12-bromine-5, 9-di-tert-butyl naphtho-indolizino phenothiazine compound as well as preparation method and anti-tumor application thereof

Abstract

The invention belongs to the field of organic synthesis and medicinal chemistry, and particularly relates to a synthesis method of a 12-bromine-5, 9-di-tert-butyl naphtho-indolizine phenothiazine compound and an anti-tumor application of the 12-bromine-5, 9-di-tert-butyl naphtho-indolizine phenothiazine compound. Through efficient and simple one-pot bromination reaction, selective functionalization of a C12 site is realized, and the first brominated derivative based on a naphtho-indolizine phenothiazine skeleton is constructed. In-vitro anti-tumor activity research shows that the target compound shows broad-spectrum anti-tumor activity: the target compound has remarkable inhibitory activity (IC50 values are respectively 1.52 mu M, 3.14 mu M and 4.19 mu M) on cervical cancer Hela, lung adenocarcinoma A549 and prostatic cancer PC-3 cells, and the activity is improved by 3.5-16.5 times compared with that of a clinical drug cis-platinum. Preliminary toxicity evaluation shows that the toxicity to normal human umbilical vein endothelial cells (HUVEC) is lower than that of tumor cells, and good selectivity is achieved. The invention provides an important lead compound for developing novel anti-cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis and medicinal chemistry, and specifically relates to a 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound, a preparation method thereof, and anti-tumor application thereof. Background Art

[0002] Phenothiazines are tricyclic conjugated compounds composed of two benzene rings fused to a central nitrogen-sulfur heterocycle. Since their antipsychotic and antihistamine activities were discovered in the mid-20th century, phenothiazines, represented by chlorpromazine and promethazine, have been widely used in clinical practice. Recent studies have confirmed that through rational structural modification, phenothiazine derivatives can exhibit a wider range of pharmacological activities, including antitumor, antiviral, antibacterial, anti-inflammatory, and antioxidant effects. [Jaszczyszyn.A., K., P., Malinka.W., K., Petrus.J., Czarnik-Matusewicz.B.Pharmacol.Rep., 2012, 64, 16-23; Zaccagnini.L., Rossetti.G., Tran.TH, Salzano.G., Gandini.A., Baldeschi.AC, Bolognesi.ML, Carloni.P., L egname.G.Eur.J.Med.Chem., 2020, 196, 112295; Salie.S., Labuschagné.A., Walters.A., Geyer.S., Jardine.A., Jacobs.M., Hsu.N.-J.Regul.Toxicol.Pharmacol., 2019, 109, 104508.]

[0003] In anti-tumor research, the 2-benzenesulfonylamino-substituted phenothiazine derivatives designed and synthesized by Ghorab's team showed significant biological activity: the inhibition rate of aromatase could reach 68.32% (IC 50 =5.67 μmol·L -1 ), compared with the positive control letrozole (IC 50 =29.5 μmol·L -1 ) increased by 5 times; the cytotoxicity (IC 50 =8.1 μmol·L -1 ) is also superior to doxorubicin (IC 50 =9.8 μmol·L -1 ) 。[Ghorab.MM, Alsaid.MS, Samir.N., Abdel-Latif.GA, Soliman.AM, Ragab.FA, Abou El Ella.DAEur.J.Med.Chem., 2017, 134, 304-315.] The N-alkyl substituted benzathiazine derivatives developed by et al. showed broad-spectrum anti-tumor activity: the inhibition strength on lymphocyte proliferation was comparable to that of cyclosporine A; the anti-proliferative activity on L1210 leukemia cells, A-431 epidermal cancer cells and CX-1 colon cancer cells (GI s0 2.28, 2.84, and 10.83 μg·mL, respectively -1 ) is comparable to cisplatin. [ M., Pluta, K., Zimecki, M., Morak- B., Artym, J., M.Eur.J.Med.Chem., 2015, 89, 411-420.] These research results highlight the important value of phenothiazine skeleton in the development of new drugs.

[0004] Current structural modification strategies are mainly focused on two directions: (1) modification of nitrogen-sulfur heterocycles: including the introduction of N-substituents (alkyl, aryl, etc.) and oxidation of sulfur atoms (sulfoxide / sulfonation); (2) modification of benzene rings: introducing substituents at positions 1-4 and 6-9, or constructing new heterocyclic systems through benzene ring substitution (homo / heteroaromatic rings, polyaromatic ring systems, nitrogen heterocycles). It is worth noting that the research on phenothiazine derivatives based on the strategy of extending the π-conjugated system is still in the exploratory stage, especially brominated derivatives with extended conjugated systems have not yet been reported, which provides a potential research direction for the development of new functional molecules. Summary of the Invention

[0005] Based on the above content, the present invention provides a 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound, a preparation method thereof, and an anti-tumor application thereof.

[0006] One of the technical solutions of the present invention is a 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound, 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 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound, comprising the following steps: using the 5,9-di-tert-butylnaphthoindolizine-phenothiazine compound represented by formula (II) as a precursor, reacting in a solvent at room temperature under the action of N-bromosuccinimide (NBS);

[0009] Structural formula (II)

[0010]

[0011] Reaction equation:

[0012]

[0013] Furthermore, the molar ratio of the 5,9-di-tert-butylnaphthoindolizine and phenothiazine to N-bromosuccinimide is 1:1.2.

[0014] Furthermore, the concentration of the 5,9-di-tert-butylnaphthoindolizine-phenothiazine compound in the solvent is 0.01 mol / L, the solvent is anhydrous dichloromethane, the temperature is room temperature, and the reaction time is 24 h.

[0015] Furthermore, after the reaction is completed, the process further includes sodium sulfite quenching, dichloromethane extraction, drying and filtration, concentration, and purification; the purification is dichloromethane washing.

[0016] The third technical solution of the present invention is the use of the above-mentioned 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound in the preparation of anti-tumor drugs.

[0017] Furthermore, the anti-tumor drug is used to treat human cervical cancer (Hela).

[0018] Furthermore, the anti-tumor drug is used to treat human non-small cell lung cancer (A549).

[0019] Furthermore, the anti-tumor drug is used to treat human prostate cancer (PC-3).

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Through precise molecular design, the present invention constructs the first brominated derivative based on the naphthoindoleazine phenothiazine skeleton. In terms of preparation technology, an efficient and simple one-pot bromination reaction was developed. Using 5,9-di-tert-butyl naphthoindoleazine phenothiazine compound as a precursor, under the action of N-bromosuccinimide (NBS), the C12 site was selectively functionalized through a free radical bromination mechanism. This method can be completed at room temperature, and the product yield after washing and purification with dichloromethane is 96%. In vitro antitumor activity studies have shown that the target compound exhibits broad-spectrum antitumor activity: against cervical cancer Hela cell lines (IC 50 =1.52μM), lung cancer A549 cell line (IC 50 =3.14 μM) and prostate cancer PC-3 cell line (IC 50 =4.19μM) showed significant proliferation inhibition. Compared with the clinical first-line chemotherapy drug cisplatin, the inhibitory activity of this compound on the above-mentioned tumor cells was increased by 12 times, 16.5 times and 3.5 times, respectively. It is worth noting that the preliminary toxicity evaluation results showed that the cytotoxicity of this compound to normal human cell lines (HUVEC) was significantly lower than that to tumor cells. The novel molecular structure, excellent anti-tumor activity and significant selectivity characteristics of this study provide a lead compound with a clear structure-activity relationship for the development of new anti-cancer drugs, and have important application value in the field of anti-tumor drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound prepared in Example 1 of the present invention;

[0023] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to specific embodiments.

[0025] Example 1

[0026] Synthesis of 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compounds

[0027] In a 250 mL pressure-resistant reaction flask, 5,9-di-tert-butylnaphthoindolizine-phenothiazine (0.35 g, 0.81 mmol) was dissolved in anhydrous dichloromethane (70 mL) at 0°C and bubbling was continued under argon for 3 minutes. NBS (0.17 g, 0.97 mmol) was added portionwise, and the mixture was slowly allowed to return to room temperature and stirred for 24 hours. After completion of the reaction, saturated sodium sulfite solution was added to quench the reaction, and dichloromethane (30 mL) was added. The mixture was extracted three times, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was washed with dichloromethane (10 mL) to obtain the desired product (0.78 g, light yellow solid, 96% yield).

[0028] 1 H NMR (600MHz, C6D6, 298K) δ8.36 (d, J=8.4Hz, 1H), 8.25 (s, 1H), 7.75 (d, J=7.8Hz, 1H), 7.72 (d, J=1.2Hz, 1H), 7.62 (d, J=1.2Hz, 1H), 7.55 (t, J=7.8Hz, 1H), 7.11 (d, J=1.8Hz, 1H), 7.09 (d, J=1.2Hz, 1H), 1.34 (s, 9H), 1.20 (s, 9H); 13 C NMR (150MHz, CDCl3, 298K) δ148.1, 147.9, 130.4, 129.6, 127.8, 127.6, 126.4, 124.9, 124.4, 123.2, 121.8, 121.3, 121. 2, 120.8, 119.9, 119.8, 118.5, 117.1, 116.4, 114.3, 112.3, 111.6, 35.6, 35.2, 32.1(3C), 31.6(3C); HRMS(ESI) m / z: [M + ]calcd.For C 30 H 26 NSBr,511.0969;found,511.0968.

[0029] Example 2

[0030] Synthesis of 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compounds

[0031] In a 250 mL pressure-resistant reaction flask, 5,9-di-tert-butylnaphthoindolizine-phenothiazine (0.42 g, 0.97 mmol) was dissolved in anhydrous dichloromethane (80 mL) at 0°C and bubbling was continued under argon for 3 minutes. NBS (0.2 g, 1.16 mmol) was added portionwise, and the mixture was slowly allowed to return to room temperature and stirred for 24 hours. After the reaction was completed, saturated sodium sulfite solution was added to quench the reaction. Dichloromethane (30 mL) was added, and the mixture was extracted three times. The mixture was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was washed with dichloromethane (10 mL) to obtain the desired product (0.94 g, light yellow solid, 96% yield).

[0032] 1 H NMR (600MHz, C6D6, 298K) 68.36 (d, J = 8.4Hz, 1H), 8.25 (s, 1H), 7.75 (d, J = 7.8Hz, 1H), 7.72 (d, J = 1.2Hz, 1H), 7.62 (d, J=1.2Hz, 1H), 7.55 (t, J=7.8Hz, 1H), 7.11 (d, J=1.8Hz, 1H), 7.09 (d, J=1.2Hz, 1H), 1.34 (s, 9H), 1.20 (s, 9H); 13 C NMR (150MHz, CDCl3, 298K) δ148.1, 147.9, 130.4, 129.6, 127.8, 127.6, 126.4, 124.9, 124.4, 123.2, 121.8, 121.3, 121. 2, 120.8, 119.9, 119.8, 118.5, 117.1, 116.4, 114.3, 112.3, 111.6, 35.6, 35.2, 32.1(3C), 31.6(3C); HRMS(ESI) m / z: [M + ]calcd.For C 30 H 26 NSBr,511.0969;found,511.0968.

[0033] Example 3

[0034] Synthesis of 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compounds

[0035] In a 250 mL pressure-resistant reaction flask, 5,9-di-tert-butylnaphthoindolizine-phenothiazine (0.53 g, 1.22 mmol) was dissolved in anhydrous dichloromethane (90 mL) at 0°C and bubbling was continued under argon for 3 minutes. NBS (0.26 g, 1.46 mmol) was added portionwise, and the mixture was slowly allowed to return to room temperature and stirred for 24 hours. After completion of the reaction, saturated sodium sulfite solution was added to quench the reaction, and dichloromethane (30 mL) was added. The mixture was extracted three times, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was washed with dichloromethane (10 mL) to obtain the desired product (1.17 g, light yellow solid, 96% yield).

[0036] 1 H NMR (600MHz, C6D6, 298K) δ8.36 (d, J=8.4Hz, 1H), 8.25 (s, 1H), 7.75 (d, J=7.8Hz, 1H), 7.72 (d, J=1.2Hz, 1H), 7.62 (d, J=1.2Hz, 1H), 7.55 (t, J=7.8Hz, 1H), 7.11 (d, J=1.8Hz, 1H), 7.09 (d, J=1.2Hz, 1H), 1.34 (s, 9H), 1.20 (s, 9H); 13 C NMR (150MHz, CDCl3, 298K) δ148.1, 147.9, 130.4, 129.6, 127.8, 127.6, 126.4, 124.9, 124.4, 123.2, 121.8, 121.3, 121. 2, 120.8, 119.9, 119.8, 118.5, 117.1, 116.4, 114.3, 112.3, 111.6, 35.6, 35.2, 32.1(3C), 31.6(3C); HRMS(ESI) m / z: [M + ]calcd.For C 30 H 26 NSBr,511.0969;found,511.0968.

[0037] Example 7

[0038] The anticancer activity of 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound against human cervical cancer cells (Hela), human non-small cell lung cancer cells (A549) and human prostate cancer cells (PC-3) was tested. The specific experimental methods are as follows:

[0039] The cytotoxicity of 5,9-di-tert-butylnaphthoindole phenothiazine compounds to human cervical cancer cells (Hela), human non-small cell lung cancer cells (A549), human prostate cancer cells (PC-3) and normal human umbilical vein endothelial cells (HUVEC) was studied by MTT thiazolyl blue colorimetry. 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 37°C incubator with a volume fraction of 5% carbon dioxide and saturated humidity, 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 cells were further cultured in the incubator for 4 h. The supernatant was removed, and then 200 μL of DMSO was added to each well. After sufficient shaking for 15 min, 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 12-bromo-5,9-di-tert-butylnaphthoindolizine and phenothiazine compound on Hela, A549, PC-3, and HUVEC cells was calculated by formula (1). Finally, the IC value was obtained by origin analysis software. 50 .

[0040] m=1-n=1-OD 样 / OD 空白 (1)

[0041] m: inhibition rate n: cell survival rate

[0042] The results of the anticancer activity test of 12-bromo-5,9-di-tert-butylnaphthoindolizine and phenothiazine compound against Hela, A549 and PC-3 are shown in Table 1:

[0043] Table 1. Cytotoxicity of 12-bromo-5,9-di-tert-butylnaphthoindolizine and phenothiazine compounds and positive control drug cisplatin (DDP) to Hela, A549, PC-3, HUVEC (data in the table are IC 50 value): Compound Hela A549 PC-3 HUVEC This patent 1.52 3.14 4.19 37.16 DDP 18.58 51.92 14.51 26.48

[0044] As shown in Table 1, the 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compounds Hela, A549, and PC-3 synthesized in this patent exhibited varying degrees of inhibitory effects, with significant effects that were superior to the positive control drug cisplatin (DDP), and had low toxicity, indicating potential for the development of anti-tumor drugs.

Claims

1. A 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound, characterized in that: The structural formula is shown in formula (I):

2. The method for preparing 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound according to claim 1, wherein: The following steps are involved: The 5,9-di-tert-butyl naphthoindolizine-phenothiazine compound represented by formula (II) is used as a precursor, and reacts in a solvent at room temperature under the action of N-bromosuccinimide (NBS); Structural formula (II) 3. The method for preparing 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound according to claim 2, wherein: The molar ratio of the 5,9-di-tert-butylnaphthoindolizine and phenothiazine compound to N-bromosuccinimide is 1:1.

2.

4. The method for preparing 12-bromo-5,9-di-tert-butylnaphthoindolizine-phenothiazine compound according to claim 2, wherein: The concentration of the 5,9-di-tert-butylnaphthoindolizine-phenothiazine compound in the solvent is 0.01 mol / L, the solvent is anhydrous dichloromethane, the temperature is room temperature, and the reaction time is 24 h.

5. Use of the 12-bromo-5,9-di-tert-butylnaphthoindolizinophethiazine compound according to claim 1 in the preparation of antitumor drugs.

6. Use of the 12-bromo-5,9-di-tert-butylnaphthoindolizinophethiazine 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).

7. Use of the 12-bromo-5,9-di-tert-butylnaphthoindolizinophethiazine 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).

8. Use of the 12-bromo-5,9-di-tert-butylnaphthoindolizinophethiazine 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).

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