WO2004111071A1 - 5'-aminocarbonylphosphonates d4t utilises comme inhibiteurs de la reproduction du virus de l'immunodeficience humaine - Google Patents

5'-aminocarbonylphosphonates d4t utilises comme inhibiteurs de la reproduction du virus de l'immunodeficience humaine Download PDF

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Publication number
WO2004111071A1
WO2004111071A1 PCT/RU2003/000513 RU0300513W WO2004111071A1 WO 2004111071 A1 WO2004111071 A1 WO 2004111071A1 RU 0300513 W RU0300513 W RU 0300513W WO 2004111071 A1 WO2004111071 A1 WO 2004111071A1
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Prior art keywords
hiv
compound
cells
compounds
inhibitors
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PCT/RU2003/000513
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English (en)
Russian (ru)
Inventor
Andrei Georgievich Pokrovsky
Tatyana Rudolfovna Pronyaeva
Nina Vladimirovna Fedyuk
Marina Konstantinovna Kukhanova
Elena Anatolevna Shirokova
Maksim Vladimirovich Yasko
Anastasia Lvovna Khandazhinskaya
Dmitry Vasilevich Yanvarev
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Gosudarstvenny Nauchny Tsentr Virusologii I Biotekhnologii 'vektor'
Institut Molekulyarnoy Biologii Im Vaengelgardta Ran
Original Assignee
Gosudarstvenny Nauchny Tsentr Virusologii I Biotekhnologii 'vektor'
Institut Molekulyarnoy Biologii Im Vaengelgardta Ran
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Publication of WO2004111071A1 publication Critical patent/WO2004111071A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the invention relates to the field of molecular biology, 5 virology and medicine, namely to the use of new derivatives of nucleosides, namely d4T amides to suppress the reproduction of the human immunodeficiency virus.
  • the new compounds are highly effective inhibitors of the reproduction of the human immunodeficiency virus type 1 in the culture of vivo transfused lymphocytes MT-4, and also protect cells from the cytopathogenic effect of the virus.
  • nucleosides 15 nucleosides, protease inhibitors and the so-called non-nucleoside inhibitors.
  • nucleoside derivatives the most known and used are Z'-azido-3'-deoxythymidine (AZT), 2'3'-dideoxyxycytidine, 2'3'-dideoxyxyinosine, 2'3'-dideoxyxy-2'3'-didehydrothymidine and 2 ' 3'-dideoxy-3'-thiotimidine [1-2].
  • the closest analogue of the claimed compound is a compound belonging to the group of nucleoside inhibitors of 2'3'-dideoxy-2'3'-didehydrothymidine (d4T) [2]. With moderate toxicity for uninfected transplanted cells of the MT-4 lymphoid line, this compound has high anti-HIV activity.
  • the technical result of the invention is the creation of new highly effective anti-HIV modified nucleoside derivatives that are able to penetrate into cells that are resistant to dephosphorylation enzymes and have selective activity in inhibiting DNA synthesis catalyzed by HIV reverse transcriptase.
  • the specified technical result is achieved by the fact that according to the invention, new compounds of 5'aminocarbonylphosphonates - derivatives of 2'3'-dideoxy-2'3'-didehydrothymidine (d4T prototype, [2]) of the general formula:
  • R Alkul which are low toxic inhibitors of human immunodeficiency virus.
  • 2'3'-didehydrothymidine (d4T prototype, [2]) with moderate toxicity for uninfected transplanted cells of the MT-4 lymphoid line has high anti-HIV activity, which made it possible to conduct directed synthesis of new effective derivatives on its basis.
  • FIG. Figure 1 shows dose-dependent curves for calculating the quantitative characteristics of the inhibition of HIV-1 by the studied compounds (using compound 3 b as an example) while being introduced with the virus. Similar plots were obtained for compounds 3a; 3 sec
  • FIG. Figure 2 shows dose-dependent curves for calculating the quantitative characteristics of the inhibition of HIV-1 by the studied compounds (for example, compound 3 b) when introduced after adsorption of the virus. Similar plots were obtained for compounds 3a; 3 sec
  • Target 5'aminocarbonylphosphonates were prepared according to the following scheme:
  • the starting compound was 2 ', 3'-dideoxy-2', 3'-dehydro-thymidine-5'-ethoxycarbonylphosphonate (1) (or the corresponding 5'-methoxycarbonylphosphonate) synthesized previously.
  • Amination of ethoxycarbonylphosphonate (1) with appropriate amines leads to amidophosphonates (2) that are alkylated with alcohols in the presence of triisopropylsulfonyl chloride (TPSCl) or under the Mitsunobu reaction conditions.
  • TPSCl triisopropylsulfonyl chloride
  • Triphenylphosphine (130 mg, 0.5 mmol) and ethanol (100 ⁇ l) were added to a solution of compound 1 (120 mg, 0.5 mmol) in DMF (2 ml), the solution was cooled to 4 ° C and a solution of diethyl azodicarboxylate (0.5) was added to it. mmol, 77 ⁇ l) in THF (1 ml). The reaction mixture was kept at room temperature for 5 h, evaporated, the residue was chromatographed on a silica gel column (2.5 x 30 cm), eluting with a methanol gradient in chloroform (0-> 10%). 45 mg (60%) of compound Za were obtained.
  • Example 2. 2 ', 3'-Dideoxy-2', 3'-dihydro-thymidine-5 '- (N-methylaminocarbonyl) phosphonate (compound 2b) (R' Me).
  • a study of the inhibition of HIV reproduction includes the cultivation of primary infected lymphoid cells of the MT-4 line in the presence of the test compounds, final concentrations which in the culture medium are 0.0001-100 ⁇ g / ml, during one passage - for 4 days.
  • the cytotoxicity of the drug is assessed by adding dilutions in serum-free RPMI-1640 medium to the MT-4 cell suspension, placed in the wells of a 96-night plate (Cell-Cult, Epglapd), to final concentrations of 0.001-100 ⁇ g / ml (three wells each) per dose), followed by cultivation at 37 ° C for 4 days [7].
  • the inoculum concentration is 0.5 x 10 b cell particles per milliliter. Cells are used as control without the addition of a drug, instead of which the same amount of serum-free medium is added.
  • 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, OxIO 6 cell particles per milliliter and a viability of at least 90% is placed in the wells of a 96-night plate ("Orpge") immediately after application of the virus-containing material and the test compounds diluted in RPMI-medium are immediately added 1640 serum-free, 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 (0.5 x 10 6 per milliliter) with RPMI-1640 culture medium supplemented with 10% fetal KPC serum pre-inactivated by heating at 56 ° C for 30 minutes, ZOOmg / ml b-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.
  • 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 the virus-specific protein p24 by direct enzyme immunoassay, as described in [8], and dose-dependent curves are constructed (Fig. 1.2), which are used to calculate the concentrations that inhibit growth by 50 and 90% viral antigen (ID 50 and ID 90 ).
  • the therapeutic index, or selectivity index (IS) is considered as the ratio of the 50% toxic concentration of the compound to its 50% effective dose (the results are presented in the table).
  • the therapeutic indices of the studied compounds are comparable to IS d4T with simultaneous administration of the drug and the virus and are 2-5 times higher than IS d4T under post-adsorption conditions. This suggests the promise of new d4T derivatives for further research with the goal of creating dosage forms for the treatment of HIV / AIDS.
  • Table 1 The therapeutic indices of the studied compounds (IS) are comparable to IS d4T with simultaneous administration of the drug and the virus and are 2-5 times higher than IS d4T under post-adsorption conditions. This suggests the promise of new d4T derivatives for further research with the goal of creating dosage forms for the treatment of HIV / AIDS. Table 1.
  • % inhibition (Px-Po) / (P-Po) * 100, where Px is the concentration of p24 protein on the 4th day of cultivation in the presence of the drug, Po is the concentration of p24 after 1 hour incubation of HIV-1 with cells without drug addition (“O” point), P - p24 concentration on day 4 of cultivation of HIV-1 in cells without drug addition (virus control).
  • the therapeutic index, or selectivity index (IS), 5 is defined as the ratio of the concentration of the drug, toxic to 50% of the cells (CD 5 o), to the concentration, 50% inhibiting the growth of viral antigen (ID 5 o) '.
  • inventive compounds have pronounced inhibitory activity against the human immunodeficiency virus and may be potential candidates for the preparation of antiviral agents used in medicine and the pharmaceutical industry.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • AIDS & HIV (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Cette invention relève du domaine de la biologie moléculaire, de la virologie et de la médecine et concerne plus précisément l'utilisation de nouveaux dérivés de 2'3'-didésoxy-2'3'-didéhydrothymidine (d4T)-aminocarbonylphosphonates représentés par la formule générale (I) dans laquelle R' désigne H, alkyle ou aryle; R'' désigne H, alkyle ou aryle; R', R'' désignent alkyle cyclique et R désigne alkyle, et utilisés comme inhibiteurs du virus de l'immunodéficience humaine (VIH). Ces composés présentent en particulier la capacité de supprimer la reproduction du virus de l'immunodéficience humaine de type 1 dans une culture par passages de lymphocytes MT-4. L'activité anti-virale de ces nouveaux composés par rapport au VIH est supérieure à l'activité des d4T.
PCT/RU2003/000513 2003-06-16 2003-11-21 5'-aminocarbonylphosphonates d4t utilises comme inhibiteurs de la reproduction du virus de l'immunodeficience humaine Ceased WO2004111071A1 (fr)

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RU2003118102/04A RU2247124C1 (ru) 2003-06-16 2003-06-16 5`-аминокарбонилфосфонаты d4т - ингибиторы репродукции вируса иммунодефицита человека

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0306597A2 (fr) * 1985-03-16 1989-03-15 The Wellcome Foundation Limited Nucléosides antiviraux
RU2130942C1 (ru) * 1997-07-11 1999-05-27 "С энд Ти Сайенс энд Текнолоджи Инк" Способ получения 3'-азидо-2', 3'-дидезокситимидина
RU2188203C2 (ru) * 2000-10-05 2002-08-27 Государственный научный центр вирусологии и биотехнологии "Вектор" 2',3'-дидегидро-2',3'-дидезокситимидин-5'[(этоксикарбонил)(этил)фосфонат]- ингибитор репродукции вируса иммунодефицита человека

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0306597A2 (fr) * 1985-03-16 1989-03-15 The Wellcome Foundation Limited Nucléosides antiviraux
RU2130942C1 (ru) * 1997-07-11 1999-05-27 "С энд Ти Сайенс энд Текнолоджи Инк" Способ получения 3'-азидо-2', 3'-дидезокситимидина
RU2188203C2 (ru) * 2000-10-05 2002-08-27 Государственный научный центр вирусологии и биотехнологии "Вектор" 2',3'-дидегидро-2',3'-дидезокситимидин-5'[(этоксикарбонил)(этил)фосфонат]- ингибитор репродукции вируса иммунодефицита человека

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