阅读说明：本技术 化合物JNc-463的制备方法及其应用 (Preparation method and application of compound JNc-463 ) 是由 马鑫 毛爱琴 唐春雷 于 2021-10-11 设计创作，主要内容包括：本发明公开了化合物JNc-463的制备方法及其应用。本发明所述制备方法包括如下步骤：(1)将色胺与乙酸溶解于甲醇中,加入3,4-二苄氧基苯甲醛,搅拌得到混合溶液Ⅰ；(2)加入NaBH-(3)CN搅拌,得到混合溶液Ⅱ；将步骤(2)配制的混合溶液Ⅱ浓缩,得到粗品；将步骤(3)得到的粗品经硅胶层析后得到化合物JNc-463。本发明所述方法制备的化合物JNc-463用于制备治疗抗高血压药物,可以很好的实现特异性靶向TRPV4-eNOS耦联复合体,克服了非专一性靶点以及副作用较多等问题,满足日益增长的临床需求。(The invention discloses a preparation method and application of a compound JNc-463. The preparation method comprises the following steps: (1) dissolving tryptamine and acetic acid in methanol, adding 3, 4-dibenzyloxybenzaldehyde, and stirring to obtain a mixed solution I; (2) adding NaBH 3 CN is stirred to obtain a mixed solution II; concentrating the mixed solution II prepared in the step (2) to obtain a crude product; and (4) carrying out silica gel chromatography on the crude product obtained in the step (3) to obtain a compound JNc-463. The compound JNc-463 prepared by the method is used for preparing antihypertensive drugs, can well realize specific targeting of TRPV4-eNOS coupled complex, overcomes the problems of non-specific target spots, more side effects and the like, and meets the increasing clinical requirements。)

1. A process for the preparation of compound JNc-463, characterized in that it comprises the following synthetic route:

2. the method of claim 1, comprising the steps of:

(1) dissolving tryptamine and acetic acid in methanol, adding 3, 4-dibenzyloxybenzaldehyde, and stirring to obtain a mixed solution I;

(2) adding NaBH₃CN is stirred to obtain a mixed solution II,

(3) concentrating the mixed solution II prepared in the step (2) to obtain a crude product;

(4) and (4) carrying out silica gel chromatography on the crude product obtained in the step (3) to obtain a compound JNc-463.

3. The production method according to claim 2, wherein in the step (1), the methanol is anhydrous methanol; the molar concentration of tryptamine in the mixed solution I is 0.29-0.32mol/L, the molar concentration of acetic acid is 0.29-0.32mol/L, and the molar concentration of 3, 4-dibenzyloxybenzaldehyde is 0.29-0.32 mol/L.

4. The method according to claim 2, wherein in step (2), NaBH is added to the mixed solution II₃The molar concentration of CN is 0.29-0.32 mol/L; the stirring time is 4-6 h.

5. The method according to claim 2, wherein the yield of the compound JNc-463 in step (4) is 20-22%.

6. The method of claim 1, comprising the steps of:

(1) dissolving 3.12mmol of tryptamine and 3.12mmol of acetic acid in 10mL of methanol solution, adding 3.12mmol of 3, 4-dibenzyloxybenzaldehyde, and stirring for 1h to obtain a mixed solution I;

(2) 3.12mmol of NaBH was added₃CN, stirring for 5 hours to obtain a mixed solution II;

(3) concentrating the mixed solution II obtained in the step (2) to obtain a crude product;

(4) performing chromatography on the crude product obtained in the step (3) on silica gel to obtain a compound JNc-463; the yield of compound JNc-463 was 21%.

7. The use of compounds JNc-463 prepared by the process according to any one of claims 1-6, wherein compounds JNc-463 are used for the preparation of a medicament for the treatment of hypertension.

8. The use of claim 7, wherein said medicament is in the form of a tablet, pill, or granule, said tablet comprising compound JNc-463 and an excipient; the auxiliary material comprises one or more of disintegrant and starch.

9. The use of claim 7, wherein the medicament is in the form of a capsule.

10. The use of claim 7, wherein said compound JNc-463 is formulated as an injection solution in physiological saline; the molar concentration of the compound JNc-463 in the injection is 1-30 mu mol/L.

Technical Field

The invention relates to the technical field of medical application, in particular to a preparation method and application of a compound JNc-463.

Background

Antihypertensive drugs including diuretics, beta-blockers, Ca²⁺Channel blockers, angiotensin converting enzyme inhibitors and angiotensin receptor blockers, are the main means of treating hypertension. This is achieved byThese drugs mainly affect sympathetic nerve activity or cardiac output, resulting in a decrease in blood pressure. However, due to their systemic activity, there are side effects during treatment. Therefore, in the development of hypertension, it is important to develop new means specifically directed to the essential components to avoid unnecessary side effects.

Transient receptor potential channel V4(TRPV4) is a nonselective cation channel protein that is widely expressed on cells of epithelial cells, macrophages, airway smooth muscle, etc. of the respiratory tract, can be activated by various stimuli, such as PH, temperature, arachidonic acid, osmotic pressure, etc., while the function of endothelial cells is of great interest, and is involved in various vascular functions, including responding to blood flow shear force, regulating vascular tension, mechanical signaling, angiogenesis, etc.

In large ductal vessels, Nitric Oxide (NO) selectively mediates endothelium-dependent relaxation. Loss of NO raises blood pressure, and dysfunction of the european community is often accompanied by a decrease in nitric oxide production and bioavailability, and a decrease in arterial baroreflexivity and compliance further raises blood pressure, leading to vicious circulation. The main source of NO is NO synthase (eNOS) in vascular endothelial cells. It is well known that physiological stimuli such as blood flow shear forces regulate the production of NO through phosphorylation of eNOS, which contributes greatly to the vasodilation response. Studies have shown that TRPV4 is critical for NO-related vasodilatory responses. Under normal conditions, TRPV4 forms a coupling complex with eNOS, TRPV4 is activated to cause calcium ion influx, thereby stimulating eNOS to produce NO, so that blood vessels relax, but under the pathological condition of hypertension, TRPV4 is reduced in coupling with eNOS, so that NO production is reduced, and blood pressure is increased. In recent years, hypotensor with better curative effect, less side effect and good bioavailability is urgently needed in clinic.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method and application of compounds JNc-463. The preparation method is simple and low in cost, and the prepared compound JNc-463 serving as an antihypertensive drug can well realize specific targeting of a TRPV4-eNOS coupled complex, overcomes the problems of non-specific targets, more side effects and the like, and meets the increasing clinical requirements.

The technical scheme of the invention is as follows:

a process for the preparation of compound JNc-463, said process comprising the following synthetic route:

the preparation method specifically comprises the following steps:

(1) dissolving tryptamine and acetic acid in methanol, adding 3, 4-dibenzyloxybenzaldehyde, and stirring to obtain a mixed solution I;

(2) adding NaBH₃CN is stirred to obtain a mixed solution II,

(3) concentrating the mixed solution II prepared in the step (2) to obtain a crude product;

(4) and (4) carrying out silica gel chromatography on the crude product obtained in the step (3) to obtain a compound JNc-463.

Further, in the step (1), the methanol is anhydrous methanol; the molar concentration of tryptamine in the mixed solution I is 0.29-0.32mol/L, the molar concentration of acetic acid is 0.29-0.32mol/L, and the molar concentration of 3, 4-dibenzyloxybenzaldehyde is 0.29-0.32 mol/L.

Further, in the step (2), NaBH is contained in the mixed solution II₃The molar concentration of CN is 0.29-0.32 mol/L; the stirring time is 4-6 h.

Further, in step (4), the yield of the compound JNc-463 was 20-22%.

Further, the preparation method comprises the following steps:

(1) dissolving 3.12mmol of tryptamine and 3.12mmol of acetic acid in 10mL of methanol solution, adding 3.12mmol of 3, 4-dibenzyloxybenzaldehyde, and stirring for 1h to obtain a mixed solution I;

(2) 3.12mmol of NaBH was added₃CN, stirring for 5 hours to obtain a mixed solution II;

(3) concentrating the mixed solution II obtained in the step (2) to obtain a crude product;

(4) performing chromatography on the crude product obtained in the step (3) on silica gel to obtain a compound JNc-463; the yield of compound JNc-463 was 21%.

An application of the compound JNc-463 prepared by the preparation method, wherein the compound JNc-463 is used for preparing a medicine for treating hypertension and hyperlipidemia.

Further, the medicament is formulated into a tablet, pill or granule, the tablet comprising compound JNc-463 and an excipient; the auxiliary material comprises one or more of disintegrant and starch.

Further, the medicine is prepared into capsules.

Further, the compound JNc-463 is dissolved in physiological saline to be prepared into an injection; the molar concentration of the compound JNc-463 in the injection is 1-30 mu mol/L.

The eluent of the chromatography is CH₂Cl₂With MeOH, CH₂Cl₂Volume ratio to MeOH was 20: 1.

the compound JNc-463 prepared by the method of the invention is dissolved in normal saline to prepare a solution with the molar concentration of 8-12 mu mol/L, and the solution is administrated by tail vein injection according to the mass ratio of the solution to the body weight of a human body of 0.8-1.2 mg/kg.

The beneficial technical effects of the invention are as follows:

(1) the invention firstly searches the interaction site structural domain of TRPV4 protein and eNOS by a structural domain site mutation method, and then screens a compound capable of enhancing the spatial coupling degree of a TRPV4-eNOS complex, wherein the compound acts on a hypertension mouse model and has good curative effect on hypertension; the vasodilation degree of the blood vessel of the hypertension model mouse is improved by 25 to 30 percent; the blood pressure of the hypertension model mouse is reduced by 18-26 mmHg.

(2) The small molecular drug capable of specifically targeting the TRPV4-eNOS coupled complex is found by acting on a hypertension mouse model, has the effect of inhibiting hypertension by enhancing the spatial coupling degree of the TRPV4-eNOS complex, overcomes the problems of non-specific targets, more side effects and the like, meets the growing clinical requirements, and has great significance for the research and development of hypertension drugs.

Drawings

FIG. 1 is a schematic representation of the effect of compounds JNc-463 prepared in example 1 of the present invention on the efficiency of TRPV4 and eNOS interaction.

FIG. 2 is a graph showing the effect of compounds JNc-463 prepared in example 1 of the present invention on the aortic relaxation in hypertensive mice raised with high salt and induced by AngII.

FIG. 3 is a graph showing the effect of compounds JNc-463 prepared in example 1 of the present invention on the hypotensive effect of high-salt fed and AngII-induced hypertensive mice.

FIG. 4 is a graph of the effect of different concentrations of compounds JNc-463 on TRPV4-eNOS coupling.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.

Example 1: preparation method of compound JNc-463

Tryptamine (500mg, 3.12mmol) and AcOH (187mg, 3.12mmol) were dissolved in 10mL MeOH, 3, 4-dibenzyloxybenzaldehyde (994mg, 3.12mmol) was added to the solution, and the mixture was stirred at room temperature for 1 hour. Then NaBH is added₃CN (196mg, 3.12mmol), and after addition, the mixture was stirred at room temperature for a further 5 hours. The mixture was concentrated to give the crude product on silica gel (CH)₂Cl₂Chromatography on MeOH 20/1) afforded compound JNc-463 as a white solid (300mg, yield: 21%).

Ms (esi): mass is calculated as C₃₁H₃₀N₂O₂462.2, M/z 463.2[ M + H ]]⁺。¹H NMR(400MHz，DMSO)δ10.78(s，1H)，7.50-7.28(m，12H)，7.11-7.04(m，3H)，7.02-6.93(m，2H)，6.83-6.80(m，1H)，5.07(d，J＝8.0Hz，4H)，3.65(s，2H)，2.83(t，J＝6.0Hz，2H)，2.75(t，J＝6.0Hz，2H)。

Example 2

Example 1: preparation method of compound JNc-463

Dissolving tryptamine 2.9mmol and 2.9mmol AcOH in 10mL MeOH, adding 2.9mmol 3, 4-dibenzyloxybenzaldehyde to the solution, and stirring the mixed solution at room temperatureStirring for 1 h. Then 2.9mmol of NaBH was added₃CN, after addition, the mixture was stirred at room temperature for a further 4 h. Freeze-concentrating the mixed solution to obtain crude product on silica gel (CH)₂Cl₂MeOH 20/1) gave compound JNc-463 as a white solid.

Example 3

After 3.2mmol of tryptamine and 3.2mmol of AcOH were dissolved in 10mL of MeOH, 3.2mmol of 3, 4-dibenzyloxybenzaldehyde was added to the solution, and the mixture was stirred at room temperature for 1 hour. Then 3.2mmol of NaBH was added₃CN, after addition, the mixture was stirred at room temperature for a further 6 h. Freeze-concentrating the mixed solution to obtain crude product on silica gel (CH)₂Cl₂MeOH 20/1) gave compound JNc-463 as a white solid.

Application example:

compound JNc-463 prepared in example 1 was dissolved in 0.9% physiological saline to prepare a solution having a molar concentration of 10. mu. mol/L, and administered by tail vein injection in an amount of 1.0mg/kg of the mass of the solution to the mass of the mouse body. The administration is performed once a day until the blood pressure returns to normal value, and each continuous administration is performed for no more than 14 days.

Application example 2:

compound JNc-463 prepared in example 1 was dissolved in 0.9% physiological saline to prepare a solution having a molar concentration of 1. mu. mol/L, and administered by tail vein injection in an amount of 0.8mg/kg of the mass of the solution to the mass of the mouse body. The administration is performed once a day until the blood pressure returns to normal value, and each continuous administration is performed for no more than 14 days.

Application example 3:

the compound JNc-463 prepared in example 1 was dissolved in 0.9% physiological saline (0.9% physiological saline was used in the present invention) to prepare a solution having a molar concentration of 30. mu. mol/L, and the solution was administered by tail vein injection in an amount such that the mass ratio of the solution to the body weight of the mouse was 1.2 mg/kg. The administration is performed once a day until the blood pressure returns to normal value, and each continuous administration is performed for no more than 14 days.

Test example 1: TRPV4 and eNOS coupling degree test

A hypertension mouse model of high-salt breeding and AngII (potent vasoconstrictor) induction is constructed, the compound JNc-463 prepared in example 1 is dissolved in physiological saline according to the method of application example 1, the solution is injected into a mouse body through a tail vein (1mg/kg), the physiological saline is directly injected into the mouse body through the tail vein according to the amount of 1.0mg/kg of the mass ratio of the physiological mass to the body weight of the mouse to serve as a control group, the aorta of the mouse is respectively taken after 2h, and the interaction (coupling degree) of TRPV4 and eNOS is detected by fluorescence energy resonance transfer (FRET).

As shown in fig. 1, compound JNc-463 acted on both the high-salt fed and AngII-induced hypertensive mice, the degree of TRPV4 and eNOS coupling was significantly increased compared to the control group (saline was directly injected into the tail vein), indicating that the compound had a significant effect of enhancing complex coupling in the hypertension model.

Test example 2: vasodilatory conditions

A hypertension mouse model with high-salt feeding and AngII induction was constructed, the compound JNc-463 prepared in example 1 was dissolved in physiological saline by the method of application example 1, injected into the mouse body through the tail vein (1mg/kg), the physiological saline was directly injected into the mouse body through the tail vein in an amount of 1.0mg/kg of the mass ratio of the physiological mass to the body weight of the mouse as a control group, the aorta of the mouse was taken after 2 hours, and the diastolic condition of the thoracic aorta of the mouse was detected by a myograph (myograph).

As shown in figure 2, after the compounds JNc-463 act on the high-salt fed and AngII induced hypertensive mice, the relaxation degree of thoracic aorta of the mice is obviously enhanced compared with that of the control group, which indicates that the compounds have obvious vasodilation effect on the hypertension model and can improve the vasodilation degree of the blood vessels of the hypertension model mice by 25% -30%.

Test example 3: test for hypotensive Effect

A hypertension mouse model with high-salt feeding and AngII induction was constructed, the compound JNc-463 prepared in example 1 was dissolved in physiological saline by the method of application example 1, injected into the mouse body through the tail vein (1mg/kg) once a day for 7 days, and the physiological saline was directly injected into the mouse body through the tail vein in an amount of 1.0mg/kg of the ratio of the physiological mass to the body weight of the mouse as a control group, and the change in blood pressure of the mice in the experimental group and the control group into which the compound JNc-463 was injected was detected.

As shown in FIG. 3, when compounds JNc-463 act on the hypertension mice raised in high salt and induced by AngII, the blood pressure of the mice is obviously reduced compared with the control group, which shows that the compounds have obvious blood pressure reducing effect on the hypertension model and can reduce the blood pressure of the hypertension model mice by 18-26 mmHg.

Test example 4: effect of Compound JNc-463 concentration on TRPV4-eNOS coupling

Primary isolated C57BL/6J mouse aortic endothelial cells were cultured in NaCl (60mmol) containing medium in a cell incubator for 24h, then JNc-463 (1. mu. mol/L, 3. mu. mol/L, 10. mu. mol/L, 30. mu. mol/L) at different doses, JNc-463 at different doses were prepared with physiological saline, and after further culturing for 24h, the cells were fixed with 4% paraformaldehyde in PBS. Cells were blocked, permeabilized in blocking solution (5% BSA and 0.1% Triton X-100 in PBS), incubated overnight with primary antibody at 4 degrees, washed three times, and then incubated with secondary antibody at room temperature for 2 h. As primary antibodies, coat anti-TRPV4 and rabbitanti-eNOS were used, and secondary antibodies Alexa Fluor 568 and Alexa Fluor 488. And detecting the coupling condition of the two proteins before and after mutation by using a FRET AB mode of a laser confocal microscope.

As shown in FIG. 4, JNc-463 enhanced the TRPV4-eNOS interaction in a dose-dependent manner in high-salt primary cultured mouse aortic endothelial cells, and at a dose of 10. mu. mol/L, the effect of JNc-463 concentrations on TRPV4-eNOS interaction was reduced.

The present invention is not limited to the specific embodiments, but can be modified without inventive contribution as required by those skilled in the art after reading the present specification, and all changes of equivalent structures or equivalent processes that can be made by using the content of the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of patent protection of the present invention.