CN113861258A - Method for synthesizing sargasterol - Google Patents

Abstract

The invention discloses a synthesis method of sargasterol, which takes hyodeoxycholic acid as raw material to carry out esterification to generate hyodeoxycholate or directly takes hyodeoxycholate as raw material to generate 3, 6-disulfonyl hyodeoxycholate through sulfonylation reaction, and then generates 3 through nucleophilic substitution-elimination reactionβ-hydroxychole-5-en-24-oic acid ester, followed by reaction withN,O‑Preparing weinreb amide by dimethyl hydroxylamine reaction, and finally obtaining sargasterol by two-step Greenia reaction. The synthesis method disclosed by the invention is mild in reaction conditions, short in reaction time, low in prices of raw materials hyodeoxycholic acid and reaction reagents, high in final yield and suitable for large-scale preparation of gulfweed sterol.

Description

Method for synthesizing sargasterol

Technical Field

The invention relates to a synthesis method of phytosterol, in particular to a synthesis method of sargasterol.

Background

Sargasterol is one of main active ingredients in Hizikia fusiforme, and has various physiological effects of inhibiting lipase, resisting depression, obesity, malaria and tuberculosis. Chinese patent application with publication number CN 102861023A discloses application of sarasterol in preparation of Liver X Receptor (LXRs) agonists, in particular 24: (LXRs)S) -sarasterol as LXRβUse of an agonist to promote reverse cholesterol transport by up-regulating the expression of ABCA1 and ABCG 1.

LXRs are steady regulators of cholesterol metabolism of organisms, have become hot drug targets of diseases related to cholesterol metabolism in recent years, and LXRs agonists have important value in the research and development of drugs for treating Alzheimer's disease, metabolic syndrome, tumors, tuberculosis and the like. LXRs haveαAndβtwo subtypes, whereinβSelective agonists avoid fatty liver side effects with high safety, therefore, 24: (S) -sarasterol as LXRβSelective agonist, has great development prospect.

The sargasterol is usually from Cyrtymenia SparsaSargassum fusiforme (Harvey.) Setchell was obtained by fractionation. Rongwei et al first separated sargasterol from Cyrtymenia Sparsa by normal phase preparative liquid chromatography to obtain sargasterol 24SAnd 24RIsomers (Chinese herbal medicine, 2008, vol.39, 5 th, page 657-661). However, the sargasterol has a low content in seaweed (such as sargassum fusiforme), and is difficult to meet the requirements by extraction and separation, and the cost of extraction and separation preparation using crude drugs as raw materials is extremely high.

Currently, few reports are made on studies on the chemical synthesis of sargasterol. During the course of fucosterol synthesis by Sucrow and Rauechel in 1970, 24-ketocholesterol was reacted with vinylmagnesium bromide to obtain sargasterol (Sucrow, W., Rauechel, B., 1970. Die Synthesis von (24(28) E) -Stigmastadien- (5.24(28)) -ol- (3.24 (28)))β) und (24(28)E) -5α-Stigmastadien-(7.24(28))-ol-(3β) Chemische Berichte, 103, 2711-2717). Le et al synthesized 3 in 1982βIn the process of 29-dihydroxystimasta-5, 24(28) (E) -dien-7-one, 20-bis (dinorcolenaldehydide) is taken as a raw material, firstly, the raw material is subjected to Wittig reaction with phosphate to generate dienone, and then 10 percent Pd/BaSO₄Catalytic hydrogenation to obtain 24-ketocholesterol-3β-acetate, followed by reaction with vinylmagnesium bromide grignard reagent to obtain gulfwesterols and 3 thereofβA mixture of acetates (Le, P.H., Preus, M.W., McMorris, T.C., 1982. Synthesis of 3 β, 29-Dihydrotystigmasta-5, 24(28) (E) -dien-7-one. J Org Chem 47, 2163-. 2001W ä chter et al use natural fucosterol as raw material, and introduce oxygenUnder the condition of gas, irradiating for 26 h under a 500W tungsten halogen lamp at 5 ℃, adding acetic acid and zinc powder, and finally obtaining gulfwestersterol (W ä chter, G.A., Franzblau, S.G., Montenego, G., Hoffmann, J.J., Maiese, W.M., Timmermann, B.N., 2001. Inhibition of Mycobacterium tuberculosis Growth by Saringosterol free Lessonia nigrescens, J Nat Prod, 64, 1463-. Castro Navas et al in 2018 produced by using i-stigmasterol methyl ester as raw material through phosphoylide reactionα，β-Unsaturated ketone, then through catalytic hydrogenation, Grignard reaction addition, and finally with pTosOH & H₂The reaction of the O reagent produces sargasterol (Castro Navas, F.F., Giorgi, G., Magdioni, D., Pacciarini, M., Russo, V., Marinozzi, M., 2018. C24-hydroxylated stimastane derivatives as Liver X Receptor agonists, Chem Phys Lipids, 212, 44-50.).

However, the existing synthesis methods generally have the problems of high raw material cost, harsh reaction conditions, long reaction time and the like.

Disclosure of Invention

In view of the above problems, the present invention aims to provide an economical, efficient, simple and convenient method for synthesizing sargasterol.

The technical scheme of the invention is as follows:

a process for synthesizing sargasterol from hyodeoxycholic acid includes such steps as esterifying hyodeoxycholic acid to obtain hyodeoxycholic acid ester, sulfonating to obtain 3, 6-disulfonyl hyodeoxycholic acid ester, nucleophilic substitution-elimination reaction to obtain 3β-hydroxychole-5-en-24-oic acid ester to build sterol mother nucleus, followed by reaction withN,O-Dimethyl hydroxylamine is used for preparing weinreb amide, and the sargasterol is finally obtained through two-step Grignard reaction, wherein the structure of the sargasterol is shown as a formula 1.

1。

The general synthetic route of the sargasterol is as follows:

reagents and conditions: a) CH (CH)₃OH，H₂SO₄，8h，95℃；b) TsCl，Py，24h，r.t.；c) ①AcOK，DMF，H₂O，7h，105℃；② 2% KOH-CH₃OH, 3h, r.t.; d) an isopropyl-type magnesium chloride, and a magnesium chloride,N,O-dimethylhydroxylamine hydrochloride, THF, 12h, 0 ℃ -r.t.; e) isopropyl magnesium chloride, THF, 18h, 0 ℃ -r.t.; f) vinyl magnesium bromide, THF, 12h, 0-r.t. Overall yield of the above six-step reaction: 27.1 percent.

Separating the synthesized sargasterol (1) by chromatography (semi-preparative HPLC) to obtain 24: (S) Gulasterol (1 a) and 24: (a)R) -sargasterol (1 b).

1a；

1b。

The invention has the advantages that: compared with other reported methods, the synthesis method disclosed by the invention is mild in reaction conditions, short in reaction time, low in prices of starting raw materials hyodeoxycholic acid and reaction reagents, high in final yield and suitable for large-scale preparation of gulfweisterol.

Drawings

FIG. 1 shows the LXR response of different sargasterol in human embryonic kidney cell HEK293αRXR and LXRβInfluence of/RXR transcriptional Activity.

FIG. 2 shows the LXR response of different sargasterol in human hepatoma cells HepG2αRXR and LXRβInfluence of/RXR transcriptional Activity.

FIG. 3 shows the LXR response of different sarasterols in human astrocytoma CCF-STTG1αRXR and LXRβInfluence of/RXR transcriptional Activity.

FIG. 4 shows the LXR response of different sargasterol in human microglia CHME3αRXR and LXRβ/RXRGraph of the effect of transcriptional activity.

FIG. 5 shows the LXR response of different sargasterol in human neuroblastoma SH-SY5YαRXR and LXRβInfluence of/RXR transcriptional Activity.

Wherein, 1-synthesis of sargasterol, 1 a-synthesis of 24: (S) Gulasterol, 1 b-Synthesis 24: (R) -gulfwosterol; N1-Natural gulosterol, N1 a-Natural 24 (C)S) Gulasterol, N1 b-native 24 (C)R) -gulfwosterol.

In the figure, P <0.001, P <0.01, compared to the blank group; p < 0.05.

Detailed Description

The invention will be described in detail below with reference to the attached drawings by way of specific embodiments, which are intended to facilitate a better understanding of the invention and do not limit the scope of the invention.

Example 1: synthesis of sargasterol (1):

synthesis of hyodeoxycholic acid methyl ester (3):

3

compound 2 (100.0 g, 0.25 mol) was weighed into a 2L three-necked flask, methanol (1.2L, 29.7 mol) was added, dissolved by magnetic stirring, concentrated sulfuric acid (50 mL, 0.92 mol) was added dropwise, and the mixture was heated under reflux at 95 ℃ for 8 hours. TLC detection, after the reaction is completed, decompression concentration, pouring into 500 mL water, saturated NaHCO₃Neutralization to pH =8, at which time a large white viscous precipitate was produced. The solution was extracted with ethyl acetate (500 mL. times.3), and the precipitate was dissolved in ethyl acetate. The organic phases were combined, concentrated to a small volume, washed with saturated NaCl solution (250 mL), and the organic phase was anhydrous Na₂SO₄Drying and concentration under reduced pressure gave compound 3 (pale yellow solid, 92.2 g) in 89.0% yield.

Compound 3: positive ion ESI-MSm/z 429 [M+Na]⁺, 389 [M-H₂O+H]⁺, 371 [M-2H₂O+H]⁺；¹H NMR (500 MHz, CDCl₃) δ 4.05 (1H, m, H-6), 3.66 (3H, s, 24-OCOCH₃), 3.61 (1H, m, H-3), 0.91 (3H, d, J H-21), 0.90 (3H, s, H-19), 0.63 (3H, s, H-18). Compared with the literature, the compound is identified as Hyodeoxycholate (Methyl hyodesoxycholate).

(II) synthesis of 3, 6-di-p-toluenesulfonyl hyodeoxycholic acid methyl ester (4):

4

compound 3 (50.0 g, 123 mmol), 4-dimethylaminopyridine (10.0 g, 82 mmol) and p-toluenesulfonyl chloride (188.0 g, 990 mmol) were weighed into a 2L three-necked flask, and triethylamine (51 mL, 367 mmol) and pyridine (300 mL) were added. And (3) introducing nitrogen for protection, adding magnetons, stirring at room temperature for reaction, monitoring the reaction process by TLC, and completely reacting for about 24 hours. The pyridine was removed by rotary evaporation, the solid was redissolved in 1000 mL of ethyl acetate, 2M hydrochloric acid (500 mL) was added, a large amount of precipitate was precipitated, washed to neutrality with water, filtered off with suction, and dried under vacuum overnight. After separation of the filtrates, the aqueous layer was extracted with ethyl acetate (200 mL. times.2), the organic phases were combined, then saturated NaHCO was used₃The solution (400 mL. times.2), the saturated NaCl solution (400 mL. times.2) was washed, and Na was added₂SO₄And (5) drying. The solvent was dried by spinning, the solids were combined, washed with methanol to remove p-toluenesulfonyl chloride, and dried in vacuo to give a crude white product. The crude product was isolated by silica gel reduced pressure column chromatography (petroleum ether-dichloromethane =30:70, v/v) to give compound 4 (white solid, 68.5 g) in 77.9% yield.

Compound 4: positive ion ESI-MS:m/z 737 [M+Na]⁺; ¹H NMR (500 MHz, CDCl₃): δ7.78 (2H, d, J = 8.0 Hz, 3α- and 6α-C₆H₄CH₃), 7.72 (2H, d, J = 8.0 Hz, 3α- and 6α-C₆H₄CH₃), 7.34 (4H, t, J = 9.0 Hz, 3α- and 6α-C₆H₄CH₃), 4.78 (1H, m, H-6), 4.30 (1H, m, H-3), 3.66 (3H, s, 24-OCOCH₃), 2.46 (6H, s, 3α- and 6α-C₆H₄CH₃), 2.33 (1H, m), 2.20 (1H, m), 0.88 (3H, d, J h-21), 0.80 (3H, s, H-19), 0.59 (3H, s, H-18). Compared with the literature, the product is identified as 3, 6-di-p-toluenesulfonyl hyodeoxycholic acid Methyl ester (Methyl 3)α,7β-ditosyloxy-5β-cholan-24-oate）。

(III) 3β-synthesis of methyl hydroxychole-5-en-24-oate (5):

5

compound 4 (20.0 g, 28.0 mmol), potassium acetate (29.3 g, 299 mmol) were weighed into a 500 mL three-necked flask, water (14 mL) and DMF (160 mL) were added, dissolved by magnetic stirring, and reacted at 105 ℃ under reflux for 7 h. The reaction mixture was cooled to room temperature, poured into ice-cold 5% HCl solution (1000 mL), filtered under reduced pressure, and the precipitate was washed with water to neutrality, redissolved in 2% KOH-MeOH (100 mL), and stirred at room temperature for 4 h. Then poured into ice of 10% HCl solution (1000 mL), the precipitate was filtered, washed with water to neutrality, and the obtained crude product was recrystallized from silica gel by column chromatography under reduced pressure (dichloromethane-methanol =100:0 → 50:50, v/v), dichloromethane-methanol (1: 1) to obtain compound 5 (10.518 g) with a yield of 96.7%.

Compound 5: ESI-MS:m/z 411 [M+Na]⁺, 389 [M+H]⁺, 371 [M-H₂O+H⁺]; ¹H NMR (500 MHz, CDCl₃): δ 5.35 (1H, br d, H-6), 3.66 (3H, s, 24-OCOCH₃), 3.52 (1H, m, H-3), 1.00 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.67 (3H, s, H-18). Compared with the literature, is identified as 3β-Hydroxychole-5-en-24-oic acid Methyl ester (Methyl 3)β-hydroxychol-5-en-24-oate）。

(IV) 3β-hydroxy-N-methoxy-N-synthesis of methylcholest-5-en-24-amide (6):

6

weighing 11.6 g (30 mmol) of compound 5,N,ODimethylhydroxylamine hydrochloride (3.5 g, 35.9 mmol) was placed in a 1L dry three-necked flask, and a magneton was added and sealed under nitrogen. After the solid was dissolved in THF (200 mL) at 0 ℃ in an ice bath, isopropyl magnesium chloride solution (60 mL, 120 mmol) was added dropwise via a dropping funnel, and the mixture was stirred overnight at room temperature, followed by TLC detection, and the starting material disappeared after 12 hours. Cooling to 0 deg.C, adding saturated NH into the bottle₄Cl solution (200 mL), stirred for 30 min, completely quenched the Grignard reagent. The reaction was concentrated by rotary evaporation, extracted with ethyl acetate (3X 200 mL), and then saturated NaHCO₃The ester layer was washed with a solution (3X 600 mL), a saturated NaCl solution (3X 600 mL), and anhydrous Na was added₂SO₄Drying, evaporating solvent, and subjecting the crude product to silica gel reduced pressure column chromatography (petroleum ether-ethyl acetate =85: 15)

25:75, v/v) to give compound 6 (white solid, 10.668 g) in 85.5% yield.

Compound 6: ESI-MS:m/z 857 [2M+Na]⁺, 835 [2M+H]⁺, 440 [M+Na]⁺, 418 [M+H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 5.32 (1H, br d, H-6), 3.67 (3H, s, -OCH₃), 3.49 (1H, m, H-3), 3.15 (3H, s, -NCH₃), 0.99 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.665 (3H, s, H-18). Compared with the literature, is identified as 3β-hydroxy-N-methoxy-N-methylcholest-5-en-24-ylamide (3)β- Hydroxy-N-methoxy-N-methylchol-5-en -24-amide)。

(V) Synthesis of 24-keto-Cholesterol (7):

7

compound 6 (8.0 g, 19.2 mmol) was weighed into a dry three-necked flask, and then magneton was added and nitrogen gas was introduced to seal. THF (100 mL) was added to the flask at 0 ℃ in an ice bath, and after dissolving the solid, an isopropyl magnesium chloride solution (9) was added dropwise from a dropping funnel0 mL, 180.0 mmol), stirred at room temperature overnight, checked by TLC, and the starting material disappeared after 18 h. Cooling to 0 deg.C, adding saturated NH into the bottle₄Cl solution (50 mL), stirred for 30 min, completely quenched the Grignard reagent. The reaction was concentrated by rotary evaporation, extracted with ethyl acetate (3X 200 mL), and then saturated NaHCO₃The ester layer was washed with a solution (3X 600 mL), a saturated NaCl solution (3X 600 mL), and anhydrous Na was added₂SO₄Drying, evaporating solvent, and subjecting the crude product to silica gel reduced pressure column chromatography (petroleum ether-ethyl acetate =90: 10)

50:50, v/v) to give compound 7 (white solid, 4.025 g) in 52.0% yield.

Compound 7: ESI-MS:m/z 423 [M+Na]⁺, 401 [M+H]⁺, 383 [M-H₂O+H]⁺, 365 [M-2H₂O+H]⁺ ; ¹H NMR (500 MHz, CDCl₃ ): δ 5.35 (1H, br d, J = 3.5 Hz, H-6), 3.52 (1H, m, H-3), 2.61 (1H, m, H-25), 1.09 (6H, d, J = 6.9 Hz, H-26 and H-27), 1.00 (3H, s, H-19), 0.91 (3H, d, J = 6.6 Hz, H-21), 0.67 (3H, s, H-18). Compared with the literature, 24-keto-cholesterol (24-keto-cholestrol) is identified.

(VI) synthesis of sargasterol (1):

1

compound 7 (3.2 g, 7.9 mmol) was weighed into a 250 mL dry three-necked flask, and then magneton was added and sealed with nitrogen. After the solid was dissolved in THF (50 mL) at 0 ℃ in an ice bath, vinylmagnesium bromide solution (30 mL, 30.0 mmol) was added dropwise via a dropping funnel, the mixture was stirred overnight at room temperature, and the starting material disappeared after 12 hours by TLC detection. Cooling to 0 deg.C, adding saturated NH into the bottle₄Cl solution (50 mL), stirred for 30 min, completely quenched the Grignard reagent. The reaction was concentrated by rotary evaporation, extracted with ethyl acetate (3X 50 mL), and then saturated NaHCO₃The ester was washed with solution (3X 150 mL), saturated NaCl solution (3X 150 mL)Layer, adding anhydrous Na₂SO₄Drying, evaporating solvent, and subjecting the crude product to silica gel flash column chromatography (petroleum ether-ethyl acetate =90: 10)

50:50, v/v) to give compound 1 (white solid, 3.063 g) in 90.8% yield.

Compound 1: APCI-MS:m/z 411 [M-H₂O+H]⁺, 393 [M-2H₂O+H]⁺; ¹H NMR (500 MHz, CDCl₃): δ 5.86-5.75 (1H, m, H-28), 5.35 (1H, br d, H-6), 5.22-5.16 (1H, m, H-29), 5.16-5.11 (1H, m, H-29), 3.52 (1H, m, H-3), 1.00 (3H, s, H-19), 0.92 (3H, m, H-21), 0.89 (3H, m, H-27), 0.87 (3H, d, J = 6.9 Hz, H-26), 0.67 (3H, s, H-18). Comparing with the literature, the sargasterol is identified.

Example 2: 24(S)Gulasterol (1 a) and 24: (a)R)Isolation of sargasterol (1 b)

Subjecting compound 1 to semi-preparative high performance liquid chromatography, eluting with methanol-acetonitrile-water (85: 1:14, v/v/v) at flow rate of 4.0 mL/min, t _R And the reaction lasts for 31.6 min, so that the compound 1a is obtained. APCI-MS:m/z 411 [M-H₂O+H]⁺, 393 [M-2H₂O+H]⁺; ¹H NMR (500 MHz, CDCl₃): δ 5.796 (1H, dd, J = 17.4, 10.9 Hz, H-28), 5.350 (1H, br d, H-6), 5.184 (1H, dd, J = 17.4, 1.0 Hz, H-29), 5.131 (1H, d, J = 10.9, 1.0 Hz, H-29), 3.523 (1H, m, H-3), 1.005 (3H, s, H-19), 0.919 (3H, d, J = 6.6 Hz, H-21), 0.899 (3H, d, J = 6.8 Hz, H-27), 0.872 (3H, d, J = 6.9 Hz, H-26), 0.672 (3H, s, H-18). Compared with the literature, is identified as 24: (S) -sargasterol (24)S-saringosterol)。

Method for separating and purifying compound 1b and compounds 1a, t _R =32.7 min。APCI-MS: m/z 411 [M-H₂O+H]⁺, 393 [M-2H₂O+H]⁺; ¹H NMR (500 MHz, CDCl₃): δ5.810 (1H, dd, J = 17.4, 10.9 Hz, H-28), 5.349 (1H, br d, H-6), 5.190 (1H, dd, J = 17.4, 1.0 Hz, H-29), 5.137 (1H, d, J = 10.9,1.0 Hz, H-29), 3.524 (1H, m, H-3), 1.005 (3H, s, H-19), 0.924 (3H, d, J = 6.4 Hz, H-21), 0.891 (3H, d, J = 6.8 Hz, H-27), 0.871 (3H, d, J = 7.0 Hz, H-26), 0.671 (3H, s, H-18). Compared with the literature, is identified as 24: (R)-sargasterol (24)R-saringosterol)。

Example 3: activity test of sargasterol for agonizing liver X receptor

The transcription activation effect of sargasterol on liver X receptor is measured by adopting a dual-luciferase reporter gene measuring system. Respectively taking human embryonic kidney cell HEK293, human liver cancer cell HepG2, human brain astrocytoma cell CCF-STTG1, human microglial cell CHME3 and human neuroblastoma cell SH-SY5Y at a ratio of 0.8 × 10⁵Each cell was inoculated in 24-well plates at a density of 10% FCS and 1% P/S in DMEM/F-12 and incubated at 37 ℃ for 24 hours. Adding 1mg LXR alpha or LXR beta expression plasmid, 1mg RXR expression plasmid, 4mg LXRE (containing firefly luciferase gene) expression plasmid and 1mg Renilla luciferase plasmid into a blank DMEM/F-12 culture medium, wherein the ratio of FuGENE 6 transfection reagent to DNA is 2.5: 1, shaking lightly and mixing uniformly to obtain 500 mu L of transfection complex liquid. The blank plasmid control group replaced LXR α/β and RXR with an equal amount of empty pcDNA3.1/V5-HisA vector. And adding 20 mu L of composite transfection solution into each hole for transfection. 24 hours after transfection, DMEM/F-12 medium (containing no phenol red, 10% activated carbon-treated fetal calf serum and 1% P/S) containing sargasterol compounds (2.5, 5.0, 7.5. mu.M), positive drugs T0901317 (1. mu.M) and GW3965 (5. mu.M) from different sources was added, and after 24 hours of incubation, 100. mu.l of lysate was added to each well, and the mixture was left for 10min at room temperature and transferred to a total white 96-well microplate for assay.

As can be seen from fig. 1-5, the activation of LXRs by synthetic sargasterol (1, 1a and 1b) was comparable to that of natural sargasterol (N1, N1a and N1b), suggesting that the synthetic product was effective. At the same time, it can be found thatS) The agonism of sargasterol on LXRs is stronger than 24: (R) -gulfwosterol.

It should be noted that the above mentioned embodiments are only preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, many modifications and decorations can be made without departing from the inventive concept, and these modifications and decorations should not be excluded from the scope of the present invention.

Claims (7)

1. A process for synthesizing sargasterol includes esterifying hyodeoxycholic acid to obtain hyodeoxycholic acid ester, sulfonylating to obtain 3, 6-disulfonyl hyodeoxycholic acid ester, nucleophilic substitution-elimination reaction to obtain 3β-hydroxychole-5-en-24-oic acid ester, followed by reaction withN,O-Preparing weinreb amide by dimethyl hydroxylamine reaction, and finally obtaining sargasterol by two-step Greenia reaction.

2. The method for synthesizing gulosterol according to claim 1, wherein the hyodeoxycholic acid ester is hyodeoxycholic acid methyl ester.

3. The method of claim 1, wherein the step of chromatographically separating the resulting sargasterol comprises the step of obtaining 24 (a)S) -sarasterols and 24: (a)R) -gulfwosterol.

4. The method of claim 3, wherein the chromatography is semi-preparative HPLC.

5. The method of synthesizing sargasterol as claimed in claim 1, wherein preparation of weinreb amide is catalyzed by isopropyl magnesium chloride.

6. The method of synthesizing sargasterol according to claim 1, wherein said sulfonylating agent is p-toluenesulfonyl chloride.

7. The method of synthesizing sargasterol as claimed in claim 1, wherein both of said two grignard reactions occur on the 24-keto group.

Images