TWI692467B - Myocardial regeneration promoting compounds, preparation method thereof, pharmaceutical composition, and their use - Google Patents

Abstract

The present invention discloses a novel 3-aryl-2-propen-1-one series derivative and the synthesis processes thereof. Besides, the present invention also discloses the said series derivative as a pharmaceutical composition and their use for promoting myocardial regeneration.

Description

Compound for promoting regeneration of myocardium, its preparation method, medicine and its use

The present invention relates to a novel compound, its preparation method and use, especially a novel 3-aryl-2-propen-1-one series derivative, its preparation method and its use for promoting myocardial regeneration.

Myocardial infarction (myocardial infarction, MI) is a clinically common acute heart disease, the cause of which is the sudden interruption of blood circulation of part of the myocardium, and damage to the myocardium due to insufficient oxygen. It is generally believed that once a patient has a myocardial infarction, the necrotic myocardial cells cannot be regenerated, and the damaged area of the myocardium can only form fibrous plaques for repair. The fibrous plaques formed after repair are stiff scar tissue and do not have the function of contraction, although It can help the heart maintain the structural integrity, but it will further affect the heart remodeling and cause dysfunction, and even lead to heart failure, arrhythmia, cardiogenic shock and other symptoms.

At present, the treatment methods for myocardial infarction mainly include interventional therapy and drug administration. Among them, interventional therapy such as balloon dilatation, stent placement and coronary artery bypass surgery, etc., and the treatment drugs are mainly thrombolytic drugs, calcium ions Blockers, nitrates, angiotensin-converting enzyme inhibitors, beta-sympathetic blockers, and narcotic analgesics.

However, no matter in the field of clinical therapy or basic medical research, currently developed interventional therapy or drug therapy cannot promote the recovery of damaged myocardial tissue, and are more likely to cause other side effects. There is no other effective method until it recovers on its own.

Therefore, there is still a need to develop a related technology to promote myocardial regeneration to provide drugs that can effectively cure myocardial infarction in clinical treatment or basic medical research.

In view of the shortcomings of the prior art, the object of the present invention is to provide a novel compound that can promote the regeneration and repair of myocardial cells, and thus provide a non-invasive and therapeutic treatment strategy for the treatment of myocardial infarction.

其中，X₁為氫或未經取代之C₁-C₆之烷基；X₂為氧(-O-)或胺基(-NH-或-NX₄-)；X₃為氫(-H)、羥基(-OH)或前述以-X₂(CO)-(Y)_p-Z所示之取代基；Y為C₁-C₆之伸烷基(C₁-C₆ alkylene group)、C₂-C₁₂之伸烯基(C₂-C₁₂ alkenylene group)、C₇-C₁₈之伸烷基芳基(C₇-C₁₈ arylenealkylene group)或C₆-C₁₈之伸芳基(C₆-C₁₈ arylene group)；Z為-COOH、-COOCH₃、-OCOCH₃、-NHCOOC(CH₃)₃、-F、-Cl、-Br或-I；且p為0或1。 To achieve the foregoing objective, the present invention provides a novel compound as shown in formula (I):

Among them, X ₁ is hydrogen or unsubstituted C ₁ -C ₆ alkyl; X ₂ is oxygen (-O-) or amine (-NH- or -NX ₄ -); X ₃ is hydrogen (-H ), hydroxyl (-OH) or in the -X ₂ (CO) - shown in the substituent group (Y) _p -Z; Y is alkylene of C ₁ -C _{6 (C 1 -C 6 alkylene group)} , C ₂ -C ₁₂ alkenylene group (C ₂ -C ₁₂ alkenylene group), C ₇ -C ₁₈ alkylene aryl group (C ₇ -C ₁₈ arylenealkylene group) or C ₆ -C ₁₈ alkylene group ( C ₆ -C ₁₈ arylene group); Z is -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ , -F, -Cl, -Br, or -I; and p is 0 or 1.

Preferably, in the chemical formula (I), the Z is -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ or -Cl.

In the present specification, the “C ₁ -C ₆ alkyl group” may be a linear or branched alkyl group, which means that the overall substituent represented by X ₁ has 1 to 6 total carbon numbers. Preferably, the C ₁ -C ₆ alkyl group represented by X ₁ may be methyl, ethyl, propyl, or isopropyl, but it is not limited thereto.

。 Preferably, in the chemical formula (I), X ₂ is -NX ₄ -, and X ₄ may be the aforementioned substituent represented by -X ₂ -(CO)-(Y) _p -Z; more preferably , X ₄ may be a substituent

.

In this specification, the "C ₁ -C ₆ alkylene extension" may be a linear or branched alkylene extension, which means that the overall substituent represented by Y has 1 to 6 total carbon numbers; the same In this specification, the "C ₂ -C ₁₂ alkenyl group" may be a linear or branched alkenyl group, which means that the overall substituent represented by Y has a total carbon number of 2 to 12 . In this specification, the "C ₇ -C ₁₈ alkylene aryl group" refers to an aryl group-bonded alkylene group, which means that the overall substituent represented by Y has 7 to 18 total The carbon number, that is, the total carbon number of the aryl and alkylene groups is 7 to 18. In this specification, the "C ₆ -C ₁₈ aryl group" may be a single aromatic ring or multiple aromatic rings bonded or fused to each other to form a polyaromatic ring, which means the overall substitution represented by Y The base has 6 to 18 total carbon numbers.

Preferably, in the chemical formula (I), the C ₁ -C ₆ alkylene group represented by Y may be a methylene group (-CH ₂ -), an ethylene group (-CH ₂ CH ₂ -), ethylidene group (-CH(CH ₃ )-), propylene group (-CH ₂ CH ₂ CH ₂ -), isopropylidene group (-C(CH ₃ ) ₂ -), butylene group (-CH ₂ CH ₂ CH ₂ CH ₂ -), etc., but not limited to this; the C ₂ -C ₁₂ alkylene group represented by Y may be vinylidene ( vinylene group, -CH=CH-), propenylene group (-CH ₂ CH=CH- or -CH=CHCH ₂ -), butene group (-CH ₂ CH ₂ -CH=CH -, -CH ₂ CH=CHCH ₂ -or -CH=CHCH ₂ CH ₂ -), etc., but not limited to this; the C ₇ -C ₁₈ alkylene aryl group represented by Y may be benzyl (cresylene) group, -CH ₂ C ₆ H ₄ -), tolylene group (-C ₆ H ₃ (CH ₃ )-) or phenylenedimethylene group (-CH ₂ C ₆ H ₄ CH ₂ -) etc., but not limited to this; the C ₆ -C ₁₈ aryl aryl group represented by Y may be phenylene group (-phenylene group, -C ₆ H ₄ -), biphenylene group (-C) ₆ H ₄ -C ₆ H ₄ -) or naphthylene group (-C ₁₀ H ₆ -), but not limited to this.

Specifically, in the formula (the I), the phenylene may be o - phenylene (ortho -phenylene group), inter - phenylene (meta -phenylene group) or p - phenylene (para - phenylene group), preferably o-phenylene. Specifically, the benzylidene group may be an ortho- cresylene group, a meta- cresylene group or a para- cresylene group, preferably Meta-benzyl.

According to the present invention, the novel compound may be any one of the following compound 1 to compound 15, but it is not limited to this:

Compound 7 Compound 8 Compound 9

Another object of the present invention is to provide a method for preparing the above novel compound, which reacts reactant A and reactant B at a temperature of 0°C to 25°C, wherein reactant A is coumaric acid derivative, coffee Acid derivatives or cinnamic acid derivatives; reactant B is an acid compound, acid anhydride compound, acetyl chloride compound or ester compound.

Preferably, the reactant A and the reactant B are reacted in an alkaline environment at a temperature of 0°C to 25°C, and the pH value in the reaction can be controlled between pH 9 and pH 12.

Preferably, the reactant A is para-Coumaric acid, Caffeic acid, Methyl caffeoate or 4-Aminocinnamic acid.

Preferably, the reactant B is Acetylsalicylic acid, 3-Chlorophenylacetic acid, n-tert-Butoxycarbonyl-gamma- aminobutyric acid), glutaric anhydride (Glutaric anhydride), adipic anhydride (Adipic anhydride), methyl adipoyl chloride (Methyl adipoyl chloride) or fumaric acid monomethyl ester (Fumaric acid monomethyl ester).

Another object of the present invention is to provide the use of 3-aryl-2-propen-1-one series derivatives for the preparation of medicinal products that promote myocardial regeneration.

其中，R₁為羥基、未經取代之C₁-C₆之烷基、未經取代之C₁-C₆之烷氧基、經取代之C₁-C₆之烷酸基、經取代之C₇-C₁₂之環烷酸基、經取代或未經取代之C₆-C₁₈之芳胺基、經取代或未經取代之C₆-C₁₈之酚基、經取代或未經取代之C₆-C₁₈之芳基、經取代或未經取代之噻吩基、經取代或未經取代之吡咯基、經取代或未經取代之吡啶基或經取代或未經取代之噻唑基；R₂為氫、羥基、未經取代之C₁-C₆之烷基、胺基(-NH₂)、乙醯氧基(-OCOCH₃)、

或-X₂(CO)-(Y)_p-Z，R₃為氫、羥基、未經取代之C₁-C₆之烷基、未經取代之C₁-C₆之烷氧基或-O-CO-(Y)_p-Z，或者，R₂以及R₃相互連接 形成

；R₄為氫、未經取代之C₁-C₆之烷氧基或

；以A表示之環狀結構為苯環、噻吩環或吡啶環； X₂為氧(-O-)或胺基(-NH-或-NX₄-)；Y為C₁-C₆之伸烷基(C₁-C₆ alkylene group)、C₂-C₁₂之伸烯基(C₂-C₁₂ alkenylene group)、C₇-C₁₈之伸烷基芳基(C₇-C₁₈ arylenealkylene group)或C₆-C₁₈之伸芳基(C₆-C₁₈ arylene group)；Z為吡啶基(-C₅H₄N)、乙醯基(-COCH₃)、-COOH、-COOCH₃、-OCOCH₃、-NHCOOC(CH₃)₃、-F、-Cl、-Br或-I；p為0或1。 In order to achieve the foregoing objective, the present invention provides the use of a compound represented by formula (II) for preparing a medicinal product that promotes myocardial regeneration;

Among them, R ₁ is hydroxy, unsubstituted C ₁ -C ₆ alkyl, unsubstituted C ₁ -C ₆ alkoxy, substituted C ₁ -C ₆ alkanoic acid, substituted C ₇ -C ₁₂ naphthenic acid group, substituted or unsubstituted C ₆ -C ₁₈ arylamine group, substituted or unsubstituted C ₆ -C ₁₈ phenol group, substituted or unsubstituted C ₆ -C ₁₈ aryl, substituted or unsubstituted thienyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, or substituted or unsubstituted thiazolyl; R ₂ is hydrogen, hydroxy, unsubstituted C ₁ -C ₆ alkyl, amine (-NH ₂ ), acetyloxy (-OCOCH ₃ ),

Or -X ₂ (CO)-(Y) _p -Z, R ₃ is hydrogen, hydroxyl, unsubstituted C ₁ -C ₆ alkyl, unsubstituted C ₁ -C ₆ alkoxy or- O-CO-(Y) _p -Z, or R ₂ and R ₃ are connected to each other to form

; R ₄ is hydrogen, unsubstituted C ₁ -C ₆ alkoxy or

; The cyclic structure represented by A is benzene ring, thiophene ring or pyridine ring; X ₂ is oxygen (-O-) or amine group (-NH- or -NX ₄ -); Y is the extension of C ₁ -C ₆ Alkyl (C ₁ -C ₆ alkylene group), C ₂ -C ₁₂ alkenylene group (C ₂ -C ₁₂ alkenylene group), C ₇ -C ₁₈ alkylene aryl group (C ₇ -C ₁₈ arylenealkylene group ) Or C ₆ -C ₁₈ arylene group (C ₆ -C ₁₈ arylene group); Z is pyridyl (-C ₅ H ₄ N), acetyl (-COCH ₃ ), -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ , -F, -Cl, -Br or -I; p is 0 or 1.

In short, the 3-aryl-2-propen-1-one series derivatives include, in addition to the novel compounds shown in the chemical formula (I) above, other commercially available cinnamic acid derivatives ( cinnamic acid derivatives).

Preferably, in the chemical formula (II), the Z is -COCH ₃ , -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ or -Cl.

、

或-NX₄，X₄可以為前述以-X₂-(CO)-(Y)_p-Z所示之取代基；更佳的，X₄可以為取代 基

。 Preferably, in the chemical formula (II), X ₂ is

,

Or -NX ₄ , X ₄ may be the aforementioned substituent represented by -X ₂ -(CO)-(Y) _p -Z; more preferably, X ₄ may be a substituent

.

、-NH₂或-O-CO-(Y)_p-Z。 Preferably, in the chemical formula (II), the R ₃ is -CH ₃ ,

, -NH ₂ or -O-CO-(Y) _p -Z.

Preferably, in the chemical formula (II), when R ₂ is -X ₂ (CO)-(Y) _p -Z, R ₁ is a hydroxyl group.

Preferably, in the chemical formula (II), when Y is an alkenyl group, Z is -COOCH ₃ .

Preferably, in the chemical formula (II), X ₂ , Y, P and Z are as described in the aforementioned chemical formula (I). In addition, in the chemical formula (II), the Z may also be pyridyl (-C ₅ H ₄ N) or acetyl (-COCH ₃ ).

Preferably, in the chemical formula (II), the R ₁ is -OH, -OCH ₃ , -OCH ₂ C ₆ H ₄ OH, -OCH(COOH)CH(OH)COOH, -O(C ₆ H ₇ )(OH) ₃ (COOH), -NHC ₆ H ₄ OH, -C ₄ H ₃ S(CH ₃ ), -C ₆ H ₄ OH, -( N )-C ₄ H ₄ N, -C ₅ H ₄ N or -C ₃ SN(NH ₂ )(CH ₃ ).

Preferably, the 3-aryl-2-propen-1-one series derivatives that can promote the regeneration and repair of cardiomyocytes may be any of the above compounds 1 to 15 and the following compounds 16 to 44, but not Only for this:

Compound 43 Compound 44

In addition, the present invention also provides a medicinal product for promoting myocardial regeneration, which comprises the above-mentioned 3-aryl-2-propen-1-one series derivatives (for example, Compound 1 to Compound 44) and pharmaceutically acceptable Carrier.

In summary, the present invention provides a novel 3-aryl-2-propen-1-one series derivative and its preparation method. The novel compound can be applied to promote the repair of damaged cardiomyocytes and the regeneration of cardiomyocytes; To provide a use of 3-aryl-2-propen-1-one series derivatives for the preparation of medicinal products that promote myocardial regeneration.

Figure 1 shows the effect of different compounds on the expression of α-MHC in cardiomyocytes.

Figure 2 shows the effect of different compounds on the repair of zebrafish myocardial trauma.

Attachment 1 is a schematic diagram of the zebrafish cryotrauma experimental procedure.

Several examples are listed below to illustrate the embodiments of the compound of the present invention and its use for preparing a medicament for promoting myocardial regeneration. Those skilled in the art can easily understand what the present invention can achieve through the contents of this manual Advantages and effects, and without departing from the spirit of the present invention, various modifications and changes are made to implement or apply the content of the present invention.

Example 1: Preparation of the compound

The compound of the present invention is prepared by reacting reactant A and reactant B at a reaction temperature of 0°C to 25°C under an alkaline environment, wherein reactant A is coumaric acid derivative, coffee Acid derivatives or cinnamic acid derivatives; reactant B is an acid compound, acid anhydride compound, acetyl chloride compound or ester compound.

Compound 1

Take 1 gram of p-coumaric acid dissolved in 8 ml of 10% by weight sodium hydroxide aqueous solution, then cool the reaction to 5 ℃ to 10 ℃, then take 0.97 g of glutaric anhydride dissolved in 5 ml of tetrahydrofuran and slowly add the solution After stirring for 30 minutes, add 40 ml of water and 40 ml of ethyl acetate and adjust the pH to 5.2. Take the organic layer and concentrate to dryness. Then add 50 ml of water to adjust the pH to 9.33, then add 1N A hydrochloric acid aqueous solution was precipitated until the acid-base value was 5.0. After stirring for another hour, the solid was filtered, and after drying it, 0.5335 g of light-skinned powder was obtained, and its HPLC purity was 100%.

The structure of Compound 1 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, DMSO- d ₆ ): δ 12.255 (s, 2H), 7.723 (d, 2H), 7.577 (d, 1H ), 7.166 (d, 2H), 6.494 (d, 1H), 2.618 (t, 2H), 2.330 (t, 2H), 1.841 (tt, 2H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₄ H ₁₅ O ₆ ; 279.0859.

Compound 2

Add 0.3 g of caffeic acid to 6 ml of acetonitrile. After mixing, add 0.46 ml of triethylamine and stir for 5 minutes. Then place the reaction in an ice bath and lower the temperature to 5°C to 10°C. Take another 0.17 g of glutaric anhydride Dissolve in 2 ml of tetrahydrofuran and slowly drop it in. After 2 hours of reaction, drain off. Then add 30 ml of ethyl acetate and 30 ml of 10% aqueous ammonium chloride solution for liquid phase extraction. After extraction, the organic layer was concentrated to dryness to obtain 0.2685 g of a white solid, which was then crystallized from isopropyl acetate and acetone. After filtration, 0.1339 g of white powder was obtained with HPLC purity of 100%.

The structure of compound 2 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.576 (d, 1H), 7.347-6.913 (m, 3H), 6.392-6.287 ( d+d, 1H), 2.705-2.669(m, 2H), 2.283-2.447(m, 2H), 2.152-1.986(m, 2H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₄ H ₁₅ O ₇ ; 295.0812.

Compound 3

Dissolve 0.25 g of caffeic acid in 5 ml of tetrahydrofuran, then add 0.76 ml of triethylamine, and stir for 7 minutes; concentrate the reaction solution to dryness, add 7 ml of tetrahydrofuran, stir until dissolved and reduce the temperature to 5°C in an ice bath At 10°C, 0.4 g of glutaric anhydride was dissolved in 3 ml of tetrahydrofuran and slowly dropped into the reaction. After the reaction for 1 hour, the solution was concentrated to dryness, and then 30 ml of ethyl acetate and 30 ml of water were added for liquid phase extraction. The organic layer was washed once with 30 ml of saturated saline solution, and finally dehydrated with anhydrous sodium sulfate, and concentrated to dryness, and then crystallized with isopropyl acetate and acetone, and filtered to obtain 0.1629 g of white powder with HPLC purity of 100%.

The structure of compound 3 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD); δ 7.645 (d, 1H), 7.537-7.513 (m, 2H), 7.278 (d, 1H), 6.482 (d, 1H), 2.703-2.663 (m, 4H), 2.463-2.425 (m, 4H), 2.009-1.969 (m, 4H). Mass spectrometry analysis: [M+NH ₄ ] ⁺ ; 426.1402.

Compound 4

Dissolve 1 g of p-coumaric acid in 10 ml of acetonitrile, add 2.5 ml of triethylamine first, then cool the reaction to 0°C to 10°C, then take 0.93 g of adipic anhydride in 5 ml of dichloromethane and slowly add During the reaction, after addition, concentrate to dryness to obtain a brown oil; add 30 ml of water and wash three times with 30 ml of dichloromethane each time, take the water layer and wash twice with 30 ml of ethyl acetate each time, and finally take the water layer The acid-base value was adjusted to 4.8, and the precipitated solid was filtered to obtain 0.2542 g of white solid with HPLC purity of 99.2%.

The structure of compound 4 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, DMSO- d ₆ ): δ 7.717 (d, 2H), 7.569 (d, 1H), 7.150 (d, 2H ), 6.488 (d, 1H), 2.583 (t, 2H), 2.250 (t, 2H), 1.656-1.558 (m, 4H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₅ H ₁₇ O ₆ ; 293.1013.

Compound 5

Take 0.333 g of p-coumaric acid (2 mmol) in a nitrogen-sealed reaction flask, add 10 ml of dehydrated and dried tetrahydrofuran and stir to dissolve. Add 0.36 grams of methyl adipate acetyl chloride (2 millimoles, 1 equivalent), then slowly add 0.3 grams of ethylenediamine (3 millimoles, 1.5 equivalents) in an ice bath, and add The reaction was stirred at room temperature for 2 to 3 hours. Concentrate in vacuo to remove the solvent, then extract with water/dichloromethane, and take the organic layer. Concentrate to dryness in vacuo and separate with reverse silica gel column. The eluent is 50% methanol aqueous solution. The product is 17 mg and the purity is 100%.

The structure of compound 5 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.675-7.629 (m, 3H), 7.150 (d, 2H), 6.463 (d, 1H), 3.667 (s, 3H), 2.620 (t, 2H), 2.401 (t, 2H), 1.751-1.736 (m, 4H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₆ H ₁₉ O ₆ ; 307.1181; [M+Na] ⁺ ; 329.1000.

Compound 6

Weigh 0.369 g of caffeic acid (2 mmol) in a nitrogen-sealed reaction flask, add 10 ml of dehydrated and dried tetrahydrofuran and stir to dissolve. Add 0.5146 grams of methyl adipate acetyl chloride (3 millimoles, 1.5 times equivalent) with a syringe, then slowly add 0.5129 grams of ethylenediamine (5 millimoles, 2.5 times equivalent) in an ice bath, and add The reaction was stirred at room temperature for 2 to 3 hours. Concentrate in vacuo to remove the solvent, then extract with water/dichloromethane, and take the organic layer. Concentrate to dryness in vacuum and separate with a reverse silica gel column. The eluent is 50% to 60% methanol in water, and 13.8 mg is isolated with a purity of 100%.

The structure of compound 6 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.635 (d, 1H), 7.523 (dd, 1H), 7.498 (d, 1H) , 7.264 (d, 1H); 6.481 (d, 1H), 3.670 (s, 6H), 2.638-2.599 (m, 4H), 2.418-2.384 (m, 4H), 1.747-1.723 (m, 8H). Mass spectrometry analysis: [M+NH4] ⁺ ; 482.2059.

Compound 7

Dissolve 1 g of p-coumaric acid in 10 ml of 10% by weight aqueous sodium hydroxide solution, stir until dissolved, and then lower the temperature by 5° C. to 10° C. Take 2.45 g of acetylsalicylic anhydride in 3 ml of tetrahydrofuran Slowly add to the reaction, stir for 30 minutes, concentrate to dryness, then add 30 ml of water and 40 ml of ethyl acetate, add 20 ml of water to the organic layer, adjust to pH 4 to 5, then take the organic layer again Wash 3 times with 5% ammonium chloride aqueous solution by weight, add 40 ml of water to the organic layer, adjust the pH to 6.01 with sodium bicarbonate, remove the water from the organic layer with anhydrous sodium sulfate, and concentrate to dryness to obtain 1.9 g of white solid. Then add 30 ml of dichloromethane, wash twice with water and adjust to pH 7.37, take the organic layer to remove water with anhydrous sodium sulfate, and concentrate to dryness to obtain 49.6 mg of white solid with HPLC purity of 89.9%.

The structure of compound 7 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 8.200 (dd, 1H), 7.741-7.682 (m, 4H), 7.459 (t, 1H), 7.259-7.241 (m, 3H), 6.500 (d, 1H), 2.264 (s, 3H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₈ H ₁₅ O ₆ ; 327.0860; [M+Na] ⁺ ; 349.0677.

Compound 8

In a 250 ml single-necked bottle, add 1.8 g of acetylsalicylic acid, 20 ml of toluene and dimethylformamide, and stir at 40°C for 3 minutes. Add 1.8 ml of chlorinated chlorite and raise the temperature to 60 ℃ for 2 hours. After the reaction is cooled to 25 ℃, use a concentrator to remove excess chlorobenzene and all the solvents. After adding 20 ml of toluene and mixing, the solvent was removed by a rotary concentrator to obtain a pale yellow liquid. The above liquid was added to 1.81 g of caffeic acid, 20 ml of tetrahydrofuran and 1.5 ml of triethylamine. After stirring at room temperature for 3 hours, 20 ml of saturated aqueous ammonium chloride solution was added to separate the organic layer. After removing the solvent with a cyclone concentrator, an off-white solid was obtained with a wet weight of 4.82 g. Purified by column chromatography (63.4 g of silica gel, column diameter 4.5 cm; length 13.5 cm, eluent is dichloromethane: methanol = 1:0 to 50:1), the main product was collected. After removing the solvent from the above collection, it was purified by column chromatography (silica gel 43.2 g, column diameter 4 cm; length 8 cm, eluent was dichloromethane: methanol = 50:1), and the main product was collected , 56 mg was obtained, and the HPLC purity was 94.6%.

The structure of compound 8 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 8.242-8.207 (m, 1H), 7.708 (t, 1H), 7.602 (d, 1H), 7.451 (t, 1H), 7.407-6.968 (m, 4H), 6.425-6.312 (d+d, 1H), 2.266 (s, 3H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₈ H ₁₅ O ₇ ; 343.0822; [M+NH ₄ ] ⁺ ; 360.1091.

Compound 9

5.0 g of 3-chlorophenylacetic acid and 10.0 ml of sulfonium chloride were mixed and heated to 79.8°C. After 1.5 hours of reaction, the temperature was lowered to room temperature and concentrated to obtain 5.1 g of yellow liquid. Under an ice bath, 0.304 g of p-coumaric acid was mixed with 2.9 ml of triethylamine and 4.0 ml of acetonitrile, and the reaction was carried out at room temperature for 1 hour. Then add water and 1,2-dichloroethane for liquid phase extraction, take the organic layer with 1N hydrochloric acid aqueous solution to adjust the pH to 6.1, stir for 0.5 hours, and filter to obtain 2.8 g of yellow solid. The silica gel column was used to flush the bank with a volume ratio of ethyl acetate and dichloromethane of 1:1.5. About 0.026 grams of white solid was collected, and the HPLC purity was 87.8%.

The structure of compound 9 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.661 (d, 1H), 7.635 (d, 2H), 7.423 (s, 1H) , 7.342 (d, 1H), 7.324-7.312 (m, 2H), 7.146 (d, 2H), 6.457 (d, 1H), 3.939 (s, 2H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₇ H ₁₄ ClO ₄ ; 317.0580.

Compound 10

In a 250-ml single-necked bottle, add 1.71 g of 3-chlorophenylacetic acid, 20 ml of toluene and 2 drops of dimethylformamide, stir at room temperature at 24°C for 5 minutes, and then add 1.5 ml of sulfonium chloride. The temperature was raised to 65°C for 1 hour, and after the reaction was cooled to 25°C, the excess chlorinated chloride and all solvents were removed with a rotary concentrator. After adding 25 ml of tetrahydrofuran and mixing, the solvent was removed by a rotary concentrator to obtain a pale yellow liquid with a wet weight of 2.18 g. After adding 1.81 g of caffeic acid, 20 ml of tetrahydrofuran and 1.4 ml of triethylamine, the mixture was stirred and reacted at room temperature for 4 hours. After adding 20 ml of saturated aqueous ammonium chloride solution, the organic layer was separated and the solvent was removed by a rotary concentrator. 5.13 g of off-white solid was obtained. Purified by column chromatography (silica gel 67.4 g, column diameter 4 cm; length 14 cm, eluent ethyl acetate: heptane = 1:3), the product was collected, HPLC purity was 97.6%.

The structure of compound 10 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.613 (d, 1H), 7.511-7.478 (m, 2H), 7.356-7.281 ( m, 6H), 7.250-7.214 (m, 3H), 6.455 (d, 1H), 3.726 (s, 2H), 3.696 (s, 2H). Mass spectrometry analysis: [M+H] ⁺ ; C ₂₅ H ₁₉ Cl ₂ O ₆ ; 485.0569.

Compound 11

In a 100 ml single-necked bottle, add 1.8 g of 3-chlorophenylacetic acid, 20 ml of toluene and 2 drops of dimethylformamide, and stir at 24°C for 10 minutes. Chlorosulfite (0.5 ml, one-time addition) was added, and the temperature was raised to 65°C after the completion of the reaction. After 1 hour of reaction, the temperature was lowered to 25°C, and the excess chlorinated chloride and all solvents were removed by a rotary concentrator. After adding 10 ml of toluene and mixing, the solvent was removed by a rotary concentrator to obtain a pale yellow liquid. 0.49 g of methyl caffeate, 10 ml of tetrahydrofuran and 0.35 ml of triethylamine were added, and the reaction was stirred at room temperature for 3 hours. After adding 10 ml of saturated aqueous ammonium chloride solution, the organic layer was separated. After removing the solvent with a cyclone concentrator, it was mixed with 10 ml of ethyl acetate, washed with 10 ml of a saturated sodium bicarbonate aqueous solution, and the organic layer was retained. After removing the solvent with a cyclone concentrator, 1.03 g of orange liquid was obtained. Purified by column chromatography (silicone 48.4 g, column diameter 3.81 cm; length 10 cm, eluent ethyl acetate: heptane = 1:5), the product was collected to obtain 27.3 mg, HPLC purity was 98.6% .

The structure of compound 11 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.618 (d, 1H), 7.503-7.476 (m, 2H), 7.344-7.266 ( m,6H),7.242-7.205(m,3H),6.490(d,1H),3.767(s,3H),3.713(s,2H),3.686(s,2H). Mass spectrometry analysis: [M+H] ⁺ ; C ₂₆ H ₂₁ Cl ₂ O ₆ ; 499.0719.

Compound 12

In a 250 ml single-necked bottle, 1.59 g of tert-butoxycarbonyl-γ-aminobutyric acid was added to protect the reaction system with dry nitrogen. Add 8 ml of tetrahydrofuran and 2.2 ml of triethylamine, and stir at -20°C for 5 minutes. Acetochloric acid solution (0.96 ml mixed with 5 ml of tetrahydrofuran) was added for 10 minutes. After completion, the temperature was returned to 0°C. After 2 hours of reaction, a caffeic acid solution (0.7 g and 8 ml of tetrahydrofuran) was added Mix), add time 2 minutes. After completion, return to room temperature and react for 44.5 hours. After adding 30 ml of saturated ammonium chloride aqueous solution, the organic layer is retained and the solvent is removed by a rotary concentrator. The residue is mixed with 30 ml of ethyl acetate and washed with water (30 ml once) After the organic layer was retained, the solvent was removed by a rotary concentrator to obtain a white solid with a wet weight of 2.13 g. Purified by column chromatography (65 g silica gel, column diameter 4 cm; length 15 cm, eluent: methylene chloride: methanol = 50:1 to 20:1), the product was collected to obtain 17.8 mg, HPLC purity 97.4%.

The structure of compound 12 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.644 (d, 1H), 7.535-7.507 (m, 2H), 7.293 (d, 1H), 6.845 (d, 1H), 3.173-3.134 (m, 4H), 2.637-2.596 (m, 4H), 1.885-1.846 (m, 4H), 1.440 (s, 18H). Mass spectrometry analysis: [M+H] ⁺ ; C ₂₇ H ₃₉ N ₂ O ₁₀ ; 551.2591; [M+Na] ⁺ ; 573.2414.

Compound 13

At room temperature, 0.201 g of 4-aminocinnamic acid and 2.0 ml of tetrahydrofuran were mixed, 0.169 g of glutaric anhydride was added for 1.5 hours, a small amount of acetonitrile, ethyl acetate and n-heptane were added, and the solid was filtered to obtain 0.336 g of rice. White powder, HPLC purity is 98.2%.

The structure of compound 13 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, CD ₃ OD): δ 7.643-7.611 (m, 3H), 7.551 (d, 2H), 6.401 (d, 1H), 2.454 (t, 2H), 2.393 (t, 2H), 1.978 (tt, 2H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₄ H ₁₆ NO ₅ ; 278.1067.

Compound 14

In a 100 ml single-necked bottle, add 0.72 g of monomethyl fumarate, 10 ml of toluene and 2 drops of dimethylformamide, and stir at 17°C for 3 minutes. Add 0.9 ml of chlorinated chlorobenzene, and after heating, heat to 60 ℃ for 3 hours, wait for the reaction to cool down to 30 ℃, and remove excess chlorobenzene and all solvents with a rotary concentrator. 0.82 g of 4-aminocinnamic acid, 10 ml of tetrahydrofuran and 1.4 ml of triethylamine were added, and the reaction was stirred at 0°C for 30 minutes and returned to room temperature. After 19 hours of reaction, 20 ml of saturated ammonium chloride aqueous solution and 5 ml of water were added, and the organic layer was separated and retained. The lower aqueous solution was extracted with dichloromethane (20 ml twice), and the remaining Aircraft layer. After combining all organic solutions, the solvent was removed with a rotary concentrator to obtain a yellow solid. Purified by column chromatography (silica gel 52.9 g, column diameter 4 cm; length 11 cm, eluent ethyl acetate: heptane = 1:3 to 1:0), the product was collected to give 22.5 mg, HPLC The purity is 94.0%.

The structure of compound 14 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, DMSO- d ₆ ): δ 12.283 (br s, 1H), 10.709 (s, 1H), 7.707 (d, 2H), 7.663 (d, 2H), 7.523 (d, 1H), 7.211 (d, 1H), 6.726 (d, 1H), 6.430 (d, 1H), 3.746 (s, 3H). Mass spectrometry analysis: [M+H] ⁺ ; C ₁₄ H ₁₄ NO ₅ ; 276.0866.

Compound 15

In a 100 ml single-necked bottle, add 1.0 g of acetosalicylic acid, 20 ml of toluene and 3 drops of dimethylformamide, and stir at 20°C for 3 minutes. Add 0.9 ml of chlorinated chlorinated chloride, and then heat to 60 ℃ for 1.5 hours. After the reaction temperature is lowered to 35 ℃, remove excess chlorinated chlorinated chloride and all solvents with a rotary concentrator. After adding 10 ml of tetrahydrofuran and mixing, the solvent was removed by a rotary concentrator to obtain 1.00 g of pale yellow liquid. 0.81 g of 4-aminocinnamic acid, 10 ml of tetrahydrofuran and 1.4 ml of triethylamine were added, and the reaction was stirred at 0°C for 1 hour and returned to room temperature. After reacting for 22 hours, after adding 25 ml of a saturated ammonium chloride aqueous solution and 5 ml of water, the organic layer was separated. After the solvent was removed by a rotary concentrator, 0.91 g of light yellow solid was obtained. Purified by column chromatography (silica gel 64.7 g, column diameter 4 cm; length 12.5 cm, eluent ethyl acetate: heptane = 1:5 to 1:1), the product was collected to give 10.7 mg, HPLC The purity is 92.7%.

The structure of compound 15 is shown in Table 1 below, and its nuclear magnetic resonance hydrogen spectrum is: ¹ H NMR (500 MHz, DMSO- d ₆ ): δ 10.523 (s, 1H), 7.948 (dd, 1H), 7.765 (td, 1H ), 7.700 (d, 2H), 7.666-7.627 (m, 3H), 7.564 (td, 1H), 7.508 (d, 1H), 7.378 (t, 1H), 7.321 (t, 1H), 7.236 (d, 2H), 6.369 (d, 1H), 2.230 (s, 3H), 2.154 (s, 3H). Mass spectrometry analysis: [M+H] ⁺ ; C ₂₇ H ₂₂ NO ₈ ; 488.1348; [M+Na] ⁺ ; 510.1171.

Example 2: Use of compounds for promoting regeneration of cardiomyocytes

在化學式(II)中，R₁為羥基、未經取代之C₁-C₆之烷基、未經取代之C₁-C₆之烷氧基、經取代之C₁-C₆之烷酸基、經取代之C₇-C₁₂之環烷酸基、經取代或未經取代之C₆-C₁₈之芳胺基、經取代或未經取代之C₆-C₁₈之酚基、經取代或未經取代之C₆-C₁₈之芳基、經取代或未經取代之噻吩基、經取代或未經取代之吡咯基、經取代或未經取代之吡啶基或經取代或未經取代之噻唑基；R₂為氫、羥基、未經取代之C₁-C₆之烷基、胺基(-NH₂)、乙醯氧基(-OCOCH₃)、

或-X₂(CO)-(Y)_p-Z，R₃為氫、羥基、未經取代之C₁-C₆之烷基、未經取代之C₁-C₆之烷氧基或-O-CO-(Y)_p-Z，或者，R₂以及R₃相互連接形成

；R₄為氫、未經取代之C₁-C₆之烷氧基或

；以A表示之環狀結構為苯環、噻吩環或吡啶環；X₂為氧(-O-)或胺基(-NH-或-NX₄-)；Y為C₁-C₆之伸烷基(C₁-C₆ alkylene group)、C₂-C₁₂之伸烯基(C₂-C₁₂ alkenylene group)、C₇-C₁₈之伸烷基芳基(C₇-C₁₈ arylenealkylene group)或C₆-C₁₈之伸芳基(C₆-C₁₈ arylene group)；Z為吡啶基(-C₅H₄N)、乙醯基(-COCH₃)、-COOH、-COOCH₃、-OCOCH₃、-NHCOOC(CH₃)₃、-F、-Cl、-Br或-I；p為0或1。 The compound of the following formula (II) can be used to promote the regeneration of cardiomyocytes and repair the injured cardiomyocytes:

In formula (II), R ₁ is hydroxy, unsubstituted C ₁ -C ₆ alkyl, unsubstituted C ₁ -C ₆ alkoxy, substituted C ₁ -C ₆ alkanoic acid Group, substituted C ₇ -C ₁₂ naphthenic acid group, substituted or unsubstituted C ₆ -C ₁₈ arylamine group, substituted or unsubstituted C ₆ -C ₁₈ phenol group, Substituted or unsubstituted C ₆ -C ₁₈ aryl, substituted or unsubstituted thienyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl or substituted or unsubstituted Substituted thiazolyl; R ₂ is hydrogen, hydroxyl, unsubstituted C ₁ -C ₆ alkyl, amine (-NH ₂ ), acetyloxy (-OCOCH ₃ ),

Or -X ₂ (CO)-(Y) _p -Z, R ₃ is hydrogen, hydroxy, unsubstituted C ₁ -C ₆ alkyl, unsubstituted C ₁ -C ₆ alkoxy or- O-CO-(Y) _p -Z, or R ₂ and R ₃ are connected to each other to form

; R ₄ is hydrogen, unsubstituted C ₁ -C ₆ alkoxy or

; The cyclic structure represented by A is a benzene ring, thiophene ring or pyridine ring; X ₂ is an oxygen (-O-) or an amine group (-NH- or -NX ₄ -); Y is an extension of C ₁ -C ₆ Alkyl (C ₁ -C ₆ alkylene group), C ₂ -C ₁₂ alkenylene group (C ₂ -C ₁₂ alkenylene group), C ₇ -C ₁₈ alkylene aryl group (C ₇ -C ₁₈ arylenealkylene group ) Or C ₆ -C ₁₈ arylene group (C ₆ -C ₁₈ arylene group); Z is pyridyl (-C ₅ H ₄ N), acetyl (-COCH ₃ ), -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ , -F, -Cl, -Br or -I; p is 0 or 1.

In addition to the above compounds 1 to 15, the compounds having the above formula (II) also include, for example, compounds 16 to 44, and the structures of compounds 16 to 44 are listed in Table 2 below.

The above compound 1 to compound 44 are used as active ingredients, and then mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition that can promote the regeneration of myocardial cells, a health food that can prevent myocardial infarction, or a health food that can maintain the prognosis of myocardial infarction.

The above compounds that can be used to promote regeneration of cardiomyocytes may be in the form of salts or esters of various compounds represented by formula (II), for example, various compounds represented by formula (II) and alkali metals or alkalis Salts of sodium, potassium, lithium, magnesium, calcium, ammonium, carbonate, nitrate, bicarbonate, hydrochloride, sulfate, phosphate, or silicate formed from earth metals ; Or esters formed by various compounds of formula (II) and alcohols, such as methyl, ethyl, propyl, butyl, pentyl, methyl acetate, ethyl acetate, butyl formate, butyl acetate , Butyl valerate, butyl propionate, methyl butyrate, ethyl butyrate and other esters.

The above "pharmaceutically acceptable carrier" or "acceptable carrier" may include pharmaceutical or food acceptable excipients or additives, such as starch, corn starch, gelatin, gum arabic, food coloring, flavor, Flavoring agents, preservatives, etc. The administration route may include oral administration, transdermal administration, intraperitoneal administration, intravenous administration, nasal administration, or ocular administration, etc., preferably oral administration.

The above-mentioned pharmaceutical composition can be administered according to the common knowledge in the technical field by the relevant medical personnel according to factors such as the patient's age, weight, health status, disease type, disease progression, affected part and other factors. The pharmaceutical composition in this case can also be administered alone or together with other medicaments. The course of administration should be carried out according to the routine methods of pharmacy according to the doctor or related person.

The above-mentioned health foods can adjust the content of the appropriate active ingredients according to the set ethnic groups, preferably the content that can be taken daily. The outer packaging can indicate the recommended amount of use, the use standard and conditions of a specific ethnic group (such as pregnant women, patients with kidney disease), or the recommended items for co-administration with other foods or medicines, so that the purchaser without the guidance of a doctor, pharmacist or relevant deacon It can be taken at home without safety concerns.

Test Example 1: Determination of the activity of compounds to promote regeneration of cardiomyocytes

Myosin is an important component of myocardial structure and function, which is composed of myosin heavy chain (myosin heavy chain), of which the myosin heavy chain α-type (α-cardiac myosin heavy chain; α-MHC) is composed of MYH6 gene The encoded protein is a cardiomyocyte-specific gene that is expressed early in the development of the heart. It is mainly expressed in the atria and plays a vital role in the contractile function of cardiomyocytes. This gene provides clues to the heart to identify transcriptional regulatory events. , To participate in the induction and maintenance of the heart cell lineage. Since α-MHC white is an important protein formed early in the myocardium, it can be used in the heart The amount of expression in myoblast H9C2 cell line is used as an index to evaluate whether the test substance has the ability to promote stem cell differentiation and maintenance after myocardial infarction.

1.1 Cultivation of H9C2 cell line

H9C2 cell line is a myoblast of rats (cardiac myoblast), which was purchased from Taiwan Food Industry Research Institute. The medium used for this cell line is 90% by volume. Dulbecco's modified Eagle's medium (Gibco ^™ , Cat. 12800017) contains 1.5g/L sodium bicarbonate (Sigma, Cat. S5761) and 10% by volume. Fetal bovine serum (Gibco ^™ , Cat. 10437028). The cell line was cultured at 37°C in a cell incubator containing 5% carbon dioxide by volume.

1.2 MTT cell survival rate test

To establish a suitable compound concentration without cytotoxicity, H9C2 cells were cultured to 90%, and then inoculated with 1.5*10 ⁴ cell/well to a 24well cell culture plate. After 24 hours of culture, each was added at a concentration of 0.2 to 30 μg/mL The above compound 1 to compound 44 and DMSO (Sigma, Cat. D4540) were used for 48 hours (each group was repeated 3 times), then washed with PBS buffer (Phosphate buffered saline) and added 0.5mg/mL MTT (Thiazoly Blue Tetrazolium Bromide; Sigma, Cat. M2128), placed in a cell incubator at 37°C containing 5% carbon dioxide by volume for 4 hours, and then purple crystals can be seen under the microscope. After removing the supernatant, add 200 μL/well of DMSO to dissolve the crystals, leave it at room temperature and shake for 10 minutes, and finally use ELISA Reader to read the absorbance at a wavelength of 570 nm (absorbance value is the average of three repeats). Taking the absorbance value of the DMSO group as 100%, the representative cell survival rate is 100%.

In the subsequent determination of the effect of each compound on the regeneration and repair of cardiomyocytes, the concentration of the compound with a relative absorbance value in the range of 80% to 120% is selected for testing, as shown in Figure 1, so as to ensure that the cardiomyocytes are growing normally Test.

1.3 Cell disruption collection

Cultivate H9C2 cells to 90%, and then inoculate 1.5*10 ⁴ cell/well to 24well cell culture plate. After 24 hours of culture, add the above compound 1 to compound 44 and DMSO (each group is 3 repeated) After 48 hours of action (the concentration of each group of compounds is listed in Table 3 below), wash with PBS buffer and add 0.05% volume of trypsin 200μL/well for 5 minutes, then add cell culture solution to terminate the trypsin reaction, and then contain The solution of suspended cells was centrifuged at 1000 rpm for 3 minutes at 4°C, and then the pellet was re-dissolved using PBS buffer, repeating the same washing step 3 times, and finally the cells were thoroughly mixed in PBS buffer and moved to -80°C refrigerator Let stand for 30 minutes, then transfer to a 37°C water bath until thawing. Repeat freezing and thawing 3 times to cause cell disruption, and finally centrifuge at 3000 rpm for 15 minutes at 4°C. Collect the supernatant and store in a -20°C refrigerator .

1.4 Determination of α-MHC performance

Use Mouse Myosin Heavy Chain 6, Cardiac Muscle Alpha (MYH6) ELISA Kit (MyBioSource, Cat.MBS7583946) to determine the content of α-MHC, and finally use ELISA Reader to read the absorbance value at 450nm wavelength (absorbance value is the average of three repeats), Taking the absorbance of the DMSO group added with the same compound concentration as 100%, the relative α-MHC content of compound 1 to compound 44 was calculated.

The results are shown in Figure 1. In the case where H9C2 cells grow safely (that is, the cell survival rate is between 80% and 120%), the cells after adding any of Compound 1 to Compound 44 are cultured, and their α-MHC content Compared with the group added with DMSO, there is an increase in the phenomenon. It can be seen that Compound 1 to Compound 44 can indeed promote the expression level of α-MHC in H9C2 cells, which shows that the above Compound 1 to Compound 44 can promote the regeneration and repair of cardiomyocytes. Of efficacy.

Test Example 2: Evaluation of the efficacy of compounds in promoting zebrafish myocardial regeneration

The zebrafish heart can be repaired in a region (~30%) with a frozen method to repair the injury within 21 days, so the adult zebrafish was used as the experimental animal to study the damaged heart. After the zebrafish heart is damaged, the regeneration process can be divided into three stages. The initial stage is the proliferation of cardiomyocytes, the second stage is the regeneration of pericardial cells, and the final stage is the regeneration of vascular endothelial cells.

In order to easily observe whether the tested sample has the effect of promoting myocardial regeneration and repair, the transgenic gene zebrafish (Tg-fli1-eGFP) was used for experiments, and the transgenic gene zebrafish was anesthetized and subjected to cardiac cryotrauma surgery. Since this transgenic gene zebrafish can express green fluorescent protein in vascular endothelial cells, after cryotrauma to the heart, the damaged cells will not be able to continue to express fluorescence, so as to assess the size of the heart injury area.

2.1 Induction of ventricular frostbite in adult zebrafish

As shown in Annex I, the flow chart of the zebrafish cryotrauma experiment. Figure A is a photo of the cryoprobe, which has a probe with a length of about 6 mm and a width of about 0.8 mm at one end, which is used to wound the ventricle of the zebrafish. Before undergoing cryotrauma, the anesthetized adult zebrafish must be placed in the gap of the sponge with its ventral side up, as shown in Figure B. Referring again to Figure C, cut the skin and muscle between the two pectoral fins (red line) of the zebrafish, then insert scissors into the incision, and cut the skin in the direction indicated by the red arrow (Figure D), under the skin, use tweezers Gently open the small silvery subcutaneous tissue (enclosed by the blue dotted line) to enter the heart (Figure E), and then cut the incision with forceps to expose the zebrafish ventricle (green arrow in Figure F) for cryo-injury, as in Panel G The cryoprobe is gently inserted into the chest cavity to touch the heart.

When evaluating whether a compound can promote myocardial regeneration and repair, the heart of the zebrafish must be removed to assess the damage. The process of heart extraction requires a deep and long incision through the gill arch of the chest to enter the pericardial cavity. At this time, two types of heart structures can be seen: the arterial ball (white arrow in Figure H) and the ventricle (green arrow in Figure H). Then use arterial forceps to hold the arterial bulb and pull it out of the body cavity (Figure I), and then remove the entire heart from the body to complete the heart removal (green arrow in Figure J).

2.2 Efficacy evaluation of zebrafish myocardial regeneration

After anesthetizing the transgenic gene zebrafish for cardiac cryotrauma surgery, feed the fish subject for another 7 days. The daily dose is 12.5 μg/g. On the 8th day, sacrifice the fish and perform heart extraction. Observe and calculate The damaged area of the heart, and then evaluate whether the test substance has the effect of promoting the regeneration and repair of zebrafish myocardium.

Since the transgenic gene zebrafish can express green fluorescent protein in vascular endothelial cells, the area of the green fluorescent expression can be used to assess the damage or regeneration and repair of cardiomyocytes, and the area without green fluorescent expression Represents the damaged area of the myocardium. Calculate the ratio of the area of the area to the area of the entire heart to obtain the percentage of the wounded area.

Taking the test substance containing only the solvent as a blank group, after feeding zebrafish that had undergone heart trauma for 7 days, the area of heart damage was calculated, and the percentage of the damaged area was 27%. After establishing the proportion of the blank damaged area, the effect of the compound on myocardial regeneration and repair was then evaluated. After 7 days of feeding the test substances containing the above compound 1 to compound 44 (the concentration of each compound is shown in Table 3) to the zebrafish that had undergone heart trauma, the calculation of the heart injury area of each group was performed, and it was compared with the blank group. The results are listed together in Figure 2. The experimental data in Figure 2 shows that after 7 days of feeding the test substances containing the above compounds 1 to 44, the proportion of zebrafish heart trauma is significantly reduced compared with the blank group, indicating that the above compounds 1 to 44 do Promote the regeneration and repair of zebrafish myocardium.

The different modifications and changes that can be made according to the present invention will obviously not deviate from the scope and spirit of the present invention for those skilled in the art. Although the invention has described specific preferred specific facts, it must be understood that the invention should not be unduly limited to such specific specific facts. In fact, in implementing the described modes of the present invention, different amendments that are obvious to those skilled in the art are also covered by the following patent applications.

Claims (26)

A compound as shown in formula (I):

Where X ₁ is hydrogen; X ₂ is oxygen or amine; X ₃ is hydrogen, hydroxyl or the aforementioned substituent represented by -X ₂ -(CO)-(Y) _p -Z; Y is C ₂ -C ₆ Alkylene group, C ₃ -C ₁₂ alkenyl group, C ₇ -C ₁₈ alkylene group or C ₆ -C ₁₈ alkylene group; Z is -COCH ₃ , -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ , -F, -Cl, -Br or -I; p is 0 or 1. The compound according to claim 1, wherein Z is -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ or -Cl. The compound according to claim 1, wherein X ₂ is

,

Or-NX ₄ -, X ₄ is a substituent

. The compound according to claim 1, wherein Y is propylene, butylene, benzyl or phenylene. The compound according to claim 1, wherein the compound is represented by one of the following compounds 1 to 10, compounds 12 to 13 and compound 15:

A compound as shown in formula (I):

Where X ₁ is an unsubstituted C ₁ -C ₆ alkyl group; X ₂ is an oxygen or amine group; X ₃ is hydrogen, hydroxy or the aforementioned as -X ₂ -(CO)-(Y) _p -Z Substituent; Y is C ₇ -C ₁₈ alkylene aryl or C ₆ -C ₁₈ alkylene aryl; Z is -COCH ₃ , -COOH, -COOCH ₃ , -NHCOOC(CH ₃ ) ₃ ,- F, -Cl, -Br or -I; p is 0 or 1. The compound according to claim 6, wherein Z is -COOH, -COOCH ₃ , -NHCOOC(CH ₃ ) ₃ or -Cl. The compound according to claim 6, wherein X ₂ is

,

Or-NX ₄ -, X ₄ is a substituent

. The compound according to claim 6, wherein Y is benzyl or phenylene. The compound according to claim 6, wherein the compound is represented by the following compound 11:

A compound as shown in formula (I):

Where X ₁ is hydrogen; X ₂ is oxygen or amine; X ₃ is hydrogen, hydroxyl or the aforementioned substituent represented by -X ₂ -(CO)-(Y) _p -Z; Y is C ₂ -C ₁₂ Alkenyl; Z is -COCH ₃ , -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ , -F, -Cl, -Br, or -I; p is 0 or 1. The compound according to claim 11, wherein Z is -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ or -Cl. The compound according to claim 11, wherein X ₂ is

,

Or -NX ₄ -, X ₄ is a substituent

. The compound according to claim 11, wherein Y is vinylidene. The compound according to claim 11, wherein the compound is represented by the following compound 14: compound 14

A method for preparing a compound according to any one of claims 1 to 15, which comprises reacting reactant A and reactant B at a temperature of 0°C to 25°C, wherein reactant A is coumaric acid Derivatives, caffeic acid derivatives or cinnamic acid derivatives; reactant B is an acid compound, anhydride compound, acetyl chloride compound or ester compound. The method according to claim 16, wherein the reactant A is p-coumaric acid, caffeic acid, caffeic acid methyl ester or 4-aminocinnamic acid. The method according to claim 16, wherein the reactant B is acetylsalicylic acid, 3-chlorophenylacetic acid, tert-butoxycarbonyl-γ-aminobutyric acid, glutaric anhydride, adipic anhydride, methylhexyl Diacid chloride or monomethyl fumarate. The method according to any one of claims 16 to 18, wherein the reactant A and the reactant B are reacted in an alkaline environment at a temperature of 0°C to 25°C. A pharmaceutical product for promoting myocardial regeneration, which comprises the compound according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. A compound of formula (II) for the preparation of medicinal products that promote myocardial regeneration;

Where R ₁ is hydroxy, unsubstituted C ₁ -C ₆ alkyl, unsubstituted C ₁ -C ₆ alkoxy, substituted C ₁ -C ₆ acid alkyl, substituted C ₇ -C ₁₂ acid cycloalkyl, substituted or unsubstituted C ₆ -C ₁₈ arylamino group, substituted or unsubstituted C ₆ -C ₁₈ aryloxy group, substituted or unsubstituted Substituted C ₆ -C ₁₈ aryl, substituted or unsubstituted thienyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, or substituted or unsubstituted thiazole Group; R ₂ is hydrogen, hydroxy, unsubstituted C ₁ -C ₆ alkyl, amine, acetoxy,

Or -X ₂ (CO)-(Y) _p -Z, R ₃ is hydrogen, hydroxy, unsubstituted C ₁ -C ₆ alkyl, unsubstituted C ₁ -C ₆ alkoxy or- O-CO-(Y) _p -Z, or R ₂ and R ₃ are connected to each other to form

; R ₄ is hydrogen, unsubstituted C ₁ -C ₆ alkoxy or

; The cyclic structure represented by A is benzene ring, thiophene ring or pyridine ring; X ₂ is oxygen or amine group; Z is -C ₅ H ₄ N, -COCH ₃ , -COOH, -COOCH ₃ , -OCOCH ₃ , -NHCOOC(CH ₃ ) ₃ , -F, -Cl, -Br or -I; Y is C ₁ -C ₆ alkylene group, C ₂ -C ₁₂ alkylene group, C ₇ -C ₁₈ alkylene group Arylaryl or C ₆ -C ₁₈ extended aryl; p is 0 or 1. The use according to claim 21, wherein X ₂ is

,

Or-NX ₄ , X ₄ is a substituent

. The use according to claim 21, wherein R ₃ is -CH ₃ ,

, -NH ₂ or -O-CO-(Y) _p -Z. The use according to claim 21, wherein when R ₂ is -X ₂ (CO)-(Y) _p -Z, R ₁ is a hydroxyl group. The use according to claim 21, wherein when Y is an alkenyl group, Z is -COOCH ₃ . The use according to claim 21, wherein the compound represented by the chemical formula (II) is represented by one of the following compound 1 to compound 44:

Compound 13 compound 14 compound 15

Compound 28 Compound 29 Compound 30

Compound 43 Compound 44

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