Synthesis method of R- / S-hydroxypropyl tetrahydropyrantriol

Abstract

The present invention belongs to the technical field of organic synthesis, and particularly relates to a method for synthesizing R- / S-hydroxypropyl tetrahydropyrantriol. Compound 2 is synthesized by using D-xylose; three hydroxyl groups at positions 2, 3, and 4 of compound 2 are protected with acetyl groups by using acetic anhydride to obtain compound 4; compound 4 is reduced with a reducing agent at low temperature to obtain 5 and 5'; a mixture of 5 and 5' is connected with an amino acid protected by amino group Fmoc to obtain compounds 9 and 9', and the ester group is hydrolyzed with sodium hydroxide to obtain target compound molecules 3 and 3'. The present invention can synthesize high-purity R-hydroxypropyl tetrahydropyrantriol, which can be used for testing and comparison of the biological activity of hydroxypropyl tetrahydropyrantriol S- / R-isomers.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and particularly relates to a method for synthesizing R- / S-hydroxypropyl tetrahydropyrantriol. Background Art

[0002] Hydroxypropyl tetrahydropyrantriol (also known as Pro-xylane) is converted from D-xylose (which can be extracted from beech wood). It has a wide range of biological activities and can activate the synthesis of glycosaminoglycans (GAGs). It has also been shown to have in vivo activity in clinical trials and is widely used in cosmetics as a raw material with anti-wrinkle activity. Hydroxypropyl tetrahydropyrantriol can also promote the production of hyaluronic acid and collagen; as well as improve the adhesion between the dermis and the epidermis, promote the regeneration of damaged tissues by inducing the synthesis of dermal and epidermal structural components, help maintain the elasticity of the dermis, and prevent skin aging. The compound has five chiral sites, namely C2, C3, C4, C5, and C7 (Formula 1). In addition, it is a biodegradable and environmentally friendly compound.

[0003]

[0004] In 2002, patent WO 02 / 051828 Al first proposed an original preparation method of hydroxypropyl tetrahydropyrantriol (Formula 2): using D-xylose as raw material and water as solvent, heating at 90°C under the action of sodium bicarbonate for 6 hours, condensing acetylacetone and xylose, cyclizing and then cracking, and obtaining β-acetone xyloside in high yield; then using methanol as solvent, sodium borohydride was used to reduce the keto carbonyl group of β-acetone xyloside, and an isomer mixture of hydroxypropyl tetrahydropyrantriol was obtained in high yield.

[0005]

[0006] Formula 2. Original first generation scheme for the synthesis of hydroxypropyl tetrahydropyrantriol

[0007] In 2009, Bioorganic & Medicinal Chemistry Letters, 2009, 19, 845-849 Formula 3: In the first step, sodium hydroxide is used instead of sodium bicarbonate. Under the new conditions, the reaction temperature can be reduced to 50°C, the reaction time is shortened to 45 minutes, and the yield is significantly improved; the improvement of the second step reaction is to use isopropanol-acetic acid mixed solvent as the medium for the reduction reaction, which can greatly improve the selectivity of the reaction, and its C7 (S) -1 / C7 (R) -1 can reach 9 / 1. The document also proposed that the stereochemical characteristics of hydroxypropyl tetrahydropyrantriol are of great significance to its biological activity. Experiments show that the stereochemistry of the C7 hydroxyl group has an effect on the activity level of activating mucopolysaccharide (GAG) synthesis. The level of activation of mucopolysaccharide (GAG) synthesis by hydroxypropyl tetrahydropyrantriol with dr = 70:30 is greater than that of hydroxypropyl tetrahydropyrantriol with dr = 50:30 (should be dr = 50:50).

[0008]

[0009] Patent CN113735810B of 2022 obtained single S-hydroxypropyl tetrahydropyrantriol through acetal / ketal protection and reduction, and patent CN113735811B obtained single S-hydroxypropyl tetrahydropyrantriol through acylation protection and reduction. The protected hydroxypropyl tetrahydropyrantriol precursors obtained by both methods can be crystallized to remove R-hydroxypropyl tetrahydropyrantriol to obtain optically pure S-hydroxypropyl tetrahydropyrantriol.

[0010] In 2011, Richard L. Gallo published an article titled Exogenous Addition of a C-Xylopyranoside Derivative Stimulates Keratinocyte Dermatan Sulfate Synthesisand Promotes Migration, which disclosed the biological activity of hydroxypropyl tetrahydropyrantriol. Keratinocytes treated with hydroxypropyl tetrahydropyrantriol greatly increased the secretion of total sulfated GAGs (the induced GAGs were mainly chondroitin sulfate (CS) and dermatan sulfate (DS)). Hydroxypropyl tetrahydropyrantriol may promote the repair of epithelial cells by acting as a promoter of DS synthesis.

[0011] In 2012, Romain R. Vives published an article titled New C-Xyloside Induces Modifications of GAG Expression, Structure and Functional Properties, which confirmed for the first time that hydroxypropyl tetrahydropyrantriol promoted the synthesis of GAG chains, but also caused significant changes in their structure. The main changes were: the size of chondroitin sulfate (CS) and dermatan sulfate (DS) decreased, O-sulfonation increased, and the content and distribution of iduronate changed.

[0012] The synthesis of S-hydroxypropyl tetrahydropyrantriol has a clear synthetic route, that is, starting from xylose, a two-step method is used to obtain a high dr value target product, and then the isomers are purified by different purification methods. The activity research of hydroxypropyl tetrahydropyrantriol is also basically clear, including promoting GAG synthesis and DEJ repair. However, the research on different isomers of hydroxypropyl tetrahydropyrantriol is not clear enough. S- / R-hydroxypropyl tetrahydropyrantriol are two compounds that are extremely difficult to separate. The method of the present invention can promote the biological activity research of S- / R-hydroxypropyl tetrahydropyrantriol related isomers. Summary of the invention

[0013] The purpose of the present invention is to provide a highly efficient synthesis method of R- / S-hydroxypropyl tetrahydropyrantriol, wherein an amino acid or other molecule is connected to the C7 hydroxyl group to obtain a derivative of R- / S-hydroxypropyl tetrahydropyrantriol, wherein two isomers of the derivative can be quickly separated on column chromatography or preparative thin layer chromatography, and then the derivative is hydrolyzed to conveniently and efficiently obtain a target molecule.

[0014] The present invention uses D-xylose as a raw material to first synthesize 2; the three hydroxyl groups at positions 2, 3, and 4 of 2 are protected with acetyl groups using acetic anhydride to obtain a fat-soluble compound 4; compound 4 is reduced with a reducing agent at low temperature to obtain 5 and 5', and the two isomers cannot be separated by column chromatography; the mixture of 5 and 5' is connected with an amino acid protected by amino group Fmoc to obtain 9 and 9' that can be separated by column chromatography, and the ester group is hydrolyzed with sodium hydroxide to obtain target molecules 3 and 3'. The specific reaction formula is shown as follows

[0015]

[0016] The synthesis method of compound 2 is as follows: methanol, sodium carbonate, xylose and acetylacetone are added to a reaction bottle in sequence, reacted at 80° C. for 12 hours, the pH is adjusted to 7 with hydrochloric acid, and the compound 2 is obtained by spin drying.

[0017] The synthesis method of compound 4 is as follows: compound 2 is added to a reaction bottle, and dichloromethane, triethylamine, and acetic anhydride are added in sequence, reacting at 25°C for 24 hours, adding dichloromethane to dilute, extracting with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated brine respectively, drying the organic phase with anhydrous sodium sulfate, adding activated carbon for decolorization, filtering, and spinning the filtrate to obtain compound 4.

[0018] The synthesis method of 5 and 5' is as follows: tetrahydrofuran, acetyl-protected compound 4, and methanol are added to a reaction bottle, a reducing agent is added at room temperature, and the reaction is carried out at room temperature for 24 hours. After the reaction is completed, the reaction is filtered, and the filtrate is diluted with ethyl acetate, extracted three times with water, and then extracted once with saturated brine. The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is spin-dried to obtain compound 5.

[0019] The reducing agent used is: sodium triacetoxyborohydride, sodium borohydride, sodium cyanoborohydride or borane dimethyl sulfide;

[0020] The amount of compound 4 is 1 equivalent, the amount of sodium triacetoxyborohydride is 1-6 equivalents, the amount of sodium borohydride is 0.5-1 equivalents, the amount of sodium cyanoborohydride is 1-2 equivalents, and the amount of borane dimethyl sulfide is 0.3-0.5 equivalents. When sodium triacetoxyborohydride is used, methanol needs to be added as a catalyst. Generally, 1 ml is required for every 10g of substrate (compound 4).

[0021] The synthesis method of compounds 9 and 9' is as follows: dichloromethane, 5 and 5', amino acid (Fmoc-Pro-OH / Fmoc-Phe-OH / Fmoc-Val-OH), 4-dimethylaminopyridine, and N,N'-diisopropylcarbodiimide are added to a reaction bottle in sequence, stirred at room temperature for 2 hours, filtered, concentrated the filtrate, and separated by column chromatography to obtain compounds 9 and 9'.

[0022] The amount of 5 and 5' is 1 equivalent, the amount of amino acid (Fmoc-Pro-OH / Fmoc-Phe-OH / Fmoc-Val-OH) is 1.2 equivalents, the amount of 4-dimethylaminopyridine is 0.05-0.3 equivalents, and the amount of N,N'-diisopropylcarbodiimide is 1.2 equivalents.

[0023] The preparation method of the target molecule is as follows: add methanol to a reaction bottle, cool to 0°C, add sodium hydroxide, stir to dissolve at 0°C, add compounds 9 and 9', reflux the reaction system at 60°C with stirring for 8 hours, after the reaction is complete, lower the temperature to room temperature, adjust the pH to 7 with hydrochloric acid, and concentrate the reaction solution to obtain the oily target molecule.

[0024] Beneficial effects:

[0025] 1. The present invention provides a chiral separation method for hydroxypropyl tetrahydropyrantriol, and the products of different isomers obtained by reduction can be efficiently separated by column chromatography.

[0026] 2. The present invention can synthesize high-purity R-hydroxypropyl tetrahydropyrantriol, which can be used for different biological activity tests of hydroxypropyl tetrahydropyrantriol. Compared with the preparation of liquid phase, it is more convenient and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Effects of different isomers of hydroxypropyl tetrahydropyrantriol on the collagen content in fibroblasts.

[0028] Figure 2 The effect of different isomers of hydroxypropyl tetrahydropyrantriol on the upregulation rate of collagen in fibroblasts.

[0029] Figure 3 Effects of different isomers of hydroxypropyl tetrahydropyrantriol on the elastin content in fibroblasts.

[0030] Figure 4 The effect of different isomers of hydroxypropyltetrahydropyrantriol on the upregulation rate of elastin in fibroblasts. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to specific embodiments.

[0032] Example 1

[0033]

[0034] In a 500 ml reaction bottle, 150 ml of methanol, 31.8 g (0.3 mol) of sodium carbonate, 30 g (0.2 mol) of xylose, and 26 g (0.24 mol) of acetylacetone were added in sequence. After reacting at 80°C for 12 hours, the pH was adjusted to 7 with 4 mol / L hydrochloric acid, and the crude product of compound 2 was obtained by spin drying.

[0035] 2: 1 H NMR (400MHz, DMSO-d6) δ3.64(dd,J=10.9,5.4Hz,1H),3.42(td,J=9.5,2.9Hz,1H),3.23(tt,J=9.8,4.9Hz,1H),3.08(td,J=8.7,4.7H z,1H),2.97(t,J=10.7Hz,1H),2.85(td,J=9.1,5.3Hz,1H),2.75(dd,J=15.8,2.8Hz,1H),2.38(dd,J=15.8,9.4Hz,1H),2.08(s,3H). 13C NMR (101MHz, Deuterium Oxide) δ212.93,77.21,76.00,72.84,69.34,68.87,45.47,29.76.

[0036]

[0037] The crude product 2 was added to a 500 ml reaction bottle, and 150 ml of dichloromethane, 139.0 ml (1 mol) of triethylamine and 75.6 ml (0.8 mol) of acetic anhydride were added in sequence. The mixture was reacted at 25°C for 24 hours, and 200 ml of dichloromethane was added for dilution. The mixture was extracted with 1 mol / ml hydrochloric acid solution, 200 ml each time, and extracted three times. The mixture was then extracted once with 200 ml of saturated sodium bicarbonate solution and once with 100 ml of saturated brine. The organic phase was dried with 10 g of anhydrous sodium sulfate, and 1.5 g of activated carbon was added for decolorization by stirring at 50°C for 30 minutes. The mixture was filtered with suction and the filtrate was spin-dried to obtain 56.1 g of product compound 4 without further purification. The two-step yield was 88.7%.

[0038] The hydrogen spectrum of compound 4 (4: 1 H NMR (400MHz, DMSO-d6) δ5.18(t,J=9.5Hz,1H),4.83(td,J=10.4,5.6Hz,1H),4.72(t,J=9.6Hz,1H),3.98(td,J=9.6,9.0, 3.6Hz,1H),3.89(dd,J=11.0,5.6Hz,1H),3.42(t,J=10.8Hz,1H),2.64–2.48(m,2H),2.09(s,3H),1.97(d,J=9.0Hz,9H). 13 C NMR (101MHz, Chloroform-d) δ205.17,170.15,169.86,169.80,74.31,73.51,71.67,69.13,66.61,45.30,31.01,20.60.

[0039]

[0040] Add 100 ml of tetrahydrofuran to a 500 ml reaction bottle, add 8 g (25.32 mmol) of acetyl protected molecule 4, cool the reaction system to 0°C, and under nitrogen protection, add 6.33 ml (12.66 mmol) of borane dimethyl sulfide (2 mol / L) to the reaction system. The reaction is carried out in an ice bath for 1 hour. After the reaction is completed, add 10 ml of water to quench the borane, add 100 ml of ethyl acetate to dilute, extract with 0.1 mol / ml hydrochloric acid solution, use 100 ml each time, extract three times, and then extract once with 100 ml of saturated brine. The organic phase is dried with 10 g of anhydrous sodium sulfate, filtered, and the filtrate is spin-dried to obtain 7.56 g of product (5 and 5') (dr = 50:50), without further purification, with a yield of 93.9%.

[0041] 5,5': 1 H NMR(400MHz,Chloroform-d)δ5.24–5.10(m,1H),4.99(dd,J=8.9,5.7Hz,1H),4.92–4.79(m,1H),4.19–3.94(m,2H),3 .70–3.50(m,1H),3.29(td,J=10.9,7.8Hz,1H),2.14–1.94(m,9H),1.62(t,J=5.6Hz,2H),1.18(q,J=7.1,6.7Hz,3H). 13 C NMR (101MHz, Chloroform-d) δ170.35,169.85,169.69,75.66,73.43,72.18,68.95,66.68,63.85,39.89,23.90,23.34,20.69.

[0042]

[0043] 200 ml of dichloromethane, 10 g (31.45 mmol) of 5 and 5', 16.76 g (47.17 mmol) of Fmoc-Pro-OH, 383.61 mg (3.14 mmol) of 4-dimethylaminopyridine, and 7.30 ml (47.17 mmol) of N,N'-diisopropylcarbodiimide were added to a 500 ml reaction bottle in sequence. The mixture was stirred at room temperature for 2 hours, filtered, and the filtrate was concentrated and separated by column chromatography to obtain 9.86 g of product 6 and 9.78 g of product 6', with a yield of 98.0% (49.2% and 48.8% for 6 and 6', respectively).

[0044] 6: 1H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.6Hz,2H),7.63(t,J=7.3Hz,2H),7.41(q,J=7.4Hz,2H),7.32(q,J=7.4,6.7Hz,2H),5.24(dt,J=14.5,6.2Hz,2H),4.94(dd,J=10.0,5.4Hz,1H),4.86–4.75(m,1H),4.51–4.23(m,4H),3.95(dd,J=11.2,5.8Hz,1H),3.74–3.62(m,1H),3.58–3.52(m,1H),3.30(t,J=10.9Hz,1H),2.48–2.13(m,2H),2.06–1.93(m,9H),1.86(d,J=2.6Hz,2H),1.69–1.51(m,2H),1.21(dd,J=18.3,6.3Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ172.25,170.23,170.05,169.88,154.73,144.02,141.25,127.67,127.06,125.21,119.95,74.20,72.26,69.51,68.28,67.50,66.35,59.30,47.21,46.53,38.12,31.18,29.80,24.45,23.41,20.56.

[0045] 6’: 1H NMR(400MHz,Chloroform-d)δ7.76(dd,J=7.5,3.7Hz,2H),7.67–7.51(m,2H),7.40(t,J=7.5Hz,2H),7.31(t,J=7.4Hz,2H),5.13(t,J= 9.4Hz,2H),4.95(td,J=10.1,5.7Hz,1H),4.80(td,J=9.5,2.4Hz,1H),4.42(ddd,J=10.0,6.7,3.2Hz,1H),4.35(d,J=7.4Hz,1H),4.32– 4.25(m,1H),4.19(dt,J=18.2,7.3Hz,1H),4.04(ddd,J=11.1,5.7,1.6Hz,1H),3.89–3.79(m,1H),3.72–3.61(m,2H),3.58–3.48(m,1H ),3.19(td,J=11.0,5.7Hz,1H),2.38–2.09(m,2H),2.02(d,J=3.5Hz,9H),1.93–1.76(m,2H),1.29–1.28(m,2H),1.14(d,J=6.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ171.80,170.29,169.79,169.74,154.77,143.84,141.22,127.60,126.94,125. 05,75.87,73.71,72.12,69.12,67.38,66.56,59.28,47.13,46.46,37.45,31.37,30.12,24.31,23.34,20.64.

[0046]

[0047] Add 200 ml of methanol to a 500 ml reaction bottle, cool to 0°C, add 3.77 g (283.02 mmol) of sodium hydroxide, stir at 0°C to dissolve, add 10 g (15.69 mmol) of molecule 6, and reflux the reaction system at 60°C for 8 hours. After the reaction is complete, the temperature is lowered to room temperature, the pH is adjusted to about 7 with 4 mol / L hydrochloric acid, and the reaction solution is concentrated to obtain an oily substance, which is subjected to column chromatography to obtain 2.69 g of product 3, with a yield of 89.2%.

[0048]

[0049] Add 200 ml of methanol to a 500 ml reaction bottle, cool to 0°C, add 3.77 g (283.02 mmol) of sodium hydroxide, stir at 0°C to dissolve, add 10 g (15.69 mmol) of molecule 6', and reflux and stir the reaction system at 6°C for 8 hours. After the reaction is complete, lower the temperature to room temperature, adjust the pH to about 7 with 4 mol / L hydrochloric acid, concentrate the reaction solution to obtain an oily substance, and obtain 2.78 g of product 3' by column chromatography, with a yield of 92.2%.

[0050] 3: 1 H NMR(400MHz, Deuterium Oxide)δ4.01(ddd,J=9.4,6.3,3.1Hz,1H),3.95(dd,J=11.3,5.5Hz,1H),3.59(td,J=10.0,5.4Hz,1H),3.41(t,J=9.2Hz,2H), 3.27(t,J=11.0Hz,1H), 3.17(t,J=9.3Hz,1H), 2.00–1.87(m,1H), 1.52(ddd,J=14.1,10.1,3.0Hz,1H), 1.22(d,J=6.3Hz,3H). 13 C NMR (101MHz, Deuterium Oxide) δ77.43,76.99,73.57,69.59,68.81,63.89,40.19,22.77.

[0051] 3': 1 H NMR(400MHz, Deuterium Oxide)δ4.05(p,J=6.4,5.7Hz,1H),3.94(dd,J=10.3,4.6Hz,1H),3.64–3.54(m,1H),3.36(dt,J=22.3,9.3Hz,2H),3 .26(t,J=10.0Hz,1H),3.19(t,J=9.2Hz,1H),1.93(dd,J=14.4,7.2Hz,1H),1.73–1.60(m,1H),1.21(d,J=6.4Hz,3H). 13 CNMR(101MHz,Deuterium Oxide)δ78.37,77.34,73.64,69.49,68.82,65.61,39.99,21.56.

[0052] Example 2

[0053]

[0054] Add 100 ml of tetrahydrofuran, 8 g (25.32 mmol) of acetyl protected compound 4, 1 ml of methanol, and 32 g (1.52 mol) of sodium triacetoxyborohydride to a 500 ml reaction bottle at room temperature. The reaction is carried out at room temperature for 24 hours. After the reaction is completed, filter, dilute the filtrate with 100 ml of ethyl acetate, extract three times with water, each time with 100 ml, and then extract once with 100 ml of saturated brine. The organic phase is dried with 10 g of anhydrous sodium sulfate, filtered, and the filtrate is spin-dried to obtain 7.81 g of product (5 and 5') (dr = 60:40), without further purification, with a yield of 97.0%.

[0055] Example 3

[0056]

[0057] Add 100 ml of tetrahydrofuran to a 500 ml reaction bottle, add 8 g (25.32 mmol) of acetyl protected molecule 4, cool the reaction system to 0°C, add 479.0 mg (12.66 mmol) of sodium borohydride, and react in an ice bath for 1 hour. After the reaction is completed, add 10 ml of water to quench the sodium borohydride, add 100 ml of ethyl acetate to dilute, extract with 0.1 mol / ml hydrochloric acid solution, use 100 ml each time, extract three times, and then extract once with 100 ml of saturated brine. The organic phase is dried with 10 g of anhydrous sodium sulfate, filtered, and the filtrate is spin-dried to obtain 3.31 g of product (5 and 5') (dr = 80:20), without further purification, with a yield of 41.2%.

[0058] Example 4

[0059]

[0060] 200 ml of dichloromethane, 10 g (31.45 mmol) of 5 and 5' (prepared in Example 1), 18.27 g (47.17 mmol) of Fmoc-Phe-OH, 383.61 mg (3.14 mmol) of 4-dimethylaminopyridine, and 7.30 ml (47.17 mmol) of N,N'-diisopropylcarbodiimide were added to a 500 ml reaction bottle in sequence. The mixture was stirred at room temperature for 2 hours, filtered, and the filtrate was concentrated and separated by column chromatography to obtain 10.49 g of product 7 and 10.51 g of product 7', with a yield of 95.6% (48.5% and 47.1% for 7 and 7', respectively).

[0061] 7: 1H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.5Hz,2H),7.58(dd,J=7.5,3.4Hz,2H),7.40(t,J=7.4Hz,2H),7.31(dd,J=13.0,7.4Hz,6H),7.14(d,J=7.4Hz,2H),5.28(d,J=7.9Hz,1H),5.16(t,J=9.3Hz,2H),4.92(td,J=10.1,5.7Hz,1H),4.76(t,J=9.5Hz,1H),4.59(q,J=6.6Hz,1H),4.44(dd,J=10.6,7.1Hz,1H),4.40–4.29(m,1H),4.22(t,J=7.1Hz,1H),3.97(dd,J=11.2,5.7Hz,1H),3.75–3.55(m,1H),3.36(dd,J=7.7,3.3Hz,1H),3.23–3.02(m,3H),2.06–1.92(m,9H),1.18(d,J=6.3Hz,2H),1.16–1.09(d,3H). 13 C NMR(101MHz,Chloroform-d)δ171.18,170.23,169.94,169.82,155.57,143.75,141.29,135.64,129.39,128.58,127.72,127.18,127.07,125.05,119.98,73.73,72.15,69.31,68.64,67.00,66.53,60.37,54.85,47.14,38.01,29.67,22.67,21.02,20.68.

[0062] 7’: 1H NMR(400MHz,Chloroform-d)δ7.76(d,J=7.5Hz,2H),7.56(t,J=6.6Hz,2H),7.40(t,J=7.5Hz,2H),7.36–7.23(m,6H),7.13(d,J= 6.8Hz,2H),5.26(d,J=8.4Hz,1H),5.14(t,J=9.4Hz,1H),5.07(q,J=6.5Hz,1H),4.95(td,J=10.4,5.7Hz,1H),4.80(t,J=9.5Hz,1 H),4.60(q,J=6.6Hz,1H),4.43(dd,J=10.6,7.1Hz,1H),4.31(dd,J=10.7,6.9Hz,1H),4.20(d,J=14.4Hz,1H),4.06(dd,J=11.2, 5.7Hz,1H),3.88–3.78(m,1H),3.40–3.33(m,1H),3.23–2.99(m,3H),2.03(d,J=10.8Hz,9H),1.22(d,J=6.3Hz,2H),1.13(s,3H). 13 C NMR(101MHz,Chloroform-d)δ171.80,170.29,169.79,169.74,154.77,144.09,141.22,127.60,126.94,125.05,1 19.88,73.71,72.14,69.12,67.38,66.56,59.28,47.13,42.22,37.45,31.37,30.12,24.31,23.34,20.64,19.92.

[0063]

[0064] Add 200 ml of methanol to a 500 ml reaction bottle, cool to 0°C, add 3.77 g (283.02 mmol) of sodium hydroxide, stir at 0°C to dissolve, add 10 g (14.54 mmol) of compound 7, and reflux and stir the reaction system at 60°C for 8 hours. After the reaction is complete, the temperature is lowered to room temperature, the pH is adjusted to about 7 with 4 mol / L hydrochloric acid, and the reaction solution is concentrated to obtain an oily substance, which is subjected to column chromatography to obtain 2.44 g of product 3, with a yield of 87.4%.

[0065]

[0066] Add 200 ml of methanol to a 500 ml reaction bottle, cool to 0°C, add 3.77 g (283.02 mmol) of sodium hydroxide, stir at 0°C to dissolve, add 10 g (14.54 mmol) of compound 7', and reflux and stir the reaction system at 60°C for 8 hours. After the reaction is complete, lower the temperature to room temperature, adjust the pH to about 7 with 4 mol / L hydrochloric acid, concentrate the reaction solution to obtain an oily substance, and obtain 2.32 g of product 3' by column chromatography, with a yield of 83.1%.

[0067] Example 5

[0068]

[0069] 200 ml of dichloromethane, 10 g (31.45 mmol) of 5 and 5' (prepared in Example 1), 16.01 g (47.17 mmol) of Fmoc-Val-OH, 383.61 mg (3.14 mmol) of 4-dimethylaminopyridine, and 7.30 ml (47.17 mmol) of N,N'-diisopropylcarbodiimide were added to a 500 ml reaction bottle in sequence. The mixture was stirred at room temperature for 2 hours, filtered, and the filtrate was concentrated and separated by column chromatography to obtain 8.97 g of product 8 and 9.16 g of 8', with a yield of 96.2% (47.6% and 48.6% for 8 and 8', respectively).

[0070] 8: 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.5Hz,2H),7.62(d,J=7.5Hz,2H),7.41(t,J=7.6Hz,2H),7.32(td,J =7.5,1.2Hz,2H),5.31(d,J=8.8Hz,1H),5.27–5.09(m,2H),4.98–4.88(m,1H),4.78(t,J=9.6Hz,1H),4.49–4. 34(m,2H),4.27–4.22(m,1H),3.99(dd,J=11.2,5.7Hz,1H),3.42(t,J=10.0Hz,1H),3.20(t,J=10.9Hz,1H),2 .19–2.12(m,1H),2.12–1.88(m,9H),1.65–1.55(m,2H),1.35(d,J=15.5Hz,3H),0.96(dd,J=24.4,6.9Hz,6H). 13CNMR(101MHz,Chloroform-d)δ171.68,170.22,169.95,169.83,156.21,143.75,141.25,127.69,127.03,125.10,119.96,73.69,72.16,69.27,68.26,67.03,66.51,59.01,47.13,38.00,31.46,31.16,29.65,20.60,18.94,17.58.

[0071] 8’: 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.6Hz,2H),7.61(dd,J=7.5,3.3Hz,2H),7.40(t,J=7.5Hz,2H),7.32(td,J=7.5,1.4Hz,2H),5.36–5.30(m,1H),5.22–5.08(m,2H),4.99–4.91(m,1H),4.83(td,J=9.6,4.5Hz,1H),4.41(td,J=10.1,9.6,7.2Hz,2H),4.26(dd,J=10.4,5.9Hz,1H),4.08(dd,J=11.3,5.7Hz,1H),3.42(td,J=9.4,2.7Hz,1H),3.20(t,J=10.9Hz,1H),2.18(m,1H),2.05–2.01(m,9H),1.68(dt,J=7.0,3.5Hz,2H),1.35(d,J=15.5Hz,3H),0.94(dd,J=30.9,6.8Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ171.29,170.34,169.78,169.75,156.21,143.74,141.27,127.67,127.03,125.08,119.93,73.68,72.13,69.49,69.07,66.98,66.63,59.04,31.46,31.21,29.67,20.68,19.77,17.35.

[0072]

[0073] Add 200 ml of methanol to a 500 ml reaction bottle, cool to 0°C, add 3.77 g (283.02 mmol) of sodium hydroxide, stir at 0°C to dissolve, add 10 g (15.63 mmol) of compound 7, and reflux the reaction system at 60°C for 8 hours. After the reaction is complete, the temperature is lowered to room temperature, the pH is adjusted to about 7 with 4 mol / L hydrochloric acid, and the reaction solution is concentrated to obtain an oily substance, which is subjected to column chromatography to obtain 2.36 g of product 3, with a yield of 78.6%.

[0074]

[0075] Add 200 ml of methanol to a 500 ml reaction bottle, cool to 0°C, add 3.77 g (283.02 mmol) of sodium hydroxide, stir at 0°C to dissolve, add 10 g (15.63 mmol) of compound 7, and reflux and stir the reaction system at 60°C for 8 hours. After the reaction is complete, the temperature is lowered to room temperature, the pH is adjusted to about 7 with 4 mol / L hydrochloric acid, and the reaction solution is concentrated to obtain an oily substance, which is subjected to column chromatography to obtain 2.52 g of product 3', with a yield of 83.9%.

[0076] Example 6

[0077] The purpose of Example 6 is to determine the activity of R- / S-hydroxypropyl tetrahydropyrantriol in promoting the production of type I collagen and elastin in fibroblasts in vitro:

[0078] Test cells: human skin fibroblasts (the cell line passed the cell line quality control test, and the STR genotyping results were qualified)

[0079] Experimental instruments: carbon dioxide incubator, multifunctional enzyme-linked detector, biological safety cabinet, biological microscope, 8-channel pipette.

[0080] Test reagents: penicillin-streptomycin solution, trypsin-ethylenediaminetetraacetic acid solution, transforming growth factor-β1, fetal bovine serum, high-glucose DMEM medium, human type I collagen enzyme-linked immunosorbent assay kit, human elastase enzyme-linked immunosorbent assay kit.

[0081] Experimental conditions: 1. Blank control group: complete culture medium; 2. Positive control group: 100 ng / ml TGF-β1; 3. 0.003% S-hydroxypropyl tetrahydropyrantriol sample (obtained using proline); 4. 0.003% R-hydroxypropyl tetrahydropyrantriol sample (obtained using proline); 5. 0.003% hydroxypropyl tetrahydropyrantriol sample with an isomer ratio of S:R=7:3.

[0082] Steps:

[0083] 1) Cell preparation: After stable passage of frozen cell culture twice, the cells were seeded into 96-well plates, and the plates were placed in a carbon dioxide incubator for overnight culture.

[0084] 2) Drug administration: Discard the culture medium in the 96-well plate and carry out drug administration. 100 μl per well. After drug administration, place the 96-well plate in a carbon dioxide incubator for a period of time.

[0085] 3) Protein detection method: After the incubation, the detection was performed according to the instructions of the human type I collagen enzyme-linked immunosorbent assay kit and the human elastase enzyme-linked immunosorbent assay kit, and the absorbance value of each sample well was measured at 450 nm using an ELISA reader.

[0086] Test results:

[0087] Under normal culture conditions, the content and up-regulation rate of type I collagen secreted by fibroblasts were shown in Table 1. Figure 1 and Figure 2 Compared with the blank control group, the three groups of tested fibroblasts had significantly increased type I collagen content, with the upregulation rates being 37.79%, 25.64% and 28.42% respectively. It can be clearly seen that S-hydroxypropyl tetrahydropyrantriol promotes the synthesis of type I collagen in fibroblasts to a greater extent than the R isomer.

[0088] Under normal culture conditions, the 0.003% S-hydroxypropyl tetrahydropyrantriol sample group; the 0.003% R-hydroxypropyl tetrahydropyrantriol sample group; the 0.003% hydroxypropyl tetrahydropyrantriol sample group with an isomer ratio of S:R=7:3 had an effect on the elastin secretion content and the up-regulation rate of fibroblasts as shown in Table 2. Figure 3 and Figure 4 Compared with the blank control group, the elastin content of the fibroblasts in the three groups increased significantly, with the upregulation rates being 41.67%, 39.80% and 40.20% respectively. It can be clearly seen that S- / R-hydroxypropyl tetrahydropyrantriol has no significant difference in promoting the synthesis of elastin in fibroblasts.

[0089] Table 1 Effects of different isomers of hydroxypropyl tetrahydropyrantriol on collagen content and upregulation rate in fibroblasts

[0090]

[0091] Table 2 Effects of different isomers of hydroxypropyl tetrahydropyrantriol on collagen content and upregulation rate in fibroblasts

[0092]

[0093] Comparative Example 1

[0094]

[0095] 200 ml of dichloromethane, 10 g (31.45 mmol) of 5 and 5', 20.0 g (47.17 mmol) of Fmoc-Gln-OtBu, 383.61 mg (3.14 mmol) of 4-dimethylaminopyridine, and 7.30 ml (47.17 mmol) of N,N'-diisopropylcarbodiimide were added to a 500 ml reaction bottle in sequence. The mixture was stirred at room temperature for 2 hours, filtered, and the filtrate was concentrated and separated by column chromatography to obtain 21.6 g of products 10 and 10' in total, with a yield of 94.0%.

[0096] 10,10': 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=7.2Hz,2H),7.64–7.58(m,2H),7.40(t,J=7.5Hz,2H),7.31(t,J=7.4Hz,2H),5.48(dd,J=25.0, 8.2Hz,1H),5.19(t,J=9.4Hz,1H),5.14–5.08(m,1H),5.00–4.91(m,2H),4.88–4.75(m,2H),4.42(t,J=6.7Hz,1H),4.23(td,J=7.0,2.9 Hz,1H),4.05(dd,J=11.2,5.7Hz,2H),3.94–3.86(m,1H),3.44(td,J=9.3,2.7Hz,1H),3.30(t,J=10.9Hz,1H),2.68(dd,J=16.5,8.9Hz, 1H), 2.46 (dd, J=16.5, 3.0Hz, 1H), 2.03 (t, J=2.8Hz, 9H), 1.82 (ddd, J=14.8, 9.0, 6.4Hz, 2H), 1.49 (d, J=3.1Hz, 9H), 1.26–1.22 (m, 3H). 13C NMR(101MHz,Chloroform-d)δ205.16,171.99,170.98,169.78,155.85,143.70,141.19,127.61,126.97,125.04,119.90,82.41, 74.29,73.49,71.97,71.65,69.11,66.59,53.69,47.06,45.28,38.03,37.29,31.00,30.38,29.59,27.90,23.39,20.60,19.78.

[0097] The side chain carboxyl group of glutamic acid was connected to 5 or 5', but the hydroxypropyl tetrahydropyrantriol isomers were not successfully separated. This may be because the Fmoc on the amino group at the α position of the amino acid is a large molecular structure. When 5 or 5' is connected to the amino acid main chain carboxyl group (Fmoc-Pro-OH / Fmoc-Phe-OH / Fmoc-Val-OH), Fmoc is closer to the C7 position, and the obtained isomer products have a better rigid structure, a greater difference in physical properties, and are easier to separate by column chromatography. When 5 or 5' is connected to the side chain carboxyl group of glutamic acid (Fmoc-Glu-OtBu), Fmoc is farther away from the C7 position, so the obtained isomer products are relatively lacking in rigid structure, have smaller differences in physical properties, and are more difficult to separate by column chromatography.

Claims

1. A method for synthesizing R- / S-hydroxypropyl tetrahydropyrantriol, characterized in that: The synthesis method comprises: synthesizing compound 2 by using D-xylose; protecting three hydroxyl groups at positions 2, 3 and 4 of compound 2 with acetyl groups by using acetic anhydride to obtain fat-soluble compound 4; reducing compound 4 with a reducing agent at low temperature to obtain 5 and 5'; connecting a mixture of 5 and 5' with an amino acid protected by amino group Fmoc to obtain compounds 9 and 9'; and hydrolyzing the ester group with sodium hydroxide to obtain target compound molecules 3 and 3'; 2. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 1, characterized in that, The synthesis method of compound 2 is as follows: methanol, sodium carbonate, xylose and acetylacetone are added to a reaction bottle in sequence, reacted at 80° C. for 12 hours, then the pH is adjusted to 7 with hydrochloric acid, and the compound 2 is obtained by spin drying.

3. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 1, characterized in that, The synthesis method of compound 4 is as follows: compound 2 is added to a reaction bottle, and dichloromethane, triethylamine, and acetic anhydride are added in sequence, reacting at 25°C for 24 hours, adding dichloromethane to dilute, extracting with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated brine respectively, drying the organic phase with anhydrous sodium sulfate, adding activated carbon for decolorization, filtering, and spinning the filtrate to obtain compound 4.

4. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 1, characterized in that, The synthesis method of 5 and 5' is as follows: tetrahydrofuran, acetyl-protected compound 4, and methanol are added to a reaction bottle, a reducing agent is added at room temperature, and the reaction is carried out at room temperature for 24 hours. After the reaction is completed, the reaction is filtered, and the filtrate is diluted with ethyl acetate, extracted three times with water, and then extracted once with saturated brine. The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is spin-dried to obtain compound 5.

5. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 4, characterized in that, The reducing agent is: sodium triacetoxyborohydride, sodium borohydride, sodium cyanoborohydride or borane dimethyl sulfide; when the amount of compound 4 is 1 equivalent, the amount of sodium triacetoxyborohydride is 1-6 equivalents, the amount of sodium borohydride is 0.5-1 equivalents, the amount of sodium cyanoborohydride is 1-2 equivalents, and the amount of borane dimethyl sulfide is 0.3-0.5 equivalents.

6. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 1, characterized in that, The synthesis method of compounds 9 and 9' is as follows: dichloromethane, 5 and 5', Fmoc-Pro-OH / Fmoc-Phe-OH / Fmoc-Val-OH, 4-dimethylaminopyridine, and N,N'-diisopropylcarbodiimide are added to a reaction bottle in sequence, stirred at room temperature for 2 hours, filtered, concentrated the filtrate, and separated by column chromatography to obtain compounds 9 and 9'.

7. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 6, characterized in that, The amount of 5 and 5' is 1 equivalent, Fmoc-Pro-OH / Fmoc-Phe-OH / Fmoc-Val-OH is 1.2 equivalents, 4-dimethylaminopyridine is 0.05-0.3 equivalents, and N,N'-diisopropylcarbodiimide is 1.2 equivalents.

8. The synthetic method of R- / S-hydroxypropyl tetrahydropyrantriol according to claim 1, characterized in that, The preparation method of the target molecule is as follows: methanol is added to a reaction bottle, cooled to 0°C, sodium hydroxide is added, stirred at 0°C to dissolve, compounds 9 and 9' are added, the reaction system is refluxed and stirred at 60°C for 8 hours, after the reaction is complete, the temperature is lowered to room temperature, the pH is adjusted to 7 with hydrochloric acid, and the reaction solution is concentrated to obtain the oily target molecule R- / S-hydroxypropyl tetrahydropyrantriol.

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