CN103948604A - Application of hederagenin compound serving as medicament for treating liver cancer and preparation of hederagenin compound - Google Patents

Abstract

The invention provides application of a hederagenin compound serving as a medicament for treating liver cancer and a preparation of the hederagenin compound. The hederagenin compound of a liver cancer treatment medicament is successfully prepared from hederagenin and derivatives thereof by separating purification hydrolysis and chemical synthesis methods from plants. The hederagenin compound provided by the invention is subjected to anti-hepatoma activity screening, and screening results discover that the hederagenin compound has remarkable anti-hepatoma activity, and can be used for treating patients with liver cancer after being prepared into medicaments for treating liver cancer. Reports on the hederagenin and a series of derivatives with the compound frame serving as medicaments for treating liver cancer are not found until now by means of searching.

Description

Helexin compounds is as application and the preparation thereof of Hepatoma therapy medicine

Technical field

The preparation and the structure of modification method that the present invention relates to effective ingredients in plant in pharmaceutical industry, particularly a kind of helexin compounds is as application and the preparation thereof of Hepatoma therapy medicine.

Background technology

Hepatocarcinoma is called as " cancer king " in medical circle, incidence rate is high, treats very difficult.At present, the therapeutic scheme of hepatocarcinoma depend on disease by stages, remaining liver function and patient's overall health of patients.If cancer is early stage and residue liver is healthy, carrying out liver transplantation after excision or excision is the unique healing chance of liver cancer patient, but the most of liver cancer patients of China have all arrived middle and advanced stage, at present basic " pasting medical help " while finding.Finding now that some conventional medicaments have certain inhibitory action to hepatoma carcinoma cell as Herba Houttuyniae, Radix Bupleuri, Herba Artemisiae Scopariae etc., therefore from Chinese herbal medicine and ethnic drug, find the medicines resistant to liver cancer of specially good effect, is the urgent task that current medicine sector faces.

Summary of the invention

The object of the invention is for above-mentioned technical Analysis, application and the preparation thereof of a kind of helexin compounds as Hepatoma therapy medicine are provided, this preparation method is isolated the different strain slender acanthopanax of a kind of active component first glycosides from nigella glandulifera Freyn, to making helexin after its acid hydrolysis, then helexin has been carried out to structure of modification, through Pharmacodynamics screening, found that helexin compounds has good antihepatocarcinoma effect.

Technical scheme of the present invention:

A kind of helexin compounds is as the application of preparing Hepatoma therapy medicine, described helexin compounds comprises the derivant of helexin (Hederagenin) and helexin, it is characterized in that: the derivant of helexin and helexin is used as to the medicine of preparing Hepatoma therapy medicine, the chemical structural formula of described helexin (Hederagenin) is:

The chemical structural formula of the derivant of described helexin is:

In above-mentioned all chemical structural formulas: R ₁with R ₂substituent group is identical, for-COCH ₃,-COCH ₂cH ₃,-COCH ₂cH ₂cH ₃or-COCH ₂cH ₂cH ₂cH ₃; R ₃substituent group is-CH ₃,-CH ₂cH ₃,-CH ₂cH ₂cH ₃or-CH ₂cH ₂cH ₂cH ₃.

The preparation method step of described helexin is as follows:

1) by the sub-cake of the nigella glandulifera Freyn pressing the oil, by 95v/v% ethanol water reflux, extract, twice, then use 50v/v% ethanol water reflux, extract, twice, then water reflux, extract, twice, merge extractive liquid,, concentrating under reduced pressure obtains total extractum;

2) above-mentioned total extractum is suspended in water, uses according to this petroleum ether, ethyl acetate, n-butanol extraction, remaining water layer, obtains condensed cream after concentrating under reduced pressure;

3) above-mentioned condensed cream is mixed to sample by silica gel column chromatography, take methylene chloride-methanol-water system as eluant column chromatography repeatedly, obtain the different strain slender acanthopanax of compound first glycosides;

4) H through 5v/v% by above-mentioned different strain slender acanthopanax first glycosides ₂sO ₄after solution hydrolysis, can make helexin compounds.

The preparation method of the derivant of described helexin adopts the chemical synthesis route shown in following formula:

R wherein ₁with R ₂substituent group is identical, for-COCH ₃,-COCH ₂cH ₃,-COCH ₂cH ₂cH ₃or-COCH ₂cH ₂cH ₂cH ₃; R ₃substituent group is-CH ₃,-CH ₂cH ₃,-CH ₂cH ₂cH ₃or-CH ₂cH ₂cH ₂cH ₃.

Reaction condition in synthetic route is respectively:

A. helexin is at K ₂cO ₃under catalysis, in DMF solution, carry out esterification with idoalkane, obtain product 1;

B. product 1 is dissolved in oxolane, under DMAP catalysis, carries out acylation reaction with anhydrides reagent, obtain product 2;

C. product 2 is dissolved in anhydrous chloroform, adds after m-CPBA lucifuge under room temperature to place, carry out oxidation reaction, obtain product 3;

D. product 3 is dissolved in the hot ethanol containing anhydrous sodium acetate, with oxammonium hydrochloride. back flow reaction, obtains product 4;

E. product 4 is dissolved in dry pyridine, under condition of ice bath, adds phosphorus oxychloride solution, after Beckmann rearrangement, obtain product 5;

F. product 5 is dissolved in dry benzene, adds lawesson reagent to reflux, carry out vulcanization reaction, obtain product 6.

Advantage of the present invention is: the present invention has successfully made serial helexin compounds by the method for separation and purification preparation and chemosynthesis from plant, and through the screening of mtt assay resisting liver cancer activity, find all to have obvious resisting liver cancer activity (IC with helexin compounds provided by the invention ₅₀value is less than 50 μ M), make the treatment that can be used for hepatocarcinoma patient after the medicine of Hepatoma therapy, through retrieval, have no so far helexin and there is the series derivates of this compound skeleton as the report of the medicine of Hepatoma therapy.

The specific embodiment

Embodiment 1:

Helexin compounds is as a preparation method for Hepatoma therapy medicine, and step is as follows:

Get the Semen Nigellae cake 18kg pressing the oil, through 95v/v% ethanol water reflux, extract, 2 times, 50v/v% ethanol water reflux, extract, 2 times, water reflux, extract, 2 times, each 3h, merge extractive liquid,, concentrating under reduced pressure obtains total extractum 2630g.Gained extractum is scattered in the warm water of 3 times of volumes, uses according to this petroleum ether, ethyl acetate, n-butanol extraction, reclaim solvent and obtain petroleum ether part 840g, ethyl acetate part 80g and n-butyl alcohol part 560g.The concentrated 1100g that obtains of residue water layer, by this water section through silica gel column chromatography, the methylene chloride-methanol mixed liquor eluting that the volume ratio of take is 10:1, through TLC combining data detection, obtain 15 parts of flow points, wherein the 8th part of methylene chloride-methanol-water mixed liquid that flow point is 12:4:0.5 by volume ratio carries out column chromatography, obtains the different strain slender acanthopanax of compound first glycosides 1.5g; Get the different strain slender acanthopanax of 30.1g first glycosides and be positioned in 250mL round-bottomed flask, add the H of 5v/v% ₂sO ₄solution 200mL, reflux 6h, cooling after completion of the reaction.After cooling, add Na ₂cO ₃in solution and remaining acid, be extracted with ethyl acetate again organic facies, gained organic facies anhydrous magnesium sulfate drying, filter, distilling under reduced pressure obtains the crude product of faint yellow solid, methanol-dichloromethane the mixed liquor that is 1:10 by volume ratio again carries out column chromatography as eluant, and separation obtains helexin 8.80g, yield 93.12%.

The chemical constitution of obtained compound is measured to confirmation as follows:

MP>300℃。

${\left[\mathrm{\α}\right]}_D^{20}:+76.7(c0.14,\mathrm{MeOH}).$

ESI-MS(M+H) ⁺:m/z473.4

IR(KBr)cm ^-1:3455(OH)，1699(C＝O)，1466，1389。

¹H?NMR(400MHz,C ₅D ₅N):δ _H5.51(1H,br.s,H-12)，4.19(2H,br?s,H-23)，3.73(1H,d,J＝10.0Hz,H-3)，3.31(1H,d,J＝11.6Hz,H-18)，1.24,1.01,0.98,0.93(12H,4×s,4×-CH ₃)，1.06(6H,s,2×-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):179.6(C-28)，144.3(C-13)，122.0(C-12)，72.9(C-3)，67.5(C-23)。

This product, according to existing pharmaceutical technology, is made into as medicine, can be for clinical treatment hepatocarcinoma.

Embodiment 2:

Helexin compounds is as a preparation method for Hepatoma therapy medicine, and step is as follows:

1) by helexin 7.10g (15mmol), anhydrous K ₂cO ₃5.18g (37.50mmol) and dry DMF (50mL) join respectively in 100mL round-bottomed flask, slowly add CH under stirring ₃i1.88mL (30mmol).Under room temperature, stir 6h, distinguish after completion of the reaction dilute with water, the salt acid elution of 1.0M is to subacidity, add and be washed to neutrality, finally use saturated NaCl solution washing, gained is anhydrous magnesium sulfate drying, filtration for organic facies, distilling under reduced pressure obtains the crude product of faint yellow solid, ethyl acetate-petroleum ether the mixed liquor that is 1:5 by volume ratio again carries out column chromatography as eluant, and separation obtains white solid O-16.30g, yield 86.20%.

It is as follows that the chemical constitution of the compound that the present invention is obtained is measured confirmation:

MP:232-234℃。

${\left[\mathrm{\α}\right]}_D^{20}:+64.4(c0.50,\mathrm{MeOH}).$

HR-ESI-MS(M+H) ⁺:m/z487.3771(calcd487.3787for?C ₃₁H ₅₁O ₄)

IR(KBr)cm ^-1:3460(OH)，1698(C＝O)，1478，1382。

¹H?NMR(400MHz,C ₅D ₅N):δ _H5.43(1H,br.s,H-12)，4.21(2H,m,H-23)，3.76(3H,s,COOMe)，3.74(1H,d,J＝13.2Hz,H-3)，3.11(1H,dd,J＝4.0,14.0Hz,H-18)，1.20,1.08,1.02,0.95,0.92,0.89(18H,6×s,6×-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):177.8(C-28)，143.8(C-13)，122.5(C-12)，73.0(C-3)，67.4(C-23)，51.3(-COO Me)。

2) DMAP (1.25mmol) of the O-1 of 6.21g (12.5mmol) and 0.15g is dissolved in the THF of 2mL, under room temperature, stirs 30min, then add the Ac of 3mL ₂o stirs 2h, after completion of the reaction, THF is removed in distilling under reduced pressure, gained solid acetic acid ethyl dissolution, ethyl acetate solution water and saturated sodium-chloride washing, for organic facies, anhydrous magnesium sulfate drying, filtration, distilling under reduced pressure remove desolventizing and obtain thick product, the ethyl acetate-petroleum ether mixed liquor column chromatography that is 1:10 by volume ratio, separated product white solid O-26.26g, the yield 86.0% of obtaining.

It is as follows that the chemical constitution of the compound that the present invention is obtained is measured confirmation:

MP:160-163℃。

${\left[\mathrm{\α}\right]}_D^{20}:+48.4(c0.50,\mathrm{MeOH}).$

HR-ESI-MS(M+H) ⁺:m/z571.3990(calcd571.3999for?C ₃₅H ₅₅O ₆)

IR(KBr)cm ^-1:2940，1711(C＝O)，1457,1035。

¹H?NMR(400MHz,C ₅D ₅N):δ _H5.39(1H,t,J＝3.2Hz,H-12)，5.08(1H,dd,J＝5.2,12.0Hz,H-3)，4.05(2H,m,H-23)，3.72(3H,s,COOMe)，3.11(1H,dd,J＝4.0,14.0Hz,H-18)，2.05(3H,s,-OOCMe)，2.03(3H,s,-OOCMe),1.24,0.95,0.87,0.85(12H,4×s,4×-CH ₃)，0.92(6H,s,2×-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):177.4(C-28)，170.0and169.8(-O COMe?in?C-3and?C-23)，143.6(C-13)，122.1(C-12)，74.0(C-3)，65.2(C-23)，51.0(-COO Me)，?20.5and20.1(-OCO Me?in?C-3and?C-23)。

3) O-2 of 6.00g (10.5mmol) is first dissolved in the anhydrous chloroform of 50mL, separately m-CPBA3.62g (21mmol) is dissolved in the anhydrous chloroform of 50mL, then above-mentioned two kinds of solution are joined in 250mL round-bottomed flask, under room temperature, lucifuge is placed 2 days.Reactant liquor is used respectively the FeSO of 5w/v% ₄solution, Na ₂cO ₃solution, HCl solution and water washing, washing times 2 times.Organic facies anhydrous magnesium sulfate drying, filters, and distilling under reduced pressure removes desolventizing and obtains thick product, the ethyl acetate-petroleum ether mixed liquor column chromatography through being 1:6 by volume ratio, separated white crystal O-34.38g, the yield 71.0% of obtaining.

It is as follows that the chemical constitution of the compound that the present invention is obtained is measured confirmation:

MP:158-160℃。

${\left[\mathrm{\α}\right]}_D^{20}:+2.4(c0.50,\mathrm{MeOH}).$

HR-ESI-MS(M+H) ⁺:m/z587.3942(calcd587.3948for?C ₃₅H ₅₅O ₇)

IR(KBr)cm ^-1:2953,2861,1745and1723(C＝O，CH ₃COO?and?COOCH ₃),1244。

¹H?NMR(400MHz,C ₅D ₅N):δ _H5.03(1H,dd,J＝4.6,11.8Hz,H-3)，4.03(2H，br.s,H-23)，3.74(3H,s,-COOMe)，3.16(1H,br.d,J＝13.0Hz,H-18)，2.91(1H,d,J＝3.6Hz,H-13)，2.06(3H,s,-OOCMe)，2.05(3H,s,-OOCMe),1.06,1.01,0.98,0.92,0.84,0.81(18H,6×s,6×-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):210.0(C-12)，177.8(C-28)，170.0and169.8(-O COMe?in?C-3and?C-23)，73.7(C-3)，65.0(C-23)，51.4(-COO Me)，51.3(C-17)，49.5(C-13)，20.4and20.1(-OCO Me?in?C-3and?C-23)。

4) O-3 of 4.10g (7mmol) is dissolved in hot 50mL alcoholic solution, then adds oxammonium hydrochloride. 2.43g (35mmol) and anhydrous sodium acetate 4.60g (56mmol), by reaction liquid reflux 3h.React after complete question response liquid cooling but, pour in 525mL aqueous solution, slowly add the 1mol/LHCl solution of 2mL, regulate reactant liquor to subacidity.In reactant liquor, there is precipitation to generate, with decompression funnel, filter, and by massive laundering, wash after 2 times faint yellow solid O-43.21g, the yield 76.3% of weighing to obtain to be dried.

It is as follows that the chemical constitution of the compound that the present invention is obtained is measured confirmation:

MP:145-147℃。

${\left[\mathrm{\α}\right]}_D^{20}:+9.6(c0.50,\mathrm{MeOH}).$

HR-ESI-MS(M+H) ⁺:m/z602.4054(calcd602.4057for?C ₃₅H ₅₆NO ₇)

IR(KBr)cm ^-1:3462(OH，N-OH)，1745and1723(C＝O，CH3COO?and?COOCH ₃)。

¹H?NMR(400MHz,C ₅D ₅N):δ _H12.56(1H,s,N-OH)，4.02(2H，br.s,H-23)，3.76(3H,s,-COOMe)，3.52(1H,dd,J＝5.0,17.2Hz,H-3)，3.38(1H,br.d,J＝?13.2Hz,H-18)，2.54(1H,d,H-13)，2.054(3H,s,-OOCMe)，2.049(3H,s,-OOCMe),1.06,1.03,0.93,0.88,0.84,0.83(18H,6×s,6×-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):178.3(C-28)，170.1and169.8(-O COMe?in?C-3and?C-23)，156.7(C-12)，73.9(C-3)，65.1(C-23)，51.2(-COO Me)，47.9(C-17)，47.8(C-13)，20.5and20.1(-OCO Me?in?C-3and?C-23)。

5) O-4 of 3.00g (5mmol) is dissolved in dry 50mL pyridine solution, then under condition of ice bath, dropwise adds phosphorus oxychloride 2.30g15mmol) solution, after mixing, under reactant liquor room temperature, place 24h.After completion of the reaction, reactant liquor is poured in 525ml aqueous solution, slowly added the 1.0M HCl solution of 3mL, regulate reactant liquor to subacidity.In reactant liquor, there is precipitation to generate, with decompression funnel, filter, and by massive laundering, wash after 2 times the faint yellow solid that weighs to obtain to be dried.Ethyl acetate-petroleum ether the mixed liquor that is 1:5 by volume ratio again carries out column chromatography as eluant, and separation obtains white solid O-52.10g, yield 70.0%.

It is as follows that the chemical constitution of the compound that the present invention is obtained is measured confirmation:

MP:130-133℃。

${\left[\mathrm{\α}\right]}_D^{20}:-2.4(c0.50,\mathrm{MeOH}).$

HR-ESI-MS(M+H) ⁺:m/z602.4061(calcd602.4057for?C ₃₅H ₅₆NO ₇).

IR (KBr) cm ^-1: 3462 (N-H), 1745and1723 (C=O, CH ₃cOO and COOCH ₃), 1670 (C=O, lactam).

¹H?NMR(400MHz,C ₅D ₅N):δ _H6.40(1H,d,J＝6.0Hz；N-H,lactam)，5.01(1H,dd,J＝4.4,11.6Hz,H-3)，4.26(1H,t,J＝6.0Hz；H-13)，4.04and3.99(1H?each,AB,J＝11.6Hz,H-23)，3.83(3H,s,COOMe)，2.07(3H,s,-OOCMe)，2.03(3H,s,-OOCMe),1.07,1.02,0.91(9H,3×s,3×-CH ₃)，0.80(6H,s,2×-CH ₃)，0.78(3H,s,-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):177.2(C-28)，175.7(C-12)，170.1and169.8(-O COMe?in?C-3and?C-23)，73.7(C-3)，64.9(C-23)，51.5(-COO Me)，50.3(C-13)，47.1(C-17)，20.5and20.1(-OCO Me?in?C-3and?C-23)。

6) O-5 of 1.20g (2mmol) is dissolved in dry 50mL benzole soln, then adds lawesson reagent 0.80g (2mmol) to mix rear reactant liquor reflux 90min.After completion of the reaction, question response liquid cooling but, is used respectively 5w/v%K ₂cO ₃solution and water washing 2 times, for organic facies, anhydrous magnesium sulfate drying, filtration, distilling under reduced pressure remove desolventizing and obtain thick product, the ethyl acetate-petroleum ether mixed liquor column chromatography through being 1:10 by volume ratio, separated solid O-60.52g, the yield 42.3% of obtaining.

It is as follows that the chemical constitution of the compound that the present invention is obtained is measured confirmation:

MP:85-87℃。

${\left[\mathrm{\α}\right]}_D^{20}:+9.6(c0.50,\mathrm{MeOH}).$

HR-ESI-MS(M+H) ⁺:m/z618.3765(calcd618.3828for?C ₃₅H ₅₆NO ₆S)

IR (KBr) cm ^-1: 3462 (N-H), 1745and1723 (C=O, CH ₃cOO and COOCH ₃), 1469 (C=S).

¹H?NMR(400MHz,C ₅D ₅N):δ _H8.49(1H,d,J＝4.8Hz；N-H,lactam)，5.00(1H,dd,J＝4.7,11.8Hz,H-3)，4.46(1H,t,J＝5.8Hz；H-13)，4.01and3.98(1H?each,AB,J＝11.6Hz,H-23)，3.85(3H,s,COOMe)，2.09(3H,s,-OOCMe)，2.05(3H,s,-OOCMe),1.07,1.02,0.93,0.89,0.88,0.86(18H,6×s,6×-CH ₃)。

¹³C?NMR(100MHz,C ₅D ₅N):208.1(C-12)，177.4(C-28)，170.3and170.0(-O COMe?in?C-3and?C-23)，73.8(C-3)，65.0(C-23)，55.1(C-13)，51.7(-COO Me)，47.3(C-17)，22.8and21.5(-OCO Me?in?C-3and?C-23)。

Six products obtained above are made into respectively to medicine according to common process, can be used for clinical treatment of cancer.The compound providing has obvious resisting liver cancer activity, and this activity is confirmed by following experimental result: the effect of anti-hepatocarcinoma.

Materials and methods:

1) vitro human hepatoma model: HepG2

2) mtt assay detects the inhibitory action of sample on cell proliferation

3) positive drug contrast is 5-fluorouracil

Experimental result:

In embodiment 1 in prepared helexin and embodiment 26 helexin derivants of chemosynthesis by mtt assay, detect the inhibition of HepG2 cell lines propagation active, the IC of each compound of gained ₅₀value is in Table 1:

Table 1

Compound	IC 50(μM)
		Helexin	16.04
O-1	8.60
		O-2	18.61
O-3	3.04
		O-4	1.88
0-5	3.40
		O-6	6.14
5-fluorouracil	16.42

Experimental result shows: above-mentioned 7 helexin compounds, through the screening of mtt assay resisting liver cancer activity, are found its IC ₅₀value is all less than 50 μ M, with positive drug 5-fluorouracil activity quite or stronger, there is obvious resisting liver cancer activity.

Claims (3)

1. a helexin compounds is as the application of preparing Hepatoma therapy medicine, described helexin compounds comprises the derivant of helexin (Hederagenin) and helexin, it is characterized in that: the derivant of helexin and helexin is used as to the medicine of preparing Hepatoma therapy medicine, the chemical structural formula of described helexin (Hederagenin) is:

The chemical structural formula of the derivant of described helexin is:

in above-mentioned all chemical structural formulas: R ₁with R ₂substituent group is identical, for-COCH ₃,-COCH ₂cH ₃,-COCH ₂cH ₂cH ₃or-COCH ₂cH ₂cH ₂cH ₃; R ₃substituent group is-CH ₃,-CH ₂cH ₃,-CH ₂cH ₂cH ₃or-CH ₂cH ₂cH ₂cH ₃.

2. helexin compounds, as the application of Hepatoma therapy medicine, is characterized in that according to claim 1: the preparation method step of described helexin is as follows:

1) by the sub-cake of the nigella glandulifera Freyn pressing the oil, by 95v/v% ethanol water reflux, extract, twice, then use 50v/v% ethanol water reflux, extract, twice, then water reflux, extract, twice, merge extractive liquid,, concentrating under reduced pressure obtains total extractum;

2) above-mentioned total extractum is suspended in water, uses according to this petroleum ether, ethyl acetate, n-butanol extraction, remaining water layer, obtains condensed cream after concentrating under reduced pressure;

3) above-mentioned condensed cream is mixed to sample by silica gel column chromatography, take methylene chloride-methanol-water system as eluant column chromatography repeatedly, obtain the different strain slender acanthopanax of compound first glycosides;

4) H through 5v/v% by above-mentioned different strain slender acanthopanax first glycosides ₂sO ₄after solution hydrolysis, can make helexin compounds.

3. helexin compounds, as the application of Hepatoma therapy medicine, is characterized in that according to claim 1: the preparation method of the derivant of described helexin adopts the chemical synthesis route shown in following formula:

R wherein ₁with R ₂substituent group is identical, for-COCH ₃,-COCH ₂cH ₃,-COCH ₂cH ₂cH ₃or-COCH ₂cH ₂cH ₂cH ₃; R ₃substituent group is-CH ₃,-CH ₂cH ₃,-CH ₂cH ₂cH ₃or-CH ₂cH ₂cH ₂cH ₃.

Reaction condition in synthetic route is respectively:

A. helexin is at K ₂cO ₃under catalysis, in DMF solution, carry out esterification with idoalkane, obtain product 1;

B. product 1 is dissolved in oxolane, under DMAP catalysis, carries out acylation reaction with anhydrides reagent, obtain product 2;

C. product 2 is dissolved in anhydrous chloroform, adds after m-CPBA lucifuge under room temperature to place, carry out oxidation reaction, obtain product 3;

D. product 3 is dissolved in the hot ethanol containing anhydrous sodium acetate, with oxammonium hydrochloride. back flow reaction, obtains product 4;

E. product 4 is dissolved in dry pyridine, under condition of ice bath, adds phosphorus oxychloride solution, after Beckmann rearrangement, obtain product 5;

F. product 5 is dissolved in dry benzene, adds lawesson reagent to reflux, carry out vulcanization reaction, obtain product 6.