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WO2008140345A1 - Di- et/ou trinicotinates d'acide glycyrrhizique et inhibiteur de propagation du virus de l'immunodéficience humaine - Google Patents

Di- et/ou trinicotinates d'acide glycyrrhizique et inhibiteur de propagation du virus de l'immunodéficience humaine Download PDF

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Publication number
WO2008140345A1
WO2008140345A1 PCT/RU2007/000241 RU2007000241W WO2008140345A1 WO 2008140345 A1 WO2008140345 A1 WO 2008140345A1 RU 2007000241 W RU2007000241 W RU 2007000241W WO 2008140345 A1 WO2008140345 A1 WO 2008140345A1
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WO
WIPO (PCT)
Prior art keywords
glycyrrhizic acid
trinicotinates
reproduction
virus
human immunodeficiency
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/RU2007/000241
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English (en)
Russian (ru)
Inventor
Nariman Faridovich Salachutdinov
Andrei Georgievish Pokrovskii
Genrih Aleksandrovich Tolstikov
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Zakritoe Aktsionernoe Obschestvo 'sibpharmakon'
Original Assignee
Zakritoe Aktsionernoe Obschestvo 'sibpharmakon'
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Application filed by Zakritoe Aktsionernoe Obschestvo 'sibpharmakon' filed Critical Zakritoe Aktsionernoe Obschestvo 'sibpharmakon'
Priority to PCT/RU2007/000241 priority Critical patent/WO2008140345A1/fr
Publication of WO2008140345A1 publication Critical patent/WO2008140345A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the invention relates to biologically active substances derived from glycyrrhizic acid, as well as substances capable of inhibiting the reproduction of the human immunodeficiency virus.
  • glycyrrhizic acid is the main ingredient in the extract of the roots of licorice (Glusurrhiza glabara) and Ural licorice (Gl. Uralepsis), has high biological activity and is highly effective in the treatment of various diseases, for example, viral hepatitis.
  • Glycyrrhizic acid interacts with the structures of viruses, changing various phases of the viral cycle, which is accompanied by irreversible inactivation of viral particles that are in a free state outside the cells, blocks the introduction of active viral particles into the cell, impairs the ability of the virus to induce the synthesis of new viral particles, and induces the formation of interferon, which is one of the components of the antiviral effect, inactivates these viruses in concentrations that are non-toxic to normally functioning cells.
  • Virus strains, mutant and non-mutant are also highly sensitive to glycyrrhizic acid.
  • glycerrhizic acid Like glycerrhizic acid itself, its derivatives have high biological activity. For example, they are used to inhibit the reproduction of Marburg virus [RF Patent N ° 2088232], and glycyrrhizic acid glycopeptide has anti-AIDS activity [RF Patent N ° 2024546], which indicates the high potential of glycyrrhizic acid derivatives for medicine, pharmacology and biology. By this Therefore, the urgent task is to study the known and obtain new substances - derivatives of glycyrrhizic acid, which can be used in the treatment of various diseases, for example, such a serious illness as HIV infection.
  • azidothymidine monotherapy is not recommended by the World Health Organization, since it leads to the appearance of drug-resistant mutants of the virus.
  • azidothymidine is used mainly as a constituent component in the so-called “cocktails” in combination with 2-4 other drugs that act on other targets of the virus reproduction cycle [Schmit J.-C, Weber B. ⁇ Recent Advapes and Pharmaceuticals and HIV infection monitoring. " Itervirologu, 1997, V. 40, N ° 5/6, p. 304-321], typically HIV protease inhibitors, or non-nucleoside HIV reverse transcriptase inhibitors. Despite the effectiveness of some of these "cocktails", the search for new combinations and combinations of azidothymidine with drugs of a different nature and a different mechanism of action remains relevant, while being quite effective and cheap.
  • glycyrrhizic acid the mechanism of action of which is not fully understood, as some authors attribute it to the inhibition of protein kinase, which provides phosphorylation of the T4 receptor necessary for virus binding [Ito M., Sato A., Nirubaushi K., Tanabe F., Shigeta S., Baba M., De Clercq E., Nakashima H., Yamato N. “Mechanism of Inhibit Effects of Glusur on replication of human immunodeficiency viruses (HIV). " Antiviral Res, 1988, V. 10, p. 289–298], and others with direct blocking of the binding of the virus to the cell [Field AP, Bednarik P.
  • glycyrrhizic acid derivatives have demonstrated the effectiveness of their anti-HIV action in cell culture [RF Patent N ° 2199547 and RF Patent N ° 2198177].
  • the monoammonium salt of glycyrrhizic acid (glycyram) is not an antagonist with azidothymidine and, in combination with it, effectively blocks HIV reproduction in cell culture in both acute and chronic infections with an AZT: glycyr ratio of 1: 10000 [Plyasunova OA , Egoricheva I.H., Fedyuk N.V. and others. "The study of anti-HIV activity of ⁇ -glycyrrhizic acid.” Questions of Virology, 1992, N ° 5-6, p. 235-238].
  • the invention solves the problem of obtaining new derivatives of glycyrrhizic acid, as well as obtaining a new highly active inhibitor of reproduction of the human immunodeficiency virus.
  • a new substance is proposed, which is a derivative of glycyrrhizic acid, namely, di - and / or trinicotinates of glycyrrhizic acid of the general formula:
  • R is or H, and two and / or three of these R-
  • nicotinic acid is dissolved in an organic aprotic solvent by heating the reaction mixture to form a suspension, to which phosphorus pentachloride is also added while heating, then the suspension is cooled.
  • glycyrrhizic acid is added and then heated again.
  • ice and concentrated hydrochloric acid are added to it until a slightly acidic reaction.
  • Glycyrrhizic acid di- and trinicotinates precipitate as a mixture of them. If necessary, dinicotinates can be separated from trinicotinates and are thus isolated separately.
  • di- and trinicotinates of glycyrrhizic acid can be carried out, for example, in accordance with examples 1–3 below. Studies of the toxicity of di- and trinicotinates of glycyrrhizic acid have shown that they are not toxic.
  • the cytotoxicity of glycyrrhizic acid di- and trinicotinates was assessed by adding their dilutions in serum-free RPMI-1640 medium to a MT-4 cell suspension, placed in wells of a 96-night plate (Cell, Epglapd), to final concentrations of 0.001-100 ⁇ g / ml (three wells per dose), followed by cultivation at 37 ° C for 4 days.
  • the inoculum concentration was 0.5 x 10 cell particles per milliliter.
  • the cells served as control without the addition of the drug, instead of which the same amount of serum-free medium was added.
  • the aforementioned di - and trinicinates of glycyrrhizic acid are in the mixture obtained during their synthesis, but they can be isolated separately.
  • di - and trinicotinates of glycyrrhizic acid actively influence the reproduction of HIV, which is confirmed by the following examples 1-3. Their activity is significantly higher than that of other derivatives of glycyrrhizic acid, including the previously mentioned glycyrrhizic acid penta-nicotinate.
  • Mentioned studies on the inhibition of HIV reproduction include the cultivation of primary infected lymphoid cells of the MT-4 line in the presence of these compounds, the final concentrations of which in the culture medium are 0.0001-100 ⁇ g / ml for one passage for 4 days. Inhibition of HIV reproduction in a culture of sensitive cells is evidenced by a decrease in the accumulation of virus-specific protein p24 (according to enzyme-linked immunosorbent assay), as well as an increase in cell viability in the presence of the drug compared to the control determined on the 4th day of cultivation.
  • ID 50 0.134 ⁇ M
  • the obtained quantitative indicators of inhibition confirm the high degree of suppression of HIV-1 replication in MT-4 cell culture by glycyrrhizic acid di- and trinicotinates, which is, for example, 5-15 times higher than that of glycyrrhizic acid pentanicotinate.
  • di- and trinicotinates of glycyrrhizic acid inhibit the reproduction of type 1 immunodeficiency virus in MT cell culture -four.
  • the therapeutic indices of glycyrrhizic acid di- and trinicotinates (IS) are 10-100 times greater than for other glycyrrhizic acid derivatives.
  • new chemical compounds glycyrrhizic acid di- and trinicotinates, have high biological activity, which is manifested at least in that they are highly active inhibitors of the reproduction of the human immunodeficiency virus and can be used in pure form or as the basis for new highly effective dosage forms for AIDS treatment.
  • Example 1 The best embodiment of the invention is shown in example 1, and for a better understanding of the essence of the invention, examples 2 and 3 are also given here.
  • Example 1
  • DMF organic aprotic solvent
  • nicotinic acid 500 g
  • the reaction mixture is heated to 40-45 ° C with stirring (hereinafter, the bath temperature is indicated), then without stopping mixing, add to the resulting suspension 265.5 g of phosphorus pentachloride.
  • the mixture is heated to bO ⁇ 3 ° C and stirred at this temperature for 2 hours (after about 30-40 minutes the light color of the reaction mixture becomes dark brown).
  • the flask with the reaction mixture was placed in an ice bath, and the reaction mixture was stirred for ⁇ 20 min. until it cools and add with stirring 150 g of glycyrrhizic acid.
  • the flask with the reaction mixture was placed in a bath with glycerin and heated to 55 ⁇ 5 0 C, without stopping stirring, and kept at this temperature for 2 hours and 50 minutes. When heated, a significant portion of the precipitate dissolves.
  • the bath is removed and the flask is allowed to cool down to -30-40 ° C, then the resulting suspension is poured equally into 2 glasses of 5 L capacity and ⁇ 1.5 L of crushed ice prepared from distilled water is added to each glass to transfer the remaining on the walls Sediment flasks use distilled water.
  • the contents of the glasses are mixed and concentrated hydrochloric acid ( ⁇ 1.3 L in each glass) is added to them until a weak acidity (pH ⁇ 6) is achieved, while an additional precipitate is observed. Leave the glasses for several hours to completely precipitate.
  • the brown precipitate formed in both glasses is filtered off.
  • the precipitates combined from both glasses are washed with distilled water (4 x l) and dried thoroughly on a filter. In the process of drying the precipitate, large conglomerates are formed, which are crushed to increase the drying efficiency.
  • the weight of the dried light brown precipitate is ⁇ 300 g.
  • the precipitate is washed with 150 ml of diethyl ether (three times 50 ml), dried on a filter. The weight of the precipitate was -165 g. 150 ml of ethyl alcohol was added to the obtained precipitate, stirred, filtered, dried on a filter. The weight of the dried light brown precipitate is ⁇ 150 g (yield -60%).
  • the resulting substance is a mixture of di- and tri-nicotinates of glycyrrhizic acid in a ratio of 20:80, respectively.
  • a study of the antiviral activity of compounds against HIV-1 is carried out on the transplantable line of sensitive cells MT-4.
  • the supernatant of infected cells stored in liquid nitrogen is used, the multiplicity of infection is 0.2-0.5 infectious units per cell.
  • a suspension of MT-4 cells with a concentration of 2.0 x 10 b cell particles per milliliter and a viability of at least 90% is placed in the wells of a 96-night plate ("Orappe") immediately after the introduction of the virus-containing material and di-and trinocotinates of glycyrrhizic acid are added in the ratio of 20: 80, diluted in serum-free RPMI-1640 medium, to a final concentration of 0.0001-100 ⁇ g / ml (three wells per dose).
  • the controls are HIV-1 infected MT-4 cells without drug addition (instead of the drug, the same amount of RPMI-1640 medium without additives is added) and uninfected cells.
  • the plate is incubated for one hour at 37 ° C to adsorb the virus, then the cells are diluted to a seed concentration (0.5x10 in milliliter) with RPMI-1640 culture medium supplemented with 10% fetal KPC serum, previously inactivated by heating at 56 ° C for 30 minutes , ZOOmg / ml L-glutamine and 100 ⁇ g / ml gentamicin. Then the tablet is placed in a thermostat at 37 0 C in an atmosphere of 5% CO 2 . On the 4th day of cultivation, the concentration and viability of the cells are calculated by the formazan method. According to the data obtained, graphs of the dependence of the growth of cell viability relative to control under the influence of increasing doses of drugs are constructed determine the ability of drugs to protect infected cells from the cytopathogenic effect of the virus.
  • Evaluation of the anti-HIV activity of the compounds is carried out using a quantitative determination of virus-specific protein p24 by direct Enzyme-linked immunosorbent assay, as described in Filds AP, Vedparik PP, Ness A., Mau WS "Human ii-virus virus and its virus-receptor receptor.” Nature (London), 1988, V. 333, p. 278-280, and the dose-dependent curves, according to which the concentrations are calculated, inhibit the growth of the viral antigen by 50 and 90% (ID 50 and ID 90 ).
  • the therapeutic index, or selectivity index (IS) is considered as the ratio of a 50% toxic concentration of a compound to its 50% effective dose.
  • DMF organic aprotic solvent
  • the bath was removed, and the flask with the reaction mixture was placed in an ice bath, the reaction mixture was stirred for ⁇ 20 min to cool and 120 g of glycyrrhizic acid was added with stirring.
  • the cooling bath is removed, and the flask with the reaction mixture is placed in a glycerin bath and heated to 50 ⁇ 5 0 C without stopping stirring and maintained at this temperature for 2 hours. When heated, a significant part of the precipitate dissolves.
  • the weight of the dried light brown precipitate is ⁇ 160 g (yield ⁇ 65%).
  • the obtained substance is a mixture of glycyrrhizic acid di- and trinicotinates in a ratio of 50:50, respectively.
  • DMF organic aprotic solvent
  • the bath is removed, and the flask with the reaction mixture is placed in an ice bath, and the reaction mixture is stirred for ⁇ 20 min until cooling, and 100 g of glycyrrhizic acid are added with stirring.
  • the cooling bath is removed, and the flask with the reaction mixture is placed in a bath of glycerin and heated to 40 ⁇ 5 0 C without stopping stirring and kept at this temperature for 3 hours and 50 minutes. When heated, part of the precipitate dissolves.
  • reaction mixture Further processing of the reaction mixture is carried out as in example 1.
  • the weight of the dried light brown precipitate is ⁇ 120 g (yield ⁇ 45%).
  • the resulting substance is a mixture of di- and trinicotinates of glycyrrhizic acid in a ratio of 80:20, respectively.
  • Di - and trinicotinates of glycyrrhizic acid being biologically active substances, like other derivatives of glycyrrhizic acid, can be used in medicine, virology, and pharmacology. In particular, they can be used as inhibitors of human immunodeficiency virus reproduction in its pure form or as a basis for new highly effective dosage forms for the treatment of AIDS.

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  • Health & Medical Sciences (AREA)
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  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Oncology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Communicable Diseases (AREA)
  • AIDS & HIV (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
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Abstract

L'invention concerne des matériaux bioactifs se présentant comme des dérivés d'acide glycyrrhizique et des substances capables d'inhiber la propagation du virus de l'immunodéficience humaine. On propose des di- et des trinicotinates d'acide glycyrrhizique possédant la formule générale suivante (Formule I) dans laquelle R est (Formule II) ou H, deux et/ou trois des R étant (Formule III), ainsi qu'un inhibiteur de propagation du virus de l'immunodéficience humaine comprenant des di- et/ou trinicotinates d'acide glycyrrhizique.
PCT/RU2007/000241 2007-05-16 2007-05-16 Di- et/ou trinicotinates d'acide glycyrrhizique et inhibiteur de propagation du virus de l'immunodéficience humaine Ceased WO2008140345A1 (fr)

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PCT/RU2007/000241 WO2008140345A1 (fr) 2007-05-16 2007-05-16 Di- et/ou trinicotinates d'acide glycyrrhizique et inhibiteur de propagation du virus de l'immunodéficience humaine

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PCT/RU2007/000241 WO2008140345A1 (fr) 2007-05-16 2007-05-16 Di- et/ou trinicotinates d'acide glycyrrhizique et inhibiteur de propagation du virus de l'immunodéficience humaine

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2088232C1 (ru) * 1995-02-14 1997-08-27 Государственный научный центр вирусологии и биотехнологии "Вектор" Ингибитор репродукции вируса марбург
RU2254861C1 (ru) * 2003-12-01 2005-06-27 Государственный научный центр вирусологии и биотехнологии "Вектор" Индуктор гамма-интерферона
RU2304145C1 (ru) * 2006-03-17 2007-08-10 Закрытое акционерное общество "Сибфармакон" Ди- и триникотинаты глицирризиновой кислоты и ингибитор репродукции вируса иммунодефицита человека

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2088232C1 (ru) * 1995-02-14 1997-08-27 Государственный научный центр вирусологии и биотехнологии "Вектор" Ингибитор репродукции вируса марбург
RU2254861C1 (ru) * 2003-12-01 2005-06-27 Государственный научный центр вирусологии и биотехнологии "Вектор" Индуктор гамма-интерферона
RU2304145C1 (ru) * 2006-03-17 2007-08-10 Закрытое акционерное общество "Сибфармакон" Ди- и триникотинаты глицирризиновой кислоты и ингибитор репродукции вируса иммунодефицита человека

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