N, N-diphenyl aniline compound with naphtho-indolizino-phenothiazine skeleton and preparation method and application of N, N-diphenyl aniline compound

Abstract

The invention belongs to the field of organic synthesis and medicinal chemistry, and particularly provides an efficient synthesis method of an N, N-diphenyl aniline compound based on a naphtho-indolizino-phenothiazine skeleton and anti-tumor application of the N, N-diphenyl aniline compound based on the naphtho-indolizino-phenothiazine skeleton. Through Suzuki reaction, functional coupling based on a naphtho-indolizino-phenothiazine skeleton and an N, N-diphenyl aniline group is realized for the first time. In-vitro anti-tumor evaluation shows that the compound has remarkable inhibitory activity on various human tumor cells, especially has the strongest effect on breast cancer MCF-7 cells (IC50 = 0.15 mu M), and the activity is improved by 266 times compared with that of cis-platinum; prostate cancer PC-3, liver cancer HepG2, lung adenocarcinoma A549 and cervical cancer Hela cells are also remarkably inhibited. The toxicity to normal umbilical vein endothelial cells (HUVEC) is far lower than that of tumor cells, and the selectivity is excellent. The research provides a lead compound with great potential for developing high-efficiency and low-toxicity targeted anti-tumor drugs.

Description

Technical field:

[0001] The present invention belongs to the technical field of organic synthesis and medicinal chemistry, and specifically relates to a highly efficient synthesis method of an N,N-diphenylaniline compound based on a naphthoindole-1,1-diphenylaniline skeleton, and systematically evaluates its antitumor activity. Background technology:

[0002] Cancer remains a leading cause of death worldwide and a major public health concern: in 2020, over 19 million new cancer cases were diagnosed worldwide, and over 9 million deaths were reported. While cancer deaths increased significantly during the 20th century, the cancer mortality rate has declined by approximately 1.5% annually since 1991, reaching an overall decrease of 27% by 2016. Over the past two decades, the decline in mortality rates for the four major cancers (lung, breast, prostate, and colorectal) has been primarily driven by advances in early detection technologies and the development of existing treatments, such as surgery, radiotherapy, chemotherapy, and biological therapies. Chemotherapy, in particular, has become an important treatment modality, improving patient survival. Despite this, current drug treatments are associated with numerous limiting side effects, and the development of drug resistance can lead to cancer recurrence; therefore, saving lives remains a daunting task. [R.L. Siegel, K.D. Miller, A. Jemal, C.A. Cancer J. Clin., 2019, 69, 7-34.]

[0003] These are key reasons why medicinal chemists are committed to developing new chemotherapeutic agents with the potential for greater potency, lower toxicity, and multi-target activity. Among known anticancer molecules based on heterocyclic skeletons, phenothiazines have attracted considerable attention due to their wide range of medical applications and rich chemical properties. [MC Posso, FC Domingues, S. Ferreira, S. Silvestre, Molecules, 2022, 27, 276.] In addition to their primary use as antipsychotics for the treatment of schizophrenia, phenothiazine-containing compounds have also shown activity in treating nausea and vomiting during anesthesia, anxiety, acute and intermittent porphyria, and methemoglobinemia. Other biological activities exhibited by phenothiazines include antihistamine, immunosuppressive, antitrypanosomal, antifungal, antiparkinsonian, antifilar, antimalarial, antimalarial, antibacterial, antiviral, anti-inflammatory, anticonvulsant, analgesic, and antioxidant properties. [C. Gopi, MD Dhanaraju, Rev. J. Chem. 2019, 9, 95-126.]

[0004] The current structural optimization strategies for phenothiazine derivatives are mainly focused on the following directions: (1) modification of nitrogen-sulfur heterocycles: including functional design of substituents (alkyl / aryl) attached to nitrogen atoms (N) and regulation of the oxidation state of sulfur atoms (S) (such as sulfoxide and sulfonation); (2) modification of aromatic ring systems: by introducing different substituents at positions 1-4 and 6-9 of the parent nucleus, or adopting strategies such as homoaromatic / heteroaromatic ring substitution, construction of polyaromatic ring fusion systems, and nitrogen heterocycle fusion, the diversity of molecular structures is expanded. It is worth noting that the research on new derivatives based on the expansion of π-conjugated systems (such as N,N-diphenylaniline-substituted phenothiazine derivatives) is still in the exploratory stage. This type of design provides an innovative research direction for the development of functional molecules with unique pharmacological activities. Summary of the invention:

[0005] Based on the above content, the present invention provides an N,N-diphenylaniline compound based on a naphthoindole azino phenothiazine skeleton, a synthesis method thereof, and an anti-cancer application thereof.

[0006] One of the technical solutions of the present invention is an N,N-diphenylaniline compound based on a naphthoindole azino-phenothiazine skeleton, the structural formula of which is shown in formula (I):

[0007]

[0008] The second technical solution of the present invention is a method for preparing the above-mentioned N,N-diphenylaniline compound with a naphthoindole azinothiazine skeleton, comprising the following steps: using a 12-bromo-5,9-di-tert-butylnaphthoindole azinothiazine compound represented by formula (II) and 4-(diphenylamino)phenylboronic acid as raw materials, heating the reaction in a solvent in the presence of tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2′,6′-dimethoxybiphenyl, and anhydrous potassium carbonate to obtain the compound;

[0009] Structural formula (II)

[0010]

[0011] Reaction equation:

[0012]

[0013] Furthermore, the molar ratio of the 12-bromo-5,9-di-tert-butylnaphthoindolizine and phenothiazine compound, 4-(diphenylamino)phenylboric acid, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2′,6′-dimethoxybiphenyl, and anhydrous potassium carbonate is 1:3:0.2:0.4:3.

[0014] Furthermore, the concentration of the 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound in the solvent is 0.02 mol / L, the solvent is toluene and water, the volume ratio of toluene and water is 10:1, the temperature is 100° C., and the reaction time is 24 h.

[0015] Furthermore, after the reaction is completed, the process further includes water washing, dichloromethane extraction, drying and filtration, concentration, and purification; the purification is performed by silica gel column chromatography (eluent is n-hexane: dichloromethane = 50 / 1, V / V).

[0016] The third technical solution of the present invention is the use of the above-mentioned N,N-diphenylaniline compound with a naphthoindole azinophenothiazine skeleton in the preparation of anti-tumor drugs.

[0017] Furthermore, the anti-tumor drug is used to treat human breast cancer (MCF-7).

[0018] Furthermore, the anti-tumor drug is used to treat human prostate cancer (PC-3).

[0019] Furthermore, the anti-tumor drug is used to treat human liver cancer (HepG2).

[0020] Furthermore, the anti-tumor drug is used to treat human non-small cell lung cancer (A549).

[0021] Furthermore, the anti-tumor drug is used to treat human cervical cancer (Hela).

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Through precise molecular design, the present invention has achieved for the first time the functionalized coupling of naphthoindole azinothiazine skeleton and N,N-diphenylaniline group. The synthesis method uses 12-bromo-5,9-di-tert-butylnaphthoindole azinothiazine compound and 4-(diphenylamino)phenylboronic acid as raw materials, and constructs the target molecule through efficient Suzuki-Miyaura coupling reaction under the weak alkaline conditions of tris(dibenzylideneacetone)dipalladium / 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl catalytic system and anhydrous potassium carbonate. The reaction conditions are mild and the yield is as high as 90%. In vitro antitumor activity evaluation showed that the compound exhibited differentiated proliferation inhibitory effects on a variety of human tumor cell lines: breast cancer MCF-7 cells (IC 50 =0.15μM) showed nanomolar potent inhibitory activity against prostate cancer PC-3 (IC 50 =5.52μM), HepG2 (IC 50 =9.74 μM), lung adenocarcinoma A549 (IC 50=18.13 μM) and cervical cancer Hela (IC 50 =33.79μM) cells. Compared with the clinical first-line drug cisplatin, except for the slightly lower inhibitory activity on cervical cancer Hela cells, its inhibitory activity on the other tumor cell lines mentioned above is significantly improved, especially the inhibitory activity on breast cancer MCF-7 cells is increased by 266 times. Preliminary toxicity evaluation showed that the cytotoxicity of this compound to normal human umbilical vein endothelial cells (HUVEC) was significantly lower than that to tumor cells, showing excellent selective inhibitory properties. The results of this research have dual innovative value: on the one hand, it develops an efficient construction strategy for a new polycyclic aromatic hydrocarbon skeleton, providing a new idea for the synthesis of complex heterocyclic compounds; on the other hand, it reveals the significant tumor-selective inhibitory effect of this structural type of compound, and its specific killing effect on breast cancer cells is particularly prominent, providing a highly potential lead compound for the development of highly efficient and low-toxic targeted anti-tumor drugs. Description of the drawings:

[0024] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the N,N-diphenylaniline compound with a naphthoindole azinophenothiazine skeleton prepared in Example 1 of the present invention;

[0025] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the N,N-diphenylaniline compound with a naphthoindole azinophenothiazine skeleton prepared in Example 1 of the present invention. Specific implementation method:

[0026] The present invention is described in detail below with reference to specific embodiments.

[0027] Example 1

[0028] Synthesis of N,N-diphenylaniline Compounds Containing a Naphthoindolizine-Phenothiazine Skeleton

[0029] In a 120 mL pressure-resistant reaction flask, 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine (0.1 g, 0.2 mmol), 4-(diphenylamino)phenylboronic acid (0.17 g, 0.6 mmol), tris(dibenzylideneacetone)dipalladium (0.04 g, 0.04 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (0.03 g, 0.08 mmol), and anhydrous potassium carbonate (0.08 g, 0.6 mmol) were added sequentially. Under argon protection, anhydrous toluene (10 mL) and water (1 mL) were added, and bubbling was continued for 3 minutes. The mixture was stirred at 100°C for 24 hours. After the reaction, the mixture was cooled to room temperature, dichloromethane (50 mL) was added, and the mixture was washed with water three times. The mixture was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was separated and purified by silica gel column chromatography (eluent: n-hexane:dichloromethane = 50 / 1, V / V) to give the desired product (0.12 g, yellow solid, yield 90%).

[0030] 1 H NMR (600MHz, C6D6, 298K) δ8.23 (d, J=7.8Hz, 1H), 8.00 (s, 1H), 7.85 (d, J=7.8Hz, 1H), 7.81 (t, J=2.4Hz, 2H), 7.54 (d, J=8.4Hz, 2H), 7.49 (t, J=7.8Hz, 1H), 7.33 (d, J=7.8Hz, 2H), 7.29 (d, J=7.2Hz, 5H), 7.14 (d, J=7.8Hz, 5H), 6.91 (t, J=7.2Hz, 2H), 1.36 (s, 9H), 1.22 (s, 9H); 13 C NMR (150MHz, C6D6, 298K) δ148.6, 147.7, 147.5, 147.3, 136.8, 131.5, 131.2, 130.6, 129.8, 128.6, 128.5, 128.3, 128.0, 127.3, 126.8, 124. 7, 123.8, 123.2, 122.9, 122.7, 122.5, 122.2, 121.7, 120.5, 118.4, 11 7.4, 116.6, 116.2, 114.6, 112.4, 35.5, 34.9, 32.1(3C), 31.4(3C); [M + ]calcd.For C 48 H 40 N2S, 676.2912; found, 676.2914.

[0031] Example 2

[0032] Synthesis of N,N-diphenylaniline Compounds Containing a Naphthoindolizine-Phenothiazine Skeleton

[0033] In a 120 mL pressure-resistant reaction flask, 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine (0.15 g, 0.3 mmol), 4-(diphenylamino)phenylboronic acid (0.26 g, 0.9 mmol), tris(dibenzylideneacetone)dipalladium (0.06 g, 0.06 mmol), 2-dicyclohexylphosphine-2′,6′-dimethoxybiphenyl (0.05 g, 0.12 mmol), and anhydrous potassium carbonate (0.12 g, 0.9 mmol) were added sequentially. Under argon protection, anhydrous toluene (15 mL) and water (1.5 mL) were added, and bubbling was continued for 3 minutes. The mixture was stirred at 100°C for 24 hours. After the reaction, the mixture was cooled to room temperature, and dichloromethane (50 mL) was added. The mixture was washed with water three times, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was separated and purified by silica gel column chromatography (eluent: n-hexane:dichloromethane = 50 / 1, V / V) to give the desired product (0.18 g, yellow solid, yield 90%).

[0034] 1 H NMR (600MHz, C6D6, 298K) δ8.23 (d, J=7.8Hz, 1H), 8.00 (s, 1H), 7.85 (d, J=7.8Hz, 1H), 7.81 (t, J=2.4Hz, 2H), 7.54 (d, J=8.4Hz, 2H), 7.49 (t, J=7.8Hz, 1H), 7.33 (d, J=7.8Hz, 2H), 7.29 (d, J=7.2Hz, 5H), 7.14 (d, J=7.8Hz, 5H), 6.91 (t, J=7.2Hz, 2H), 1.36 (s, 9H), 1.22 (s, 9H); 13 C NMR (150MHz, C6D6, 298K) δ148.6, 147.7, 147.5, 147.3, 136.8, 131.5, 131.2, 130.6, 129.8, 128.6, 128.5, 128.3, 128.0, 127.3, 126.8, 124. 7, 123.8, 123.2, 122.9, 122.7, 122.5, 122.2, 121.7, 120.5, 118.4, 11 7.4, 116.6, 116.2, 114.6, 112.4, 35.5, 34.9, 32.1(3C), 31.4(3C); [M + ]calcd.For C 48 H 40 N2S, 676.2912; found, 676.2914.

[0035] Example 3

[0036] Synthesis of N,N-diphenylaniline Compounds Containing a Naphthoindolizine-Phenothiazine Skeleton

[0037] To a 120 mL pressure-resistant reaction flask, add 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine (0.2 g, 0.4 mmol), 4-(diphenylamino)phenylboronic acid (0.34 g, 1.2 mmol), tris(dibenzylideneacetone)dipalladium (0.08 g, 0.08 mmol), 2-dicyclohexylphosphine-2′,6′-dimethoxybiphenyl (0.06 g, 0.16 mmol), and anhydrous potassium carbonate (0.16 g, 1.2 mmol). Under argon, add anhydrous toluene (20 mL) and water (2 mL), and continue bubbling for 3 minutes. Stir at 100°C for 24 hours. After the reaction, cool to room temperature, add dichloromethane (50 mL), wash with water three times, dry over anhydrous magnesium sulfate, filter, and concentrate the filtrate to dryness. The crude product was separated and purified by silica gel column chromatography (eluent: n-hexane:dichloromethane = 50 / 1, V / V) to give the desired product (0.24 g, yellow solid, yield 90%).

[0038] 1 H NMR (600MHz, C6D6, 298K) δ8.23 (d, J=7.8Hz, 1H), 8.00 (s, 1H), 7.85 (d, J=7.8Hz, 1H), 7.81 (t, J=2.4Hz, 2H), 7.54 (d, J=8.4Hz, 2H), 7.49 (t, J=7.8Hz, 1H), 7.33 (d, J=7.8Hz, 2H), 7.29 (d, J=7.2Hz, 5H), 7.14 (d, J=7.8Hz, 5H), 6.91 (t, J=7.2Hz, 2H), 1.36 (s, 9H), 1.22 (s, 9H); 13 C NMR (150MHz, C6D6, 298K) δ148.6, 147.7, 147.5, 147.3, 136.8, 131.5, 131.2, 130.6, 129.8, 128.6, 128.5, 128.3, 128.0, 127.3, 126.8, 124. 7, 123.8, 123.2, 122.9, 122.7, 122.5, 122.2, 121.7, 120.5, 118.4, 11 7.4, 116.6, 116.2, 114.6, 112.4, 35.5, 34.9, 32.1(3C), 31.4(3C); [M + ]calcd.For C 48 H 40N2S, 676.2912; found, 676.2914.

[0039] Example 4

[0040] The anti-tumor activity of the naphthoindole azino-phenothiazine skeleton N,N-diphenylaniline compound on human breast cancer (MCF-7), human prostate cancer cells (PC-3), human liver cancer (HepG2), human non-small cell lung cancer cells (A549), and human cervical cancer cells (Hela) was tested. The specific experimental methods are as follows:

[0041] The MTT thiazolyl blue colorimetric method was used to study the cytotoxicity of N,N-diphenylaniline compounds with naphthoindole azino-phenothiazine skeleton to human breast cancer (MCF-7), human prostate cancer cells (PC-3), human liver cancer (HepG2), human non-small cell lung cancer cells (A549), human cervical cancer cells (Hela) and normal human umbilical vein endothelial cells (HUVEC). The cells to be tested in the logarithmic growth phase were prepared into 10 5 A single cell suspension of 100 μL / mL was inoculated on a 96-well culture plate, with 100 μL per well. After culturing for 24 h in a 5% carbon dioxide, saturated humidity, and 37°C incubator, 100 μL of the sample to be tested at different concentrations was added to the culture plate, with 6 replicates for each concentration. After culturing for 48 h, 100 μL (1 mg / mL) of MTT staining solution was added to each well, and the plate was cultured for another 4 h in the incubator. The supernatant was removed, and then 200 μL of DMSO was added to each well. The plate was fully shaken for 15 min. After 1 minute, the OD sample value was measured at a wavelength of 595 nm on a microplate reader. The blank group of the experiment was 100 μL of serum-free DMEM culture medium with DMSO reagent added instead of the sample. The absorbance value at this time was the OD blank value. The inhibition rate of the N, N-diphenylaniline compound with a naphthoindole phenyl phenothiazine skeleton on MCF-7, PC-3, HepG2, Hela, A549, Hela, and HUVEC cells was calculated by formula (1). Finally, the IC 50 .

[0042] m=1-n=1-OD 样 / OD 空白 (1)

[0043] m: inhibition rate n: cell survival rate

[0044] The results of the antitumor activity experiments of the naphthoindole azino-phenothiazine skeleton N, N-diphenylaniline compound on MCF-7, PC-3, HepG2, Hela, A549, and Hela are shown in Table 1:

[0045] Table 1. Cytotoxicity test of naphthoindole azino-phenothiazine skeleton N,N-diphenylaniline compounds and positive control drug cisplatin on MCF-7, PC-3, HepG2, Hela, A549, Hela, and HUVEC (data in the table are IC 50 value):

[0046]

[0047] As shown in Table 1, the N,N-diphenylaniline compound with a naphthoindole phenothiazine skeleton synthesized in this patent exhibited varying degrees of inhibitory effects on MCF-7, PC-3, HepG2, Hela, A549, and Hela, with significant effects. Among them, the inhibitory effects on MCF-7, PC-3, HepG2, and A549 were significantly better than those of the positive control drug cisplatin, and the toxicity was relatively low, indicating that the compound has the potential to be developed into an anti-tumor drug.

Claims

1. A naphthoindole azino-phenothiazine skeleton N, N-diphenylaniline compound, characterized in that, The structural formula is shown in formula (I):

2. The method for preparing the N,N-diphenylaniline compound of naphthoindole azino thiazine skeleton according to claim 1, characterized in that: The following steps are involved: The 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound represented by formula (II) and 4-(diphenylamino)phenylboronic acid are used as raw materials, and tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl and anhydrous potassium carbonate are reacted in a solvent under heating to obtain the product. Structural formula (II) 3. The method for preparing the N,N-diphenylaniline compound of naphthoindole azino thiazine skeleton according to claim 2, characterized in that: The molar ratio of the 12-bromo-5,9-di-tert-butylnaphthoindolizine and phenothiazine compound, 4-(diphenylamino)phenylboric acid, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl and anhydrous potassium carbonate is 1:3:0.2:0.4:

3.

4. The method for preparing the N,N-diphenylaniline compound having a naphthoindole azino-phenothiazine skeleton according to claim 2, wherein: The concentration of the 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound in the solvent is 0.02 mol / L, the solvent is toluene and water, the volume ratio of toluene to water is 10:1, the temperature is 100° C., and the reaction time is 24 h.

5. Use of the N,N-diphenylaniline compound having a naphthoindole azinophenothiazine skeleton according to claim 1 in the preparation of antitumor drugs.

6. Use of the naphthoindole azinophenothiazine skeleton N,N-diphenylaniline compound according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is used for treating human breast cancer (MCF-7).

7. Use of the naphthoindole azinophenothiazine skeleton N,N-diphenylaniline compound according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is used for treating human prostate cancer (PC-3).

8. Use of the naphthoindole azinophenothiazine skeleton N,N-diphenylaniline compound according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is used for treating human liver cancer (HepG2).

9. Use of the naphthoindole azinophenothiazine skeleton N,N-diphenylaniline compound according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is used to treat human non-small cell lung cancer (A549).

10. Use of the naphthoindole azinophenothiazine skeleton N,N-diphenylaniline compound according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is used for treating human cervical cancer (Hela).

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