RU2624861C2 - Nuflein bisanhydrochloride, with cytotoxic activity related to human tumour cells - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention is application of a Nuflein bisanhydrohydrodehyde chloride compound of formula I

,

which has selective cytotoxic activity on human cervical cancer cells. The invention allows application of a Nuflein bisanhydrohydrodehyde chloride compound preparation based on plant material for a new purpose.

EFFECT: preparation has the lowest cytotoxic effect with respect to healthy cells.

3 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of creating highly effective antitumor preparations of plant origin, namely to nouflein bisanhydrodichloride having selective, cytotoxic activity on human cervical cancer cells. The invention may find application in medicine for the treatment of malignant tumors of the female genital area.

One of the strategies for treating malignant neoplasms is drug therapy using various cytostatic agents, including herbal origin.

) [Д.А. Муравьева, И.А. Самылина, Г.П. Яковлев. Фармакогнозия, 2002, стр. 413]. Кубышка желтая распространена по всей России, за исключением горных районов и Арктики, растет зарослями в прудах, озерах, по берегам речек с тихим течением. Препарат «Лютенурин» впервые был разработан во Всероссийском научно-исследовательском институте лекарственных и ароматических растений, представляет собой сумму гидрохлоридов димерных серосодержащих фуранохинолизидиновых алкалоидов из корневищ кубышки желтой (Nuphar luteae (L.) Smith syn. Nuphar luteum. L.) семейства кувшинковых. Антимикробная активность препарата обусловлена основным компонентом суммы алкалоидов - бисангидродихлоридом нуфлеина. [М.Е. Перельсон, Т.Н. Ильинская, О.Н. Толкачев. Структура нуфлеина - алкалоида Nuphar Luteum // Химия природных соединений, 1975, №6, стр. 768-770]A fairly well-studied alkaloid is Lutenurin, obtained from a yellow capsule (lat. -

) [YES. Muravyova, I.A. Samylina, G.P. Yakovlev. Pharmacognosy, 2002, p. 413]. The yellow capsule is widespread throughout Russia, with the exception of mountainous regions and the Arctic, growing in thickets in ponds, lakes, along the banks of rivers with a quiet course. The drug “Lutenurin” was first developed at the All-Russian Scientific Research Institute of Medicinal and Aromatic Plants; it is the sum of hydrochlorides of dimeric sulfur-containing furanoquinolizidine alkaloids from the rhizomes of the yellow egg capsule (Nuphar luteae (L.) Smith syn. Nuphar luteum. L.) of the water lily family. The antimicrobial activity of the drug is due to the main component of the amount of alkaloids - nuflein bisanhydrodichloride. [M.E. Perelson, T.N. Ilyinskaya, O.N. Tolkachev. The structure of nuflein - alkaloid Nuphar Luteum // Chemistry of Natural Compounds, 1975, No. 6, pp. 768-770]

The drug Lutenurin is a mixture of hydrochlorides, mainly dimeric sulfur-containing quinolysidine alkaloids: thiobinufaridine, neothiobinufaridine, nuflein. The active principle of the drug is the alkaloid nuflein (dihydroxy-thiobinufaridine) (T. N. Ilyinskaya, A. D. Kuzovkov, T. G. Monakhova. Chemical study of yellow capsule alkaloids, in the book: "Medicinal Plants. Chemistry", Volume 15, VILAR, under the editorship of A.I. Bankovsky, Kolos, Moscow, 1969, p. 322-333). The well-known drug Lutenurin (ed. Certificate of the USSR No. 145858, FS 42-948-75) has a high antibacterial activity and is used to treat trichomonas urogenital diseases and as a contraceptive.

There are various methods of obtaining the drug Lutenurin developed at the All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR).

So, for example, the method of obtaining the drug Lutenurin consists in the fact that the dry extract of the sum of alkaloids of the rhizome of the egg capsule is dissolved in ethanol, a 20% solution of methylene-bis-salicylic acid is added, or dissolved in 10% hydrochloric acid, filtered from tar impurities , add a 10% solution of sodium perchlorate and the resulting lutenurin - base (or lutenurin perchlorate) is treated by known methods (ed. certificate. USSR No. 145184).

Also known is a method of producing lutenurin from the rhizomes of the yellow capsule (Nuphar Lutea (L.), which consists in extracting raw materials, extracting alkaloids and obtaining hydrochlorides by saturating the alkaloids with hydrogen chloride, characterized in that the raw materials are extracted at the head stage with 20-60% aqueous an aliphatic alcohol, preferably ethyl or isopropyl acidified with 5% phosphoric acid, is filtered, the organic solvent is removed, the aqueous bottoms after distillation of the alcohol are treated with butanol, which is then It is separated, the aqueous acid phase after neutralization to pH 8 is treated with calcium carbonate to remove phosphate ions, the precipitate is filtered, washed with acetone and the filtrate is evaporated to dryness, the residue is dissolved in diluted sulfuric acid, alkalized to pH 8, the alkaloids are extracted with toluene, the extractant is evaporated, the extractant is evaporated and hydrochlorides are obtained (US Pat. RF No. 2292218).

A known method for producing lutenurin, which consists in the following: dried and crushed rhizomes of yellow capsules containing 0.4% of the total alkaloids, is treated with ammonium hydroxide, extracted with dichloroethane, then extracted with sulfuric acid, the aqueous phase is made alkaline, with ammonium hydroxide, and the alkaloids are extracted with ether, after alkaloids are extracted with ether drying the extract with potash it is concentrated, impurities are precipitated with petroleum ether, and then hydrochlorides are obtained by saturating the alkaloids solution with hydrogen chloride, the target product is separated by washing petroleum ether, dried and sieved through a sieve ("Medicinal substances from plants and methods for their production", Tashkent, FAN Uz SSR, 1980, S. 10-12).

The active principle of the drug Lutenurin is the alkaloid nuflein (dihydroxy-thiobinofaridine) of the formula 1 (T. N. Ilyinskaya, A. D. Kuzovkov, T. G. Monakhova. Chemical study of yellow capsule alkaloids, in the book: "Medicinal plants. Chemistry", Volume 15, VILAR, under the editorship of A.I. Bankovsky, "Kolos", Moscow, 1969, p. 322-333).

Formula 1. Nuflein bisanhydrodihydrochloride

As a result of the studies conducted by the authors, a high cytotoxic activity of nouflein bisanhydrodichloride of formula 1, which is the subject of this study, was found.

The main objective of this invention is the creation of a new drug based on plant materials with cytotoxic activity on human cervical cancer cells, high selective activity against tumor cells and the least cytotoxic effect on healthy cells.

Alkaloids obtained from medicinal plants may possess such properties.

This goal is achieved through the use of the compound nuflein bisanhydrohydrochloride of the formula I

C30H40N2C12 mm 563.6

possessing selective, cytotoxic activity on human cervical cancer cells.

Nuflein bisanhydro dihydrochloride of formula 1 is 100 times more effective than foreign cisplatin for the same purpose. The mechanism of cytotoxic activity of nouflein bisanhydrodihydrochloride is associated with the dissociation of oxidative phosphorylation in mitochondria, the induction of the mitochondrial apoptosis pathway.

The invention is illustrated by the following examples.

Example 1. Obtaining Nuflein bisanhydrohydrochloride

Nuflein is obtained in a known manner, which consists in the extraction of raw materials, the extraction of alkaloids and the production of hydrochlorides by saturating the alkaloids solution with hydrogen chloride. The resulting preparation "Lutenurin" is dissolved in a solution of chloroform, which is treated preferably with a 10% solution of hydrochloric acid. The alkaloids hydrochlorides formed are distributed by solubility between chloroform and aqueous acid salts; as a result, up to 45% of side alkaloids hydrochlorides remain in chloroform, and nouflein with accompanying alkaloids are completely converted into acid solutions, which, after alkalization, are extracted with chloroform. The residue after evaporation in vacuo is chromatographed on a silica gel column (chloroform is used as eluent), the nouflein-containing fractions are taken, evaporated, and dissolved in diethyl ether, from which the target product is removed with a 5% hydrochloric acid solution. Acidic solutions are treated with sodium perchlorate solution, get nouflein perchlorate, crystallize and check its purity, then get nouflein bisanhydrodichloride.

The purity of the obtained substance of nouflein bisanhydrodichloride is confirmed by the absence of signals from other substances in the 1H NMR spectrum (see Fig. 1).

Explanation of Fig. one

In the ¹ H NMR spectrum (Fig. 1) of a solution of nouflein in a mixture of solvents benzene: chloroform: trifluoroacetic acid (2: 1: 3) the chemical shifts of the signals have the following values (in ppm): 0.69 and 0.67 doublets 3H (1.1 'methyl groups), 2.47 and 2.62 (doublets ² J = 15.2 Hz) - protons of the methylene group in the five-membered ring in the β-position to the sulfur atom, 2.76 and 3.11 (doublets J = 12.3 Hz) - protons of the methylene group in the α-position to the sulfur atom; 3.44 (2H) multiplets with broadened components - protons at positions 1 and 1 ', broadened singlets 4.11, 4.17, 4.75 and 4.80 belong to protons 10.10' and 4, 4 ', respectively. In furan rings, the signals are 6.26 and 6.36 ppm. (β-protons of furan rings), 7.30 (2H), 7.38, 7.52 and 7.58 ppm. (α-protons of furan rings), 7.58 and 7.83 ppm. (protons of the ammonium fragment).

Example 2. Determination of the cytotoxic activity of nuflein

The cytotoxic activity of nouflein bisanhydrodichloride was evaluated on cultures of tumor cells and normal cells. As a model of tumor cells, human cervical cancer cells HeLa, sensitive to cytostatics, were used. This cell culture is a standard object for evaluating the cytotoxic effect of antitumor compounds (Federal Guidelines for Preclinical Testing of Medicines, 2014). Fast dividing rat skin fibroblasts were used as normal cells. The effect of nuflein bisanhydrodichloride on energy supply parameters of the cell was studied on isolated mitochondria obtained from rat liver. The cytotoxic activity of nouflein bisanhydrodichloride and the cisplatin comparison drug was determined by MTT spectrophotometric method. HeLa cells were incubated at 37 ° C, under conditions of 5% CO ₂ in standard DMEM culture medium and 10% fetal calf with test substances in a concentration range of 10 ^-7 -10 ^-4 M for 48 h, after which they were 3 h, 3- (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide (MTT) was added to a final concentration of 0.5 mg / ml. Then, the liquid from the wells was removed and 150 μl of dimethyl sulfoxide were added for 30 min. MTT is restored due to the action of dehydrogenases of living cells to water-insoluble crystals of formazan. The amount of formazan formed (determined by the colorimetric method after it is dissolved in organic solvents) and characterizes the intensity of the redox processes in tumor cells and can be used as a criterion for the cytotoxic effect of the studied compounds. The optical density of the samples was recorded at a wavelength of 530 nm using the UNIPLAN ELISA enzyme-linked immunosorbent assay AIFR-01 (Russia). The optical density of the control samples was taken as 100% cell survival.

Nuflein bisanhydrodichloride has been shown to have a cytotoxic effect 100 times greater than the cytotoxic effect of Cisplatin. So, IC ₅₀ - the concentration that causes 50% cell death - for cisplatin was 6.2 * 10 ^-6 M, and for nuflein - 2.89 * 10 ^-8 M (see Fig. 2). In Fig. Figure 2 shows the effect of nuflein bisanhydrodichloride on the viability of HeLa tumor cells, incubation for 48 hours.

Bisanhydrodichloride had almost 100 times less pronounced cytotoxic effect on normal, transformed skin fibroblasts of rat skin than on tumor cells (see Fig. 3. The effect of bisanhydrodichloride nuflein on the viability of rat skin fibroblasts).

The mechanisms of the cytotoxic effect of nouflein bisanhydrodichloride are associated with the effect on mitochondria - key cellular organelles involved both in the processes of energy supply of the cell and in the processes of apoptosis.

Mitochondria were obtained from the liver of adult male Wistar rats by standard differential centrifugation [Fedotcheva N.I., Teplova V.V., Fedotcheva T.A., Rzheznikov V.M., Shimanovskii N.L. Effect of progesterone and its synthetic analog on the activity of mitochondrial permeability transition pore in isolated rat liver mitochondria // Biochemical Pharmacology. 2009. V. 78. No. 8. P. 1060-1068]. The liver was homogenized in ice-cold 10 mM HEPES-Tris (pH 7.4) buffer containing 220 mM mannitol, 70 mM sucrose, 1 mM EGTA. The homogenate was centrifuged at 600 g for 7 min and 4 ° C, then the supernatant was centrifuged at 9,000 g for 10 min to obtain a precipitate with mitochondria. Mitochondria were washed twice with the above buffer without EGTA. The final mitochondrial pellet was suspended in wash buffer to obtain 60-80 mg protein / ml. Mitochondria were incubated in a medium containing 120 mM KCl, 1.5 mM KH2PO4, 10 mM HEPES (pH 7.25). Control samples with solvent were carried out in each series of experiments.

The effect of nouflein bisanhydrodichloride on the induction of the mitochondrial pore by calcium ions was determined using selective electrodes to measure the membrane potential and calcium capacity of mitochondria. Determination of calcium capacity is based on measuring the concentration of Ca2 + required to irreversibly reduce the membrane potential and the release of calcium ions from mitochondria with successive Ca2 + additions in concentrations of 20-50 μM. Mitochondria were incubated in a medium containing 120 mM KCl, 1.5 mM KH2PO4, 10 mM HEPES (pH 7.25). The concentration of TPP + in the cell was 1 μM. All measurements were carried out with constant stirring in a thermostatic cuvette with a volume of 1 ml at 26 ° C.

On isolated rat liver mitochondria, it was shown that nuflein bisanhydrodichloride (20-50 μM) causes dissociation of oxidative phosphorylation - mitochondria absorb oxygen, but ATP synthesis does not occur. In addition, nuflein bisanhydrodichloride induces the opening of the mitochondrial pore, which is subsequently accompanied by the release of cytochrome C from the mitochondria and the induction of apoptosis. At concentrations of 20-50 μM nouflein, bisanhydrodichloride inhibits mitochondrial respiration.

The inventive compound - nouflein bisanhydrodichloride - has a high cytostatic activity against human cervical cancer cells, superior to the activity of cisplatin. In relation to normal cells, nuflein exhibits a moderate (1000 times smaller) cytotoxic effect, which is very valuable for increasing the selectivity of tumor chemotherapy. An apoptotic component and mitochondrial respiration inhibition are present in the mechanism of the cytotoxic effect of nuflein. Currently undergoing clinical trials of nuflein.

Claims (3)

The use of the compound Nuflein bisanhydrohydrochloride formula I

, possessing selective, cytotoxic activity on human cervical cancer cells.

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