o and 2a-2h through electrophilic reaction and epoxide ring opening.

[0020] Preparation of intermediate 1

[0021] Preparation of 9-(oxirane-2-ylmethyl)-9H-carbazole

[0022]

[0023] Carbazole (59.09 mmol) was dissolved in DMF (30 mL), then potassium carbonate (59.09 mol) and epichlorohydrin (70.91 mmol) were added in turn. The mixture was stirred at 60 °C for 6 h to complete the reaction. Then the reaction mixture was extracted with ethyl acetate and dried with anhydrous sodium sulfate. Further purification was performed by silica gel column chromatography using a mixture of ethyl acetate and petroleum ether in a ratio of 20: 1 to obtain intermediate 1 as the final product. White solid, yield 82%; melting point 126-127 °C. 1 HNMR (400 MHz, CDC13) δ 8.09 (d, J = 7.8 Hz, 2H), 7.52 - 7.39 (m, 4H), 7.27 - 7.23 (m, 2H), 4.61 (dd, J = 15.9, 3.4 Hz, 1H), 4.38 (dd, J = 15.9, 4.8 Hz, 1H), 3.33 (dt, J = 7.3, 3.6 Hz, 1H), 2.81 - 2.76 (m, 1H), 2.55 (dd, J = 4.8, 2.6 Hz, 1H); 13C NMR (101 MHz, CDC13) δ 140.75, 125.93, 123.06, 120.39, 119.42, 108.81, 50.61, 45.38, 44.62; HRMS (ESI): m / z calcd for C 15 H 13 NO[M+H] + 223.09971, found: 223.09934.

[0024] Synthesis of target compounds 1a-1o (example of synthesis of compound 1a)

[0025]

[0026] Intermediate 1 (2.22 mmol) was dissolved in acetonitrile (4 mL) and then K2CO3(2.22 mmol) and hydroxyaldehyde (2.22 mmol) were added sequentially. The reaction was carried out at 75 °C for 8 h. Upon completion, the solvent was removed under vacuum and the product was purified by silica gel column chromatography using a mixture of ethyl acetate and diethyl ether (ratio 25:1) for elution.

[0027] Target compound 1a

[0028]

[0029] White solid, yield 32%; melting point 118-119 °C. 1 H NMR (400 MHz, CDC13) δ 10.38 (s, 1H), 8.07 (d, J = 7.8 Hz, 2H), 7.79 (dd, J = 7.7, 1.8 Hz, 1H), 7.48 - 7.39 (m, 5H), 7.24 - 7.20 (m, 2H), 7.07 (t, J = 7.5 Hz, 1H), 6.77 (d, J = 9.3 Hz, 1H), 4.65 - 4.47 (m, 3H), 4.12 - 3.97 (m, 2H), 3.13 (s, 1H); 13 C NMR (101 MHz, CDC13) δ 189.90, 159.98, 140.66, 136.06, 131.01, 126.02, 125.18, 123.08, 121.60, 120.45, 119.47, 113.23, 108.79, 70.09, 68.84, 45.63; HRMS (ESI): m / z calcd for C 25 H 18 NO3[M+Na] + 368.13649, found: 368.12592.

[0030] Target compound 1b

[0031]

[0032] White solid, yield 43%; melting point 141-142 °C. 1 H NMR (400 MHz, CDC13) δ 10.33 (d, J = 2.4 Hz, 1H), 8.06 (dd, J = 7.8, 1.1 Hz, 2H), 7.46 (dd, J = 8.1, 3.3 Hz, 1H), 7.44 - 7.39 (m, 4H), 7.24 (dd, J = 3.5, 1.6 Hz, 1H), 7.23 - 7.20 (m, 1H), 7.15 (ddd, J = 9.1, 7.5, 3.3 Hz, 1H), 6.73 (dd, J = 9.1, 3.9 Hz, 1H), 4.60 - 4.46 (m, 3H), 3.99 (tdd, J = 9.8, 7.0, 2.9 Hz, 2H); 13 C NMR (101 MHz, CDC13) δ 188.39, 156.42, 140.62, 126.05, 123.12, 122.76, 122.52, 120.51, 119.56, 116.00, 115.76, 114.92, 108.71, 70.79, 68.94, 45.66; 19 F NMR (376 MHz, CDC13) δ -121.07; HRMS (ESI): m / z calcd for C 22 H 17 F N03[M + Na] + 386.11629, found 386.11653.

[0033] Target compound 1c

[0034]

[0035] White solid, yield 78%; melting point 107-108 °C. 1H NMR (400 MHz, CDC13) δ 10.42 (s, 1H), 8.06 (dt, J = 7.7, 0.9 Hz, 2H), 7.45 (dt, J = 8.4, 1.0 Hz, 2H), 7.42 - 7.35 (m, 3H), 7.21 (ddd, J = 7.9, 6.9, 1.2 Hz, 2H), 6.79 - 6.71 (m, 1H), 6.48 (d, J = 8.4 Hz, 1H), 4.61 (dd, J = 14.6, 7.4 Hz, 1H), 4.55 - 4.45 (m, 2H), 3.97 (dd, J = 9.3, 3.7 Hz, 1H), 3.89 (dd, J = 9.3, 4.2 Hz, 1H); 13 C NMR (101 MHz, CDC13) δ 186.99, 166.75, 164.16, 159.88, 140.61, 136.50, 125.98, 123.03, 120.39, 119.39, 109.42, 109.21, 108.82, 70.77, 68.36, 45.28; 19 F NMR (376 MHz, CDC13) δ -118.05; HRMS (ESI): m / z calcd for C 22 H 17 F NO3 [M + Na] + 386.11629, found 386.11608.

[0036] Target compound 1d

[0037]

[0038] White solid, yield 62%; melting point 114-115 °C. 1 H NMR (400 MHz, CDC13) δ 10.32 (s, 1H), 8.08 (dt, J = 7.7, 1.0 Hz, 2H), 7.75 (d, J = 2.8 Hz, 1H), 7.45 - 7.39 (m, 5H), 7.25 - 7.22 (m, 2H), 6.74 (d, J = 8.9 Hz, 1H), 4.61 - 4.50 (m, 3H), 4.07 - 3.98 (m, 2H), 2.82 (s, 1H); 13 C NMR (101 MHz, CDC13) δ 188.26, 158.53, 140.60, 135.54, 129.89, 127.19, 126.06, 123.11, 120.51, 119.57, 114.72, 108.69, 70.48, 68.84, 45.65, 29.72; HRMS (ESI): m / z calcd for C22 H 17 ClNO3[M+Na] + 402.08674, found 402.08638.

[0039] Target compound 1e

[0040]

[0041] White brown solid, yield 60%; m.p. 108-109 °C. 1 H NMR (400 MHz, CDC13) δ 10.50 (s, 1H), 8.04 (dt, J = 7.8, 1.0 Hz, 3H), 7.47 - 7.34 (m, 5H), 7.24 (d, J = 8.2 Hz, 1H), 7.19 (ddd, J = 8.0, 6.9, 1.2 Hz, 2H), 7.02 (dd, J = 8.1, 0.9 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 5.25 (s, 1H), 4.63 - 4.53 (m, 1H), 4.52 - 4.37 (m, 3H), 3.93 - 3.76 (m, 4H); 13 C NMR (101 MHz, CDC13) δ 160.01, 140.62, 139.04, 135.25, 125.98, 123.59, 123.02, 122.29, 120.39, 119.38, 112.90, 108.85, 70.77, 68.39, 60.47, 53.50, 45.23, 21.09, 14.24; HRMS (ESI): m / z calcd for C 22 H 17 ClNO3[M+Na] + 402.08674, found 402.08676.

[0042] Target compound 1f

[0043]

[0044] White brown solid, yield 60%; m.p. 108-109 °C. 1H NMR (400 MHz, CDC13) δ 10.35 (s, 1H), 7.99 (dt, J = 7.7, 0.9 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.33 (ddd, J = 8.2, 7.0, 1.2 Hz, 2H), 7.18 - 7.11 (m, 4H), 6.59 (dd, J = 8.0, 1.2 Hz, 1H), 4.54 (dd, J = 14.9, 8.0 Hz, 1H), 4.49 - 4.43 (m, 1H), 4.41 - 4.36 (m, 1H), 3.89 (dd, J = 9.3, 3.6 Hz, 1H), 3.81 (dd, J = 9.3, 4.2 Hz, 1H); 13 C NMR (101 MHz, CDC13) δ 192.08, 159.96, 140.59, 135.43, 128.17, 127.03, 125.98, 123.37, 123.04, 120.41, 119.38, 113.88, 108.79, 70.87, 68.38, 45.19. HRMS (ESI): m / z calcd for C 19 H 22 04N2[M+H] + 343.16443, found: 343.16523.

[0045] Target compound 1g

[0046]

[0047] Brown oil, yield 90%. 1 H NMR (400 MHz, CDC13) δ 10.31 (s, 1H), 8.08 (dt, J = 7.8, 1.0 Hz, 2H), 7.89 (d, J = 2.6 Hz, 1H), 7.55 (dd, J = 8.8, 2.6 Hz, 1H), 7.46 - 7.40 (m, 4H), 7.27 - 7.23 (m, 2H), 6.69 (d, J = 8.9 Hz, 1H), 4.63 - 4.49 (m, 3H), 4.03 (tdd, J = 9.4, 8.3, 7.2, 3.2 Hz, 2H), 2.87 (s, 1H); 13 C NMR (101 MHz, CDC13) δ 188.13, 158.98, 140.59, 138.43, 133.00, 126.41, 126.07, 123.12, 120.52, 119.59, 115.12, 114.27, 108.67, 70.42, 68.85, 45.64; HRMS (ESI): m / z calcd for C 22H 17 BrNO3[M+Na] + 446.03623, found 446.03599.

[0048] Target compound 1h

[0049]

[0050] White oil, yield 49%. 1 H NMR (400 MHz, CDC13) δ 10.33 (s, 1H), 8.09 (dt, J = 7.7, 1.0 Hz, 2H), 7.66 (d, J = 8.3 Hz, 1H), 7.48 - 7.40 (m, 4H), 7.27 - 7.22 (m, 5H), 7.01 (d, J = 1.6 Hz, 1H), 4.62 - 4.52 (m, 3H), 4.13 - 4.08 (m, 2H), 4.07 - 4.02 (m, 1H); 13 C NMR (101 MHz, CDC13) δ 188.63, 160.19, 140.60, 131.69, 130.63, 125.09, 124.03, 123.12, 120.51, 119.57, 116.84, 70.47, 68.77, 60.44, 45.70, 31.95, 29.38, 22.71, 21.06, 14.14; HRMS (ESI): m / z calcd for C 22 H 17 BrNO3[M+Na] + 446.03623, found 446.03593.

[0051] Target compound 1i

[0052]

[0053] Clear oil liquid, yield 87%. 1H NMR (400MHz, CDCl3) δ10.14(s,1H),8.07(dt,J=7.8,1.0Hz,2H),7.59(dd,J=7.7,1.9 Hz,1H),7.49(dt,J=8.3,1.0Hz,2H),7.44(ddd,J=8.2,6.9,1.2Hz,2H),7.38(ddd,J=7 .5,1.9,0.9Hz,1H),7.23(ddd,J=7.9,7.1,1.2Hz,2H),7.15(t,J=7.6Hz,1H),4.57–4 .44(m,3H),4.02–3.97(m,1H),3.96–3.90(m,1H),3.77(d,J=3.9Hz,1H),2.16(s,3H); 13 C NMR (101MHz, CDCl3) δ191.43,158.77,140.74,138.04,132.46,130.66,128.81,125 .93,124.61,123.09,120.41,119.37,109.01,69.40,45.83,16.36; HRMS(ESI):m / z calcd for C 23 H 20 NO3[M+Na] + 382.14137, found 382.14138.

[0054] Target compound 1j

[0055]

[0056] White solid, yield 64%; melting point 73-74℃. 1 H NMR (400MHz, CDCl3) δ10.28–10.23(m,1H),8.10–7.96(m,2H),7.63(dt,J=7.8,1.6Hz,1H),7.46–7.32(m,4H),7.20(ddd,J=7.9,6.6,1.4H z,2H),6.87–6.77(m,1H),6.51(dd,J=4.7,2.4Hz,1H),4.60–4.40(m,3H),3.96(dddd,J=13.6,9.6,5.6,2.0Hz,2H),2.27(d,J=1.7Hz,3H); 13C NMR (101 MHz, CDC13) δ 189.53, 159.98, 147.64, 140.67, 131.28, 126.01, 123.09, 122.61, 120.44, 119.45, 114.02, 108.81, 70.07, 68.81, 45.63, 29.73, 22.22; 19 F NMR (376 MHz, CDC13) δ -112.54; HRMS (ESI): m / z calcd for C 23 H 20 NO3[M + Na] + 382.14137, found 382.14132.

[0057] Target compound 1k

[0058]

[0059] A white solid, yield 86%; m.p. 111-112 °C. 1 H NMR (400 MHz, CDC13) δ 10.36 (s, 1H), 8.07 (dt, J = 7.8, 1.0 Hz, 2H), 7.59 (d, J = 2.3 Hz, 1H), 7.49 - 7.37 (m, 5H), 7.29 - 7.18 (m, 4H), 6.69 (d, J = 8.4 Hz, 1H), 4.65 - 4.57 (m, 1H), 4.55 - 4.48 (m, 3H), 4.01 (qd, J = 9.5, 4.1 Hz, 3H), 3.09 (d, J = 5.0 Hz, 1H), 2.31 (s, 4H); 13 C NMR (101 MHz, CDC13) δ 189.99, 158.07, 140.67, 136.65, 131.20, 131.10, 126.01, 124.94, 123.09, 120.44, 119.46, 113.43, 108.78, 70.27, 68.92, 45.64, 20.31; HRMS (ESI): m / z calcd for C 23 H 20 NO3[M + Na] + 382.14137, found 382.14132.

[0060] Target compound 1l

[0061]

[0062] White solid, yield 86%; melting point 111-112 °C. 1 H NMR (400 MHz, CDC13) δ 10.24 (s, 1H), 8.11 (s, 1H), 8.09 (s, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.43 (dd, J = 8.2, 1.2 Hz, 1H), 7.26 (s, 2H), 7.24 (s, 1H), 6.27 (d, J = 2.2 Hz, 1H), 4.67 - 4.62 (m, 1H), 4.62 - 4.53 (m, 2H), 4.08 - 4.05 (m, 1H), 4.04 - 4.01 (m, 1H), 3.78 (s, 3H). 13 C NMR (101 MHz, CDC13) δ 189.44, 154.80, 154.23, 140.67, 126.02, 125.43, 123.17, 123.07, 120.45, 119.48, 115.11, 112.51, 108.80, 70.83, 69.01, 55.84, 45.68. HRMS (ESI): m / z calcd for C 23 H 20 NO4[M+Na] + 398.13628, found 398.13798.

[0063] Target compound 1m

[0064]

[0065] Pure white solid, yield 49%; melting point 132-133 °C. 1 H NMR (400 MHz, CDC13) δ 10.38 (s, 1H), 8.06 (dt, J = 7.8, 1.0 Hz, 2H), 7.46 - 7.38 (m, 4H), 7.28 (d, J = 3.2 Hz, 1H), 7.22 (ddd, J = 8.0, 6.2, 1.7 Hz, 2H), 7.02 (dd, J = 9.0, 3.2 Hz, 1H), 6.73 (d, J = 9.0 Hz, 1H), 4.61 - 4.45 (m, 3H), 3.98 (qd, J = 9.5, 4.1 Hz, 2H), 3.78 (s, 3H), 2.98 (d, J = 4.8 Hz, 1H); 13C NMR (101 MHz, CDC13) δ 189.44, 154.80, 154.23, 140.67, 126.02, 125.43, 123.17, 123.07, 120.45, 119.48, 115.11, 112.51, 108.80, 70.83, 69.01, 55.84, 45.68; HRMS (ESI): m / z calcd for C 23 H 20 NO4[M + Na] + 398.13628, found 398.13798. Target compound In

[0066]

[0067] Brown solid, yield 61%; melting point 101-102 °C. 1 H NMR (400 MHz, CDC13) δ 11.43 (s, 1H), 9.71 (s, 1H), 8.09 (d, J = 7.7 Hz, 2H), 7.48 - 7.39 (m, 5H), 7.24 (td, J = 6.9, 5.9, 2.0 Hz, 3H), 6.54 (dd, J = 8.6, 2.3 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.55 (dt, J = 23.0, 9.7 Hz, 3H), 4.11 - 3.93 (m, 2H); 13 C NMR (101 MHz, CDC13) δ 165.18, 164.35, 140.63, 135.50, 126.05, 123.12, 120.49, 119.56, 115.71, 108.71, 108.28, 101.65, 69.49, 68.91, 45.55; HRMS (ESI): m / z calcd for C 22 H 18 NO4[M + Na] + 384.12063, found 384.12047.

[0068] Target compound lo

[0069]

[0070] White solid, yield 57%; melting point 125-126 °C. 1H NMR (400MHz, CDCl3) δ10.17(d,J=2.1Hz,1H),8.09–8.03(m,4H),7.68(d,J=8.6Hz,1H),7.43–7.35(m,7H),7.20(dddd,J=9.9,7.0, 3.7,1.6Hz,4H),6.48(ddd,J=8.7,3.4,2.2Hz,1H),6.16(dd,J=4.7,2.2Hz,1H),5.29(s,2H),4.59–4.38(m,6H),4.01–3.76(m,4H); 13 CNMR(101MHz, CDCl3)δ188.48,164.44,161.67,140.60,133.47,126.01,123.08,120.49,119.74,119.54, 119.48,119.35,108.78,107.40,107.27,99.96,70.11,69.45,68.78,68.62,53.45,45.48; HRMS(ESI):m / z calcd forC 37 H 31 N₂O₅[M+Na] + 607.22034, found 607.22194.

[0071] Preparation of intermediate 2

[0072] Preparation of 3-bromo-9-(ethylene-2-ylmethyl)-9H-carbazole

[0073]

[0074] 3-Bromocarbazole (20.15 mmol) was dissolved in DMF (15 mL), followed by the sequential addition of potassium carbonate (20.15 mmol) and epichlorohydrin (24.18 mmol). The mixture was stirred at 75 °C for 8 h to complete the reaction. The reaction mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. Further purification was performed using silica gel column chromatography with a mixture of ethyl acetate and petroleum ether in a ratio of 30:1 to give intermediate 2, a brown solid, as the final product, in 61% yield, with a melting point of 132–133 °C. 1HNMR (400 MHz, CDC13) δ 8.12 (d, J = 1.9 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.51 - 7.40 (m, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.27 - 7.18 (m, 2H), 4.51 (dd, J = 15.9, 3.0 Hz, 1H), 4.19 (dd, J = 15.9, 5.0 Hz, 1H), 3.27 - 3.20 (m, 1H), 2.75 - 2.69 (m, 1H), 2.44 (dd, J = 4.8, 2.6 Hz, 1H). 13 CNMR (101 MHz, CDC13) δ 140.99, 139.39, 128.54, 126.69, 124.73, 123.06, 121.97, 120.59, 119.86, 112.23, 110.44, 109.07, 50.54, 45.14, 44.66. HRMS (ESI): m / z calcd for C 15 H 12 BrNO[M+Na] + 324.00001, found: 324.00036.

[0075] Synthesis of target compounds 2a-2d (synthesis of compound 2a as an example)

[0076]

[0077] Illustrated by 2a. Intermediate 2 (1.65 mmol) was dissolved in acetonitrile (4 mL), followed by the addition of K2CO3(1.65 mmol) and salicylaldehyde (1.65 mmol) in sequence. The reaction was carried out at 80 °C for 8 h. After completion, the solvent was removed under vacuum, and the product was purified by silica gel column chromatography using a mixture of ethyl acetate and diethyl ether (ratio 30:1) for elution.

[0078] Target compound 2a

[0079]

[0080] Brown solid, yield 43%; melting point 121-122 °C. 1H NMR (400 MHz, CDC13) δ 10.28 (s, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.50 - 7.38 (m, 5H), 7.29 (d, J = 8.6 Hz, 1H), 7.21 (dt, J = 7.9, 4.0 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 4.53 - 4.41 (m, 2H), 4.08 (q, J = 7.2 Hz, 1H), 4.00 (d, J = 5.6 Hz, 1H), 3.93 (d, J = 5.8 Hz, 1H). 13 C NMR (101 MHz, CDC13) δ 190.15, 159.84, 140.96, 139.40, 136.23, 131.57, 128.65, 126.81, 125.63, 123.21, 121.78, 120.70, 119.94, 113.31, 112.29, 110.43, 109.12, 70.05, 68.79, 45.75, 21.18, 14.29. HRMS (ESI): m / z calcd for C 22 H 18 BrNO3[M+Na] + 446.03623, found 446.03676.

[0081] Target compound 2b

[0082]

[0083] Off-white solid, yield 52%; melting point 123-124 °C. 1 H NMR (400 MHz, CDC13) δ 10.32 (d, J = 2.4 Hz, 1H), 8.15 (d, J = 2.1 Hz, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.49 - 7.42 (m, 3H), 7.31 (d, J = 8.7 Hz, 1H), 7.25 (s, 1H), 6.78 (dd, J = 9.1, 3.9 Hz, 1H), 4.55 - 4.48 (m, 2H), 4.10 (s, 1H), 4.08 (s, 1H), 3.99 (d, J = 4.4 Hz, 0H). 13C NMR (101 MHz, CDC13) δ 188.59, 171.41, 140.94, 139.38, 128.69, 126.84, 123.27, 122.69, 122.10, 120.75, 120.03, 116.32, 114.95, 112.37, 110.37, 109.05, 70.74, 68.89, 61.26, 45.81, 21.17, 14.28. HRMS (ESI): m / z calcd for C 22 H 17 BrFNO3[M+Na] + 464.026806, found 464.02727.

[0084] Target compound 2c

[0085]

[0086] Brown-white solid, yield 57%; melting point 116-117 °C. 1 H NMR (400 MHz, CDC13) δ 10.34 (s, 1H), 8.18 (d, J = 1.9 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.60 (s, 1H), 7.47 (t, J = 5.9 Hz, 2H), 7.37 - 7.28 (m, 2H), 6.72 (d, J = 8.5 Hz, 1H), 4.62 (dd, J = 16.2, 8.4 Hz, 1H), 4.53 (dd, J = 11.7, 4.6 Hz, 1H), 4.06 (dd, J = 9.5, 4.0 Hz, 1H), 3.99 (dd, J = 9.5, 4.3 Hz, 1H), 2.33 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 190.16, 159.35, 141.35, 139.85, 137.35, 130.26, 128.29, 128.25, 126.82, 124.55, 123.13, 121.62, 121.16, 119.69, 113.91, 112.36, 111.36, 110.48, 71.11, 68.25, 46.42, 20.28. HRMS (ESI): m / z calcd for C 23 H 20 BrNO3[M+Na] + 460.05188, found 460.05145.

[0087] Target compound 2d

[0088]

[0089] Brown solid, yield 48%; melting point 114-115 °C. 1 H NMR (400 MHz, CDC13) δ 10.39 (s, 1H), 8.19 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 6.6 Hz, 1H), 7.46 (d, J = 4.0 Hz, 2H), 7.37 - 7.28 (m, 3H), 7.07 (dd, J = 9.1, 3.2 Hz, 1H), 6.79 (d, J = 9.1 Hz, 1H), 4.60 (dd, J = 16.4, 8.3 Hz, 1H), 4.55 - 4.49 (m, 2H), 4.05 (dd, J = 9.4, 3.8 Hz, 1H), 3.99 (dd, J = 9.5, 4.4 Hz, 1H), 3.81 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 189.91, 155.88, 153.74, 141.35, 139.85, 128.29, 126.83, 125.18, 124.54, 123.49, 123.14, 121.62, 121.16, 119.70, 115.85, 112.37, 111.35, 110.77, 110.49, 71.70, 68.32, 56.02, 46.43. HRMS (ESI): m / z calcd for C 23 H 20 BrNO4[M+Na] + 476.04679, found 476.04694.

[0090] Method for testing antifungal and anticancer biological activities of target compounds

[0091] Preparation of test materials and culture medium

[0092] Plant fungi: P. trachicarpicola (Pestalotiopsis trachicarpicola) and C. camelliae (Colletotrichum camelliae) were provided by Guizhou Tea Research Institute. Carboxin (analytical standard, Aralab Reagent Co., Ltd. Shanghai).

[0093] Cancer cells: MC38 (mouse colon cancer cancer cell line), B16 (mouse melanoma cell line), A549 (human non-small cell lung cancer cell line), and PC9 (human non-small cell lung cancer cell line carrying EGFR sensitive mutation) were obtained from ATCC (American Type Culture Collection).

[0094] Test of the activity of the compounds on plant fungi

[0095] 1) Preparation of the drug solution and PDA medium

[0096] Preparation of the PDA medium: 800 g of peeled potatoes were weighed, boiled to extract the juice, and then filtered. Agar 80 g and glucose 80 g were added, mixed and dissolved, and then transferred to 200 mL conical flasks at 90 mL per flask. After sealing, the medium was autoclaved at 120°C for 30 min, and then cooled for storage.

[0097] 10 mg of the compound to be tested was weighed and dissolved in 1.0 mL of DMSO. The solution was transferred to a 15 mL centrifuge tube containing 9.0 mL of sterile Tween water in a sterile clean bench, and then added to the sterilized 90 mL PDA medium and mixed to obtain a final concentration of 100 μg / mL. The medium was evenly poured into 9 culture dishes and cooled for storage. An equal amount of DMSO in Tween water was used as a blank control, and a commercial drug, iprodione, was used as a control drug.

[0098] 2) Test of the activity of the compounds on plant fungi

[0099] The inhibitory activity of the target compounds on 8 plant pathogenic fungi was determined by the mycelial growth rate method. The edge of the pre-activated fungus was punched to form a 4.0 mm diameter fungus cake, which was transferred to the center of the drug-containing medium with a sterile inoculation needle and incubated in a constant temperature incubator at 28°C for 2-6 days. When the mycelial diameter of the blank control group reached about 6.0 cm, the mycelial diameter was measured by the cross method with a ruler. The inhibition rate was calculated according to the following formula, where I is the inhibition rate, C is the measured diameter of the blank control mycelium, and T is the measured diameter of the drug-treated group.

[0100] I (%) = [(C-T) / (C-0.4)] x 100

[0101] 3) Analysis of the activity data of the target compounds against plant fungi

[0102] The inhibitory activity of the target compounds on tea tree and tea tree anthracnose was tested by the mycelial growth rate method, and the results are shown in Table 1.

[0103] Table 1. Test results of the activity of compounds 1a-2d on five plant fungi at a concentration of 100 μg / mL

[0104] Table 1. Test results of the activity of compounds 1a-2d on five plant fungi at a concentration of 100 μg / mL

[0105]

[0106] "a" each group of experiments was repeated three times.

[0107] The analysis of Table 1 shows that the series of salicylaldehyde carbazole compounds have certain inhibitory activity on tea anthracnose and tea cercospora. First, the inhibitory activity of the series of compounds on tea cercospora is Cl > F > Br, and the activity of the benzene ring connected with the electron-donating group is greater than that connected with the electron-withdrawing group. The inhibitory activity on tea anthracnose is F > Cl > Br, and the activity of the benzene ring connected with the electron-donating group is greater than that connected with the electron-withdrawing group. Finally, the compound 1o with carbazole double substitution shows the best antibacterial activity on tea cercospora and tea anthracnose.

[0108] Anti-cancer activity test of compounds

[0109] 1) Anti-cancer activity test of compounds

[0110] MC38 or B16 cells were cultured in DMEM medium containing 5% FBS, and then treated with compounds 1a-1o or 2a-2d at concentrations of 0, 2.5, 5, 10, 20 or 40 μM for 48 hours, respectively. DMSO was used as a negative control, and 5-fluorouracil was used as a positive control. Cell viability was determined using a CCK-8 kit. The inhibition efficiency at 10 μM and the 50% inhibition concentration (IC 50 value) of each compound on MC38 or B16 were calculated using Graphpad Prism version 9.5.0.

[0111] 2) Analysis of activity data of target compounds on colon cancer and melanoma

[0112] The inhibitory activity of the target compounds on cancer cells was determined using a CCK-8 kit, and the results are shown in Table 2.

[0113] Table 2. Inhibition rate and IC 50 value of compounds 1a-1o and 2a-2d on colon cancer cells and melanoma cells.

[0114]

[0115]

[0116] a. Average of three replicates; b. Commercial anticancer agent 5-fluorouracil was used as a positive control.

[0117] The anti-cancer activity of the series of compounds against MC38 and B16 was determined using CCK8 assay, and the results are shown in Table 2. The inhibition rates of 1a-1o and 2a-2d against MC38 and B16 at 10 mM were 19.01% to 90.43% and 11.60% to 93.53%, respectively. The IC 50 values of the compounds against MC38 and B16 were 2.04 to 19.08 and 2.9 to 37.37, respectively. For MC38, the activity was better when -R2 was an electron-donating group compared to an electron-withdrawing group (1n, 1m > 1a, 1f); the activity was best when the electron-donating group was at the para position (1k > 1j > 1i); enhancing the electron-donating ability of the group helped to improve the anti-cancer activity (1n > 1m > 1k). For B16, the activity was better when -R2 was an electron-withdrawing group compared to an electron-donating group (1e, 1g > 1m, 1n); the activity was best when the electron-withdrawing group was at the para position (1g > 1e > 1f); the activity order of the electron-withdrawing group was -Br > -Cl > -F (1g > 1d > 1b). When the -R1 substituent was -Br instead of -H, the target compounds observed showed better anti-cancer activity (2a, 2b > 1a, 1b). Finally, the target compound 1o with a disubstituted carbazole showed the best anti-cancer activity (when -R1 = H) with IC 50 values of 2.04 mM and 2.9 mM, which were 1.73 mM and 0.12 mM lower than the IC 50 values of the positive control drugs 5-fluorouracil (3.77 mM and 3.02 mM), respectively.

[0118] 3) Analysis of the activity data of the target compounds against human lung cancer cells

[0119] Table 3 Inhibition rates and IC 50 values of compounds 1a-1o and 2a-2d against lung cancer cells A549 and PC9.

[0120]

[0121]

[0122] a. Mean of triplicate; b. Commercial anti-cancer agents osimertinib and 5-fluorouracil were used as positive controls.

[0123] The anti-cancer activity of the series of compounds against A549 and PC9 was determined using CCK8 assay, and the results are shown in Table 3. The inhibition rates of 1a-1o and 2a-2d against A549 and PC9 at 10 mM were 6.19% to 45.45% and 17.21% to 86.94%, respectively. The IC 50The effective concentrations ranged from 10.6 μM to 26.92 μM and from 3.27 μM to 22.77 μM, respectively. For A549, the activity was better when -R2 was an electron-donating group than the electron-withdrawing group (1m > 1a); the activity was optimal when the electron-donating group was in the para position. For PC9, the activity was better when -R2 was an electron-withdrawing group than the electron-donating group (1c > 1m); the activity was optimal when the electron-withdrawing group was in the ortho position; the activity order of the electron-withdrawing groups was -Br > -F > -Cl (1g > 1b > 1d). For both cell lines, the target compounds showed better anticancer activity when the -R1 substituent was -Br instead of -H (2a, 2b > 1a, 1b).

[0124] Compound 1o inhibits the growth and proliferation of cancer cells.

[0125] 1) Cell proliferation assay method

[0126] Use MC38 or B16 at 2×10 per hole 4 Cells were seeded and, after adhesion, stimulated with DMSO (negative control), 5-fluorouracil (positive control), or compound 1O at concentrations of 5, 10, or 20 μM. Viable cell counts were determined using a CCK-8 assay at 0, 24, 48, and 72 hours. The proliferation index was calculated by setting the cell count at 0 hours to 1.

[0127] 2) Analysis of cell proliferation results

[0128] Colorectal cancer cells (MC38) or melanoma cells (B16) were cultured with compound 1o at 5 μM, 10 μM, or 20 μM, with 5-fluorouracil as a positive control. Cell viability was measured using CCK-8 assay every 24 hours. At low concentrations, compound 1o (5 μM) significantly inhibited the growth of colorectal cancer cells (MC38, B16, and C16). Figure 1 (A) and melanoma cells ( Figure 1 It inhibits the growth of tumor cells (D) and thus suppresses tumor cell proliferation. Furthermore, the inhibitory efficiency of compound 1o is comparable to that of the standard drug 5-fluorouracil. Figure 1 (A, D) and it is dose-dependent ( Figure 1 The presence of AF indicates that compound 1o has anti-cancer potential.

[0129] Compound 1o triggers apoptosis and necrosis in cancer cells.

[0130] 1) Experimental methods for apoptosis assay

[0131] MC38 or B16 cells were seeded in ultra-low attachment plates (Corning) and treated with DMSO (as control) or Compound 1o at concentrations of 5, 10 or 20 mM in DMEM medium containing 5% FBS for 24 h at 37 °C. After incubation, cells were stained with Annexin V FITC and propidium iodide (Invitrogen). Flow cytometry (BD FACSCelesta) was used to quantify apoptotic and necrotic cell death and data were analyzed using flow Jo software (TreeStar, Inc).

[0132] 2) Analysis of the results of cell apoptosis

[0133] Resistance to cell apoptosis is a common feature of cancer cells. The potential of Compound 1o to induce cell apoptosis in MC38 and B16 cells after 24 h of treatment at concentrations of 5, 10 or 20 mM was analyzed. Cells were stained with Annexin V FITC and propidium iodide and differentiated into live cells, early apoptotic cells, late apoptotic cells and necrotic cells using flow cytometry. Compound 1o induced cell apoptosis to a greater extent in MC38 and B16 cells than the control group. After incubation with Compound 1o, the proportion of live cells in MC38 decreased slightly at 5 and 10 mM, but significantly at 20 mM (Fig. 2A). Compound 1o induced a greater proportion of late apoptosis and necrosis in cells in a dose-dependent manner compared to 5-fluorouracil (Fig. 2B). This phenomenon can be due to differences in the duration of drug action. For B16 cells, Compound 1o decreased the percentage of viable cells and increased the number of apoptotic and necrotic cells, similar to the effect observed with 5-fluorouracil (Fig. 2C, D). The data indicate that Compound 1o induces cell death in MC38 and B16 cells, in part by activating cell apoptosis. Figure 2 Figure 2 Figure 2 Figure 4

[0134] Compound 1o inhibits cancer cell glycolysis to block ATP production

[0135] 1) Experimental method for ECAR analysis

[0136] MC38 cells were seeded at 3 x 105cells per well in 6-well plates and treated with DMSO (as control) or Compound 1o at concentrations of 5, 10 or 20 mM in DMEM medium containing 5% FBS for 24 h at 37 °C. After incubation, cells were washed with PBS and incubated with 10 mM glucose in Krebs-Ringer buffer (pH 7.4) for 1 h at 37 °C. Extracellular acidification rate (ECAR) was measured using a Seahorse XF96 Extracellular Flux Analyzer (Agilent) and data were analyzed using Seahorse XF96 software (Agilent). 4 ​​​​Cells were seeded in XF-96 plates. Cells were treated with DMSO (as control) or Compound 1o at concentrations of 5, 10 or 20 mM, then incubated in DMEM medium containing 5% FBS at 37 °C for 24 h. Extracellular acidification rate (ECAR) was measured in a Seahorse XF Extracellular Flux Analyzer (Agilent Technologies) according to the manufacturer’s instructions. Glucose (10 mM, Sigma), oligomycin (1 mM, Sigma) and 2-deoxyglucose (2-DG, 50 mM, Sigma) were used. Data were analyzed using Wave Desktop software version 2.6 (Agilent Technologies).

[0137] 2) Medium lactate assay experimental method

[0138] MC38 or B16 cells were incubated in DMEM medium containing 0.5% FBS, then treated with or without Compound 1o at 37 °C for 24 h. Medium lactate concentration was determined using a lactate assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s protocol.

[0139] 3) Cell ATP assay experimental method

[0140] MC38 or B16 cells were incubated in DMEM medium containing 0.5% FBS, then treated with or without Compound 1o at 37 °C for 24 h. After incubation, cells were lysed and cell ATP was determined using a cellTiter Glo luminescent cell viability assay kit (Promega) according to the manufacturer’s protocol.

[0141] 4) Compound 1o inhibits cancer cell glycolysis to block ATP production

[0142] Aerobic glycolysis is a hallmark of cancer cells, which produces a large amount of lactate regardless of the availability of oxygen. Therefore, when measuring the extracellular acidification rate (ECAR) of MC38 cells after Compound 1o treatment, it was found that the ECAR of cells incubated with Compound 1o was significantly reduced compared to the control after the addition of glucose (Figure 2A). Therefore, treatment of MC38 and B16 cells with Compound 1o reduced lactate production compared to the control (Figure 2B), indicating a decrease in glycolysis. In addition, cell ATP production was reduced after Compound 1o treatment (Figure 2C). Figure 3 Figure 3 Figure 3

[0143] Compound 1o inhibits mTOR signaling and downregulates the glycolysis pathway

[0144] 1) Real-time quantitative PCR (qPCR)

[0145] ​​​MC38 cells were cultured in DMEM medium containing 5% FBS, then treated with PBS or 5 mM of compound 10 for 24 h. RNA was extracted using TRNzol reagent, then reverse transcribed into cDNA for qPCR to detect the expression level of related genes (Tiangen). The primers used for qPCR are listed in Table 4. Actin was used as an internal control, and the relative expression level of genes was calculated by AACt method.

[0146] 2) List of primers used for qPCR

[0147] Table 4. Primers used for qPCR

[0148]

[0149]

[0150] 3) Compound 10 inhibits mTOR signaling to downregulate glycolysis pathway

[0151] Under EGFR signaling, the PI3K / Akt / mTOR pathway is highly dysregulated in human cancers and plays a crucial role in regulating glucose uptake and metabolism. To test whether the inhibition of cancer cell glycolysis by compound 10 is related to mTOR signaling, the gene expression of related key molecules was measured. The results showed that compound 10 effectively inhibited the expression of EGFR Figure 4 Fig. 4A). In addition, it was found that compound 10 did not affect the expression of PI3K, but significantly inhibited the expression of Akt and mTOR Figure 4 Fig. 4C, Figure 4 Fig. 4D). Akt / mTOR signaling promotes glycolysis by increasing the expression of glucose transporters and glycolytic enzymes. Therefore, the mRNA expression of genes required for glycolysis was measured, and it was found that the mRNA abundance of hexokinase 2 (HK2, phosphorylates glucose to glucose-6-phosphate, a key rate-limiting enzyme in glycolysis) and lactate dehydrogenase A (Ldha, converts pyruvate to lactate, allowing cancer cells to maintain glycolysis) was reduced, while the expression of glucose transporter 1 (Glut1, responsible for glucose uptake) did not change after compound 10 treatment, consistent with previous results Figure 4 Fig. 4D-F). These data suggest that compound 10 has the ability to effectively block mTOR signaling and the glycolysis pathway, possibly disrupting the dependence of cancer cells on aerobic glycolysis and ultimately leading to cell death.

Claims

1. A class of salicylaldehyde-carbazole compounds, characterized in that: Its structural formula is as follows: Where R1 is hydrogen or bromine; R2 is ortho, meta, or para hydrogen, hydroxyl, halogen, methyl, or methoxy, or the compound is .

2. The salicylaldehyde-carbazole compound according to claim 1, characterized in that: R1 is hydrogen; R2 is fluorine or methoxy, or the compound is... .

3. The method for preparing a class of salicylaldehyde-carbazole compounds as described in claim 1 or 2, characterized in that: The reaction formula is as follows: .

4. The application of a class of salicylaldehyde-carbazole compounds as described in any one of claims 1-2 in the preparation of agents for controlling plant fungi.

5. The use of a class of salicylaldehyde-carbazole compounds as described in any one of claims 1-2 in the preparation of anticancer agents.

6. The application according to claim 5, characterized in that: The cancers mentioned include colon cancer and lung cancer.

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