Inhibitors of cytochrome p450 enzyme 3a4 and methods of making and using the same

Abstract

The present application provides a cytochrome P450 enzyme 3A4 inhibitor, which has the following structural formula: The present application also provides a preparation method and use of the inhibitor. The present application can effectively inhibit the activity of human CYP3A4, prolong the metabolic half-life of CYP3A4 substrate drugs, slow down the first-pass metabolism of CYP3A4 substrate drugs, and further improve the pharmacological activity of CYP3A4 substrate drugs. In vitro activity determination finds that the IC 50 can reach 0.78 micromole to 0.04 micromole. In addition, the compound has the advantages of simple preparation process, high synthesis yield, etc., indicating that the compound has good development prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to an aza-chalcone cytochrome P450 enzyme, in particular to a cytochrome P450 enzyme 3A4 inhibitor and a preparation method and application thereof. BACKGROUND

[0002] Cytochrome P450 enzymes (CYP) are a class of monooxygenases widely distributed in various organisms, with ferriheme as the catalytic center, which catalyzes the oxidative metabolism of many endogenous substances and exogenous compounds, and further regulates the in vivo effects of drugs / toxins, endogenous substance metabolism balance, and the occurrence and development of various diseases. Among the many human P450 enzymes, CYP3A4 has been the focus of pharmacological and toxicological researchers due to its high expression in liver, intestine and kidney, and its wide substrate spectrum and diverse functions.

[0003] CYP3A4 can catalyze the metabolic clearance of many clinical drugs (such as cardiovascular drugs, antitumor drugs and antiviral drugs). CYP3A4 inhibitors have various pharmacological effects: 1) CYP3A4 inhibitors can slow down the first-pass metabolism of CYP3A4 substrate drugs in vivo, prolonging the half-life of the drugs. For example, some combination drugs containing enhancers have been developed in the clinic, such as the anti-coronavirus drug Paxlovid and the anti-AIDS drug Kaletra, which contain nirmatrelvir or lopinavir (active ingredients) and ritonavir (CYP3A4 inhibitor). 2) CYP3A4 is also involved in the metabolism of many endogenous substances such as steroids (such as testosterone, progesterone, cholesterol, deoxycholic acid, lithocholic acid, etc.), vitamin D, arachidonic acid, etc. Regulating the expression / function of CYP3A4 can improve hormone / bile acid metabolism abnormalities and intervene in the occurrence and development of metabolic diseases such as osteoporosis, inflammation and tumors. 3) CYP3A4 is also a key protein that triggers resistance to many chemical drugs (such as platinum and taxanes), and CYP3A4 is highly expressed in many tumor-resistant cells. Strong inhibition of this enzyme can effectively increase the intracellular concentration of chemotherapeutic drugs and drug sensitivity, thereby reversing tumor drug resistance. Therefore, it is of great significance to develop new CYP3A4 inhibitors with good safety and in vivo effectiveness. SUMMARY

[0004] In view of the above technical problems in the prior art, the present application provides a cytochrome P450 enzyme 3A4 inhibitor and a preparation method and application thereof, which solve the technical problem that the effect of the compound in the prior art on inhibiting CYP3A4 is not good.

[0005] The present application provides a cytochrome P450 enzyme 3A4 inhibitor, which has the following structure:

[0006]

[0007] wherein R 1 ,R 2 ,R 4 ,R 5 is any one of hydrogen, methoxy, hydroxyl; R 3 is any one of hydrogen, methoxy, amino, dimethylamino, pyrrole.

[0008] Further, the cytochrome P450 enzyme 3A4 inhibitor is any one of the following structural formulae,

[0009] The application further provides a preparation method of the (E)-3-(isoquinoline-6-yl)-1-phenylprop-2-en-1-one compound, which uses isoquinoline-6-formaldehyde and phenylacetone derivative as raw materials, reacts under alkaline conditions for 6-8 hours, and then separates (E)-3-(4-phenyl)-1-(isoquinoline-6-yl)prop-2-en-1-one compound through fast column chromatography.

[0010] The application further provides application of the compound in preparation of a medicine for inhibiting cytochrome P450 enzyme 3A4.

[0011] Further, the dosage form is any one of enteric agent, tablet, powder, suspension, injection, enema, emulsion, film, suppository, capsule, infusion, dripping pill or injection.

[0012] Further, the administration route includes any one of oral administration, local administration or injection administration.

[0013] The compound of the application can strongly inhibit the activity of CYP3A4 in mammalian cells, thereby prolonging the metabolic half-life of CYP3A4 substrate drugs and slowing down the first-pass metabolism of CYP3A4 substrate drugs. 50 The IC50 of the compound in inhibiting CYP3A4 can reach 0.78 micromole to 0.04 micromole. In addition, the compound has the advantages of simple preparation process, high synthesis yield and the like, and indicates that the compound has good development prospect.

[0014] The compound has the advantages of simple preparation process, high synthesis yield and the like, and indicates that the compound has good development prospect. 50 The IC50 of the compound in inhibiting CYP3A4 can reach 0.78 micromole to 0.04 micromole. In addition, the compound has the advantages of simple preparation process, high synthesis yield and the like, and indicates that the compound has good development prospect. Figure 5It is indicated that compound 7 can occupy the catalytic pocket of human CYP3A4 and form coordination with the iron porphyrin ring, and meanwhile, the carbonyl group thereof can closely interact with arginine 105 through hydrogen bond, which can better explain the strong inhibition effect of the compound on CYP3A4. In addition, the compound has the advantages of simple preparation process, high synthesis yield and the like, and indicates that the compound has good development prospect.

[0015] Compared with the prior art, the technical effect of the present application is positive and obvious:

[0016] 1. Simple preparation process: the CYP3A4 inhibitor provided by the present application takes isoquinoline-6-formaldehyde and acetophenone derivatives as raw materials, is obtained through one-step chemical synthesis, and has simple and easy synthesis process and high yield.

[0017] 2. Strong inhibition capacity: the CYP3A4 inhibitor has a half-inhibition concentration IC 50 of nM level on cytochrome P450 enzyme 3A4 in human tissue microsomes.

[0018] 3. Good cell membrane permeability: the CYP3A4 inhibitor can rapidly penetrate the cell membrane, enter the cell and combine with intracellular CYP3A4, and then play a CYP3A4 inhibition effect.

[0019] 4. Good metabolic stability: the CYP3A4 inhibitor has good metabolic stability in human liver microsomes, and can play a long-acting inhibition effect on CYP3A4 in vivo.

[0020] 5. High safety: the CYP3A4 inhibitor shows good safety in mice.

[0021] 6. Oral effectiveness: after oral administration, the CYP3A4 inhibitor can inhibit CYP3A4 in the intestinal tract and liver, and then slow down the first-pass metabolism and in-vivo metabolism of CYP3A substrate drugs, prolong the half-life thereof, and increase the plasma exposure thereof. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Structural general formula of the (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compound.

[0023] Figure 2 Structural formula of the (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compound.

[0024] Figure 3 Synthetic route of the (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compound.

[0025] Figure 4 Dose-inhibition curve of (E)-1-(4-(dimethylamino)phenyl)-3-(isoquinolin-6- yl)prop-2-en-1-one inhibiting CYP3A4.

[0026] Figure 5 Molecular docking of (E)-1-(4-(dimethylamino)phenyl)-3-(isoquinolin-6- yl)prop-2-en-1-one with CYP3A4. DETAILED DESCRIPTION

[0027] The following examples will further illustrate the present application but do not limit the present application.

[0028] Example 1.

[0029] Synthesis and purification of (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compounds

[0030] [Experimental materials] isoquinoline-6-carboxaldehyde, acetophenone derivatives, basic reagent, thin layer plate

[0031] [Experimental procedure]

[0032] The organic solvent includes but is not limited to ethanol, dichloromethane, 1,2-dichloroethane, acetone, tetrahydrofuran, diethyl ether, toluene, etc. The base is a basic reagent, including but not limited to sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate, cesium carbonate, etc.

[0033] The acetophenone derivative (2.00 mmol) and isoquinoline-6-carboxaldehyde (1 eq) were mixed in an organic solvent, stirred at room temperature, and then a basic reagent was slowly added. The reaction mixture was stirred at room temperature overnight, and monitored by thin layer chromatography (TLC). After the reaction was completed, hydrochloric acid aqueous solution was added dropwise to the reaction mixture to adjust the pH value to neutral. The aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed once with water, once with saturated brine, dried over anhydrous sodium sulfate, the solvent was rotary evaporated, and column chromatography was used for purification, with a yield of 80-90%.

[0034] [Experimental results]

[0035] The nuclear magnetic resonance spectrum of (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one (compound 1) is as follows:

[0036]

[0037] 1H NMR (500 MHz, Chloroform-d) δ 9.31 (s, 1H), 8.61 (s, 1H), 8.15 - 8.05 (m, 2H), 8.03 (d, J = 8.3 Hz, 2H), 8.00 - 7.89 (m, 2H), 7.76 - 7.67 (m, 2H), 7.59 (dt, J = 42.2, 7.3 Hz, 3H). 13 C NMR (125 MHz, Chloroform-d) δ 189.64, 151.74, 143.48, 143.02, 137.41, 136.22, 135.47, 132.65, 128.28, 128.19, 128.11, 127.95, 127.89, 127.62, 124.84, 123.87. HRMS (ESI): C 18 H 14 NO[M+H] + Calculated for: 260.107, Found: 260.1069.

[0038] The nuclear magnetic resonance spectra of (E)-1-(2-hydroxyphenyl)-3-(isoquinolin-6- yl)prop-2-en-1-one (Compound 2) are as follows:

[0039]

[0040] 1 H NMR (500 MHz, DMSO-d6) δ 12.25 (s, 1H), 9.59 (s, 1H), 8.63 (d, J = 6.0 Hz, 1H), 8.55 (s, 1H), 8.44 - 8.40 (m, 1H), 8.37 (d, J = 8.7 Hz, 1H), 8.31 - 8.22 (m, 2H), 8.14 (d, J = 6.0 Hz, 1H), 8.04 - 7.96 (m, 1H), 7.62 - 7.56 (m, 1H), 7.04 (t, J = 7.3 Hz, 2H). 13 C NMR (125 MHz, DMSO-d6) δ 193.75, 162.07, 150.35, 143.06, 139.23, 137.25, 137.04, 131.48, 129.94, 129.34, 128.62, 127.99, 126.54, 123.30, 121.58, 119.81, 118.26. HRMS (ESI): C 18 H 14 NO2[M+H] + Calculated for: 276.1019, Found: 276.102.

[0041] The nuclear magnetic resonance spectrum of (E)-3-(isoquinolin-6-yl)-1-(2- methoxyphenyl)prop-2-en-1-one (Compound 3) is as follows:

[0042]

[0043] 1 H NMR (500 MHz, Chloroform-d) δ 9.27 (s, 1H), 8.57 (d, J = 5.6 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 1.4 Hz, 1H), 7.87 (dd, J = 8.6, 1.6 Hz, 1H), 7.79 (d, J = 15.8 Hz, 1H), 7.70 - 7.66 (m, 2H), 7.57 (d, J = 15.9 Hz, 1H), 7.52 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H), 7.07 (td, J = 7.5, 1.0 Hz, 1H), 7.04 (dd, J = 8.4, 0.9 Hz, 1H), 3.94 (s, 3H). 13 C NMR (125 MHz, Chloroform-d) δ 191.89, 157.84, 151.76, 143.45, 141.07, 136.59, 135.48, 132.85, 130.09, 128.82, 128.46, 127.79, 127.66, 124.90, 120.42, 120.28, 111.21, 55.34. HRMS (ESI): C 19 H 16 NO2[M+H] + calculated for: 290.1176, found: 290.1178.

[0044] The nuclear magnetic resonance spectrum of (E)-3-(isoquinolin-6-yl)-1-(3- methoxyphenyl)prop-2-en-1-one (Compound 4) is as follows:

[0045]

[0046] 1H NMR (500 MHz, Chloroform-d) δ 9.30 (s, 1H), 8.60 (s, 1H), 8.05 (d, J = 9.2 Hz, 2H), 7.99 - 7.92 (m, 2H), 7.74 (d, J = 5.9 Hz, 1H), 7.70 (d, J = 15.7 Hz, 1H), 7.65 (dt, J = 7.7, 1.3 Hz, 1H), 7.59 (dd, J = 2.7, 1.6 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.18 (ddd, J = 8.2, 2.7, 0.9 Hz, 1H), 3.91 (s, 3H). 13 C NMR (125 MHz, Chloroform-d) δ 189.80, 160.04, 151.78, 143.36, 139.24, 137.13, 129.74, 128.59, 128.36, 125.61, 124.64, 121.15, 119.65, 113.00, 55.56. HRMS (ESI): C 19 H 16 NO2[M+H] + Calculated for: 290.1176, Found: 290.1179.

[0047] The nuclear magnetic resonance spectra of (E)-3-(isoquinolin-6-yl)-1-(4- methoxyphenyl)prop-2-en-1-one (Compound 5) are as follows:

[0048]

[0049] 1 H NMR (500 MHz, Chloroform-d) δ 9.29 (s, 1H), 8.59 (d, J = 5.7 Hz, 1H), 8.12 - 8.07 (m, 2H), 8.06 - 8.00 (m, 2H), 7.98 - 7.91 (m, 2H), 7.76 - 7.70 (m, 2H), 7.04 - 6.99 (m, 2H), 3.92 (s, 3H). 13 C NMR (125 MHz, Chloroform-d) δ 187.73, 163.24, 151.40, 142.90, 142.05, 136.74, 135.65, 130.47, 130.30, 127.97, 127.64, 125.06, 123.91, 120.52, 113.51, 55.08. HRMS (ESI): C 19 H 16 NO2[M+H] +HRMS (ESI): C

[0050] The nuclear magnetic resonance spectrum of (E)-1-(4-aminophenyl)-3-(isoquinolin-6- yl)prop-2-en-1-one (Compound 6) is as follows:

[0051]

[0052] 1 H NMR (600 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.70 (d, J = 6.4 Hz, 1H), 8.65 (s, 1H), 8.60 - 8.52 (m, 2H), 8.40 (d, J = 6.4 Hz, 1H), 8.30 (d, J = 15.6 Hz, 1H), 8.12 (d, J = 9.0 Hz, 2H), 7.86 (d, J = 15.5 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 3.08 (s, 6H). 13 C NMR (125 MHz, DMSO-d6) δ 185.88, 154.71, 149.61, 140.57, 140.02, 137.98, 137.67, 131.89, 130.03, 128.30, 128.24, 127.22, 125.50, 123.61, 113.29, 49.06. HRMS (ESI): C 18 H 15 N2O [M+H] + calculated for: 275.1179, found: 275.118.

[0053] The nuclear magnetic resonance spectrum of (E)-1-(4-(dimethylamino)phenyl)-3-(isoquinolin-6- yl)prop-2-en-1-one (Compound 7) is as follows:

[0054]

[0055] 1 H NMR (600 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.70 (d, J = 6.4 Hz, 1H), 8.65 (s, 1H), 8.60 - 8.52 (m, 2H), 8.40 (d, J = 6.4 Hz, 1H), 8.30 (d, J = 15.6 Hz, 1H), 8.12 (d, J = 9.0 Hz, 2H), 7.86 (d, J = 15.5 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 3.08 (s, 6H). 13C NMR (125 MHz, DMSO-d6) δ 185.84, 151.56, 148.26, 141.87, 139.71, 138.46, 135.27, 131.67, 130.68, 129.04, 128.27, 127.99, 127.94, 124.90, 124.68, 111.58, 47.85, 25.41. HRMS (ESI): C22H21N2O [M+H] 20 H 19 N2O[M+H] + calculated for:303.1492,found:303.1489.

[0056] The NMR spectra of (E)-3-(isoquinolin-6-yl)-1-(4-(pyrrolin-1-yl)phenyl)prop-2-en-1-one (Compound 8) are as follows:

[0057]

[0058] 1 H NMR (500 MHz, DMSO-d6) δ 9.77 (s, 1H), 8.68 (d, J = 6.3 Hz, 1H), 8.61 (s, 1H), 8.57 - 8.48 (m, 2H), 8.34 (d, J = 6.3 Hz, 1H), 8.28 (d, J = 15.5 Hz, 1H), 8.11 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 6.65 (d, J = 8.8 Hz, 2H), 3.39 (d, J = 12.9 Hz, 4H), 2.05 - 1.97 (m, 4H). 13 C NMR (125 MHz, DMSO-d6) δ 185.84, 151.56, 148.26, 141.87, 139.71, 138.46, 135.27, 131.67, 130.68, 129.04, 128.27, 127.99, 127.94, 124.90, 124.68, 111.58, 47.85, 25.41. HRMS (ESI): C22H21N2O [M+H] + calculated for:329.1648,found:329.1653.

[0059] The NMR spectra of (E)-1-(2,4-dimethoxyphenyl)-3-(isoquinolin-6-yl)prop-2-en-1-one (Compound 9) are as follows:

[0060]

[0061] 1H NMR (600 MHz, Chloroform-d) δ 9.23 (s, 1H), 8.60-8.50 (m, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.93 (s, 1H), 7.85 (dd, J = 8.5, 1.6 Hz, 1H), 7.83-7.77 (m, 2H), 7.69 (d, J = 15.7 Hz, 1H), 7.65 (d, J = 5.6 Hz, 1H), 6.57 (dd, J = 8.7, 2.3 Hz, 1H), 6.50 (d, J = 2.3 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H). 13 C NMR (150 MHz, Chloroform-d) δ 189.90, 164.57, 164.52, 160.65, 152.21, 143.88, 140.49, 137.42, 136.01, 133.14, 129.55, 128.20, 127.91, 125.44, 121.90, 120.76, 105.43, 98.68, 55.84, 55.62. HRMS (ESI): C 20 H 18 NO3[M+H] + calculated for: 320.1281, found: 320.1283.

[0062] The nuclear magnetic resonance spectra of (E)-1-(2,3-dimethoxyphenyl)-3-(isoquinolin-6- yl)prop-2-en-1-one (Compound 10) are as follows:

[0063]

[0064] 1 H NMR (500 MHz, Chloroform-d) δ 9.27 (s, 1H), 8.58 (s, 1H), 8.02-7.93 (m, 2H), 7.87 (dt, J = 8.5, 1.6 Hz, 1H), 7.79-7.73 (m, 1H), 7.67 (d, J = 5.8 Hz, 1H), 7.56 (dd, J = 15.9, 1.3 Hz, 1H), 7.19 (dd, J = 19.6, 7.8, 1.5 Hz, 3H), 7.11 (dt, J = 8.0, 1.6 Hz, 1H), 3.94 (s, 3H), 3.87 (s, 3H). 13CNMR (125 MHz, Chloroform-d) δ 192.82, 153.01, 152.17, 148.21, 143.82, 142.42, 136.89, 135.97, 134.16, 129.12, 128.38, 128.21, 125.50, 124.48, 121.22, 117.48, 115.46, 62.14, 56.21, 56.06. HRMS (ESI): C 20 H 18 NO3[M+H] + calculated for: 320.1281, found: 320.1285.

[0065] The nuclear magnetic resonance spectra of (E)-1-(3,4-dimethoxyphenyl)-3-(isoquinolin-6-yl)prop-2-en-1-one (Compound 11) are as follows:

[0066]

[0067] 1 H NMR (600 MHz, Chloroform-d) δ 9.24 (s, 1H), 8.55 (d, J = 5.7 Hz, 1H), 8.00 - 7.96 (m, 2H), 7.92 (d, J = 15.6 Hz, 1H), 7.89 (dd, J = 8.5, 1.7 Hz, 1H), 7.74 - 7.67 (m, 2H), 7.67 - 7.64 (m, 1H), 7.63 (d, J = 2.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.96 (d, J = 2.6 Hz, 6H). 13 C NMR (150 MHz, Chloroform-d) δ 188.13, 153.59, 152.27, 149.41, 144.03, 142.68, 136.86, 135.96, 131.04, 128.92, 128.33, 128.18, 125.33, 123.96, 123.24, 120.75, 110.79, 110.01, 56.15, 56.10. HRMS (ESI): C 20 H 18 NO3[M+H] + calculated for: 320.1281, found: 320.1283.

[0068] The nuclear magnetic resonance spectra of (E)-1-(2-hydroxy-6-methoxyphenyl)-3-(isoquinolin-6-yl)prop-2-en-1-one (Compound 12) are as follows:

[0069]

[0070] 1 H NMR (500 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.65 (s, 1H), 8.62 (d, J = 6.2 Hz, 1H), 8.44 (s, 1H), 8.37 (d, J = 8.7 Hz, 1H), 8.26 (dd, J = 8.7, 1.7 Hz, 1H), 8.19 (d, J = 6.2 Hz, 1H), 7.52 (d, J = 16.1 Hz, 1H), 7.44 (d, J = 16.1 Hz, 1H), 7.28 (t, J = 8.3 Hz, 1H), 6.58 (dd, J = 13.8, 8.3 Hz, 2H), 3.74 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 194.67, 158.58, 157.30, 149.45, 141.91, 137.78, 132.79, 132.52, 130.40, 128.75, 128.31, 128.07, 123.97, 116.09, 109.47, 102.85, 56.32. HRMS (ESI): C 19 H 16 NO3[M+H] + calculated for: 306.1125, found: 306.1128.

[0071] The nuclear magnetic resonance spectra of (E)-1-(2-hydroxy-4,5-dimethoxyphenyl)-3- (isoquinolin-6-yl)prop-2-en-1-one (Compound 13) are as follows:

[0072]

[0073] 1 H NMR (500 MHz, DMSO-d6) δ 12.42 (s, 1H), 9.80 (s, 1H), 8.64 (d, J = 6.4 Hz, 1H), 8.45 (dd, J = 14.7, 7.6 Hz, 2H), 8.32 (s, 1H), 8.10 - 8.04 (m, 1H), 7.28 (s, 1H), 6.54 (s, 1H), 5.47 (dd, J = 8.3, 4.2 Hz, 1H), 3.80 (s, 3H), 3.72 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 201.80, 159.23, 156.91, 154.32, 147.70, 142.19, 138.49, 133.46, 130.50, 129.64, 126.88, 124.90, 124.00, 112.61, 112.33, 100.87, 69.26, 56.67, 56.45, 47.85. HRMS (ESI): C 20 H 18 NO4[M+H] + Calculated for: 336.123, Found: 336.1232.

[0074] The NMR spectra of (E)-3-(Isoquinolin-6-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (Compound 15) are as follows:

[0075]

[0076] 1 H NMR (600 MHz, Chloroform-d) δ 14.18 (s, 1H), 9.23 (s, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.03 (d, J = 15.6 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.95 - 7.82 (m, 3H), 7.67 (d, J = 5.7 Hz, 1H), 6.11 (d, J = 2.3 Hz, 1H), 5.97 (d, J = 2.4 Hz, 1H), 3.94 (s, 3H), 3.84 (s, 3H). 13 C NMR (150 MHz, Chloroform-d) δ 192.21, 168.54, 166.58, 162.53, 152.13, 143.77, 140.81, 137.52, 136.06, 130.14, 128.82, 128.28, 128.05, 125.39, 120.85, 106.36, 93.87, 91.43, 55.99, 55.68. HRMS (ESI): C 20 H 18 NO4[M+H] + Calculated for: 336.123, Found: 336.1232.

[0077] The NMR spectra of (E)-3-(Isoquinolin-6-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (Compound 15) are as follows:

[0078]

[0079] 1 H NMR (600 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.68 (d, J = 6.2 Hz, 1H), 8.61 (s, 1H), 8.58 - 8.53 (m, 1H), 8.49 (d, J = 8.7 Hz, 1H), 8.31 - 8.24 (m, 2H), 7.96 (d, J = 15.6 Hz, 1H), 7.50 (s, 2H), 3.93 (s, 6H), 3.79 (s, 3H). 13 CNMR (150 MHz, DMSO-d6) δ 188.23, 153.49, 149.23, 142.87, 142.31, 137.97, 133.02, 130.35, 129.23, 128.57, 128.34, 126.76, 124.19, 106.94, 60.73, 56.80. HRMS (ESI): C 21 H 20 NO4[M+H] + calculated for: 350.1387, found: 350.1389.

[0080] The nuclear magnetic resonance spectrum of (E)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)-3-(isoquinolin-6-yl)prop-2-en-1-one (Compound 16) is as follows:

[0081]

[0082] 1 H NMR (500 MHz, Chloroform-d) δ 13.47 (s, 1H), 9.39 (s, 1H), 8.57 (d, J = 5.9 Hz, 1H), 8.21 - 8.13 (m, 2H), 8.11 (s, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 5.9 Hz, 1H), 7.93 (d, J = 15.6 Hz, 1H), 6.33 (s, 1H), 3.95 (d, J = 17.3 Hz, 6H), 3.86 (s, 3H). 13C NMR (125 MHz, Chloroform-d) δ 192.13, 162.98, 160.92, 154.88, 140.02, 137.66, 135.39, 131.29, 129.55, 128.17, 127.85, 127.32, 123.03, 108.68, 96.69, 62.03, 61.37, 56.26. HRMS (ESI): C 21 H 20 NO5[M+H] + Calculated for: 366.1336, Found: 366.1338.

[0083] Example 2.

[0084] Evaluation of the inhibitory ability of (E)-3-(4-phenyl)-1-(isoquinolin-6-yl)prop-2-en-1-one compound on CYP3A4 [Experimental materials] Human liver microsomes, NEN, phosphate buffer, magnesium chloride, NADP + , glucose-6-phosphate, glucose-6-phosphate dehydrogenase.

[0085] [Experimental procedure]

[0086] The IC50of (E)-3-(4-phenyl)-1-(isoquinolin-6-yl)prop-2-en-1-one compound on human cytochrome P450 enzyme 3A4 inhibitory ability was determined by using the probe reaction of NEN metabolism and the in vitro incubation system of liver microsomes. 50

[0087] a. 200 microliters of in vitro metabolic reaction system contained phosphate buffer with pH of 7.4, 4 mM of magnesium chloride, 1 mM of NADP + , 10 mM of glucose-6-phosphate, 1 unit / ml of glucose-6-phosphate dehydrogenase, and the final concentration of liver microsomal protein was 0.05 mg / ml;

[0088] b. Pre-incubated for 3 minutes at 37°C, added NEN (final concentration of 10 mM) to start the reaction, and incubated the reaction on the enzyme marker for 30 minutes;

[0089] c. Quantitative detection of NEHN (metabolite of NEN in HLMs) was carried out at excitation wavelength of 450 nm and emission wavelength of 558 nm.

[0090] [Experimental results]

[0091] As Figure 4 ​, As shown in Table 1, the (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compound can effectively inhibit human cytochrome P450 enzyme 3A4 and the half-inhibitory effective concentration IC 50 Below 100 nM.

[0092] Table 1. Inhibition of cytochrome P450 enzyme 3A4 by (E)-3-(isoquinolin-6-yl)-1- phenylprop-2-en-1-one compound

[0093] SEQ ID NO CYP3A4-IC 50 ]]> SEQ ID NO CYP3A4-IC 50 ]]> 1 102.10 nM 9 70.03 nM 2 115.70 nM 10 225.90 nM 3 159.40 nM 11 94.11 nM 4 90.00 nM 12 187.20 nM 5 77.59 nM 13 254.10 nM 6 75.87 nM 14 83.56 nM 7 43.93 nM 15 784.70 nM 8 55.19 nM 16 102.10 nM

[0094] Example 3.

[0095] Evaluation of the inhibitory ability of (E)-3-(4-phenyl)-1-(isoquinolin-6-yl)prop-2-en-1- one compound on CYP3A4 in living cells

[0096] [Experimental materials] PBS, Ham's F-12 medium, fetal bovine serum, trypsin, cell counting plate, puromycin, NEN, acetonitrile, 96-well cell culture plate.

[0097] [Experimental steps]

[0098] Using the incubation system of CHO-3A4 cells as a probe reaction for NEN metabolism, the inhibitory ability of (E)-3-(4-phenyl)-1-(isoquinolin-6-yl)prop-2-en-1-one compound on cytochrome P450 enzyme 3A4 was evaluated:

[0099] a. The CHO-3A4 stably transfected cell line was cultured in Ham's F-12 medium containing 10% fetal bovine serum at 37°C in a humidified atmosphere (95% air and 5% CO2).

[0100] b. The CHO-3A4 stably transfected cell line was inoculated in a 96-well plate, and when the cells in the 96-well plate reached about 60% density, ketokonazole or (E)-3-(4- phenyl)-1-(isoquinolin-6-yl)prop-2-en-1-one compound was used for treatment for 1 hour. Then the cells were treated with NEN (10 μM, final concentration) for 30 minutes, and 100 μL of acetonitrile was added to terminate the reaction.

[0101] c. Centrifugation at 20,000 g, 4°C for 30 minutes, and the supernatant was subjected to liquid chromatography-fluorescence detection analysis for quantitative detection of metabolites.

[0102] [Experimental results]

[0103] As shown in Table 2, the (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compounds have good cell membrane permeability and can effectively inhibit CYP3A4 in cells.

[0104] Table 2 Inhibition of cytochrome P450 enzyme 3A4 by (E)-3-(isoquinolin-6-yl)-1-phenylprop-2-en-1-one compounds in CHO-3A4 cell incubation system

[0105]

[0106] Example 6. Effect of compounds 7, 8 on the pharmacokinetics of CYP3A substrate drug midazolam

[0107] [Experimental materials] C57 mice, 0.5% carboxymethylcellulose sodium solution, midazolam, acetonitrile.

[0108] [Experimental steps]

[0109] a. 108 mice were raised in a controlled environment (22±2℃; 40%-80% relative humidity; 12h light / dark cycle) for one week, with free access to food and water. The rats were fasted overnight before administration, but could drink water freely, and food was provided after the end. 108 mice were randomly divided into three groups, 36 in each group. Then 36 mice were immediately divided into 6 cages, 6 in each cage, and each mouse was bled at two time points. Group 1: CMC-Na+midazolam; Group 2: 7(100mg / kg)+midazolam; Group 3: CMC-Na+midazolam; Group 3: 8(50mg / kg)+midazolam.

[0110] b. 30 minutes after gavage of 7, 8 or CMC-Na, midazolam was administered again by gavage. Then blood samples were collected at 5, 10, 20, 30, 60, 90, 120, 180, 240, 360 minutes after administration of midazolam, respectively. Then the blood samples were centrifuged at 8000rpm, 4℃ for 10 minutes, and the supernatant was stored at-80℃ until analysis.

[0111] c. Before LC-MS / MS analysis, the plasma samples were mixed with acetonitrile at a ratio of 1:5, then centrifuged at 20,000xg at 4℃ for 30min. The supernatant was used for LC-MS / MS analysis.

[0112] [Experimental results]

[0113] Compounds 7, 8 can effectively increase the plasma exposure and metabolic half-life of midazolam in mice by inhibiting CYP3A in mice.

[0114] Table 1 Pharmacokinetic parameters of compound 7 and midazolam in mice

[0115]

[0116] Table 2 Pharmacokinetic parameters of compound 8 and midazolam in mice

[0117]

Claims

1. An inhibitor of cytochrome P450 enzyme 3A4, characterized in that: Its structural formula is as follows: or 。 2. The use of the cytochrome P450 enzyme 3A4 inhibitor of claim 1 in the preparation of a drug that slows down the first-pass metabolism of a CYP3A4 substrate drug in vivo and prolongs the drug's half-life, wherein the CYP3A4 substrate drug is midazolam.

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