阅读说明：本技术 4,5-二氢萘并异噁唑类衍生物及其在抗肿瘤药物中的应用 (4, 5-dihydronaphthoisoxazole derivatives and application thereof in antitumor drugs ) 是由 杨金飞 于 2021-06-25 设计创作，主要内容包括：本发明属于药物合成技术领域,尤其涉及本发明属于药物化学技术领域,尤其涉及4,5-二氢萘并异噁唑类衍生物和制备方法,及其作为CYP1B1抑制剂在抗肿瘤药物中的应用。本发明的提供一种通式(I)所示的新型4,5-二氢萘并异噁唑类衍生物,及其几何异构体或其药学上可接受的盐、水合物、溶剂化物或前药；本发明所述CYP1B1酶抑制剂与传统的抗肿瘤药物联合用药可以提高药物的抗肿瘤活性,提高特异性和有效性,具有更为广阔的发展前景。本发明通过MTT实验显示,本课题组合成的4,5-二氢萘并异噁唑类衍生物具有开发抗肿瘤药物增敏剂的前景。(The invention belongs to the technical field of drug synthesis, particularly relates to a 4, 5-dihydronaphthoisoxazole derivative, a preparation method thereof and application thereof as a CYP1B1 inhibitor in antitumor drugs, and belongs to the technical field of pharmaceutical chemistry. The invention provides a novel 4, 5-dihydronaphthoisoxazole derivative shown in a general formula (I), and a geometric isomer thereof or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; the CYP1B1 enzyme inhibitor and the traditional antitumor drug combined drug can improve the antitumor activity of the drug, improve the specificity and the effectiveness, and have wider development prospect. MTT experiments show that the 4, 5-dihydronaphthoisoxazole derivatives synthesized by the subject have a prospect of developing anti-tumor drug sensitizers.)

The 4, 5-dihydronaphthoisoxazole derivative is characterized by having the following structural formula:

wherein, R is₁Or R₂Selected from hydrogen, halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkyl radical, C₁-C₆Cycloalkyl, alkenyl, alkynyl or aryl;

x is selected from N or C;

y is selected from hydrogen, halogen and C₁-C₆Alkyl, alkenyl or alkynyl.

2. The 4, 5-dihydronaphthoisoxazole derivative according to claim 1, wherein R is₁Or R₂Selected from hydrogen, halogen, C₁-C₆Alkoxy or C₁-C₆An alkyl group.

3. The 4, 5-dihydronaphthoisoxazole derivative according to claim 1, wherein Y is selected from hydrogen and C₁-C₆An alkyl group.

4. The 4, 5-dihydronaphthoisoxazole derivative according to claim 1, which is selected from the group consisting of:

5. the process for producing a 4, 5-dihydronaphthoisoxazole derivative according to any one of claims 1 to 3, which comprises: condensing different substituted 1-tetralone and diethyl oxalate under LiHMDS condition to obtain an intermediate 2, performing ring closing reaction on the intermediate 2 and hydroxylamine hydrochloride to obtain an intermediate 3, hydrolyzing with sodium hydroxide to obtain an intermediate 4, and finally performing amide reaction on the intermediate 4 and 5-aminoisoxazole to obtain a target product;

route 1 is as follows:

route 1 reagents and conditions: (a) diethyl oxalate, LiHMDS; (b) hydroxylamine hydrochloride, EtOH, reflux,2 h; (c) NaOH, MeOH/H2O, r.t., 7H; (d) EDCI, HOBt, DIEA, r.t.,7h.

6. The 4, 5-dihydronaphthoisoxazole derivatives as claimed in any one of claims 1 to 3 can be used as antitumor drugs, in particular as a sensitizer for CYP1B1 inhibitor to enhance paclitaxel or doxorubicin, which are first-line clinical antitumor drugs.

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a 4, 5-dihydronaphthoisoxazole derivative, a preparation method thereof, and application thereof as a CYP1B1 inhibitor in antitumor drugs.

Background

Cytochrome P450 enzymes, originally present in rat liver microsomes, contain heme and have a strong spectral absorption band at 450nm, and are therefore called cytochrome P450 enzymes. Currently, the identified human cytochrome P450 family members are CYP1a1, CYP1a2 and CYP1B 1. Among them, CYP1B1 is a key P450 enzyme involved in exogenous and endogenous substrate metabolism. Research shows that CYP1B1 metabolizes precancerogen such as polycyclic aromatic hydrocarbon and the like, and is likely to cause activation of carcinogenic compounds. It is worth mentioning that CYP1B1 is involved in estrogen metabolism in endogenous substrates, and interestingly, women show higher CYP1B1 expression than men. CYP1B1 is induced by the transcription of TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) or dioxin and is regulated by several key transcription factors such as Estrogen Receptor (ER) and aromatic hydrocarbon receptor (AhR). In addition to playing a role in xenogeneic metabolism, CYP1B1 is also involved in the biological activation of carcinogens and appears to play a role in the metabolism of certain anticancer agents used in hormone-induced cancer therapy. In clinical and preclinical studies, overexpression of CYP1B1 was associated with reversible resistance to the anticancer drug docetaxel or doxorubicin.

In recent years, research and development of CYP1B1 inhibitors become a hotspot in the field of drug research and development, and a plurality of researches show that the CYP1B1 inhibitor can enhance the chemotherapy effect of anticancer drugs docetaxel or adriamycin on cancer cell lines of lung cancer, breast cancer, liver cancer, prostate, colorectal cancer, gastric cancer, leukemia and the like. More importantly, some inhibitors have been proved to overcome the drug resistance of cancer cell lines with docetaxel or adriamycin resistance. However, the mechanistic role of CYP1B1 in mediating the chemosensitization of these compounds is complex and yet uncertain. The chemosensitization of the inhibitors is often attributed to other mechanisms, including AMPK activation to promote apoptosis, regulation of miR-520B/ATG7, miR-101/Nrf2 pathway, FZD 7/beta-catenin pathway, down-regulation of p-glycoprotein expression, PTEN/Akt pathway and the like, and inhibition of CYP1B1 possibly interacts with these pathways to cause chemosensitization.

In conclusion, the research on the novel CYP1B1 inhibitor with stronger specificity is very important for the clinical treatment of tumor drug patients and the reversal of the multi-drug resistance phenomenon of chemotherapeutic drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel 4, 5-dihydronaphthalene isoxazole derivative; and a preparation method of the derivative and application of the derivative as a CYP1B1 enzyme inhibitor in antitumor drugs.

In order to achieve the purpose, the invention adopts the technical scheme that: the invention provides a novel 4, 5-dihydronaphthoisoxazole derivative shown in a general formula (I), and a geometric isomer thereof or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof;

the R is₁Or R₂Selected from hydrogen, halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkyl radical, C₁-C₆Cycloalkyl, alkenyl, alkynyl or R₃Substituted aryl, wherein R₃Selected from hydrogen, halogen, C₁-C₆Alkoxy radical, C₁-C₆An alkyl group.

And X is selected from N or C.

Y is selected from hydrogen, halogen and C₁-C₆Alkyl, alkenyl or alkynyl.

Preferably, said R is₁Or R₂Selected from hydrogen, halogen, C₁-C₆Alkoxy or C₁-C₆An alkyl group.

Preferably, Y is selected from hydrogen, C₁-C₆An alkyl group.

The 4, 5-dihydronaphthoisoxazole derivative shown in the general formula (I) is selected from the following compounds:

the invention also includes prodrugs of the derivatives of the invention. Prodrugs of the derivatives of the invention are derivatives of formula I which may themselves have poor or no activity, but which, upon administration, are converted under physiological conditions (e.g., by metabolism, solvolysis, or otherwise) to the corresponding biologically active form.

The compounds of formula I can be synthesized according to the method of the scheme 1, and are prepared by condensing different substituted 1-tetralone and diethyl oxalate under the condition of LiHMDS to obtain an intermediate 2, performing a ring-closing reaction on the intermediate 2 and hydroxylamine hydrochloride to obtain an intermediate 3, hydrolyzing with sodium hydroxide to obtain an intermediate 4, and finally performing an amide reaction on the intermediate 4 and various substituted arylmethylamines to obtain a target product.

Synthetic scheme 1 reagents and conditions: (a) diethyl oxalate, LiHMDS; (b) hydroxylamine hydrochloride, EtOH, reflux,2 h; (c) NaOH, MeOH/H2O, r.t., 7H; (d) EDCI, HOBt, DIEA, r.t.,7h.

The 4, 5-dihydronaphthoisoxazole derivative can be used as an antitumor drug, in particular as a sensitizer for a CYP1B1 inhibitor to enhance a first-line clinical antitumor drug paclitaxel or adriamycin.

The tumor cell of the invention can be MCF-7/ADM, MCF-7/Taxol or A549/TAX.

The invention has obvious technical effect.

The research and development of CYP1B1 enzyme inhibitor drugs are one of the main research hotspots of the current anti-tumor drugs. The discovery of the drugs brings new hope for the treatment of tumor patients, and opens up a new place for reversing the multi-drug resistance phenomenon of clinical antitumor drugs. The CYP1B1 enzyme inhibitor and the traditional antitumor drug are combined to be used, so that the antitumor activity of the drug can be obviously improved, the specificity and the effectiveness are improved, and the development prospect is wider. MTT experiments show that the 4, 5-dihydronaphthoisoxazole derivatives synthesized by the subject have a prospect of developing anti-tumor drug sensitizers.

Detailed Description

The following examples are intended to illustrate but not limit the scope of the invention. The nuclear magnetic resonance hydrogen spectrum of the compound is measured by BrukeraRx-400, and the mass spectrum is measured by Agilent 1100 LC/MS; all reagents used were analytically or chemically pure.

Example 1

The synthesis steps of the compound are as follows:

step 1, synthesis of an intermediate 2:

dissolving 1-tetralone (2.0g,13.7mmol) in tetrahydrofuran, adding diethyl oxalate (3.0g,20.5mmol), cooling the reaction temperature to 0 ℃ in an ice bath under the protection of nitrogen, then slowly dropwise adding LiHMDS solution (20.5mL of 1M in THF, 20.5mmol), heating to 40 ℃ after dropwise adding, reacting for 4h, completing TLC detection reaction, and evaporating the solvent under reduced pressure to obtain an intermediate 2 which is directly used for the next reaction without purification.

Step 2, synthesis of an intermediate 3(4, 5-dihydronaphtho [2,1-d ] isoxazole-3-ethyl formate):

intermediate 2 was dissolved in 60mL of glacial acetic acid, hydroxylamine hydrochloride (1.4g,20.5mmol) was added and the temperature was raised to 80 ℃. After 10 hours of reaction, TLC was carried out to detect completion of the reaction, and 100mL of water was poured into the reaction solution, followed by extraction with ethyl acetate, washing of the organic layer with saturated brine, and addition of Na₂SO₄Dry overnight. The drying agent was filtered off, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give 2.10g of a white solid with a yield of 63.1%.

Step 3, synthesis of an intermediate 4(4, 5-dihydronaphtho [2,1-d ] isoxazole-3-carboxylic acid):

4, 5-dihydronaphtho [2,1-d ]]Isoxazole-3-carboxylic acid ethyl ester (1.8g, 7.4mmol) was dissolved in 40mL of methanol, and then 2N NaOH solution (10mL) was added to the reaction solution. After stirring at room temperature for 4h, the methanol was evaporated under reduced pressure, the pH was adjusted to 5-7 with 1N hydrochloric acid to precipitate a white solid, which was filtered and dried to obtain 1.49g of intermediate 4 with a yield of 93.6%.¹H-NMR(600MHz,DMSO-d₆)δ7.63(d,J＝6.5Hz,1H),7.14–7.08(m,2H),7.03(d,J＝6.8Hz,1H),2.64(t,J＝7.0Hz,2H),2.32–2.27(m,2H).

Step 4, synthesis of example 1.

4, 5-dihydronaphtho [2,1-d ]]Isoxazole-3-carboxylic acid (0.5g,2.3mmol) was dissolved in 30mL DMF and EDCI (0.53g,2.8mmol) and HOBt (0.38g,2.8mmol) were added sequentially. After stirring at room temperature for 1h benzylamine (0.30g,2.8mmol) and DIEA (0.60mg,4.6mmol) were added and the reaction was allowed to warm to 70 ℃ in an oil bath for 8 h. And (3) stopping heating after TLC detection reaction is finished, cooling the temperature to room temperature, pouring the reaction liquid into 60mL of water, separating out a solid, filtering, and purifying the residue by silica gel column chromatography to obtain 0.48g of white solid with the yield of 67.9%.¹H-NMR(600MHz,DMSO-d₆)δ9.22(s,1H),7.63(d,J＝6.5Hz,1H),7.35(t,J＝8.0Hz,2H),7.30-7.27(m,3H),7.14–7.08(m,2H),7.03(d,J＝7.0Hz,1H),4.11(s,2H),2.64(t,J＝7.0Hz,2H),2.32–2.27(m,2H).ESI-MS m/z:305.1[M+H]+.

Examples 2-11 were prepared according to the procedure of example 1, using substituted 1-tetralones as starting materials, respectively, via four-step reactions of condensation, ring closure, hydrolysis, and condensation.

Example 2.

¹H-NMR(600MHz,DMSO-d₆)δ9.24(s,1H),7.64(d,J＝7.5Hz,1H),7.38(d,J＝8.0Hz,2H),7.14–7.06(m,4H),7.01(d,J＝6.8Hz,1H),4.12(s,2H),2.65(t,J＝7.1Hz,2H),2.33–2.26(m,2H).ESI-MS m/z:323.3[M+H]⁺.

Example 3.

¹H-NMR(600MHz,DMSO-d₆)δ9.20(s,1H),7.61(d,J＝7.0Hz,1H),7.39–7.34(m,4H),7.15–7.09(m,2H),7.02(d,J＝7.4Hz,1H),4.16(s,2H),2.63(t,J＝7.4Hz,2H),2.31–2.25(m,2H).ESI-MS m/z:339.1[M+H]⁺.

Example 4.

¹H-NMR(600MHz,DMSO-d₆)δ9.21(s,1H),7.60(d,J＝6.8Hz,1H),7.19(d,J＝8.0Hz,2H),7.15–7.08(m,4H),7.01(d,J＝6.6Hz,1H),4.06(s,2H),2.62(t,J＝7.3Hz,2H),2.30–2.24(m,2H).ESI-MS m/z:319.1[M+H]⁺.

Example 5.

¹H-NMR(600MHz,DMSO-d₆)δ9.21(s,1H),7.69-7.63(m,3H),7.26(dd,J＝2.4,8.0Hz,2H),7.14–7.08(m,3H),7.01(d,J＝7.2Hz,1H),4.21(s,2H),2.66(t,J＝7.0Hz,2H),2.34–2.29(m,2H).ESI-MS m/z:323.1[M+H]⁺.

Example 6.

¹H-NMR(600MHz,DMSO-d₆)δ9.24(s,1H),8.56(d,J＝7.8Hz,2H),7.62(d,J＝7.0Hz,1H),7.35(d,J＝7.8Hz,2H),7.13–7.07(m,2H),7.01(d,J＝7.0Hz,1H),4.83(s,2H),2.65(t,J＝7.0Hz,2H),2.32–2.27(m,2H).ESI-MS m/z:306.1[M+H]⁺.

Example 7.

¹H-NMR(600MHz,DMSO-d6)δ9.23(s,1H),8.56(d,J＝7.8Hz,2H),7.77(d,J＝6.5Hz,1H),7.35(d,J＝7.8Hz,2H),7.08(d,J＝2.2,1H),7.03(d,J＝6.8Hz,1H),4.83(s,2H),3.70(s,3H),2.93(t,J＝7.9Hz,2H),2.86–2.76(m,2H).ESI-MS m/z:336.1[M+H]⁺.

Example 8.

¹H-NMR(600MHz,DMSO-d6)δ9.26(s,1H),8.56(d,J＝7.8Hz,2H),7.92(d,J＝6.5Hz,1H),7.45(d,J＝2.2,1H),7.35(d,J＝7.8Hz,2H),7.28(d,J＝6.8Hz,1H),4.83(s,2H),2.99(t,J＝7.9Hz,2H),2.87–2.78(m,2H).ESI-MS m/z:385.2[M+H]⁺.

Example 9.

¹H-NMR(600MHz,DMSO-d6)δ9.28(s,1H),8.56(d,J＝7.8Hz,2H),7.77(d,J＝6.5Hz,1H),7.35(d,J＝7.8Hz,2H),7.08(d,J＝2.2,1H),7.03(d,J＝6.8Hz,1H),4.97-4.94(m,1H),3.70(s,3H),2.93(t,J＝7.9Hz,2H),2.86–2.76(m,2H),1.48(d,6.6Hz,3H).ESI-MS m/z:350.1[M+H]⁺.

Example 10.

¹H-NMR(600MHz,DMSO-d6)δ9.21(s,1H),7.77(d,J＝6.5Hz,1H),7.52-7.47(m,4H),7.41(d,J＝7.4Hz,2H),7.11(d,J＝6.9Hz,2H),7.08(d,J＝2.2,1H),6.93(d,J＝6.8Hz,1H),4.13(s,2H),3.70(s,3H),2.93(t,J＝7.9Hz,2H),2.86–2.76(m,2H).ESI-MS m/z:429.1[M+H]⁺.

Example 11.

¹H-NMR(600MHz,DMSO-d₆)δ9.22(s,1H),7.77(d,J＝6.5Hz,1H),7.47(d,J＝7.4Hz,2H),7.40-7.35(m,4H),7.15(d,J＝6.9Hz,2H),7.08(d,J＝2.2,1H),6.93(d,J＝6.8Hz,1H),4.12(s,2H),3.70(s,3H),2.93(t,J＝7.9Hz,2H),2.86–2.76(m,2H),2.33(s,3H).ESI-MS m/z:425.2[M+H]⁺.

Pharmacological research of partial product of the invention

1. CYP1B1/CYP1A1 enzyme inhibitory Activity assay (EROD assay)

The test principle is as follows: the 7-ethoxy-3H-phenoxazin-3-one deethyl (EROD) assay is commonly used to evaluate the activity of CYP1A1 and CYP1B1 enzymes. The 7-ethoxy-3H-phenoxazin-3-one is a substrate for CYP1A1 and CYP1B1 enzymes, and is converted into a metabolite, 7-hydroxy-3H-phenoxazin-3-one, through an enzyme-mediated O-deethylation reaction. The latter emits fluorescence with wavelength of 590nm under excitation wavelength of 544nm, and is read by a microplate reader. The inhibitory activity on CYP1A1 and CYP1B1 is judged by the intensity of the compound inhibiting the EROD.

The test method comprises the following steps: the reaction system (200. mu.L) contained 10fmol CYP1A1 or 20fmol CYP1B1 enzyme, varying concentrations of test compound, NADPH regenerating system (1.3mM NADP +,3.3mM glucose-6-phosphate, 0.5U/ml glucose-6-phosphate dehydrogenase), 3.3mM MgCl₂And 150nmol 7-ethoxy-3H-phenoxazin-3-one. The reaction buffer was 50mM Tris-HCl (pH 7.4) buffer containing 1% BSA. After the reaction system is preheated for L0 min at 37 ℃, an NADPH regeneration system is added to start the reaction, and 100 mu L of precooled acetonitrile is added to stop the reaction after the reaction is finished. The excitation wavelength and the emission wavelength are 544nm and 590nm respectively by using a multifunctional microplate reader for detection.

TABLE 1 CYP1B1/CYP1A1 enzyme inhibition assay results.

The experimental results show that the compounds of examples 1-11 have obvious CYP1B1 inhibitory activity, and the optimal compound has the activity equivalent to that of the positive control drug alpha-naphthoflavone.

2. Antitumor cell activity and reverse drug resistance activity

Examples 1-11 compounds were screened separately by combination with the anticancer drug paclitaxel on sensitive beads and paclitaxel resistant beads using the CCK8 method (cell lines: MCF-7 and MCF-7/Taxol).

To confirm that the enhanced cytotoxicity of the compounds of examples 1-11 was due to pharmacodynamic effects and not to the cytotoxicity of the compounds themselves, we first determined the inhibitory activity of the compounds of the examples on MCF-7 and MCF-7/Taxol cells.

MCF-7 and MCF-7/Taxol cells in logarithmic growth phase at 6X 10³One/well inoculation in 96-well plates at 37 ℃ and 5% CO₂Culturing for 24h under the condition; then, the compound of example was added to each well to a final concentration of 5. mu.M, 3 wells were set, and after 48 hours of incubation, 10. mu.L of CCK-8 reagent was added to each well, and the incubation was continued for 1 hour, and the absorbance at a wavelength of 450nm was measured for each well, and the survival rate of the compound against tumor cells was calculated. The results of the experiment show that the test compound has no obvious toxicity to the test cells when the concentration of the test compound is 5 mu M.

Table 2 survival of tumor cells at 5.0 μ M for the compounds of examples 1-11.

Compound (I)	MCF-7	MCF-7/Taxol	Compound (I)	MCF-7	MCF-7/Taxol
						Example 1	92.7	91.2	Example 7	85.2	92.7
Example 2	88.2	>99.0	Example 8	86.3	75.2
						Example 3	82.1	>99.0	Example 9	94.7	>99.0
Example 4	91.7	91.2	Example 10	89.4	92.1
						Example 5	94.5	72.6	Example 11	>99.0	>99.0
Example 6	95.2	>99.0

According to the experimental data in table 1, the concentration of the test compound is selected to be 5.0 μ M, and the research experiment of the test compound reversing drug resistance of drug-resistant cells (the cells are MCF-7/Taxol) is carried out, specifically: MCF-7/Taxol cells at 6X 10³One/well inoculated in 96-well plates at 37 ℃ and 5% CO₂Culturing for 24h under the condition; then adding compound to be tested (5.0 μ M) and paclitaxel with different concentrations into each well, incubating, setting 3 multiple wells, incubating for 48 hr, adding 10 μ L CCK-8 reagent into each well, culturing for 1 hr, and measuring absorbance value at 450nm wavelength, IC₅₀The concentration of inhibitor at which cell growth was inhibited by half, and the results are shown in Table 3.

Table 3 experimental results for reversal of drug resistant cells by the compounds of examples 1-11.

Compound (I)	IC50/Tax	RF	Compound (I)	IC50/Tax	RF
						Example 1	30.1	2.87	Example 7	39.9	2.16
Example 2	39.9	2.16	Example 8	23.3	3.71
						Example 3	42.8	2.01	Example 9	27.6	3.13
Example 4	59.8	1.44	Example 10	30.2	2.86
						Example 5	43.2	2.00	Example 11	36.5	2.37
Example 6	39.8	2.17	Paclitaxel	86.4

Experimental results show that the compound of the examples 1-11 can obviously enhance the anti-tumor activity of the paclitaxel when being used together with the paclitaxel.