A large-scale preparation method of natural alkaloids and its application in removing harmful substances from cigarettes

Abstract

The invention discloses a large-scale preparation method of a natural sulfur-containing alkaloid and its application in the removal of harmful substances in cigarettes. The large-scale preparation method of the target compound macathiohydantoin I is as follows: using benzyl isothiocyanate, alanine and 1-(chloromethyl)-3-methoxybenzene as starting materials, and obtaining its corresponding artificial synthetic product through "one-pot" biomimetic synthesis, and its synthesis route is as follows: macathiohydantoin I prepared by the invention is added to acetate fiber tow to prepare a cigarette functional filter rod, and compared with the control cigarette, the contents of total particulate matter, carbon monoxide, nicotine, tar and benzo[a]pyrene in the mainstream smoke of the cigarette are significantly reduced. The raw materials in the invention are simple and easy to obtain, the synthesis method is concise and efficient, and the later purification steps are simple, so that the large-scale preparation of macathiohydantoin I can be realized. The invention can significantly reduce the release of harmful substances in cigarettes, and the cigarette smoke and the original taste of the cigarette are well coordinated, and the irritation and aftertaste are slightly improved, and the invention has potential application value.

Description

Technical Field

[0001] The invention belongs to the technical field of reducing the release of harmful substances in cigarettes by natural organic compounds and tobacco additives, and particularly relates to a large-scale artificial synthesis preparation method of macathiohydantoin I, a natural sulfur-containing alkaloid in the medicinal plant maca, and its application in removing harmful substances from cigarettes. Background Art

[0002] The composition of cigarette smoke is extremely complex, with up to thousands of components. During the combustion process, tobacco will produce a large number of compounds, including 13 types of compounds with a total of 44 harmful components in cigarette smoke. The main harmful substances can be roughly divided into: (1) Nicotine: alkaloids contained in tobacco, also known as nicotine. After nicotine enters the human body with smoke, most of it stays in the lungs, and only a small part enters the blood. It can reach the brain in a very short time, thereby inhibiting the sympathetic nerves. However, excessive nicotine content in cigarettes will also increase the irritation of smoke. (2) Carbon monoxide: During the combustion of cigarettes, there is a lack of oxygen in the combustion zone, which easily leads to the formation of carbon monoxide. Studies have shown that symptoms such as hypoxia poisoning and cardiovascular disorders in the human body are closely related to carbon monoxide, and excessive carbon monoxide may even cause organic lesions. (3) Tar: The composition is extremely complex, consisting of a variety of hydrocarbons and hydrocarbon oxides, sulfides and nitrides. Tar contains both the unique aroma substances of cigarettes and carcinogens or cancer promoters that are extremely harmful to the human body, such as benzo[a]pyrene and other polycyclic aromatic hydrocarbons, aromatic amines and nitrosamines, chlorinated hydrocarbons and other substances.

[0003] How to selectively reduce harmful components in smoke, improve cigarette safety, and maintain the original quality of cigarettes has become a major issue facing tobacco practitioners. With the development of tar reduction and harm reduction technology, tobacco practitioners have developed new materials including nanomaterials and medicinal plant additives to selectively reduce harmful components in cigarette smoke. The medicinal plant maca (Latin name Lepidium meyenii Walp) is a 1-2 year old herbaceous medicinal plant of the genus Lepidium in the family Cruciferae. It is native to the Andes Mountains in South America (3500-4500 meters above sea level). It is a commonly used medicinal plant and edible material in the local area and is known as the "Peruvian ginseng". my country introduced it in the early 1990s, with Lijiang and Shangri-La in Yunnan as the main planting areas. It is also widely planted in Huize, Zhaotong, Yuxi and other areas in Yunnan, and the scale of planting is constantly expanding. Qiu et al. studied the chemical composition and biological activity of the plant and found that the plant contains a novel sulfur-containing alkaloid macathiohydantoin I with a 5-methyl-2-thioimidazole-4-one mother nucleus, suggesting that the component may be one of the main active ingredients of maca as a medicinal and edible plant. However, the low content and difficulty in separation of naturally occurring novel compounds limit its further biological activity research. In order to prepare enough compounds and carry out in-depth research on pharmacological activity and its mechanism of action, the present invention has carried out artificial synthesis research on macathiohydantoin I, and found a simple and efficient "one-pot method" to achieve the total synthesis and large-scale preparation of the natural product. The thiocarbonyl group at position 2, the oxycarbonyl group at position 4 and the lone pair of electrons on N in the compound macathiohydantoin I may help adsorb harmful substances in cigarettes, making it a medicinal plant tobacco additive with potential application prospects. Summary of the invention

[0004] The first purpose of the present invention is to provide a large-scale preparation method of natural sulfur-containing alkaloid macathiohydantoin I; the second purpose is to provide the application of the target compound Imacathiohydantoin I in the removal of harmful substances in cigarettes.

[0005] The first object of the present invention is achieved in this way. The large-scale preparation method is as follows: the natural known sulfur-containing alkaloid macathiohydantoin I isolated from the medicinal plant maca is used as the target compound, and the biogenic precursors of the compound, benzyl isothiocyanate, alanine and 1-(chloromethyl)-3-methoxybenzene are used as starting materials to obtain the corresponding artificial synthetic product through a "one-pot" biomimetic synthesis. The product obtained by this synthesis method has spectral information that is completely consistent with that of natural macathiohydantoin I, and its structure is composed of a 5-methyl-2-thioimidazole-4-one nucleus and two substituents (one 3-methoxybenzyl and one benzyl), and the molecular formula is C 19 H 20 N2O2S, its structural formula is:

[0006]

[0007] The method for purifying the reaction product is to obtain a crude sample of macathiohydantoin I by forward and reverse silica gel column chromatography, and to obtain the target product macathiohydantoin I with a purity of more than 95% by preparative high performance liquid chromatography and semi-preparative high performance liquid chromatography. The specific operation is as follows:

[0008] A. Enrich the crude product by forward and reverse silica gel column chromatography: dissolve with 5-500 ml of organic solvent, add 80-200 mesh silica gel to mix the sample, apply the sample to a reverse silica gel column after mixing, pack the column with 200 mesh silica gel, gradient elute with a mixed organic solvent, detect by silica gel thin layer chromatography, combine the points with the same color development and separation coefficient, and obtain the crude product of the target compound macathiohydantoin I;

[0009] B. Preparative high performance liquid chromatography to purify the crude product: using 70% methanol as the mobile phase, a flow rate of 12 mL / min, a 21.2×250 mm, 5 μm Zorbax PrepHT GF reverse phase preparative column as the stationary phase, and a UV detector with detection wavelengths of 203 and 306 nm. Inject 100 μL each time, collect the chromatographic peaks of 10 to 20 min, and accumulate the same steps multiple times, and evaporate to dryness using a rotary evaporator to obtain the purified crude product of macathiohydantoin I;

[0010] C. Semi-preparative HPLC separation and purification: 85% methanol aqueous solution is used as the mobile phase, the flow rate is 3mL / min, 9.4×250mm, 10μm Zorbax SB-C18 reverse phase semi-preparative column is used as the stationary phase, the DAD detector detection wavelength is 203, 220, 254, 265 and 306nm, 25μL is injected each time, and the chromatographic peak of 20.0min is collected. After multiple accumulations of the same steps, the (±)-macathiohydantoin is evaporated to dryness using a rotary evaporator to obtain the pure product with a purity of more than 95%. The key reaction is Edman degradation reaction and microwave-assisted N-alkylation. The reaction solvent is 10 to 100mL of analytical pure anhydrous pyridine. In addition to being a reaction medium for Edman degradation and N-alkylation, the solvent is also used as an organic alkaline catalyst to catalyze the Edman degradation reaction. The specific biomimetic synthesis route is:

[0011]

[0012] The second object of the present invention is achieved in this way, specifically as follows:

[0013] A. The prepared target compound macathiohydantoin I (10-30 mg) is fully dissolved in 1 mL of triacetin, and the macathiohydantoin I is prepared into a 10-30 mg / mL solution, and the filter tip is immersed in the above solution until all the solution is absorbed by the acetate filter tip, and the filter tip is naturally dried in the shade and then inserted into the cigarette tipping paper as it is, so as to obtain the functional filter rod with reduced harmful substance release, and the filter rod includes three different specifications of slim cigarettes, medium cigarettes and regular cigarettes;

[0014] B. The contents of total particulate matter (TPM), carbon monoxide, water content and nicotine in the mainstream smoke of cigarettes were measured respectively according to the industry standards YC / T2, YC / T8 and YC / T30, and the tar content was obtained by removing the water content and nicotine content from the total particulate matter. Compared with the control cigarettes, the contents of total particulate matter (TPM), carbon monoxide, nicotine, tar and benzo[a]pyrene in the mainstream smoke of cigarettes were significantly reduced. After adding three concentrations of the compound of the present invention, the reduction rate of total particulate matter (TPM) in the smoke of three different specifications of cigarettes was between 3.31 and 14.80%, the reduction rate of carbon monoxide was between 5.77 and 13.32%, the reduction rate of nicotine was between 2.65 and 14.49%, the reduction rate of tar was between 3.60 and 17.62%, and the reduction rate of benzo[a]pyrene was between 3.99 and 11.14%. The above results show that the compound and the functional filter rod described in the present invention have a significant effect of intercepting harmful substances in mainstream cigarette smoke, and the interception capacity is significantly enhanced as the concentration of the compound added increases;

[0015] C. Toxicological evaluation and sensory evaluation showed that there was little difference in sensory evaluation results between the cigarettes containing the compounds of the present invention and the control cigarettes. The main difference was a slightly cool taste in the mouth, a slight improvement in irritation and aftertaste, and no obvious negative effects.

[0016] The artificial synthetic product obtained by the "one-pot" biomimetic synthesis of the present invention has spectral information that is completely consistent with that of natural macathiohydantoin I. The method is simple and efficient, and can achieve large-scale preparation of the compound, thereby solving the resource problem and laying a foundation for the development and application of later functional products.

[0017] Compared with the prior art, the present invention has the following outstanding advantages: (1) The target compound macathiohydantoin I of the present invention is derived from a safe and healthy medicinal and edible plant. By adding a natural compound with an adsorption function to a cigarette filter rod, harmful substances in cigarette smoke can be effectively intercepted, which is easier to achieve technically and has a low cost. (2) The compound of the present invention and its functional filter rod have a significant effect of intercepting harmful substances in mainstream cigarette smoke, and their interception capacity is significantly enhanced as the concentration of the compound of the present invention is increased. (3) The functional filter rod of the present invention is made by adding triacetin as a plasticizer to the filter rod. Since triacetin is added as a plasticizer during the molding of cigarette filter rods, the compound of the present invention has good solubility in the plasticizer, and the manufacturing process is very easy to implement on existing equipment without adding additional steps and equipment. (4) The compound used in the present invention has achieved industrial synthesis, and the source of raw materials is no longer restricted. Moreover, the compound is safe and non-toxic, and has good prospects for industrial application. (5) The compound of the present invention has a simple artificial synthesis step, easy to control conditions, and a low-cost synthetic route to achieve compound synthesis, which has the conditions for large-scale promotion and application. (6) After adding the compound of the present invention to the mouthpiece, the cigarette smoke and the original taste of the cigarette are well coordinated, with a slightly cool taste in the mouth, and a slight improvement in irritation and aftertaste. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The H NMR spectrum of macathiohydantoin I was synthesized. 1 H NMR);

[0019] Figure 2 The C NMR spectrum of macathiohydantoin I was synthesized. 13 C NMR). DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with implementation cases and drawings, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention belong to the protection scope of the present invention.

[0021] (1) The synthesis method described in the present invention is to use the natural known sulfur-containing alkaloid macathiohydantoin I isolated from the medicinal plant maca as the target compound, and the biogenic precursors of the compound benzyl isothiocyanate, alanine and 1-(chloromethyl)-3-methoxybenzene as starting materials, and obtain the corresponding artificial synthetic product through a "one-pot" biomimetic synthesis. The product obtained by the synthesis method has spectral information that is completely consistent with the natural (±)-macathiohydantoin I, and its structure is composed of a 5-methyl-2-thioimidazole-4-one nucleus and two substituents (one 3-methoxybenzyl and one benzyl), and the molecular formula is C 19 H 20 N2O2S, its structural formula is:

[0022]

[0023] (2) The artificial synthesis method is characterized by: using biogenic precursors benzyl isothiocyanate, alanine and 1-(chloromethyl)-3-methoxybenzene as starting materials, through a "one-pot" biomimetic synthesis, the key reactions of which are Edman degradation reaction and microwave-assisted N-alkylation. The specific reaction conditions are as follows: 0.089-89 g or 1.0-1000.0 mmol (1 equivalent) of alanine is dissolved in 10-100 mL of anhydrous pyridine (partial insolubility is allowed), 0.149-288 g or 1.0-2000.0 mmol (1-2 equivalents) of benzyl isothiocyanate is added, and the mixture is stirred at room temperature (10-40 degrees Celsius) for 2-5 hours. After the precipitate is completely dissolved, 0.156-156 g or 1.0-1000.0 mmol (1 equivalent) of 1-(chloromethyl)-3-methoxy is added to the reaction system, and the mixture is subjected to microwave reaction for 5-30 minutes. The reaction solution is evaporated to dryness, filtered, and dried, and then dissolved in 5-500 ml of an organic solvent, and a crude sample is enriched by forward and reverse silica gel column chromatography to obtain a crude sample, and then purified by preparative high performance liquid chromatography and semi-preparative high performance liquid chromatography to obtain a target product with a purity of more than 95%. The reaction solvent is 10-100 mL of analytically pure anhydrous pyridine. In addition to being a reaction medium for Edman degradation and N-alkylation, the solvent is also used as an organic alkaline catalyst to catalyze the Edman degradation reaction. The specific biomimetic synthesis route is:

[0024]

[0025] (3) The method for enriching a crude sample of the target compound is specifically performed as follows: dissolving the sample with 5 to 500 ml of an organic solvent, adding 80 to 200 mesh silica gel to mix the sample, applying the sample to a reverse silica gel column after mixing, loading the column with 200 mesh silica gel, gradient eluting with a mixed organic solvent, and detecting the sample by silica gel thin layer chromatography. The points with the same color development and separation coefficient are combined to obtain a crude product of the target compound macathiohydantoin I.

[0026] (4) The method for refining the target compound is as follows: first, preparative high performance liquid chromatography is used, with 70% methanol as the mobile phase, a flow rate of 12 mL / min, a 21.2×250 mm, 5 μm Zorbax PrepHT GF reverse phase preparative column as the stationary phase, and the detection wavelength of the ultraviolet detector is 203 and 306 nm. 100 μL is injected each time, and the chromatographic peak of 10 to 20 min is collected. After the same steps are accumulated multiple times, the chromatographic peak is evaporated to dryness using a rotary evaporator to obtain the refined crude product of macathiohydantoin I; secondly, the refined crude product is separated and purified by semi-preparative high performance liquid chromatography, with 85% methanol aqueous solution as the mobile phase, a flow rate of 3 mL / min, a 9.4×250 mm, 10 μm Zorbax The SB-C18 reversed-phase semi-preparative column was used as the stationary phase, the detection wavelengths of the DAD detector were 203, 220, 254, 265 and 306 nm, 25 μL was injected each time, and the chromatographic peak of 20.0 min was collected. After multiple accumulations of the same steps, the mixture was evaporated to dryness using a rotary evaporator to obtain the pure product of (±)-macathiohydantoin with a purity of more than 95%.

[0027] (5) The preparation method of the filter rod for reducing the release of harmful substances is as follows: 10 to 30 mg of the compound macathiohydantoin I is weighed and fully dissolved in 1 mL of triacetin, and the macathiohydantoin I is prepared into a 10 to 30 mg / mL solution, and the filter tip is immersed in the above solution until all the solution is absorbed by the acetate fiber filter tip, and after the filter tip is naturally dried in the shade, it is inserted into the cigarette tipping paper as it is, and the filter rod with the function of reducing the release of harmful substances can be obtained. The filter rod includes three different specifications: slim cigarettes, medium cigarettes and regular cigarettes.

[0028] (6) Determination of the content of total particulate matter (TPM) in mainstream cigarette smoke. A cigarette to which the compound of the present invention is added is subjected to cigarette smoke analysis, and a cigarette without the compound of the present invention is used as a control. The calculation formula for the determination of the total particulate matter (TPM) content in smoke is: total particulate matter (TPM) content = [(M2-M1) / N]×1000, wherein M1 is the weight of the Cambridge filter smoke collector before smoking (unit: gram), M2 is the weight of the Cambridge filter smoke collector after smoking (unit: gram), N is the number of cigarettes smoked (unit: cigarette), and the total particulate matter (TPM) is the weight of the total particulate matter (unit: mg / cigarette). The reduction rate of total particulate matter (TPM) was calculated by comparing with the control sample. After adding the compound of the present invention to filter rods of different specifications, the reduction rate of total particulate matter (TPM) in the mainstream smoke of cigarettes was between 3.31% and 14.80% compared with the control cigarettes, indicating that the compound of the present invention and the functional filter rod thereof have a significant effect of intercepting the total particulate matter (TPM) in the mainstream smoke of cigarettes, and the interception capacity is significantly enhanced with the increase of the concentration of the compound of the present invention.

[0029] (7) Determination of the content of carbon monoxide, moisture, nicotine and tar in the mainstream smoke of the cigarette. The cigarettes to which the compound of the present invention is added in the filter rod are subjected to cigarette smoke analysis. The content determination is carried out in accordance with the Chinese tobacco industry standards YC / T2, YC / T8 and YC / T30. The total particulate matter (TPM), carbon monoxide, moisture and nicotine content in the mainstream smoke of the cigarette are respectively determined, and the tar content is obtained by removing the moisture and nicotine content from the total particulate matter. The reduction rates of carbon monoxide, moisture, nicotine and tar are calculated by comparing with the control samples. After adding the compound of the present invention to filter rods of different specifications, compared with the control cigarettes, the reduction rates of carbon monoxide in the mainstream smoke of the cigarettes are between 5.77 and 13.32%, the reduction rates of nicotine are between 2.65 and 14.49%, and the reduction rates of tar are between 3.60 and 17.62%. The above results indicate that the compound of the present invention and the functional filter rod thereof have a significant effect of intercepting carbon monoxide, nicotine and tar in mainstream cigarette smoke, and the interception capacity is significantly enhanced with the increase in the concentration of the compound of the present invention.

[0030] (8) Determination of the content of benzo[a]pyrene in the mainstream smoke of the cigarette. The cigarettes to which the compound of the present invention is added in the filter rod are subjected to cigarette smoke analysis, and the content of benzo[a]pyrene in the mainstream smoke of the cigarette is determined in accordance with the national standard of the People's Republic of China GBT 27525-2011. The reduction rates of carbon monoxide, moisture, nicotine and tar are calculated by comparison with the control samples. After adding the compound of the present invention to the filter rods of different specifications, the reduction rate of benzo[a]pyrene in the mainstream smoke of the cigarette is between 3.99% and 11.14% compared with the control cigarettes. The above results show that the compound of the present invention and its functional filter rod have a significant effect of intercepting benzo[a]pyrene in the mainstream smoke of cigarettes, and its interception capacity is significantly enhanced with the increase in the concentration of the compound of the present invention.

[0031] (9) Toxicological evaluation and sensory evaluation. The smoke condensate of cigarettes to which the compounds of the present invention were added was subjected to toxicological evaluation, Ames test, and in vitro micronucleus test. The results of three in vitro toxicological tests all showed that: the cell inhibition rate produced by the total particulate matter in the smoke of the test sample and the control sample was statistically analyzed using one-way ANOVA using SPSS software. In the three toxicological experiments, there was no statistical difference between the total particulate matter in the smoke of the cigarettes to which the compounds of the present invention were added and the control cigarettes (P>0.05). The cigarettes to which the compounds of the present invention were added were also subjected to sensory evaluation, and the same cigarettes without the compounds were used as controls. The evaluation results showed that the difference in the sensory evaluation results between the cigarettes to which the compounds of the present invention were added and the control was small. The main difference was a slightly cool taste in the mouth, which slightly improved the irritation and aftertaste, and had no obvious negative effects.

[0032] The present invention is further described below with specific implementation cases:

[0033] Example 1

[0034] Alanine (8.9 g, 10.0 mmol, 1 equivalent) was dissolved in 25 mL of anhydrous pyridine (some insoluble was allowed), benzyl isothiocyanate (17.9 g, 12.0 mmol, 1.2 equivalent) was added, and stirred at room temperature (25 degrees Celsius) for 3 hours. After the precipitate was completely dissolved, 1-(chloromethyl)-3-methoxy (15.6 g, 10.0 mmol, 1 equivalent) was added to the reaction system and microwaved for 20 minutes. The reaction solution was evaporated to dryness under reduced pressure using a rotary evaporator, filtered under reduced pressure and dried in a vacuum drying oven for 6 hours to obtain a reaction mixture of about 40 g, which was dissolved in 50 mL of dichloromethane, and then 40 g of 80-mesh silica gel was added to mix the sample. After mixing the sample, it was applied to a reverse-phase silica gel column and loaded with 800 g of 200-mesh silica gel; eluted with dichloromethane, gradient eluent, collected and concentrated, and detected by silica gel thin layer chromatography, and the points with the same color development and separation factor were combined to obtain 32 g of a crude target compound with a separation factor of 0.5 on thin layer chromatography (developing solvent: dichloromethane). The crude product of macathiohydantoin I was prepared by using 70% methanol as the mobile phase, a flow rate of 12 mL / min, a 21.2×250 mm, 5 μm Zorbax PrepHT GF reverse phase preparative column as the stationary phase, and a UV detector with detection wavelengths of 203 and 306 nm. 100 μL was injected each time, and the chromatographic peak of 12 min was collected. After multiple additions of the same steps, the mixture was evaporated to dryness using a rotary evaporator to obtain 26 g of the refined crude product of macathiohydantoin I. The product was then separated and purified by semi-preparative high performance liquid chromatography, with 85% methanol aqueous solution as the mobile phase, a flow rate of 3 mL / min, a 9.4×250 mm, 10 μm Zorbax SB-C18 reverse phase semi-preparative column as the stationary phase, and a DAD detector with detection wavelengths of 203, 220, 254, 265 and 306 nm. 25 μL was injected each time, and the chromatographic peak of 20.0 min was collected. After multiple accumulations of the same steps, the product was evaporated to dryness using a rotary evaporator to obtain 22 g of the pure product of macathiohydantoin I with a purity of 95% and a yield of about 69%.

[0035] The artificial synthesis route is:

[0036]

[0037] Example 2

[0038] Macathiohydantoin I was prepared in Example 1 as a colorless oily compound. The structure was determined by nuclear magnetic resonance, combined with ultraviolet spectroscopy, infrared spectroscopy, conventional mass spectrometry and literature comparison. The specific data are:

[0039] (1) UV spectrum (solvent: methanol), λ max(logε): 252(4.52), 271(4.22)nm;

[0040] (2) Infrared spectrum (potassium bromide tablet) ν max : 3428, 1960, 1558, 1412, 733cm -1 ;

[0041] (3) ESIMS shows that the quasi-molecular ion peak of the compound of the present invention is m / z 341 [M+H] + , combined with 13 C and 1 H NMR spectrum ( Figure 1 and Figure 2 ), giving its molecular formula as C 19 H 20 N2O2S.

[0042] (4) 1 H NMR (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz) data: 1 H NMR (100 MHz, inCDCl3):δ H 7.32-7.28(m,5H,H-3a'-7a'),7.25-6.85(m,4H,H-3a',5a'-7a'),5.65and 4.38(d,J=15.1Hz,2H,H2-1a),5.07and 5.03(d,J=14.5Hz,2H,H2-1a'),3.89(q,J=7.1Hz,1H,H-5),3.76(s,3H,OMe),1.40(d,J=7.1Hz,3H,H3-6); 13 C NMR (100MHz, in CDCl3):δ C 182.0(s,C-2),173.7(s,C-4),56.8(d,C-5),15.1(q,C-6),48.3(t,C-1a),13 6.3(s,C-2a),113.7(d,C-3a),159.7(s,C-4a),113.6(d,C-5a),130.1(d,C-6a ),120.2(d,C-7a),55.3(q,OMe),45.2(t,C-1a'),136.0(s,C-2a'),128.5(d,C -3a'),128.6(d,C-4a'),127.9(d,C-5a'),128.6(d,C-6a'),128.5(d,C-7a').

[0043] Structural elucidation process: macathiohydantoin I 13 C NMR spectrum ( Figure 2 )and 1 H NMR spectrum ( Figure 1 ) shows 19 carbon signals (including 2 methyl carbons, 2 methylene carbons, 10 methine and 5 quaternary carbon signals) and 20 hydrogen signals. Among these signals, there is a typical benzyl substituent signal and a typical 3-methoxybenzyl signal. The other 4 carbon atoms belong to the resonance signals on the skeleton, including 1 thiocarbonyl 182.0 (s, C-2), 1 amide carbonyl 173.7 (s, C-4), 1 azine methine 56.8 (d, C-5) and a methyl signal 15.1 (q, C-6). These characteristics are completely consistent with the compound macathiohydantoin I reported in the literature, and the systematic name of the compound is 3-benzyl-1-(3-methoxybenzyl)-5-methyl-2-thioxoimidazolidin-4-one. The compound has a rare 5-methyl-2-thioxoimidazolidin-4-one mother nucleus composed of N-1, C-2, N-3, C-4, C-5, and C-6. The above structural identification shows that the macathiohydantoin I prepared in Example 1 is the same compound as the natural product macathiohydantoin I. Therefore, the preparation method of Example 1 is simple and efficient, and can easily achieve kilogram-level large-scale preparation.

[0044] Example 3

[0045] Example 1: Macathiohydantoin I was prepared, and a solution of 10-30 mg / mL was prepared with triacetin. The filter tip was immersed in the solution until all the solution was absorbed by the acetate filter tip. After the filter tip was naturally dried in the shade, it was inserted into the cigarette tipping paper as it was, and the functional filter rod with reduced harmful substance release was obtained. The filter rod included three different specifications: slim cigarettes, medium cigarettes and conventional cigarettes. The cigarette smoke of the cigarette to which the compound of the present invention was added was analyzed, and the contents of carbon monoxide, nicotine, tar and benzo[a]pyrene were measured respectively according to the Chinese tobacco industry standards (YC / T2, YC / T8, YC / T30) and the national standards of the People's Republic of China (GBT 27525-2011). The reference calculation formula for the determination of the total particulate matter (TPM) content in smoke is: total particulate matter (TPM) content = [(M2-M1) / N]×1000, where M1 is the weight of the Cambridge filter smoke collector before smoking (unit: gram), M2 is the weight of the Cambridge filter smoke collector after smoking (unit: gram), N is the number of cigarettes smoked (unit: cigarette), and total particulate matter (TPM) is the weight of total particulate matter (unit: mg / cigarette). On this basis, cigarettes without the addition of the compound of the present invention were used as controls, and the reduction rate of each harmful component was calculated by comparing with the control samples. The results are shown in Table-1 to Table-3. The results show that after adding three concentrations of the compound of the present invention (10 mg, 20 mg and 30 mg) to three different specifications of filter rods (conventional, medium and slim cigarettes), the contents of total particulate matter (TPM), carbon monoxide, nicotine, tar and benzo[a]pyrene in the mainstream smoke of cigarettes were significantly reduced compared with the control cigarettes. After adding three concentrations of the compound of the present invention, the reduction rate of total particulate matter (TPM) in the smoke of three different specifications of cigarettes is between 3.31 and 14.80%, the reduction rate of carbon monoxide is between 5.77 and 13.32%, the reduction rate of nicotine is between 2.65 and 14.49%, the reduction rate of tar is between 3.60 and 17.62%, and the reduction rate of benzo[a]pyrene is between 3.99 and 11.14%. The above results show that the compound of the present invention and its functional filter rod have a significant effect of intercepting harmful substances in mainstream cigarette smoke, and its interception capacity increases with the increase of the concentration of the compound of the present invention.

[0046] Table-1. Determination of the content of harmful substances in conventional cigarettes after adding the compounds of the present invention

[0047]

[0048] Table-2. Determination of the content of harmful substances in medium-sized cigarettes after adding the compounds of the present invention

[0049]

[0050]

[0051] Table-3. Determination of the content of harmful substances in slim cigarettes after adding the compounds of the present invention

[0052]

[0053] On this basis, toxicological evaluation and sensory evaluation of smoking were carried out. The results of three in vitro toxicological tests, namely, toxicological evaluation, Ames test and in vitro micronucleus test, were conducted on the smoke condensate of cigarettes with the compound of the present invention added to the filter rod. All of them showed that the cell inhibition rate produced by the total particulate matter of the cigarette smoke of the test sample and the control sample was statistically analyzed by one-way ANOVA using SPSS software. In the three toxicological experiments, there was no statistical difference between the total particulate matter of the cigarette smoke of the cigarettes with the compound of the present invention added and the control cigarettes (P>0.05). The cigarettes with the compound of the present invention added were also subjected to sensory evaluation, and the same cigarettes without the compound added were used as controls. The evaluation results showed that there was little difference in the sensory evaluation results between the cigarettes with the compound of the present invention added and the control, and the main difference was a slightly cool taste at the entrance, which had a slight improvement on irritation and aftertaste, and had no obvious negative effects.

Claims

1. Application of natural sulfur-containing alkaloids in removing harmful substances from cigarettes, characterized in that The application is to add macathiohydantoin I to acetate fiber tow to make a cigarette functional filter rod; the cigarette harmful substances are total particulate matter, carbon monoxide, nicotine, tar and benzo[a]pyrene in cigarette smoke; The specific method for preparing the functional filter rod of the cigarette is as follows: weigh 10-30 mg of the compound macathiohydantoin I and fully dissolve it in 1 mL of triacetin, prepare a solution of macathiohydantoin I at 10-30 mg / mL, immerse the filter tip in the solution until all the solution is absorbed by the acetate fiber filter tip, and after the filter tip is naturally dried in the shade, insert it into the cigarette tipping paper as it is, so as to obtain a functional filter rod with reduced harmful substance release; The macathiohydantoin I described herein uses the natural known sulfur-containing alkaloid macathiohydantoin I isolated from the medicinal plant maca as the target compound, uses the compound's biogenic precursors benzyl isothiocyanate, alanine and 1-(chloromethyl)-3-methoxybenzene as starting materials, and obtains its corresponding artificial synthetic product through a "one-pot" biomimetic synthesis; the spectral information of the artificial synthetic product is completely consistent with that of the natural macathiohydantoin I, and its structure is composed of a 5-methyl-2-thioimidazolidine-4-one parent nucleus, a 3-methoxybenzyl group and a benzyl group, and the molecular formula is C 19 H 20 N2O2S, its structural formula is: 。 2. The use of the natural sulfur-containing alkaloids in removing harmful substances from cigarettes according to claim 1, characterized in that The synthetic reaction conditions are as follows: 0.089-89 g or 1.0-1000.0 mmol of alanine is added to 10-100 mL of anhydrous pyridine, and the mixture is stirred thoroughly. 0.149-288 g or 1.0-2000.0 mmol of benzyl isothiocyanate is added, and the mixture is stirred at room temperature for 2-5 hours. After the precipitate is completely dissolved, 0.156-156 g or 1.0-1000.0 mmol of 1-(chloromethyl)-3-methoxybenzene is added to the reaction system, and the reaction is carried out under microwave for 5-30 minutes. The reaction solution is evaporated to dryness, filtered, and dried, and then dissolved in 5-500 ml of an organic solvent. The crude sample is enriched by positive and reverse phase silica gel column chromatography, and then purified by preparative high performance liquid chromatography and semi-preparative high performance liquid chromatography to obtain a target product with a purity of more than 95%. The specific biomimetic synthetic route is as follows: 。 3. The use of the natural sulfur-containing alkaloids in removing harmful substances from cigarettes according to claim 2, characterized in that The crude sample of macathiohydantoin I was enriched by positive and reverse phase silica gel column chromatography, and the target product macathiohydantoin I with a purity of more than 95% was obtained by preparative HPLC and semi-preparative HPLC. The specific operation was as follows: (1) Enrichment of crude product by normal-reverse phase silica gel column chromatography: Dissolve in 5-500 ml organic solvent, add 80-200 mesh silica gel to mix the sample, apply the sample to a reverse phase silica gel column, pack the column with 200 mesh silica gel, and gradient elute with a mixed organic solvent. Detect by silica gel thin layer chromatography, combine the points with the same color and separation coefficient to obtain the crude product of the target compound macathiohydantoin I; (2) Preparative HPLC purification of crude product: 70% methanol as mobile phase, flow rate 12 mL / min, 21.2×250 mm, 5 μ The stationary phase was a Zorbax PrepHT GF reverse phase preparative column of 100 μm. The detection wavelengths of the UV detector were 203 and 306 nm. Each injection was 100 μl. μ L, collecting the chromatographic peak of 10-20 min, accumulating the same steps for multiple times, and evaporating to dryness using a rotary evaporator to obtain the refined crude product of macathiohydantoin I; (3) Semi-preparative HPLC separation and purification: 85% methanol in water was used as the mobile phase, the flow rate was 3 mL / min, 9.4×250 mm, 10 μ The stationary phase was a Zorbax SB-C18 reversed-phase semi-preparative column with a volume of 100 μm. The detection wavelengths of the DAD detector were 203, 220, 254, 265, and 306 nm. Each injection was 25 μm. μ L, collect the chromatographic peak at 20.0 min, and after multiple accumulations in the same steps, evaporate to dryness using a rotary evaporator to obtain pure (±)-macathiohydantoin with a purity of more than 95%.

4. The use according to claim 1, characterized in that The filter rods include three different specifications: slim cigarettes, medium cigarettes and regular cigarettes.

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