CN101475458B - Mitissimol with antineoplastic activity, preparation and use thereof - Google Patents

Abstract

The invention provides Luctarius rhzomorph with anti-tumor activity. The method for preparing the Luctarius rhzomorph is to extract from higher fungi Lactarius mitissimus, Lctarius hirtipes J.Z.Ying and other fruiting bodies by one or more solvents of ethanol, methanol, chloroform, ethyl acetate, acetone and sherwood oil, and chromatographically separate the prepared extractive by a silica gel column repeatedly. The invention also provides application direction of the Luctarius rhzomorph in preparation of an anti-tumor medicine, including preparation of an anti-tumor medicine taking the Luctarius rhzomorph as an effective ingredient clinically. The Luctarius rhzomorph has stronger anti-tumor activity through in-vitro experiments, wherein the activity of Luctarius rhzomorph Mitissimol D is the strongest with half inhibition concentration IC50 to A549 (lung adenocarcinoma cell line), HCT-8 (human colon cancer cell line), Bel-7402 (human hepatoma cell line) and Hela (human cervical carcinoma cell line) of 2.5, 11.0, 3.6 and 4.6 mu g/ml respectively, and is almost equivalent to positive control drug cisplatin. Therefore, the compounds are expected to be novel anti-tumor medicines.

Description

Lactomycin with anti-tumor activity and its preparation method and application

technical field

The invention relates to a lactomycin with anti-tumor activity and its preparation method and application, and belongs to the technical field of extraction and separation of fungi and preparation of anti-tumor drugs.

Background technique

Cancer has become one of the main threats to human health, and it is showing a clear upward trend. Cancer has become the most important cause of death in the United States, and the cancer mortality rate ranks first among all causes of death in cities in my country. Therefore, my country has listed the research of anticancer drugs as one of the most important strategic issues in the development of new drugs, and researching new anticancer drugs with high efficiency, low toxicity and high selectivity has become a long-term goal and task for drug researchers.

Nature provides a wide range of specific and effective bioactive ingredients, and many organisms have developed a whole set of defense mechanisms to fight against competitors during the long-term evolution process. The "weapons" used by organisms are becoming more and more sophisticated, so it is very wise to find new active ingredients or basic structures from natural resources as drugs or pesticides.

Among various biological resources, fungi are organisms with a particularly high "creation coefficient" and contain a large number of secondary metabolites with diverse structures. By comparing artificially injured and undamaged fruiting bodies through experiments, it was found that fruiting bodies produce a large number of secondary metabolites soon after injury, and these products have various activities such as antifungal and insecticidal. A typical example is the impaired response of the inactive stearoylvelutinal (1) in Lactarious to the highly active antibiotic isovelleral (2). In recent years, higher fungi have attracted more and more attention. On the one hand, it is because compounds with novel structures and significant biological activities are constantly being discovered from higher fungi, and their structures vary greatly. This structural diversity has important implications for drug (or pesticide) discovery. On the other hand, higher fungi can not only be directly harvested as research materials, but also can be easily fermented and cultured through mycelia or spores to provide a large amount of raw material resources and provide a guarantee for possible industrial production in the future. This is also a potential Advantage. Sesquiterpene compounds in higher fungi are a large class of compounds in higher fungi, with rich structural changes and remarkable biological activities. Mainly include tremulane, caryophyllane, collybial, protoilludanes, marasmanes, lactaranes, cucumanes, fomannosanes, illudanes, isoilludanes, hirsutanes, pleurotellanes, cerapicanes, bulleranes, isolactaranes and merulanes sesquiterpenoids. The existence of Humulane sesquiterpene itself in fungi is very rare. Only the applicant's research team has discovered a series of new Humulane sesquiterpene with strong anti-tumor activity. There are no other reports at home and abroad. .

Contents of the invention

The object of the present invention is to provide a new class of Humulane sesquiterpene compounds lactomycin (mitissiols) with anti-tumor activity and its preparation from Lactarius mitissimus, Lactarius mitissimus (Lactarius mitissimus) Lactarius hirtipes J.Z.Ying) and other fruiting bodies to extract lactobacillus; at the same time, further provide the application direction of drugs for the preparation of therapeutic and preventive anti-tumor drugs with lactarius as active ingredients.

The technical scheme of the present invention is achieved in that this lactomycin with anti-tumor activity is characterized in that it comprises the lactomycin of the following structural formula (I):

Wherein: _R1 is hydrogen, carboxyl, hydroxyl or aldehyde; _R2 is hydrogen, hydroxyl, ester or amido; _R3 is hydrogen, carboxyl, hydroxyl or aldehyde; _R4 is hydrogen, carboxyl, hydroxyl or aldehyde ; R ₅ is hydrogen or hydroxyl or 5 is methylene or carbonyl; 3, 4 or 6, 7 or 8, 9 or 10, 11 forms epoxy or forms a single bond or forms a double bond.

Described lactomycin with antitumor activity comprises the structure of the hopane sesquiterpene lactomycin mitissiols A-H with general formula (I) characteristics as follows:

The lactomycin with anti-tumor activity includes the hopane sesquiterpene lactomycin 11β-19α-epoxy-4Z with the characteristics of general formula (I), and the structure of 7Z-humu1adiene-5α-ol is as follows:

The preparation method of the Lactarius mitissimus with anti-tumor activity: the Lactarius mitissimus with anti-tumor activity is obtained from the fruiting body of the higher fungus Lactarius mitissimus, and mixed with ethanol, methanol , chloroform, ethyl acetate, acetone, petroleum ether, and one or more of them are extracted, and the obtained extract is repeatedly separated by silica gel column chromatography.

The preparation method of the lactomycin with anti-tumor activity comprises the following steps:

A. Extraction: Pulverize 1.6kg of higher fungus Lactarius mitissimus, extract 3 times with 15L of 95% ethanol, then extract with 4.5L of ethyl acetate, recycle the solvent to dryness, and weigh 33.5 g;

B, into 200-300 mesh silica gel column chromatography separation:

a. Use CHCl ₃ -MeOH solvent to elute with a gradient of 100:0, 98:2, 95:5, 90:10 (vol/vol), and obtain a component in the elution section of CHCl ₃ -MeOH 100:0, further Carry out silica gel column chromatography, use petroleum ether-acetone solvent to 25: 1, 15: 1, 10: 1, 5: 1 gradient elution, at 15: 1 position, obtain the pure product mitissimol A 66mg;

b. A fraction was obtained in the elution section of CHCl ₃ -MeOH 95:5 in step a, and further subjected to silica gel column chromatography, using petroleum ether-acetone solvent at 10:1, 8:1, 6:1, 4:1, 5:1, 2:1, 1:1 gradient elution, at the 10:1 site, 8 mg of pure lactomycin mitissimol B was obtained;

c. Further silica gel column chromatography at the part of petroleum ether-acetone 2:1 in step b, petroleum ether-ethyl acetate with a gradient of 20:1, 15:1, 10:1, 5:1, 1:1, 1:5 Elution, in petroleum ether-ethyl acetate 20:1 component to obtain the pure product lactomycin mitissimol C 26mg;

d. In step c, the fraction of petroleum ether-ethyl acetate 15:1 was further purified by Sephadex LH-20 gel column chromatography with CHCl ₃ -MeOH 1:1 as the eluent to obtain 5 mg of lactomycin mitissimol D;

e, in step c petroleum ether-ethyl acetate 10: 1 component further utilizes thin-layer preparative chromatography, petroleum ether-acetone 4: 1 is developer, purifies and obtains lactomycin mitissimol E 8mg;

f, in step c petroleum ether-ethyl acetate 5: 1 component to obtain lactobacillus mitissimol F 17mg;

g. Obtain lactomycin mitissimol G 4mg in step c petroleum ether-ethyl acetate 1: 1 component;

h. Obtain 7 mg of pure lactomycin mitissimol in the petroleum ether-acetone 4:1 part in step b.

The preparation method of the Lactarius hirtipes J.Z.Ying with anti-tumor activity, the Lactarius hirtipes J.Z.Ying with ethanol, Methanol, chloroform, ethyl acetate, acetone, and petroleum ether were extracted with one or more solvents, and the obtained extract was repeatedly separated by silica gel column chromatography.

The preparation method of the lactomycin with anti-tumor activity comprises the following steps:

A. Extraction: Grind 6kg of higher fungus Lactarius hirtipes J.Z.Ying, extract 3 times with 20L of 95% ethanol, then extract with 5L of ethyl acetate, recycle the solvent to dryness, and weigh 8.2 g;

B, 200-300 mesh silica gel column chromatography separation: use petroleum ether-acetone gradient elution 50:1, 20:1, 9:1, 5:1, 1:1 to obtain 5 components, take petroleum ether-acetone The 9:1 fraction was repeatedly chromatographed on an RP-C18 column and recrystallized from petroleum ether-chloroform to obtain 22 mg of lactomycin 2β-3α-epoxy-6Z, 9Z-humuladiene-8α-ol.

The application of the lactomycin with anti-tumor activity as the preparation of anti-tumor drugs includes clinical use for the preparation of anti-tumor drugs with lactomycin as an active ingredient.

The application of the lactomycin with anti-tumor activity as the preparation of anti-tumor drugs includes clinical use in the preparation of compositions or compounds containing lactomycin as an active ingredient, and the preparation of pharmaceutically acceptable An antitumor drug in which a salt, a solvate, or a substance in a corresponding crystal form is an active ingredient, and a pharmaceutically acceptable carrier or an auxiliary ingredient is added.

Lactomycin Mitissimol D of the present invention has stronger anti-tumor activity through in vitro tests, wherein the activity of Lactomycin Mitissimol D is the strongest, to A549 (human lung adenocarcinoma cell line), HCT-8 (human colon cancer cell line) , Bel-7402 (human liver cancer cell line) and Hela (human cervical cancer cell line) have half inhibitory concentration IC ₅₀ of 2.5, 11.0, 3.6, 4.6 μg/ml, which are almost equivalent to the positive control drug cisplatin. The compound is expected to become a new antitumor drug.

Description of drawings

Fig. 1 is the crystal diffraction pattern of lactomycin Mitissimol A

Figure 2 is the chemical shift difference between lactomycin (S)-MTPA ester 1b and lactomycin (R)-MTPA ester 1a

Detailed ways

Embodiment 1: the preparation of lactobacillus mitissiols A-H

A. Extraction:

1.6 kg of higher fungus Lactarius mitiss imus collected from Ailao Mountain in Yunnan Province (its strains are preserved in the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences) was crushed and extracted 3 times with 15 L of 95% ethanol , then extracted with 4.5L of ethyl acetate, recovered the solvent to dryness, and weighed 33.5g;

B, into 200-300 mesh silica gel column chromatography separation:

a. Use CHCl ₃ -MeOH solvent to elute with a gradient of 100:0, 98:2, 95:5, 90:10 (vol/vol), and obtain a component in the elution section of CHCl ₃ -MeOH 100:0, further Carry out silica gel column chromatography, use petroleum ether-acetone solvent to 25: 1, 15: 1, 10: 1, 5: 1 gradient elution, at 15: 1 position, obtain the pure product mitissimol A 66mg;

b. A fraction was obtained in the elution section of CHCl ₃ -MeOH 95:5 in step a, and further subjected to silica gel column chromatography, using petroleum ether-acetone solvent at 10:1, 8:1, 6:1, 4:1, 5:1, 2:1, 1:1 gradient elution, at the 10:1 site, 8 mg of pure lactomycin mitissimol B was obtained;

c. Further silica gel column chromatography at the part of petroleum ether-acetone 2:1 in step b, petroleum ether-ethyl acetate with a gradient of 20:1, 15:1, 10:1, 5:1, 1:1, 1:5 Elution, in petroleum ether-ethyl acetate 20:1 component to obtain the pure product lactomycin mitissimol C 26mg;

d. In step c, the fraction of petroleum ether-ethyl acetate 15:1 was further purified by Sephadex LH-20 gel column chromatography with CHCl ₃ -MeOH 1:1 as the eluent to obtain 5 mg of lactomycin mitissimol D;

e, in step c petroleum ether-ethyl acetate 10: 1 component further utilizes thin-layer preparative chromatography, petroleum ether-acetone 4: 1 is developer, purifies and obtains lactomycin mitissimol E 8mg;

f, in step c petroleum ether-ethyl acetate 5: 1 component to obtain lactobacillus mitissimol F 17mg;

g. Obtain lactomycin mitissimol G 4mg in step c petroleum ether-ethyl acetate 1: 1 component;

h. Obtain 7 mg of pure lactomycin mitissimol in the petroleum ether-acetone 4:1 part in step b.

C. Physicochemical properties of mitissiols A-H

Materials and Instruments:

The melting point was determined by the XRC-1 micro melting point apparatus produced by Sichuan Science Instrument Factory, and the temperature was not corrected; the optical rotation was measured by the SEPA-300 instrument; the infrared was measured by the Bio-Rad FTS-135 infrared spectrometer; the ultraviolet was measured by the UV-210A ultraviolet spectrometer Determination; NMR was determined by Bruker AM-400 and Bruker AM-500 nuclear magnetic resonance instrument, tetramethylsilane (TMS) was used as internal standard; mass spectrum was determined by VGA Auto Spec-3000 mass spectrometer. Thin-layer chromatography and column chromatography silica gel are products of Qingdao Ocean Chemical Factory, and Sephadex LH-20 is a product of Pharmacia Company. Use 10% sulfuric acid-ethanol for heating and iodine vapor for color development.

Lactomycin mitissimol A:

Colorless crystals (CHCl ₃ ); mp 169-171°C; [α] _D ^25.2 -42.0 (c 0.40, CHCl ₃ ); UV(CHCl ₃ )UV(CHCl ₃ )λ _max (logε) 252(4.30)nm; IR (KBr): 3481, 2988, 2959, 2925, 2881, 1638, 1445, 1388, 1297, 1273, 1045, 968, 900cm ^-1 ; ¹ H-NMR (500MHz, CDCl ₃ ) δ: 4.22(d, 1H , J=10.0, 1-H), 5.69 (d, 1H, J=16.4, 3-H), 6.06 (d, 1H, J=16.4, 4-H), 5.90 (brd, 1H, J=10.6, 7-H), 2.47(m, 1H, 8-H _α ), 2.28(m, 1H, 8-H _βα ), 2.37(m, 1H, 9-H _α ), 2.23(m, 1H, 9-H _β ), 5.25 (brd, 1H, J=10.6, 11-H), 1.14 (s, 3H, 12-CH ₃ ), 1.14 (s, 3H, 13-CH ₃ ), 1.79 (brs, 3H, 14- CH ₃ ), 1.62(s, 3H, 15-CH ₃ ); ¹³ C NMR (CDCl ₃ , 500MHz)) δ: 75.6(d), 42.0(s), 157.0(d), 127.2(d), 203.7( s), 137.8(s), 148.1(d), 24.6(t), 39.5(t), 138.6(s), 128.0(d), 26.6(q), 17.1(q), 11.7(q), 16.0( q); FABMS (pos) m/z 235[M+1] ⁺ , 469 [2M+1] ⁺ ; HRESIM (pos) m/z 257.1516 (found) (C ₁₅ H ₂₂ O ₂ Na, theoretical 257.1517 ).

b＝9.5977(19)

c＝6.152(3)

晶胞体积：V＝418.5(5)晶胞内分子数Z＝4；晶胞密度d＝1.097Mg/m³；R_f和R_w值分别为0.0423和0.1049。用MAC-DIP-2030K面探仪收集衍射强度数据，MoKα辐射、石墨单色器。用ω扫描方式，获得独立衍射点7350和2517个。在微机上用直接法(SHELXS86)解析晶体结构。CCDC号为601319。The X-crystal diffraction structure analysis of lactomycin Mitissimol A shown in Fig. 1: C ₁₅ H ₂₂ O ₂ , M is 234.33, space group is P2(1)2(1)2(1) unit cell parameter a=9.1505( 18)

b=9.5977(19)

c=6.152(3)

Unit cell volume: V=418.5(5) The number of molecules in the unit cell is Z=4; the unit cell density d=1.097Mg/m ³ ; the values of R _f and R _w are 0.0423 and 0.1049, respectively. Diffraction intensity data were collected with MAC-DIP-2030K surface probe, MoKα radiation, graphite monochromator. Using the ω scan method, 7350 and 2517 independent diffraction points were obtained. The crystal structure was solved by the direct method (SHELXS86) on a microcomputer. The CCDC number is 601319.

Table 1. Atomic coordinates of lactomycin Mitissimol A

Table 2. Bond length and bond angle values of lactomycin Mitissimol A

Lactomycin mitissimol B:

Colorless needle crystals (CHCl ₃ ); mp 200-201°C; [α] _D ^25.5 -1.0 (c 0.59, CHCl ₃ ); UV(CHCl ₃ )λ _max (logε) 244(3.43)nm; IR(KBr ): 3507, 3011, 2995, 2962, 2924, 1651, 1641, 1466, 1386, 1294, 1271, 1061, 975, 911cm ^-1 ; ¹ H NMR (500MHz, CDCl ₃ ) δ: 3.30(d, 1H, J =9.7, 1-H), 5.93 (d, 1H, J=16.4, 3-H), 6.25 (d, 1H, J=16.4, 4-H), 6.05 (brd, 1H, J=8.7, 7- H), 2.41(m, 2H, 8-H), 2.28(ddd, 1H, J=13.4, 3.9, 2.8, 9-H _α ), 1.38(td, 1H, J=13.4, 7.1, 9-H _β ), 2.75 (d, 1H, J=9.7, 11-H), 1.16 (s, 3H, 12-CH ₃ ), 1.24 (s, 3H, 13-CH ₃ ), 1.85 (brs, 3H, 14-CH ₃ ), 1.29(s, 3H, 15-CH ₃ ); ¹³ C NMR (CDCl ₃ , 500MHz)) δ: 76.1(d), 40.4(s), 155.9(d), 129.2(d), 202.5(s ), 139.2(s), 147.0(d), 24.5(t), 38.0(t), 63.5(s), 65.8(d), 26.8(q), 17.1(q), 12.0(q), 16.7(q ); ESIMS(pos) m/z 273[M+Na] ⁺ , 523[2M+Na] ⁺ ; HRESIMS(pos) m/z 273.1474 (found) (C ₁₅ H ₂₂ O ₃ Na, theoretical 273.1466 ).

Lactomycin mitissimol C:

Colorless crystals (MeOH); mp 193-196°C; [α] _D ^25.1 +99.0 (c 0.64, MeOH); UV(MeOH)λ _max (logε) 252(4.11)nm; IR(KBr): 3424(OH ), 3037, 2962, 2928, 1647 (C=CC=OC=C), 1477, 1386, 1099, 1026, 994, 901cm ^-1 ; ¹ H NMR (400MHz, CD ₃ OD) δ: 4.19(d, 1H , J=10.7, 1-H), 5.84 (d, 1H, J=16.5, 3-H), 6.07 (d, 1H, J=16.5, 4-H), 5.73 (brd, 1H, J=10.0, 7-H), 4.61 (ddd, 1H, J=10.4, 10.0, 5.5, 8-H), 2.70 (dd, 1H, J=11.4, 5.5, 9-H _α ), 2.12 (dd, 1H, J= 11.4, 10.4, 9-H _β ), 5.30 (brd, 1H, J=10.7, 11-H), 1.11 (s, 3H, 12-CH ₃ ), 1.13 (s, 3H, 13-CH ₃ ), 1.858 (brs, 3H, 14-CH ₃ ), 1.65 (s, 3H, 15-CH ₃ ); ¹³ C NMR (CD ₃ OD, 400MHz)) δ: 76.0(d), 43.8(s), 161.4(d) , 128.0(d), 206.5(s), 140.7(s), 147.8(d), 65.6(d), 52.0(t), 135.8(s), 130.9(d), 26.9(q), 17.6(q) , 12.1(q), 17.3(q); ESIMS(pos)[M+Na] ⁺ 273, [2M+Na] ⁺ 523; HRESIMS(pos)m/z 273.1467[M+Na] ⁺ (measured value)( C ₁₅ H ₂₂ O ₃ Na, theoretical 273.1466).

Lactomycin mitissimol D:

White powder; mp193-196°C (MeOH); [α] _D ²⁵ +138.0 (c 0.51, MeOH); UV (MeOH) λmax (log ε) 230 (3.93); IR (KBr): 3338 (OH), 3039, 2961, 2928, 1665 (C=CCOC=C), 1388, 1359, 1126, 1031, 982, 909 cm ^-1 ; ¹ H NMR (500 MHz, CD ₃ OD) δ: 3.25 (d, 1H, J=9.7, 1 -H), 6.12 (d, 1H, J=16.4, 3-H), 6.28 (d, 1H, J=16.4, 4-H), 5.92 (brd, 1H, J=9.9, 7-H), 4.53 (ddd, 1H, J=12.1, 9.9, 5.3, 8-H), 2.51 (dd, 1H, J=12.7, 5.3, 8-H _α ), 1.27 (dd, 1H, J=12.7, 12.1, 8-H H _α ), 2.79 (d, 1H, J=9.7, 11-H), 1.22 (s, 3H, 12-CH ₃ ), 1.11 (s, 3H, 13-CH ₃ ), 1.95 (brs, 3H, 14 -CH ₃ ), 1.29(s, 3H, 15-CH ₃ ); ¹³ C NMR (CD ₃ OD) δ: 76, 9(d), 41.9(s), 160.1(d), 130.5(d), 205.3 (s), 142.8(s), 146.8(d), 65.2(d), 48.4(t), 61.8(s), 66.7(d), 27.2(q), 17.7(q), 12.5(q), 18.0 (q); ESI-MS (pos) m/z [M+Na] ⁺ 289, [2M + Na] ⁺ 555; HRESI-MS (pos) m/z 289.1408 (found) (calcd for C ₁₅ H ₂₂ O ₄ Na, theoretical value 289.1415).

Lactomycin mitissimol E:

White powder; mp80-82°C (MeOH); [α] _D ¹⁸ -41.5 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 233 (3.84); IR (KBr): 3442 (OH), 2967, 2929, 1687, 1637 (C=OC=C), 1467, 1389, 1365, 1055, 966, 916 cm ^-1 ; ¹ H NMR (400 MHz, CD ₃ OD) δ: 3.39 (d, 1H, J=9.8, 1 -H), 6.08 (d, 1H, J=17.2, 3-H), 6.80 (d, 1H, J=17.2, 4-H), 2.81 (d, 1H, J=9.1, 7-H), 3.77 (ddd, 1H, J=11.6, 9.1, 3.6, 8-H), 2.44 (dd, 1H, J=13.4, 3.6, 8-H _α ), 1.40 (dd, 1H, J=13.4, 11.6, 8-H H H _α ), 2.77 (d, 1H, J=9.8, 11-H), 1.20 (s, 3H, 12-CH ₃ ), 1.18 (s, 3H, 13-CH ₃ ), 1.69 (s, 3H, 14 -CH ₃ ), 1.46(s, 3H, 15-CH ₃ ); ¹³ C NMR (CD ₃ OD)δ: 76.1(d), 41.8(s), 158.9(d), 128.1(d), 200.4(s ), 67.0(s), 66.9(d), 66.0.4(d), 46.7(t), 60.6(s), 66.0(d), 27.5(q), 18.1(q), 16.7(q), 18.9 (q); ESI-MS (pos) m/z [M+Na] ⁺ 305, [2M + Na] ⁺ 587; HRESI-MS (pos) m/z 305.1365 (found) (C ₁₅ H ₂₂ O ₅ Na, theoretical value, 305.1364).

Lactomycin mitissimol F:

Colorless colloidal substance, mp148-150°C (MeOH); [α] _D ^25.1 -37.7 (c 0.55, MeOH); UV (MeOH) λ _max (log ε) 235 (3.48); IR (KBr): 3441, 2968 , 2927, 1676, 1617, 1453, ₁₃₉₅ , 1150, 1116, 1064, 994, 901 cm -1 cm ^-1 ; ¹ H NMR (CD ³ OD) δ: 3.34 (d, 1H, J=10.0, 1-H) , 6.20 (d, 1H, J=16.1, 3-H), 7.11 (d, 1H, J=16.1, 4-H), 2.52 (dd, 1H, J=13.4, 9.2, 7-H _α ), 1.23 (dd, 1H, J=13.4, 5.2, 7-H _β ), 2.43 (ddd, 1H, J=9.2, 5.2, 2.4, 8-H), 2.68 (d, 1H, J=2.4, 9-H) , 2.81 (d, 1H, J=10.0, 11-H), 1.22 (s, 3H, 12-CH ₃ ), 1.13 (s, 3H, 13-CH ₃ ), 1.40 (s, 3H, 14-CH ₃ ), 0.95(s, 3H, 15-CH ₃ ); ¹³ C NMR (CD ₃ OD) δ: 72.9(d), 43.2(s), 150.7(d), 129.8(d), 209.5(s), 79.4 (s), 44.8(t), 52.4(d), 60.3(d), 61.8(s), 62.3(d), 27.1(q), 18.3(q), 27.9(q), 11.9(q); ESI -MS (pos) m/z [M+Na] ⁺ 305, [2M + Na] ⁺ 587; HR-ESI-MS (pos) m/z 305.1373 (found value) (C ₁₅ H ₂₂ O ₅ Na, Theoretical value 305.1364).

如下：The absolute configuration of lactomycin mitissimol F was determined by the method of Mosher reagent: the Mosher method is a method developed in recent years to determine the absolute configuration of secondary alcohols by ¹ H NMR. It utilizes the R configuration and S configuration compound of 2-methoxy-2-(trifluoromethyl)-2-phenyl-acetic acid (MTPA) to form an ester with a ^secondary alcohol, according to Δ(Δ =SR) as a positive or negative value and molecular models of the possible two isomers to determine the absolute configuration of the secondary alcohol. According to the experimental results shown in Figure 2, Lactomycin (R)-MTPA ((2-methoxy-2-(trifluoromethyl)-2-phenyl-acetic acid) (2-methoxy-2- HC(3), HC(4), _CH2 (7), HC(8), Me(12), Me(13) and Me(3) of (trifluoromethyl)-2-phenyl-acetic acid)) ester 1a (14) than lactomycin (S)-MTPA ((2-methoxy-2-(trifluoromethyl)-2-phenyl-acetic acid) (2-methoxy-2-( Trifluoromethyl)-2-phenyl-acetic acid)) ester 1b at higher fields, while the chemical shifts of HC(9), HC(11) and Me(15) are at lower fields. Therefore, the absolute configuration of mitissimol F at C-1 is the S configuration. specific method

as follows:

Esterification of MTPA (2-methoxy-2-(trifluoromethyl)-2-phenyl-acetic acid) with mitissimol F: take 2.82mg of lactomycin mitissimol F, 11.2mg of (S)-MTPA in CHCl ₂ Dissolve in 10ml solution, add (S)-MTPA 11.2mg, N,N-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide (DCC) 9.9mg and 4-dimethylaminopyridine 4-dimethylaminopyridine (DMAP ) 2mg in a 50ml round bottom flask. The round bottom flask was placed on a magnetic stirrer and stirred at room temperature for 12h. The reaction solution was prepared by TLC (thin layer chromatography) to obtain 3.1 mg of lactomycin (S)-MTPA ester 1a; take 2.60 mg of lactomycin mitissimol F, and dissolve 11.2 mg of (R)-MTPA in 10 ml of CHCl ₂ , plus (R)-MTPA 10.8mg, N, N-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide (DCC) 8.7mg and 4-dimethylaminopyridine 4-dimethylaminopyridine (DMAP) 2mg in a 50ml circle in the bottom flask. The round bottom flask was placed on a magnetic stirrer and stirred at room temperature for 12h. The reaction solution was preparatively TLC (thin layer chromatography) to obtain 3.1 mg of lactomycin (R)-MTPA ester 1a and 2.6 mg of lactomycin (R)-MPTA ester 1b.

Lactomycin (R)-MTPA (2-methoxy-2-(trifluoromethyl)-2-phenyl-acetic acid) ester 1a:

Colorless amorphous powder; ¹ H NMR δ7.180 (1H, d, J = 16.3Hz, H-4), 6.231 (1H, d, J = 16.1Hz, H-3), 5.087 (1H, d, J = 10.0Hz, H-1), 2.947 (1H, d, J=10.0Hz, H-11), 2.696 (1H, d, J=2.4Hz, H-9), 2.540 (1H, dd, J=13.2, 4.9Hz, H-7α), 1.224 (1H, br d, J=9.7Hz, H-7β), 2.474 (1H, br d, J=8.2Hz, H-8), 1.403 (3H, s, H- 14), 1.244(3H, s, H-12), 1.117(3H, s, H-13), 0.896(3H, s, H-15); FABMS m/z 499

Lactomycin (S)-MTPA (2-methoxy-2-(trifluoromethyl)-2-phenyl-acetic acid)) ester 1b:

Colorless amorphous powder; ¹ H NMR δ7.149 (1H, d, J = 16.2Hz, H-4), 6.197 (1H, d, J = 16.2Hz, H-3), 5.075 (1H, d, J = 10.1Hz, H-1), 3.015 (1H, d, J=10.1Hz, H-11), 2.732 (1H, brd, J=2.4Hz, H-9), 2.538 (1H, dd, J=13.3, 5.0Hz, H-7α), 1.221 (1H, br d, J=9.2Hz, H-7β), 2.472 (1H, br d, J=8.9Hz, H-8), 1.397 (3H, s, H- 14), 1.239(3H, s, H-12), 1.112(3H, s, H-13), 0.905(3H, s, H-15); FABMS m/z499

Lactomycin mitissimol G:

Colorless colloidal substance, mp205-207℃(MeOH).[α] _D ^24.2 -2.b(c0.11, CHCl ₃ ).UV(CHCl ₃ )λ _max (logε)241(3.63); IR(KBr ): 3552, 3391, 2990, 2967, 2927, 1696, 1630, 1464, 1387, 1338, 1127, 1081, 1049, 999, 911cm ^-1 . ¹ H NMR(pyridine-d ₅ ) δ: 3.62(d, 1H , J=9.5, 1-H), 6.77 (d, 1H, J=16.2, 3-H), 7.58 (d, 1H, J=16.2, 4-H), 2.67 (brd, 1H, J=14.6, 7-H _α ), 2.57 (dd, 1H, J=14.6, 6.8, 7-H _β ), 3.80 (dd, 1H, J=9.7, 6.8, 8-H), 2.54 (brd, 1H, J=13.1 , 9-H _α ), 1.80 (dd, 1H, J=13.1, 9.7, 9-H _β ), 3.25 (d, 1H, J=9.5, 11-H), 1.43 (s, 3H, 12-CH ₃ ), 1.32(s, 3H, 13-CH ₃ ), 2.05(s, 3H, 14-CH ₃ ), 1.34(s, 3H, 15-CH ₃ ); ¹³ C NMR(pyridine-d ₅ )δ: 74.6 (d), 42.1(s), 150.8(d), 127.2(d), 204.1(s), 77.6(s), 51.6(t), 64.6(d), 52.9(t), 60.7(s), 65.1 (d), 26.7(q), 17.6(q), 22.6(q), 18.8(q); ESI-MS(pos) m/z [M+Na] ⁺ 307, [2M+Na] ⁺ 591.HR - ESI _- MS (pos) m/z _307.1522 (found) _C15H24O5Na , calc. 307.1521).

Lactomycin mitissimol H:

White powder; mp63-65°C (MeOH); [α] _D ^19.5 +3.08 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 202 (3.54); IR (KBr): 3440, 2932, 1709, 1456 , 1015, 975, 911cm ^-1 ; ¹ H-NMR (CD ₃ OD) δ: 3.35 (d, 1H, J=10.0, 1-H), 5.43 (t, 1H, J=5.6, 3-H), 1.97 (d, 1H, J = 2.67, 4-H _α ), 1.70 (overlapped, 1H, 4-H _β ), 3.01 (m, 1H, 6-H), 4.37 (d, 1H, J = 8.2, 7 -H), 3.75(m, 1H, 8-H), 2.02(d, 1H, J=1.3, 9-H _α ), 1.70(overlapped, 1H, 9-H _β ), 5.38(d, 1H, J =9.8, 11-H), 1.71 (overlapped, 3H, 12-CH ₃ ), 1.61 (brs, 3H, 13-CH ₃ ), 0.99 (brs, 3H, 14-CH ₃ ), 0.82 (s, 3H, 15-CH ₃ ); ¹³ C NMR (CD ₃ OD) δ: 60.5(d), 47.6(s), 97.9(d), 41.4(t), 215.4(s), 41.9(d), 89.8(d) , 70.5(d), 50.2(t), 135.3(s), 120.1(d), 26.2(q), 18.5(q), 17.8(q), 17.0(q); FAB-MS(pos) m/z [M+Gly]+361, [M+2Gly]+453; HR-ESI-MS (pos) _m / _z 269.1643 (found) ( _C15H25O3 , theoretical 269.1647).

Example 2: Preparation of Lactomycin 11β-19α-epoxy-4Z, 7Z-humuladiene-5α-ol

A. Extraction:

6 kg of the higher fungus Sclerotinum trichomes collected from Ailao Mountain in Yunnan Province (its strains are preserved in the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences) was crushed, extracted 3 times with 20 L of 95% ethanol, and then extracted with 5 L of ethyl acetate , the solvent was recovered to dryness and weighed to obtain 8.2g;

B, 200-300 mesh silica gel column chromatography separation: use petroleum ether-acetone gradient elution 50:1, 20:1, 9:1, 5:1, 1:1 to obtain 5 components, take petroleum ether-acetone The 9:1 fraction was repeatedly chromatographed on RP-C18 column and recrystallized from petroleum ether-chloroform to obtain lactomycin 2β-3α-epoxy-6Z, 9Z-humuladiene-8α-ol 22 mg.

C. Physicochemical properties of lactomycin 11β-19α-epoxy-4Z, 7Z-humuladiene-5α-ol

Colorless needle crystals; mp 189-191°C; [α] _D ²⁵ -21.3 (c 0.32, CHCl ₃ ); IR(KBr): 3296, 2594, 1640, 1610, 1452, 1387, 1360, 1297 ^{, 972cm - 1} ; ¹ H NMR (500 MHz, CDCl ₃ ) δ: 1.59 (d, 1H, J=14.3, 1-Hα), 1.34 (dd, 1H, J=14.3, 9.8, 1-Hβ), 5.37 (d, 1H , J=16.1, 3-H), 5.72 (dd, 1H, J=16.1, 6.9, 4-H), 4.69 (m, 1H, 5-H), 5.44 (m, 1H, 7-H), 2.46 (m, 1H, 8-Hα), 2.08 (m, 1H, 8-Hβ), 2.02, 1.16 (m, 2H, 9-2H), 2.56 (d, 1H, J=9.8, 11-H), 1.70 (s,3H,14- _CH3 ), 1.16(s,3H,13- _CH3 ), 1.02(s,3H,12- _CH3 ⁾ , 1.16(s,3H,15- _CH3 ); (CDCl ₃ , 500MHz))δ: 40.4(t), 35.1(s), 139.3(d), 132.1(d), 78.1(d), 143.1(s), 124.9(d), 22.7(t), 37.9 (t), 61.4(s), 63.3(d), 30.5(q), 16.1(q), 12.9(q), 23.0(q); HRESIMS(pos) m/z 236.1757 (found) (C ₁₅ H ₂₄ O ₂ , theoretical value 236.1776).

Attachment: Tetrazolium salt (MTT) reduction method to detect the anti-tumor activity test of the above-mentioned lactomycin

1. Experimental materials and reagents

1-1. MTT (MTT): dissolve thiazolium MTT (3-(4,5-dimrthythiazol-z-yl)2,5-diphenytetrazolium bromide, SIGMA company) concentration with 0.01mol/L phosphate buffer 5mg/ml, sterilized by filtration, and stored in the dark at 4°C after aliquoting.

1-2. Configuration of tetrazolium salt lysate: Dissolve 80g of sodium dodecylsulfonate (SDS, Huamei Bioengineering Co., Ltd.) in 200ml of N-N-dimethyldiamide (Beijing Chemical Plant), and heat in a water bath To aid dissolution, add 200ml of distilled water, and adjust the pH to 4.7 with 80% acetic acid and 1N hydrochloric acid (1:1).

1-3. Selection of cell lines: A549 human lung adenocarcinoma cell line, HCT-8 human colon cancer cell line, Bel-7402 human liver cancer cell line, Held human cervical cancer cell line

2. Experimental method

2-1. Take a bottle of cells cultured for 4-5 days and in the exponential growth phase, add 0.05% Trypsin-EDTA solution to make the adherent cells fall off, and use 10 ml of RPMI1640 containing 5% fetal bovine serum (purchased from Gibco, USA) Company) culture medium is made into suspension;

2-2. After staining with trypan blue, count the cells on a hemocytometer;

2-3. Dilute the cell suspension with cell culture medium (purchased from Gibco, USA) to prepare 10000 or 20000 cells per 100ml.

2-4. Take a 96-well plate, add 100 μl of cell suspension to each well, and place the plate in a 37°C carbon dioxide incubator for 24 hours.

2-5. The test compound is set to 5 concentration gradients, which are 6.25ug/ml, 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml;

2-6. Cultivate the plate in an oven containing 5% CO ₂ air and 100% humidity at 37°C for 2-3 days;

2-7. Tetrazolium salt was formulated with serum-free RPMI1640 (purchased from Gibco, USA) culture solution to 1 mg/ml, 50 μl was added to each well, and incubated at 37°C for 4 hours to reduce MTT to formazan (Formdzan);

2-8. Aspirate the supernatant, add 150 μl dimethyl sulfoxide to dissolve formazan, and shake well with a plate shaker;

2-9. Use a microplate reader to detect the absorbance OD value of each well (λ=570nm);

2-10. Draw the cell viability curve and calculate the IC ₅₀ value of the half inhibitory concentration. μg/ml

3. Experimental results

Above-mentioned data shows that Lactomycin of the present invention has strong inhibitory action to cancer cell through in vitro test, and tested 9 hop-type Lactomycins all have stronger antitumor activity, wherein Lactomycin Mitissimol D has the strongest activity, half inhibitory concentration IC to A549 (human lung adenocarcinoma cell line), HCT-8 (human colon cancer cell line), Bel-7402 (human liver cancer cell line), Hela (human cervical cancer cell line) ₅₀ were 2.5, 11.0, 3.6, 4.6 μg/ml, almost equivalent to the positive control drug cisplatin. This type of compound is expected to become a new anti-tumor drug, showing good application value.

In view of the above test conclusions, the present invention provides the application of the lactomycin with anti-tumor activity as the preparation of anti-tumor drugs, including the clinical use for the preparation of anti-tumor drugs with lactomycin as the active ingredient. An antineoplastic drug containing lactomycin pharmaceutically acceptable salt, solvate, or corresponding crystalline substance as an active ingredient, and adding a pharmaceutically acceptable carrier or auxiliary ingredient is prepared.

The pharmaceutical preparation with the lactomycin with anti-tumor activity of the present invention as the main active ingredient can also include adding a pharmaceutically acceptable carrier to make a pharmaceutical composition preparation form, including: tablets, sugar-coated tablets, film-coated tablets, Enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal preparations, granules, granules, pills, powders, ointments, elixirs, suspensions, solutions, injections, suppositories, ointments Potions, plasters, creams, sprays, drops, patches, sustained-release preparations, controlled-release preparations. The preparation of the present invention is preferably in the form of injection, such as: freeze-dried powder injection, injection, large infusion, small water injection, emulsion for injection and the like.

The pharmaceutical preparation with the lactomycin having anti-tumor activity of the present invention as the main active ingredient can be prepared according to the conventional technology of pharmacy.

The pharmaceutical preparation that takes the lactomycin with antitumor activity of the present invention as the main active ingredient, its oral administration preparation may contain commonly used excipients, such as binders, fillers, diluents, tableting agents, lubricating agents, etc. Agents, disintegrating agents, coloring agents, flavoring agents and wetting agents, and tablets can be coated if necessary.

Suitable fillers include cellulose, mannitol, lactose and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants, such as magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.

Solid oral compositions can be prepared by common methods such as mixing, filling, and tabletting. Repeated mixing is performed to distribute the active material throughout those compositions where large amounts of fillers are used.

The pharmaceutical preparation with the lactomycin with anti-tumor activity of the present invention as the main active ingredient can be an aqueous or oily suspension, solution, emulsion, syrup or elixir, or can be a kind of drug that can be used with water or other drugs before use. A dry product formulated with a suitable carrier. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, Emulsifiers, such as lecithin, sorbitan-oleate, or acacia; non-aqueous vehicles (they may include edible oils), such as almond oil, fractionated coconut oil, oily esters of esters such as glycerin, propylene glycol, or ethanol; preservatives agents, such as methyl or propyl paraben, or sorbic acid, and, if desired, conventional flavoring or coloring agents.

In the injection containing the lactomycin with anti-tumor activity of the present invention as the main active ingredient, the complex can be suspended or dissolved. The solution is usually prepared by dissolving the active substance in a carrier, and then putting it into a A suitable vial or ampoule is filter sterilized before sealing. Adjuvants such as a local anesthetic, preservatives and buffering agents can also be dissolved in the vehicle. To increase its stability, the composition can be frozen after filling vials and the water removed under vacuum.

The pharmaceutical preparation with the lactomycin having anti-tumor activity of the present invention as the main active ingredient can optionally be added with a pharmaceutically acceptable carrier selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, bisulfite Sodium, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, Acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose And its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, Twain 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc.

The dosage of the pharmaceutical preparation which takes the lactomycin with anti-tumor activity of the present invention as the main active ingredient is determined according to the condition of the patient.

The pharmaceutical preparation with the lactomycin with anti-tumor activity of the present invention as the main active ingredient can be in the form of a unit dosage. or its pharmaceutically acceptable salt, solvate, and corresponding crystalline substance 0.1-1000mg, accounting for 0.1-99.9% of the total weight of the unit preparation, the rest is a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is in the form of It can be 0.1-99.9% by weight of the total weight of the preparation.

The examples listed in the present invention are intended to further clarify the structure, preparation method, formulation method and application direction of this type of lactomycin with anti-tumor activity, without any limitation to the protection scope of the present invention.

Claims (4)

1. The hopane sesquiterpene lactomycin mitissimol H with anti-tumor activity has the following structure:

2. according to the preparation method of the hopane sesquiterpene lactomycin mitissimol H with antitumor activity according to claim 1, it is characterized in that it comprises the steps: A, extraction: pulverize 1.6kg of higher fungus Lactarius mitissimus, extract 3 times with 95% ethanol of 15L, then extract with 4.5L of ethyl acetate, recycle the solvent to dryness, and weigh 33.5g; B, into 200-300 mesh silica gel column chromatography separation: Use CHCl ₃ -MeOH solvent with a volume ratio of 100:0, 98:2, 95:5, 90:10 for gradient elution, and obtain a component in the elution section of CHCl ₃ -MeOH 95:5 for further silica gel column chromatography , with petroleum ether-acetone solvent at a volume ratio of 10:1, 8:1, 6:1, 4:1, 5:1, 2:1, 1:1 gradient elution, at the petroleum ether-acetone 4:1 site Obtain pure lactobacillus mitissimol H 7mg. 3. according to the application of the hopane sesquiterpene lactomycin mitissimol H with antitumor activity according to claim 1 as the preparation of antitumor drugs, it is characterized in that: it is used to prepare clinically with lactomycin as active ingredient of antineoplastic drugs. 4. according to the application of the hopane sesquiterpene lactomycin mitiSsimol H with antitumor activity according to claim 3 as the preparation of antitumor drugs, it is characterized in that: it is used to prepare clinically containing lactomycin as active ingredient Compositions or compounds for preparing anti-tumor drugs containing lactomycin pharmaceutically acceptable salts, solvates, or substances in corresponding crystal forms as active ingredients, and adding pharmaceutically acceptable carriers or auxiliary components.

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