Tryptophan derivatives and their preparation methods and applications

Abstract

The present invention relates to the technical field of synthesis and pharmaceutical application of organic tryptophan derivatives, providing novel tryptophan derivatives with dual UCP1 / AIDA activation activity, as well as methods for preparing the novel tryptophan derivatives. The invention also discloses the use of tryptophan derivatives in activating thermogenesis and preparing thermogenic drugs. The present invention proposes a tryptophan derivative that introduces an amide bond at the amino position of the tryptophan nucleus and adds benzene rings of various lengths and substituents. The different substituent groups on the benzene rings are studied in depth to develop dual-target agonist preparations that can selectively activate UCP1 and AIDA. The present invention employs a dual-target agonist strategy to obtain a more active tryptophan derivative 1-19, which plays an important role in preparing drugs for preventing or treating cold injuries.

Description

Technical Field

[0001] The present invention relates to the technical field of synthesis and pharmaceutical application of organic tryptophan derivatives, in particular to tryptophan derivatives and preparation methods and applications thereof. Background Art

[0002] With the frequent occurrence of extreme weather conditions, cold injuries pose increasingly significant challenges and problems to outdoor workers. Each year, tens of millions of people lose their ability to function or even die as a result of these injuries, resulting in significant economic losses. Tissue and organ damage caused by exposure to cold environments can be categorized as localized tissue damage and systemic cold injury. In cold environments, a decrease in core body temperature is the most direct and life-threatening factor. Primary hypothermia is a spontaneous drop in body temperature below 35°C. Therefore, addressing the hypothermia, organ damage, and even survival issues caused by cold weather are pressing issues for the well-being of people in cold-stricken regions.

[0003] Primary hypothermia is a serious life-threatening disease following cold exposure. Brown adipose tissue heat production plays an important role in body temperature regulation, so activating the body's heat production will provide a new strategy for treating primary hypothermia. UCP1 is a specific marker for adipose tissue heat production, and simply increasing its expression level does not result in efficient heat production. Recent studies have shown that AIDA on mitochondria regulates UCP1 cysteine ​​oxidation, which can act as a "switch" to start heat production, increasing the activity of "latent" UCP1 and thus causing the body to produce large amounts of heat. Therefore, activating two key proteins, UCP1 and AIDA protein, may be an effective strategy to enhance the body's heat production.

[0004] Furthermore, the number of drugs currently under development targeting UCP1 is relatively low, primarily due to the previously unclear structure of UCP1 and the lack of a known key protein pocket for its activation. Therefore, designing targeted drugs based on the latest UCP1 protein pocket structure is becoming increasingly important. Summary of the Invention

[0005] The purpose of the present invention is to provide a tryptophan derivative and its preparation method and application, so as to solve the technical problem that the prior art lacks tryptophan derivatives with UCP1 / AIDA dual activation activity.

[0006] According to a first aspect of the present invention, a tryptophan derivative or a pharmaceutically acceptable salt thereof is provided, wherein the general formula of the tryptophan derivative is shown as I:

[0007]

[0008] Wherein, R is a substituent on the end of the side chain of the tryptophan core, and R is selected from the following structures:

[0009]

[0010] According to a second aspect of the present invention, a method for preparing tryptophan derivatives 1 to 18 comprises the following steps: dissolving tryptophan methyl ester and an acid in N,N-dimethylformamide, adding a catalyst and stirring, quenching the reaction with water, extracting with ethyl acetate, and purifying by column chromatography to obtain an intermediate; wherein the acid is 4-hydroxyphenylacetic acid, 4-p-chlorophenylbutyric acid, 4-p-methoxyphenylacetic acid, 4-p-hydroxyphenylbutyric acid, cyclohexanebutyric acid, p-methylphenylbutyric acid, p-fluorophenylbutyric acid, cinnamic acid, p-bromocinnamic acid, p-fluorocinnamic acid, p-hydroxycinnamic acid, 3-hydroxy, 4-methoxycinnamic acid, p-methoxycinnamic acid, 3-nitrocinnamic acid, adipic acid, succinic acid, glutaric acid, or p-methyltryptophan; dissolving the intermediate in ethanol, adding sodium hydroxide solution, dissolving and stirring, and then adding hydrochloric acid to adjust the pH to precipitate the product, thereby obtaining tryptophan derivatives 1 to 18.

[0011] The method for preparing tryptophan derivative 19 comprises the following steps: dissolving 1-methyltryptophan methyl ester and p-methyltryptophan in N,N-dimethylformamide, adding a catalyst and stirring, quenching the reaction with water, extracting with ethyl acetate, and purifying by column chromatography to obtain an intermediate; dissolving the intermediate in ethanol, adding sodium hydroxide solution, dissolving and stirring, and then adding hydrochloric acid to adjust the pH to precipitate the product, thereby obtaining tryptophan derivative 19.

[0012] Furthermore, the catalyst is HATU and DIPEA; the equivalent ratio of tryptophan methyl ester or 1-methyltryptophan methyl ester, acid, HATU and DIPEA is 1:1.2:2.2:2.2; after adding the catalyst, stirring is carried out at room temperature for 0.5 to 3 hours; the volume of N,N-dimethylformamide is 5 to 15 ml; the concentration of ethanol is 98%; the concentration of sodium hydroxide is 1 mol / L; the volume ratio of ethanol and sodium hydroxide is 1:1 to 2:1; after adding sodium hydroxide solution, dissolving and stirring at room temperature for 6 hours; the concentration of hydrochloric acid is 0.5 mol / L; and the pH is adjusted to 3-4.

[0013] Among them, the equivalent ratio can also be called the ratio of moles.

[0014] According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned tryptophan derivatives or pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients.

[0015] Furthermore, the preparation is prepared by adding one or more pharmaceutically acceptable excipients to the above-mentioned tryptophan derivatives or pharmaceutically acceptable salts thereof, and the dosage form of the preparation is capsule, pill, tablet, granule or injection.

[0016] According to a fourth aspect of the present invention, there is also provided the use of the above-mentioned tryptophan derivatives or pharmaceutically acceptable salts thereof in the preparation of a drug for inducing thermogenesis.

[0017] According to a fifth aspect of the present invention, there is also provided use of tryptophan derivatives or pharmaceutically acceptable salts thereof in the preparation of a UCP1 activator, an AIDA activator or a UCP1 / AIDA dual activator.

[0018] Beneficial effects:

[0019] The present invention provides novel tryptophan derivatives with UCP1 / AIDA dual activation activity, a preparation method of the novel tryptophan derivatives, and also points out the use of tryptophan derivatives in activating thermogenesis and preparing thermogenic drugs.

[0020] The tryptophan derivatives proposed in the present invention introduce an amide bond at the amino position of the tryptophan nucleus and add benzene rings of various lengths and different substituents. After in-depth research on the different substituent groups on the benzene ring, a dual-target agonist preparation capable of selectively activating UCP1 and AIDA was developed.

[0021] The present invention adopts a dual-target agonistic strategy to obtain tryptophan derivatives 1-19 with better activity. Such tryptophan derivatives play an important role in the preparation of drugs for preventing or treating cold injuries.

[0022] Among them, tryptophan derivative 13 is preferred because it has a stronger heat-generating ability than other tryptophan derivatives.

[0023] Based on the implementation methods provided in the above aspects, this application can also be further combined to provide more implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 Schematic diagram of the synthesis steps of tryptophan derivative 13;

[0026] Figure 2 Schematic diagram of the effects of the control group, positive drug all-trans retinoic acid and tryptophan derivative 1-19 on the consumption of lipid droplets in brown adipocytes;

[0027] Figure 3 The control group, the positive drug all-trans retinoic acid and tryptophan derivative 13 on the changes of lipid droplets in brown adipocytes;

[0028] Figure 4The fluorescence changes on the mitochondrial membrane of brown adipocytes after adding different concentrations of tryptophan derivatives 13, the control group, the positive drug all-trans retinoic acid and the like;

[0029] Figure 5 The effects of the control group and different concentrations of tryptophan derivatives 13 on the mitochondrial membrane potential of brown adipocytes;

[0030] Figure 6 The results of statistical analysis of mitochondrial membrane potential fluorescence ratio;

[0031] Figure 7 This is the result of docking tryptophan derivative 13 with UCP1 by Schrödinger docking;

[0032] Figure 8 The results of docking tryptophan derivative 13 with AIDA by Schrödinger docking. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0034] The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) measurements using a Bruker AVANCE-300 / 500 NMR spectrometer in DMSO-d6 solvent and TMS as the internal standard.

[0035] The process of the preparation method of tryptophan derivatives 1-19 provided by the present invention is as follows:

[0036] Tryptophan methyl ester or 1-methyltryptophan methyl ester and acid are dissolved in N,N-dimethylformamide (DMF), a catalyst is added and stirred at room temperature for 1 hour, the reaction is quenched with water, and ethyl acetate is added for extraction. The intermediate is purified by column chromatography, and the intermediate is dissolved in ethanol and NaOH solution and stirred for 6 hours, and then hydrochloric acid is added to adjust the pH to precipitate the product.

[0037] Wherein, the acid is 4-hydroxyphenylacetic acid, 4-p-chlorophenylbutyric acid, 4-p-methoxyphenylacetic acid, 4-p-hydroxyphenylbutyric acid, cyclohexanebutyric acid, p-methylphenylbutyric acid, p-fluorophenylbutyric acid, cinnamic acid, p-bromocinnamic acid, p-fluorocinnamic acid, p-hydroxycinnamic acid, 3-hydroxy, 4-methoxycinnamic acid, p-methoxycinnamic acid, 3-nitrocinnamic acid, adipic acid, succinic acid, glutaric acid or p-methyltryptophan.

[0038] The specific embodiments of the present invention are described below:

[0039] Example 1

[0040]

[0041] The preparation method of tryptophan derivative 1 specifically comprises the following steps:

[0042] 500 mg of tryptophan methyl ester (1 eq) and 460 mg of 4-hydroxyphenylacetic acid (1.2 eq) were dissolved in DMF (10 ml). 1.8 g of HATU (2.2 eq) and 0.825 ml of DIPEA (2.2 eq) were added and allowed to react for 1 h. The reaction was then quenched with water, extracted with ethyl acetate, and separated via a column to afford Intermediate 1a as a white solid in 80% yield.

[0043] Intermediate 1a was dissolved in 98% ethanol (10 ml), and 1 mol / L NaOH (5 ml) was added. After stirring at room temperature for 6 h, 0.5 mol / L HCl was added until the pH of the solution was 3-4. The target tryptophan derivative 1 was obtained by filtration as a white solid with a yield of 89%. 1 H NMR (400MHz, DMSO-d6) δ10.96–10.78(m,1H),8.28(d,J=7.9Hz,1H),7.52(d,J=7 .8Hz,1H),7.34(d,J=8.1Hz,1H),7.10(d,J=2.3Hz,1H),7.07(ddd,J=8.2,6.9,1 .2Hz,1H),7.03(s,4H),6.97(ddd,J=8.0,6.9,1.1Hz,1H),4.47(td,J=8.2,5.0H z,1H),3.18(dd,J=14.6,5.0Hz,2H),3.03(dd,J=14.6,8.5Hz,1H),2.25(s,3H).

[0044] Wherein, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA is N,N-diisopropylethylamine.

[0045] Example 2

[0046]

[0047] The preparation method of tryptophan derivative 2 specifically comprises the following steps:

[0048] The experimental procedures of Example 1 were repeated, except that 4-hydroxyphenylacetic acid was replaced with 4-p-chlorophenylbutyric acid. Other conditions remained unchanged, to obtain intermediate 2a as a white solid in 81% yield.

[0049] Replacing 1a with 2a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 2 as a white solid in a yield of 72%. 1 H NMR (400MHz, DMSO-d6) δ10.82(d,J=2.4Hz,1H),7.88(d,J=7.8Hz,1H),7.53(d,J=7.8Hz,1H),7 .34–7.30(m,1H),7.30–7.26(m,2H),7.19–7.08(m,3H),7.04(ddd,J=8.2,6.9,1.2Hz,1H),6.9 5(ddd,J=8.0,6.9,1.1Hz,1H),4.43(td,J=8.2,4.7Hz,1H),3.27–3.14(m,1H),3.00(dd,J=14. 6,8.6Hz,1H),2.44(dd,J=8.6,6.7Hz,2H),2.05(td,J=7.3,4.0Hz,2H),1.68(p,J=7.5Hz,2H).

[0050] Example 3

[0051]

[0052] The preparation method of tryptophan derivative 3 specifically comprises the following steps:

[0053] The experimental procedures of Example 1 were repeated, except that 4-hydroxyphenylacetic acid was substituted with 4-p-methoxyphenylacetic acid. Other conditions remained unchanged, to obtain intermediate 3a as a white solid in a yield of 79%.

[0054] Replacing 1a with 3a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 3 as a white solid in a yield of 66%. 1H NMR (400MHz, DMSO-d6) δ10.89(d,J=2.4Hz,1H),8.21(d,J=7.8Hz,1H),7.52(d,J =7.8Hz,1H),7.34(d,J=8.1Hz,1H),7.11(d,J=2.3Hz,1H),7.09–7.01(m,3H),7. 00–6.94(m,1H),6.82–6.75(m,2H),4.45(td,J=8.2,5.0Hz,1H),3.71(s,3H),3. 35(d,J=2.9Hz,2H), 3.18(dd,J=14.6,5.0Hz,1H), 3.03(dd,J=14.6,8.4Hz,1H).

[0055] Example 4

[0056]

[0057] The preparation method of tryptophan derivative 4 specifically comprises the following steps:

[0058] The experimental procedures of Example 1 were repeated, except that 4-hydroxyphenylacetic acid was replaced with 4-p-hydroxyphenylbutyric acid. Other conditions remained unchanged, to obtain intermediate 4a as a white solid in 50% yield.

[0059] Replacing 1a with 4a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 4 as a white solid in a yield of 67%. 1 H NMR (400MHz, DMSO-d6) δ12.58(s,1H),10.83(d,J=2.5Hz,1H),9.12(s,1H),8.07(d,J=7.9Hz,1H),7.54(d,J =7.8Hz,1H),7.33(d,J=8.0Hz,1H),7.14(d,J=2.3Hz,1H),7.06(ddd,J=8.1,6.9,1.2Hz,1H),6.98(ddd,J=8. 0,6.9,1.1Hz,1H),6.94–6.87(m,2H),6.72–6.59(m,2H),4.56–4.40(m,1H),3.17(dd,J=14.6,4.9Hz,1H),3 .00(dd,J=14.7,9.0Hz,1H),2.36(dd,J=8.6,6.6Hz,2H),2.07(td,J=7.3,4.5Hz,2H),1.66(p,J=7.5Hz,2H).

[0060] Example 5

[0061]

[0062] The preparation method of tryptophan derivative 5 specifically comprises the following steps:

[0063] The experimental procedures of Example 1 were repeated, except that cyclohexanebutyric acid replaced 4-hydroxyphenylacetic acid in Example 1, with other conditions remaining unchanged, to obtain intermediate 5a as a white solid in a yield of 72%.

[0064] Replacing 1a with 5a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 5 as a white solid in a yield of 81%. 1 H NMR (400MHz, DMSO-d6) δ10.97–10.90(m,6H),8.91(d,J=7.9Hz,3H),8.47(s,1H),8.22(dd,J=7.8, 2.1Hz,3H),7.53(dd,J=7.9,3.3Hz,7H),7.33(d,J=8.0Hz,7H),7.19–7.14(m,6H),7.14–6.93(m,20 H),6.50(d,J=15.5Hz,4H),4.62–4.44(m,8H),4.29(dd,J=7.2,5.2Hz,4H),3.73(s,2H),3.23(dd, J=14.7,4.9Hz,5H),3.14(dd,J=14.5,5.5Hz,5H),3.10–2.97(m,8H),2.46(dd,J=7.2,3.2Hz,11H).

[0065] Example 6

[0066]

[0067] The preparation method of tryptophan derivative 6 specifically comprises the following steps:

[0068] The experimental procedures of Example 1 were repeated, except that p-methylphenylbutyric acid replaced 4-hydroxyphenylacetic acid in Example 1, with other conditions remaining unchanged, to obtain intermediate 6a as a white solid in a yield of 64%.

[0069] Replacing 1a with 6a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 6 as a white solid in a yield of 69%. 1H NMR (400MHz, DMSO-d6) δ10.83(d,J=2.5Hz,1H),8.06(d,J=7.9Hz,1H),7.53(d,J=7.7Hz, 1H),7.32(d,J=8.0Hz,1H),7.13(d,J=2.3Hz,1H),7.10–7.03(m,3H),7.03–6.94(m,3H), 4.48(ddd,J=9.0,7.8,4.9Hz,1H),3.17(dd,J=14.6,4.8Hz,1H),2.99(dd,J=14.7,9.0Hz ,1H),2.42(dd,J=8.6,6.7Hz,2H),2.25(s,3H),2.16–1.95(m,2H),1.68(p,J=7.5Hz,2H).

[0070] Example 7

[0071]

[0072] The preparation method of tryptophan derivative 7 specifically comprises the following steps:

[0073] The experimental procedures of Example 1 were repeated, except that p-fluorophenylbutyric acid replaced 4-hydroxyphenylacetic acid in Example 1. Other conditions remained unchanged, to obtain intermediate 7a as a white solid in a yield of 74%.

[0074] Replacing 1a with 7a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 7 as a white solid in a yield of 63%. 1 H NMR (400MHz, DMSO-d6) δ10.86(d,J=2.4Hz,1H),8.03(d,J=7.9Hz,1H),7.54(d,J=7.8Hz,1H),7 .32(d,J=8.0Hz,1H),7.13(td,J=5.6,2.6Hz,3H),7.10–7.07(m,1H),7.07–7.01(m,2H),6.97( ddd,J=7.9,6.9,1.1Hz,1H),4.54–4.40(m,1H),3.19(dd,J=14.6,4.8Hz,1H),3.00(dd,J=14.6 ,8.9Hz,1H),2.45(dd,J=8.6,6.7Hz,2H),2.07(tt,J=11.4,5.9Hz,2H),1.69(p,J=7.4Hz,2H).

[0075] Example 8

[0076]

[0077] The preparation method of tryptophan derivative 8 specifically comprises the following steps:

[0078] The experimental procedures of Example 1 were repeated, except that cinnamic acid replaced 4-hydroxyphenylacetic acid in Example 1, with other conditions remaining unchanged, to obtain intermediate 8a as a white solid in a yield of 74%.

[0079] Replacing 1a with 8a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 8 as a white solid in a yield of 63%. 1 H NMR (400MHz, DMSO-d6) δ12.73(s,1H),10.92(d,J=2.4Hz,1H),8.44(d,J=7.9Hz,1H),7.63– 7.50(m,3H),7.40(ddt,J=15.2,6.8,2.9Hz,4H),7.34(d,J=8.1Hz,1H),7.18(d,J=2.3Hz,1H ),7.06(ddd,J=8.1,6.9,1.2Hz,1H),6.98(ddd,J=8.0,6.9,1.1Hz,1H),6.78(d,J=15.9Hz,1 H), 4.63 (td, J=8.3, 4.9Hz, 1H), 3.25 (dd, J=14.7, 5.0Hz, 1H), 3.09 (dd, J=14.7, 8.7Hz, 1H).

[0080] Example 9

[0081]

[0082] The preparation method of tryptophan derivative 9 specifically comprises the following steps:

[0083] The experimental procedures of Example 1 were repeated, except that p-bromocinnamic acid replaced 4-hydroxyphenylacetic acid in Example 1, with other conditions remaining unchanged, to obtain intermediate 9a as a white solid in a yield of 72%.

[0084] Replacing 1a with 9a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 9 as a white solid in a yield of 53%. 1H NMR (400MHz, DMSO-d6) δ12.75(s,1H),10.98(s,1H),8.51(d,J=7.8Hz,1H),7.62–7.53(m, 3H),7.50–7.43(m,2H),7.38(d,J=15.8Hz,1H),7.35–7.31(m,1H),7.19(d,J=2.4Hz,1H),7 .06(ddd,J=8.1,6.9,1.2Hz,1H),6.97(ddd,J=8.0,7.0,1.1Hz,1H),6.81(d,J=15.9Hz,1H ), 4.62(td,J=8.3,4.9Hz,1H), 3.24(dd,J=14.7,4.9Hz,1H), 3.10(dd,J=14.7,8.7Hz,1H).

[0085] Example 10

[0086]

[0087] The preparation method of tryptophan derivative 10 specifically comprises the following steps:

[0088] The experimental procedures of Example 1 were repeated with the substitution of p-fluorocinnamic acid for 4-hydroxyphenylacetic acid and other conditions remaining unchanged to obtain intermediate 10a as a white solid in a yield of 66%.

[0089] Replacing 1a with 10a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 10 as a white solid in a yield of 55%. 1 H NMR (400MHz, DMSO-d6) δ12.75(s,1H),10.88(d,J=2.4Hz,1H),8.39(d,J=7.9Hz,1H),7.67–7.58(m ,2H),7.56(d,J=7.8Hz,1H),7.40(d,J=15.8Hz,1H),7.36–7.31(m,1H),7.30–7.20(m,2H),7.17(d ,J=2.3Hz,1H),7.06(ddd,J=8.1,6.9,1.2Hz,1H),6.98(ddd,J=8.1,6.9,1.1Hz,1H),6.71(d,J=15 .9Hz, 1H), 4.64 (td, J=8.3, 5.0Hz, 1H), 3.25 (dd, J=14.7, 5.0Hz, 1H), 3.09 (dd, J=14.7, 8.6Hz, 1H).

[0090] Example 11

[0091]

[0092] The preparation method of tryptophan derivative 11 specifically comprises the following steps:

[0093] The experimental procedures of Example 1 were repeated with the substitution of p-hydroxycinnamic acid for 4-hydroxyphenylacetic acid and other conditions remaining unchanged to obtain intermediate 11a as a white solid in a yield of 71%.

[0094] Replacing 1a with 11a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 11 as a white solid in a yield of 72%. 1 H NMR (400MHz, DMSO-d6) δ12.67(s,1H),10.86(d,J=2.5Hz,1H),9.89(s,1H),8.25(d,J=7.9 Hz,1H),7.56(d,J=7.8Hz,1H),7.38(d,J=8.4Hz,2H),7.37–7.25(m,2H),7.16(d,J=2.3Hz ,1H),7.11–7.02(m,1H),7.02–6.93(m,1H),6.80(d,J=8.4Hz,2H),6.52(d,J=15.7Hz,1H) ,4.62(td,J=8.3,5.0Hz,1H), 3.23(dd,J=14.7,5.0Hz,1H), 3.07(dd,J=14.7,8.7Hz,1H).

[0095] Example 12

[0096]

[0097] The preparation method of tryptophan derivative 12 specifically comprises the following steps:

[0098] The experimental procedures of Example 1 were repeated with 3-hydroxy, 4-methoxycinnamic acid replacing 4-hydroxyphenylacetic acid in Example 1, while keeping other conditions unchanged, to obtain intermediate 12a as a white solid in a yield of 63%.

[0099] Replacing 1a with 12a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 12 as a white solid in a yield of 69%. 1H NMR (400MHz, DMSO-d6) δ10.83(d,J=2.4Hz,1H),9.33(s,1H),8.20(d,J=7.9Hz,1H),7.56(d,J =7.8Hz,1H),7.32(d,J=8.1Hz,1H),7.24(d,J=15.7Hz,1H),7.15(d,J=2.3Hz,1H),7.04(ddd, J=8.1,7.0,1.2Hz,1H),7.01–6.95(m,2H),6.95–6.89(m,2H),6.53(d,J=15.7Hz,1H),4.57(t d,J=8.1,4.9Hz,1H),3.78(s,3H),3.26(dd,J=14.7,4.8Hz,1H),3.06(dd,J=14.7,8.4Hz,1H).

[0100] Example 13

[0101]

[0102] like Figure 1 As shown, the preparation method of tryptophan derivative 13 specifically includes the following steps:

[0103] The experimental procedures of Example 1 were repeated with p-methoxycinnamic acid replacing 4-hydroxyphenylacetic acid in Example 1 and other conditions remaining unchanged to obtain intermediate 13a as a white solid in a yield of 66%.

[0104] Replacing 1a with 13a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 13 as a white solid in a yield of 53%. 1 H NMR (400MHz, DMSO-d6) δ12.70(s,1H),10.89(d,J=2.4Hz,1H),8.32(d,J=7.9Hz,1H ),7.56(d,J=7.8Hz,1H),7.53–7.45(m,2H),7.40–7.30(m,2H),7.17(d,J=2.3Hz,1H ),7.11–7.02(m,1H),7.02–6.93(m,3H),6.61(d,J=15.7Hz,1H),4.63(td,J=8.3,4 .9Hz,1H),3.78(s,3H),3.24(dd,J=14.7,5.0Hz,1H),3.08(dd,J=14.7,8.7Hz,1H).

[0105] Example 14

[0106]

[0107] The preparation method of tryptophan derivative 14 specifically comprises the following steps:

[0108] The experimental procedures of Example 1 were repeated with 3-nitrocinnamic acid replacing 4-hydroxyphenylacetic acid in Example 1 and other conditions remaining unchanged to obtain intermediate 14a as a white solid in a yield of 65%.

[0109] Replacing 1a with 14a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 14 as a white solid in a yield of 66%. 1 H NMR (400MHz, DMSO-d6) δ12.81(s,1H),10.87(d,J=2.4Hz,1H),8.44(d,J=7.8Hz,1H),8.40(t ,J=2.0Hz,1H),8.21(dd,J=8.2,2.3Hz,1H),8.04–7.97(m,1H),7.71(t,J=8.0Hz,1H),7.60– 7.49(m,2H),7.34(d,J=8.0Hz,1H),7.17(d,J=2.3Hz,1H),7.11–7.02(m,1H),7.02–6.93(m, 2H), 4.65(td,J=8.1,4.9Hz,1H), 3.26(dd,J=14.7,5.0Hz,1H), 3.10(dd,J=14.7,8.5Hz,1H).

[0110] Example 15

[0111]

[0112] The preparation method of tryptophan derivative 15 specifically comprises the following steps:

[0113] The experimental procedures of Example 1 were repeated with adipic acid replacing 4-hydroxyphenylacetic acid in Example 1 and other conditions remaining unchanged to obtain intermediate 15a as a white solid in a yield of 54%.

[0114] Replacing 1a with 15a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 15 as a white solid in a yield of 63%. 1H NMR (400MHz, DMSO-d6) δ12.80–11.73(m,2H),10.83(d,J=2.5Hz,1H),8.08(d,J=7.8Hz ,1H),7.53(d,J=7.8Hz,1H),7.33(d,J=8.0Hz,1H),7.13(d,J=2.3Hz,1H),7.06(t,J=7. 5Hz,1H),6.98(t,J=7.4Hz,1H),4.47(td,J=8.3,5.1Hz,1H),3.16(dd,J=14.6,5.1Hz,1 H),2.99(dd,J=14.6,8.8Hz,1H),2.25–1.96(m,4H),1.43(dq,J=10.1,6.7,5.1Hz,4H).

[0115] Example 16

[0116]

[0117] The preparation method of tryptophan derivative 16 specifically comprises the following steps:

[0118] The experimental procedures of Example 1 were repeated with succinic acid replacing 4-hydroxyphenylacetic acid in Example 1 and other conditions remaining unchanged to obtain intermediate 16a as a white solid in a yield of 58%.

[0119] Replacing 1a with 16a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 16 as a white solid in a yield of 67%. 1 H NMR (400MHz, DMSO-d6) δ12.29(s,1H),10.83(d,J=2.5Hz,1H),8.08(d,J=7.9Hz,1H) ,7.53(d,J=7.8Hz,1H),7.33(d,J=8.0Hz,1H),7.13(d,J=2.3Hz,1H),7.11–7.03(m,1 H),6.98(t,J=7.4Hz,1H),4.47(td,J=8.3,5.0Hz,1H),3.16(dd,J=14.6,5.1Hz,1H) ,2.99(dd,J=14.6,8.8Hz,1H),2.27–1.98(m,2H),1.43(dq,J=10.2,6.7,5.1Hz,2H).

[0120] Example 17

[0121]

[0122] The preparation method of tryptophan derivative 17 specifically comprises the following steps:

[0123] The experimental procedures of Example 1 were repeated with glutaric acid replacing 4-hydroxyphenylacetic acid in Example 1 and other conditions remaining unchanged to obtain intermediate 17a as a white solid in a yield of 68%.

[0124] Replacing 1a with 17a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 17 as a white solid in a yield of 56%. 1 H NMR (400MHz, DMSO-d6) δ12.37(s,1H),10.85(d,J=2.5Hz,1H),8.09(d,J=7.8Hz,1H),7.53(d ,J=7.8Hz,1H),7.33(d,J=8.1Hz,1H),7.14(d,J=2.3Hz,1H),7.06(ddd,J=8.2,6.9,1.2Hz,1H ),6.98(ddd,J=8.0,6.9,1.1Hz,1H),4.46(td,J=8.4,5.0Hz,1H),3.17(dd,J=14.6,5.0Hz,1 H), 3.00 (dd, J=14.6, 8.8Hz, 1H), 2.11 (ddt, J=24.2, 14.6, 7.4Hz, 4H), 1.66 (p, J=7.1Hz, 2H).

[0125] Example 18

[0126]

[0127] The preparation method of tryptophan derivative 18 specifically comprises the following steps:

[0128] The experimental procedures of Example 1 were repeated by replacing 4-hydroxyphenylacetic acid in Example 1 with p-methyltryptophan and replacing tryptophan methyl ester with 5-bromotryptophan methyl ester to obtain intermediate 18a as a white solid in a yield of 45%.

[0129] Replacing 1a with 18a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain tryptophan derivative 18 as a white solid in a yield of 55%. 1H NMR (300MHz, DMSO-d6) δ11.11(d,J=2.4Hz,1H),8.31(d,J=7.9Hz,1H),7.72(d,J=1.9Hz,1H),7.55–7.44(m,2H),7.38–7.26(m,2H),6.99– 6.89(m,2H),6.58(d,J=15.7Hz,1H),4.57(td,J=8.2,5.0Hz,1H),3.76(s,3H),3.19(dd,J=14.6,5.0Hz,1H),3.04(dd,J=14.7,8.5Hz,1H).

[0130] Example 19

[0131]

[0132] The preparation method of tryptophan derivative 19 specifically comprises the following steps:

[0133] The experimental procedures of Example 1 were repeated by replacing 4-hydroxyphenylacetic acid in Example 1 with p-methyltryptophan and replacing tryptophan methyl ester with 1-methyltryptophan methyl ester to obtain intermediate 19a as a white solid in a yield of 43%.

[0134] Replacing 1a with 19a and keeping other conditions unchanged, the experimental procedures of Example 1 were repeated to obtain the tryptophan derivative 19 as a white solid in a yield of 60%. 1 H NMR(300MHz,DMSO-d6)δ8.35(d,J=7.8Hz,1H),7.56(dt,J=7.9,1.0Hz,1H),7.52–7.44( m,2H),7.37(dd,J=2.3,1.5Hz,1H),7.35–7.27(m,1H),7.14(s,1H),7.14–7.08(m,1H),7 .04–6.98(m,1H),6.98–6.91(m,2H),6.61(d,J=15.8Hz,1H),4.59(td,J=8.1,5.1Hz,1H ),3.76(s,3H),3.70(s,3H),3.20(dd,J=14.6,5.1Hz,1H),3.06(dd,J=14.7,8.4Hz,1H).

[0135] Taking tryptophan derivative 13 as an example, the effect of tryptophan derivatives on promoting thermogenesis in brown adipocytes was studied. The specific steps and experimental results are as follows:

[0136] 1) Instruments and reagents: DMEM medium (Hyclone); fetal bovine serum (Hyclone); CKX31 inverted microscope (Olympus Corporation, Philippines); constant temperature CO2 incubator (Thermo Fisher Scientific, USA).

[0137] 2) Cell Culture: Primary cultured preadipocytes were grown in DMEM complete medium containing 10% FBS. When the monolayer reached near confluence (confluence greater than 90%), the medium was replaced with differentiation medium I (DMEM complete medium supplemented with 0.5 mmol / L IBMX, 1 μmol / L dexamethasone, and 10 μg / mL insulin). This was designated as day 0 of differentiation induction, and culture continued. On day 2 of differentiation induction, i.e., 48 hours later, the medium was replaced with differentiation medium II (DMEM complete medium supplemented with 10 μg / mL insulin) and culture continued. Thereafter, the medium was replaced with DMEM every two days, and cell morphology was observed under a microscope. The cells were cultured in a 37°C incubator (5% CO2, 90% relative humidity). After the cells reached maturity through differentiation induction, the next step of the experiment was performed.

[0138] 3) Oil Red O Staining: After primary pre-brown adipocytes were induced to differentiate and mature, the corresponding concentrations of cinnamon extract were added. After 48 hours, the original culture medium was discarded and 1 mL of 10% neutral formaldehyde solution was added to each well to fix the cells. The cells were incubated at room temperature for approximately 1 hour. The fixed cells were then stained with Oil Red O solution. 1 mL of isopropanol was added to each well to extract the oil red. The cells were shaken on a microplate shaker for 10 minutes and the OD value (absorbance A) was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA). The lipid droplet consumption rate was calculated (lipid droplet consumption rate = 1 - A sample / A control). The experiment was repeated three times.

[0139] Specific results such as Figure 2-4 shown.

[0140] Figure 2 The effects of the positive drug all-trans retinoic acid (ATRA) and 19 tryptophan derivatives on the changes of lipid droplets in brown adipocytes were studied. Figure 2 Among them, ATRA (50 μmol / L) is a positive drug that increases the metabolism of brown adipocytes. Different tryptophan derivatives cause brown adipocytes to produce different lipid droplet consumption rates. Figure 2 The comparison conclusion can be seen as follows: tryptophan derivative 13 has a better ability to increase the metabolism of brown fat cells.

[0141] Figure 3 The effects of positive drugs all-trans retinoic acid and tryptophan derivative 13 on the changes of lipid droplets in brown adipocytes. Figure 3 In the present study, tryptophan derivative 13 increased the consumption of brown adipocyte lipid droplets in a dose-dependent manner; Figure 3The comparison conclusion can be seen as follows: tryptophan derivative 13 has a better ability to increase the metabolism of brown fat cells.

[0142] Figure 4 Figure 3. Changes in lipid droplets in brown adipocytes following the addition of different concentrations of tryptophan derivative 13. Oil Red O staining results show that the number of lipid droplets induced by tryptophan derivative 13 was significantly lower than that of the control group. After 48 hours of treatment, tryptophan derivatives increased oil droplet consumption compared to UCP1-activating drugs reported in the literature, with significant differences observed between the tryptophan derivative group and the positive drug group.

[0143] The results showed that tryptophan derivative 13 could increase the lipid droplet consumption rate of primary brown adipocytes compared with the control group. The results suggest that tryptophan derivative 13 may increase energy expenditure by consuming lipid droplets in brown adipose tissue.

[0144] 4) Mitochondrial membrane potential assay: After primary pre-brown adipocytes were induced to differentiate and mature, the corresponding concentrations of tryptophan derivative 13 were added and allowed to react for 48 hours before the experiment. Specific experimental procedures were performed according to the instructions for the "Mitochondrial Membrane Potential Assay Kit (JC-1)" from Beyotime Biotechnology.

[0145] Specific results such as Figure 5-6 shown.

[0146] Figure 5 The effects of different concentrations of tryptophan derivatives on the changes of mitochondrial membrane potential of brown adipocytes are shown in Figure 2. The control group is a control group with only blank culture medium added. Figure 5 The tryptophan derivative 13 reduces the membrane potential of brown adipocytes in a dose-dependent manner.

[0147] Figure 6 The results of the statistical analysis of the mitochondrial membrane potential fluorescence ratio showed that compound 13, i.e., tryptophan derivative 13, had significant differences compared with the blank at a dosage of 33-100 μmol / L, and could reduce the mitochondrial membrane potential of brown adipocytes.

[0148] Results: Compared with the control group, the CL-316243 positive drug group significantly reduced the mitochondrial membrane potential in primary brown adipocytes, and cinnamon extract reduced the mitochondrial membrane potential in cells in a concentration-dependent manner. These results suggest that tryptophan derivative 13 may alter electron transport within and outside the mitochondrial membrane.

[0149] 5) Molecular docking: This is a method for drug design that uses receptor characteristics and the interaction between receptors and drug molecules. It primarily studies intermolecular interactions (such as ligand-receptor interactions) and predicts their binding affinity. By structurally docking the recently reported UCP1 protein model 8J1N with a tryptophan derivative, the binding pattern of the tryptophan derivative with the protein was determined. The tryptophan derivative was also docked with the predicted active pocket in the C2 domain of AIDA to predict its binding mode.

[0150] Figure 7 This is the result of docking tryptophan derivative 13 with UCP1 by Schrödinger docking; Figure 8 The results of docking tryptophan derivative 13 with AIDA by Schrödinger docking.

[0151] Depend on Figure 7 and Figure 8 The results showed that the tryptophan structure interacts with UCP1 and AIDA in multiple ways. The tryptophan indole ring binds to 91ARG on UCP1, forming a cation-π interaction and a hydrogen bond with 87SER. The carboxyl group also forms hydrogen bonds with multiple amino acids, including 83 and 183ARG. Among them, ARGs at positions 91, 83, and 183 are known to be active, playing a key role in GDP binding to UCP1 and inhibiting UCP1's uncoupling function. Furthermore, these three amino acids form a bond with the positive control drug DNP.

[0152] Furthermore, the docking model of tryptophan derivative 13 with AIDA showed that the indole ring formed a hydrogen bond with 121LYS, the carboxyl group formed a salt bridge with 112LYS, 110HIE, and 143LYS, and the benzene ring formed a hydrogen bond with 110HIE. This binding mechanism is similar to the protein-protein binding mechanism predicted by alphafold for UCP1 and AIDA.

[0153] Experimental comparison shows that the tryptophan derivative 13 provided in this application has a good effect in promoting thermogenesis in brown fat cells, can be used in the preparation of thermogenic drugs, and can be further used in the preparation of UCP1 activators, AIDA activators or UCP1 / AIDA dual activators.

[0154] Through experimental comparison, other tryptophan derivatives provided in this application also have a good effect of promoting thermogenesis in brown fat cells, and can also be used in the preparation of thermogenic drugs, and can also be further used in the preparation of UCP1 activators, AIDA activators or UCP1 / AIDA dual activators. The experimental process will not be repeated here.

[0155] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A tryptophan derivative or a pharmaceutically acceptable salt thereof, characterized in that: The tryptophan derivatives are selected from the following structures:

2. A method for preparing a tryptophan derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The preparation method comprises the following steps: Tryptophan methyl ester and acid were dissolved in N,N-dimethylformamide, the catalyst was added and stirred, the reaction was quenched with water, and ethyl acetate was added for extraction, and the intermediate was obtained after purification by column chromatography; Wherein, the acid is 4-hydroxyphenylbutyric acid, cyclohexanebutyric acid, 3-hydroxy, 4-methoxycinnamic acid, 3-nitrocinnamic acid, p-methoxycinnamic acid; The intermediate is dissolved in ethanol, and then sodium hydroxide solution is added. After dissolving and stirring, hydrochloric acid is added to adjust the pH to precipitate the product, thereby obtaining the tryptophan derivative.

3. A method for preparing the tryptophan derivatives or pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The method for preparing the tryptophan derivative 18 of claim 1 comprises the following steps: 5-Bromotryptophan methyl ester and p-methoxycinnamic acid were dissolved in N,N-dimethylformamide, the catalyst was added and stirred, the reaction was quenched with water, and ethyl acetate was added for extraction. The intermediate was obtained after purification by column chromatography; The intermediate is dissolved in ethanol, and then sodium hydroxide solution is added. After dissolution and stirring, hydrochloric acid is added to adjust the pH to precipitate the product, thereby obtaining the tryptophan derivative 18.

4. A method for preparing a tryptophan derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The method for preparing the tryptophan derivative 19 of claim 1 comprises the following steps: 1-Methyltryptophan methyl ester and p-methoxycinnamic acid were dissolved in N,N-dimethylformamide, the catalyst was added and stirred, the reaction was quenched with water, and ethyl acetate was added for extraction, and the intermediate was obtained after purification by column chromatography; The intermediate is dissolved in ethanol, and then sodium hydroxide solution is added. After dissolution and stirring, hydrochloric acid is added to adjust the pH to precipitate the product, thereby obtaining the tryptophan derivative 19.

5. A method for preparing a tryptophan derivative or a pharmaceutically acceptable salt thereof according to any one of claims 2 to 4, characterized in that: The catalyst is HATU and DIPEA; The equivalent ratio of tryptophan methyl ester or 1-methyltryptophan methyl ester or 5-bromotryptophan methyl ester, acid, HATU and DIPEA is 1:1.2:2.2:2.2; After adding the catalyst, stir at room temperature for 0.5 to 3 hours; The volume of N,N-dimethylformamide is 5 to 15 ml; The concentration of ethanol is 98%; The concentration of sodium hydroxide is 1 mol / L; Add ethanol and sodium hydroxide in a volume ratio of 1:1 to 2:1; After adding sodium hydroxide solution, dissolve and stir at room temperature for 6 hours; The concentration of hydrochloric acid is 0.5 mol / L; Adjust the pH to 3-4.

6. Use of the tryptophan derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for inducing thermogenesis.

7. Use of the tryptophan derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a UCP1 activator, an AIDA activator or a UCP1 / AIDA dual activator.

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