WO2022242620A1 - Dimeric sesquiterpenoids, preparation method therefor, and use thereof - Google Patents

Abstract

The present invention provides the following dimeric sesquiterpenoids, a preparation method therefor, and a use thereof. The dimeric sesquiterpenoids have novel chemical structures and remarkable anti-malarial biological activities, have good development prospects, and may be developed into novel anti-malarial drugs without cross-drug resistance.

Description

Dimeric sesquiterpene compound, its preparation method and use technical field

The invention belongs to the field of natural medicine preparation, and relates to dimerized sesquiterpene compounds, a preparation method thereof and an application in preparation of antimalarial medicines.

Background technique

Malaria is a global acute parasitic infectious disease caused by Plasmodium and transmitted by Anopheles mosquitoes as the main vector. It is one of the three major infectious diseases in the world and seriously endangers human health, social stability and economic development. According to the World Malaria Report 2019 by the World Health Organization, there are an estimated 229 million cases of malaria worldwide, resulting in more than 400,000 deaths, of which children under the age of 5 account for as much as 67%. Among the five pathogenic malaria parasites, Plasmodium falciparum (Plasmodium.faleiparum) and Plasmodium vivax (P.vivax) are the most common, and among them, Plasmodium falciparum is the most harmful. Although the prevalence and mortality of malaria have been significantly reduced, malaria prevention and control is still a major challenge in Africa and other regions with low income and low medical level. Due to the lack of an effective vaccine, artemisinin combination therapy is currently the most effective means of treating malaria. However, as Plasmodium falciparum has developed clinical resistance to first-line antimalarial drugs including artemisinin, antimalarial work has been hindered, and new antimalarial drugs without cross-drug resistance are urgently needed.

Natural products are an important source for discovering drugs or lead structures for the treatment of major diseases. Our country has a long history of understanding and treatment of malaria. As early as 2000 AD, the "On Malaria" and "Thorn" in "Huangdi Neijing Suwen" discussed the etiology, symptoms and treatment methods of malaria. Monographs. The discovery of artemisinin has made a great contribution to the world's antimalarial work, and it also shows that starting from the use of traditional medicinal plants, the activity-oriented isolation and extraction of active ingredients is an important source of drug candidates or lead structures.

The genus Sarcandra belongs to Chloranthaceae (Chloranthaceae), and there are only three species in the world. There are two species in my country: S. glabra and S. glabra subsp. brachystachys. Hainan grass coral is a unique species in my country. It is mainly produced in Yunnan, Hainan and Guangdong, and it mostly grows in the shade of hillsides, valleys and forests.

Contents of the invention

The present inventors conducted a systematic study on the chemical components of Coral hainanensis, and found a class of dimeric sesquiterpenoids, and activity tests showed that they had significant anti-malarial effects. On the one hand, the present invention provides a class of dimeric sesquiterpenoids with novel structures, including the following 8 structurally novel dimeric sesquiterpenoids, by studying the chemical components of the ethanol extract of the plant of the genus Chrysostasis genus-Hainania chinensis. Polysesquiterpenoids:

Among them, the structure of compounds 1-6 is a dimer formed by the endo Diels-Alder cycloaddition of two molecules of bugatane-type sesquiterpene monomers, and the structure of compounds 7 and 8 is a molecule of bugatane-type sesquiterpene A dimer formed by the endo Diels-Alder cycloaddition of a monomer and a molecule of eucalyptane-type sesquiterpene monomer.

Another aspect of the present invention provides a method for preparing the dimerized sesquiterpenoids, which can be obtained by separating the dimerized sesquiterpenoids from Chrysanthemum spp. :

(1) Extract the whole herb powder of Pseudocarpus with ethanol aqueous solution at room temperature, and concentrate the extract under reduced pressure to obtain extract;

(2) adding water to the extract obtained in step (1) for dilution, then extracting with ethyl acetate, and concentrating the organic phase under reduced pressure to obtain a crude extract;

(3) Pass the crude extract obtained in step (2) through a macroporous resin column, carry out gradient elution with ethanol aqueous solution with a volume fraction of 30%, 50%, 80%, and 95%, and collect the ethanol aqueous solution with a volume fraction of 50%. The obtained component A is removed, and the collected component B obtained by eluting the ethanol aqueous solution with a volume fraction of 80%; the component A and the component B are respectively passed through the MCI column, and the volume ratio is successively 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 mixed solution of methanol and water for gradient elution, collected from component A with volume ratio of 5:5 and 6:4 methanol and water Component A2 obtained by eluting with the mixed solution of Component A, collecting Component A4 eluted from Component A with a mixed solution of methanol and water with a volume ratio of 8:2 and 9:1, and collecting Component A4 obtained from Component B with a volume ratio of 9 Component B3 obtained by eluting the mixed solution of methanol and water in 1;

(4) Component A2 is subjected to silica gel column chromatography, using a mixed solvent of chloroform and methanol with a volume ratio of 100:1, 70:1, 50:1, 20:1, and 10:1 for gradient elution, and the collection volume ratio is 70 The eluent eluting with the mixed solvent of 1 chloroform and methanol obtains component A2b;

Component A2b was purified by gel column chromatography and further purified by semi-preparative HPLC, and eluted isocratically with 35% acetonitrile aqueous solution to obtain compound 6;

(5) Component A4 was subjected to silica gel column chromatography, and gradient elution was performed using a mixed solvent of chloroform and methanol with a volume ratio of 500:1, 100:1, 70:1, 50:1, 20:1, and 10:1, Collect the eluate eluted with a mixed solvent of chloroform and methanol at a volume ratio of 50:1 to obtain component A4e;

Component A4e was purified by gel column chromatography and then purified by semi-preparative HPLC, and was eluted isocratically with 45% acetonitrile aqueous solution to obtain compounds 5 and 8;

(6) Component B3 is subjected to silica gel column chromatography, with a volume ratio of 10:1, 8:1, 6:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3 After gradient elution with a mixed solvent of petroleum ether and acetone, collect the eluent eluted with a mixed solvent of petroleum ether and acetone at a volume ratio of 2:1 and 1:1 to obtain component B3h;

Component B3h was purified by gel column chromatography, eluted with ethanol, monitored by TLC spot plate, and divided into four components B3h1～B3h4 according to the order of elution time;

Component B3h1 uses a mixed solvent of chloroform and methanol with a volume ratio of 100:1, 70:1, 50:1, 20:1, and 10:1 for gradient elution, and collects a mixed solvent of chloroform and methanol with a volume ratio of 50:1 The eluted eluate yields fraction B3h1b;

Component B3h1b was further purified by semi-preparative HPLC and eluted isocratically with 55% volume fraction of acetonitrile in water to obtain compounds 2 and 4;

Component B3h3 is eluted using a mixed solvent of chloroform and methanol with a volume ratio of 100:1, 70:1, 50:1, 20:1, and 10:1, and a mixed solvent of chloroform and methanol with a volume ratio of 70:1 is collected The eluted eluate yields fraction B3h3b;

Component B3h3b was purified by gel column chromatography, eluted with methanol, monitored by TLC spot plate, and divided into two components B3h3b1 and B3h3b2 according to the order of elution time;

Component B3h3b1 was purified by semi-preparative HPLC, and was eluted isocratically with 55% volume fraction of acetonitrile in water to obtain compounds 1 and 7;

Component B3h3b2 was purified by semi-preparative HPLC, and compound 3 was obtained by isocratic elution with 65% methanol aqueous solution.

In the above method, the aqueous ethanol solution described in step (1) can be an aqueous ethanol solution above 70% v/v, preferably an aqueous ethanol solution above 85% v/v, more particularly ethanol above 95% v/v Aqueous solution; the extraction time at room temperature is not particularly limited, for example, it can be more than 4 hours, more than 10 hours, or more than 24 hours; the extraction can be performed once or more times, such as 1, 2, 3 times or more than 3 times.

The above-mentioned compounds 1-8 were screened for antimalarial activity, and the specific activity data are shown in Table 4. The compound has a novel chemical structure and remarkable biological activity, has good development prospects, and is expected to develop into a new antimalarial drug without cross-drug resistance.

Another aspect of the present invention provides a pharmaceutical composition, which contains one or more selected from the above-mentioned dimerized sesquiterpenoids as active ingredients. Optionally, the composition may further include pharmaceutically acceptable Acceptable pharmaceutical excipients, such as carriers, excipients, adjuvants and/or diluents, etc. The pharmaceutical composition can be used as an antimalarial drug.

Another aspect of the present invention also provides the use of the dimeric sesquiterpene compound or the pharmaceutical composition in the preparation of antimalarial drugs.

Another aspect of the present invention also provides a method for treating malaria, which includes administering one or more of the above-mentioned dimerized sesquiterpenoids as antimalarial active ingredients to patients in need of the treatment, or the above-mentioned pharmaceutical composition.

The present invention has been described in detail above, but the above-described embodiments are merely illustrative in nature and are not intended to limit the present invention. Furthermore, this document is not to be bound by any theory presented in the preceding prior art or brief summary or the following examples.

Unless expressly stated otherwise, throughout this application, numerical ranges include any subranges therein and any numerical increments by the smallest subunit of a given value therein. Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations on the range including minor deviations from the given value and embodiments having about the recited value as well as having the exact value recited. Except for the working examples provided at the end of the detailed description, all numerical values of parameters (for example, amounts or conditions) in this specification (including the appended claims) should in all cases be understood as being understood by the term "about" modifier, regardless of whether "about" actually precedes the numerical value. "About" indicates that the stated value allows for some imprecision (some close to exactness in the value; about or reasonably close to the value; approximation). If the imprecision provided by "about" is not understood in the art with this ordinary meaning, then "about" as used herein at least indicates the variation that can be produced by ordinary methods of measuring and using these parameters. For example, "about" can include variations of 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less .

Detailed ways

The preparation steps and pharmacological experiment process of the compound of the present invention are further illustrated below through specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention, and those skilled in the art can make various modifications and changes to this, without departing from the spirit and scope of the present invention, all of these Modifications are encompassed within the scope of the invention as defined in the appended claims.

Experimental equipment and reagents:

Instruments and equipment: Infrared: Scientific Nicolet iS 5 Fourier transform infrared spectrometer (KBr chip); UV: shimadzu UV-2550 UV-Vis spectrophotometer; Optical rotation: Autopol VI polarimeter; NMR: Bruker Avance III 500/600 NMR chromatography Instrument, with TMS as internal standard; mass spectrometry: LR (±) ESI mass spectrometer, Bruker Daltonics Esquire 3000 plus mass spectrometer; HRESI (±) MS, Waters Q-TOF Ultima Global mass spectrometer; semi-preparative HPLC: Waters 1525 double pump , Waters 2489 detector (210nm), YMC-Pack ODS-A chromatographic column (250×10mm, S-5μm, 12nm).

Reagents and materials: prefabricated TLC silica gel plate (GF ₂₅₄ ) and chromatography silica gel: use 200-300 and 300-400 mesh silica gel as the carrier, Qingdao Ocean Chemical Co., Ltd.; D101 macroporous resin: Shanghai Hualing Resin Co., Ltd.; CHP20P MCI gel (75–150 μM): Mitsubishi Chemical Co., Ltd.; Sephadex LH-20 gel: Amersham Biosciences; analytical pure solvent: Sinopharm Chemical Reagent Co., Ltd.; HPLC grade solvent: Bailingwei Technology Co., Ltd.

Preparation of compound 1-8 in embodiment 1 of the present invention

The dried whole herb powder (8Kg) of Hainan grass coral was extracted 3 times with 95% ethanol at room temperature, and the extract was concentrated under reduced pressure to obtain 500g extract, diluted with an appropriate amount of distilled water and extracted 4 times with ethyl acetate, and the ethyl acetate part was decompressed. Obtain 250g crude extract after concentrating, after this part gradient elution through macroporous resin column (eluent is the ethanol aqueous solution that volume fraction is successively 30%, 50%, 80%, 95%), collection volume fraction is 50% Component A (60g) obtained by elution of aqueous ethanol solution, and the component B (65g) obtained by elution of aqueous ethanol solution with a volume fraction of 80% was collected, and component A and component B were eluted through MCI column gradient respectively (washing The deliquification is the mixed solution of methanol and water whose volume ratio is 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1), and the volume ratio is collected from component A with volume Component A2 (8g) was eluted with a mixed solution of methanol and water at a ratio of 5:5 and 6:4, and collected from component A eluted with a mixed solution of methanol and water at a volume ratio of 8:2 and 9:1 Component A4 (10 g) was obtained, and component B3 (15 g) obtained by eluting component B with a mixed solution of methanol and water at a volume ratio of 9:1 was collected.

Component A2 (8g) was eluted by silica gel column chromatography (200-300 mesh) (chloroform/methanol (v/v): 100:1, 70:1, 50:1, 20:1, 10:1) gradient elution Finally, collect the eluate eluted with a mixed solvent of chloroform and methanol at a volume ratio of 70:1 to obtain component A2b. Component A2b (800 mg) was purified by gel column chromatography (Sephadex LH-20, ethanol elution) and further purified by semi-preparative HPLC (volume fraction 35% acetonitrile aqueous solution isocratic elution) to obtain compound 6 (1.5 mg) .

Component A4 (10g) was subjected to silica gel column chromatography (200-300 mesh) (chloroform/methanol (v/v): 500:1, 100:1, 70:1, 50:1, 20:1, 10:1 ) gradient elution, collect the eluate eluted with a mixed solvent of chloroform and methanol at a volume ratio of 50:1 to obtain component A4e. Component A4e (400 mg) was purified by gel column chromatography (Sephadex LH-20, methanol elution) and further purified by semi-preparative HPLC (volume fraction 45% acetonitrile aqueous solution isocratic elution) to obtain compound 5 (5.1 mg) and 8 (2.0mg).

Component B3 (15g) was subjected to silica gel column chromatography (200-300 mesh) (petroleum ether/acetone (v/v): 10:1, 8:1, 6:1, 5:1, 3:1, 2: 1, 1:1, 1:2, 1:3) gradient elution, collect the eluate eluted with the mixed solvent of petroleum ether and acetone with a volume ratio of 2:1 and 1:1 to obtain component B3h. Component B3h (2g) was purified by gel column chromatography, eluted with ethanol, and divided into four components B3h1～B3h4 according to the order of elution time and monitored by TLC spot plate.

Component B3h1 (500mg) was eluted by gradient silica gel column chromatography (300-400 mesh) (chloroform/methanol (v/v): 100:1, 70:1, 50:1, 20:1, 10:1) , collecting the eluate eluted with a mixed solvent of chloroform and methanol at a volume ratio of 50:1 to obtain component B3h1b.

Component B3h1b (50 mg) was finally purified by semi-preparative HPLC (55% volume fraction of acetonitrile in water isocratic elution) to obtain compounds 2 (8.1 mg) and 4 (2.1 mg).

Component B3h3 (400mg) was eluted by silica gel column chromatography (300-400 mesh) (chloroform/methanol (v/v): 100:1, 70:1, 50:1, 20:1, 10:1) gradient elution , collecting the eluate eluted with a mixed solvent of chloroform and methanol at a volume ratio of 70:1 to obtain component B3h3b.

Component B3h3b (150 mg) was purified by gel column chromatography, eluted with methanol, and divided into two components B3h3b1 and B3h3b2 according to the order of elution time and monitored by TLC spot plate.

Component B3h3b1 (30 mg) was purified by semi-preparative HPLC and eluted isocratically with 55% acetonitrile in water to give compounds 1 (1.2 mg) and 7 (10.2 mg).

Component B3h3b2 (17 mg) was purified by semi-preparative HPLC and eluted isocratically with 65% methanol in water to give compound 3 (3.0 mg).

Some physical and chemical data of the compound are as follows:

Table 1. ¹ HNMR (CDCl ₃ ) data of compounds 1-4 in the present invention

^a Measured with a 600MHz nuclear magnetic resonance instrument; ^b Measured with a 500MHz nuclear magnetic resonance instrument.

Table 2. ¹ HNMR (600MHz, CDCl ₃ ) data of compounds 5-8 in the present invention

Table 3. ¹³ CNMR (CDCl ₃ ) data of compounds 1-8 in the present invention

^* The signals in the corresponding columns are interchangeable; ^a is measured with a 125MHz nuclear magnetic resonance instrument; ^b is measured with a 150MHz nuclear magnetic resonance instrument.

Embodiment 2 The antimalarial activity test of compound of the present invention

Experimental principle:

Anti-malarial experiment: The dose-dependent growth inhibition assay of Plasmodium falciparum (P.falciparum) chloroquine-resistant strain Dd2 is determined with reference to the method in the prior art, that is, using a slightly modified malaria-based SYBR Green I The fluorescent method was used to test the growth of malaria parasites in the presence of inhibitors for 72 hours, and artemisinin was used as a positive control. The operation method can be briefly expressed as follows: take the Plasmodium ring body stage culture medium and place it in a 96-well plate, add different concentrations of medicinal solutions to achieve a total volume of 100 μL per well, containing 1% hematocrit (erythrocyte blood) and 1% parasitaemia (parasite-infected blood). Subsequently, the 96-well plate was maintained in a mixed gas composed of 5% CO ₂ , 5% O ₂ and 90% N ₂ , and cultured at 37° C. for 72 hours. After 72 hours, the viability of Plasmodium parasites in the medium was determined by measuring the DNA content by SYBR Green I fluorescence detection. Half-maximum effective concentration (EC ₅₀ ) is a nonlinear regression curve fitting using GraphPad Prism (GraphPad Software, Inc.) software, and the reported data is the average value of at least three parallel test results, using 10 times The serial dilution method was used to test, and the standard error value (Standard Error of Mean, SEM) was given.

Table 4. Antimalarial activity results of compounds 1-8 in the present invention

Table 4 shows the antimalarial activity data of the compounds of the present invention. Among them, the antimalarial activity of compound 1 is very significant, with an EC ₅₀ of 0.0043±0.0003nM, which is 1000 times stronger than that of artemisinin (4.0±4.2nM).

Claims (6)

1. Dimeric sesquiterpenoids selected from the group consisting of:

   
2. The preparation method of dimerization sesquiterpenoid compound described in claim 1, comprises the steps:

   (1) Extract the whole herb powder of Pseudocarpus with ethanol aqueous solution at room temperature, and concentrate the extract under reduced pressure to obtain extract;

   (2) adding water to the extract obtained in step (1) for dilution, then extracting with ethyl acetate, and concentrating the organic phase under reduced pressure to obtain a crude extract;

   (3) Pass the crude extract obtained in step (2) through a macroporous resin column, carry out gradient elution with ethanol aqueous solution with a volume fraction of 30%, 50%, 80%, and 95%, and collect the ethanol aqueous solution with a volume fraction of 50%. The obtained component A is removed, and the collected component B obtained by eluting the ethanol aqueous solution with a volume fraction of 80%; the component A and the component B are respectively passed through the MCI column, and the volume ratio is successively 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 mixed solution of methanol and water for gradient elution, collected from component A with volume ratio of 5:5 and 6:4 methanol and water Component A2 obtained by eluting with the mixed solution of Component A, collecting Component A4 eluted from Component A with a mixed solution of methanol and water with a volume ratio of 8:2 and 9:1, and collecting Component A4 obtained from Component B with a volume ratio of 9 Component B3 obtained by eluting the mixed solution of methanol and water in 1;

   (4) Component A2 is subjected to silica gel column chromatography, using a mixed solvent of chloroform and methanol with a volume ratio of 100:1, 70:1, 50:1, 20:1, and 10:1 for gradient elution, and the collection volume ratio is 70 The eluent eluting with the mixed solvent of 1 chloroform and methanol obtains component A2b;

   Component A2b was purified by gel column chromatography and further purified by semi-preparative HPLC, and eluted isocratically with 35% acetonitrile aqueous solution to obtain compound 6;

   (5) Component A4 was subjected to silica gel column chromatography, and gradient elution was performed using a mixed solvent of chloroform and methanol with a volume ratio of 500:1, 100:1, 70:1, 50:1, 20:1, and 10:1, Collect the eluate eluted with a mixed solvent of chloroform and methanol at a volume ratio of 50:1 to obtain component A4e;

   Component A4e was purified by gel column chromatography and purified by semi-preparative HPLC, and was eluted isocratically with 45% acetonitrile aqueous solution to obtain compounds 5 and 8;

   (6) Component B3 is subjected to silica gel column chromatography, with a volume ratio of 10:1, 8:1, 6:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3 After gradient elution with a mixed solvent of petroleum ether and acetone, collect the eluent eluted with a mixed solvent of petroleum ether and acetone at a volume ratio of 2:1 and 1:1 to obtain component B3h;

   Component B3h was purified by gel column chromatography, eluted with ethanol, and divided into four components B3h1～B3h4 according to the order of elution time and monitored by TLC spot plate;

   Component B3h1 uses a mixed solvent of chloroform and methanol with a volume ratio of 100:1, 70:1, 50:1, 20:1, and 10:1 for gradient elution, and collects a mixed solvent of chloroform and methanol with a volume ratio of 50:1 The eluted eluate yields fraction B3h1b;

   Component B3h1b was further purified by semi-preparative HPLC and eluted isocratically with 55% volume fraction of acetonitrile in water to obtain compounds 2 and 4;

   Component B3h3 is eluted using a mixed solvent of chloroform and methanol with a volume ratio of 100:1, 70:1, 50:1, 20:1, and 10:1, and a mixed solvent of chloroform and methanol with a volume ratio of 70:1 is collected The eluted eluate yields fraction B3h3b;

   Component B3h3b was purified by gel column chromatography, eluted with pure methanol, and divided into two components B3h3b1 and B3h3b2 according to the sequence of elution time and monitored by TLC spot plate;

   Component B3h3b1 was purified by semi-preparative HPLC, and was eluted isocratically with 55% volume fraction of acetonitrile in water to obtain compounds 1 and 7;

   Component B3h3b2 was purified by semi-preparative HPLC, and compound 3 was obtained by isocratic elution with 65% methanol aqueous solution.
3. A pharmaceutical composition comprising one or more selected from the dimerized sesquiterpenoids described in claim 1 as active ingredients.
4. The pharmaceutical composition as claimed in claim 3, which further comprises pharmaceutically acceptable pharmaceutical excipients.
5. The pharmaceutical composition according to claim 3 or 4, which is an antimalarial drug.
6. Use of the dimerized sesquiterpenoid compound according to claim 1 or the pharmaceutical composition described in claim 3 or 4 for the preparation of antimalarial drugs.