SOME ASPECTS OF THE HIV REPLICATION INHIBITORS DESIGN: DEPOT FORMS AND NEW ACYCLIC NUCLEOTIDE ANALOGUES

Abstract



Depot forms of AZT and 3TC There are about 30 currently used drugs against HIV. Since the first-in-class anti-HIV drug 3’-azido-3’-deo-xythymidine (AZT) approval for treatment of HIV, nucleoside and nucleotide analogues have gained much attention, even now, with new classes of antivirals found. This conservative therapy is the most affordable; currently used drugs offer such pharmaceutical benefits as good water solubility, inexpensive synthesis, thoroughly studied mechanisms of action and resistance. Nevertheless, the topic of the past 30 years is to make nucleosides and nucleotides less toxic in the long-term treatment, that is why prodrug and depot-form approaches have been developed. We synthesized two types of prodrugs based on phosphonate and carbamate derivatives and tested them in cell cultures and animal models. The compounds revealed lower toxicity compared to AZT and L-2,’3’-dideoxy-3’-thiacytidine (3TC). Phosphonates are stable for more than a day in pH 4.5-7, but in human blood serum some of them noticeably vary. 1b R = -CH2OTs~i 1cR = -CH2I,^ 1dR = -CH2N3,»JV 1e R = -CH2OPh 2a R = -Ph, 2b R = -CH2OCH3, 2c R = -CH2OPh, 2d R = -CH2F, 2e R = -CH2I, -1 2f R = -CH2N3 ^ 4a nr2 = -NHCH3, 4b nr2 = -nhc2 :H5, 4c nr2 = -N 0, 4d nr2 = -N НИ, 4e nr2 = -nh2 Depot forms release nucleoside analogues gradually. That allows to maintain therapeutically relevant concentrations for a prolonged period of time. J* Acyclic nucleotide analogues Phosphonate moiety is a useful bypass of the limiting first phosphorylation step, besides, it does not undergo enzymatic hydrolysis as natural phosphates do. The problem of a selective phosphonate attachment may be solved by the “click”-reaction with carbonyl group forming the oximes. In our recent study, diethyl aminooxymethylphosphonate served as an accessible and easy-coupling phosphonate fragment. We designed and tested several new oximecontaining acyclic nucleotide analogues. More often than not, several viruses occur in an HIV-infected patient simultaneously, e.g., over 80% of patients suffer from herpes and other virus-induced infections. Search for substances with combined activity against different types of viruses is important. МЕДИЦИНСКИЙ АКАДЕМИЧЕСКИЙ ЖУРНАЛ, 2016 г., ТОМ 16, № 3 117 о и /-P-OEt НО OEt PhthNOH, PPh3, DIAD, THF, 0"C Mitsunobu conditions N2H4 • H20, CH2CI2, 0eC (two steps: 85%) о N-0 О О II -P-OEt 6н + ^“N,H О + PPh30 f-РЮ H'N^ 0/-РГ Partitioned away Crystallized from CH2CI2/H20 from CCI4 о II /-P-OEt H2N-0 OEt + О N-NH, Non-fluorescent form (white crystal precipitated| from CH3CN at < 0°C) Fluorescent form (yellow solution in CH3CN) CH3CN о,. ЪО ' J RO RO ^ ^ Chromatog.^ syn- 5a R = H Chromatog^ R = Et, R' = Me -1 R = Et, R' = H -і/ 1 5b R = R' = H -1 -1 R = Et 71/ 5c R = H -1 Conditions: i - Me3SiBr, DMF then NaHC03> H20. Antiviral properties and pharmacokinetics <D : С CD != U КЛ г LO uL С f nr ComBind EC*)1, цМ CC502, цМ SI3 V,h 2a ||_ Ph-P(0)(AZT)2 1 10 1 33 3.3 >24 2b ||_ СН30СН2-Р(0)(АГТ)2 0.68 31 46 >24 2c ||_ Ph0CH2-P(0)(AZT)2 0.25 37 148 >24 2d ||_ FCH2-P(0)(AZT}2 0.015 29 1933 0.78 2e ||_ ICH2-P(0)(AZT)2 11 39 3.5 >24 2f ||_ N3CH2-P(0)(AZT)2 1 0.18 1 24.5 136 12 3 IL Ph0CH2-P(0)(AZT)(3TC) 0.24 60 250 21 AZT ||_ 3'-azido-3'-deoxythymidine 1 0.037 1 142 3837 II Nikavir* (H-P(0)(AZT)(0H)) 0.131 185 1405 6 зге I .-2',3'-dideoxy-3'-thiacytidine 0.44 43.6 100 la H-P(0)(3TC)(0H) 3.4 >3410 >1000^ FCH2-P(0)(AIT)(OH) 3.32 >500 >150 lc ICH2-P(0)(AZT)(OH) 20.6 >500 >24 Id N3CH2-P(0)(AZT)(OH) 4.64 >500 >107 4a CH3-NHC(0)(AZT) 50 >6000 >120 4b C2H5-NHC(0)(AZT) 45 >6000 >133 4c OC4H8N-C(0)(AZT) 35 >6000 >171 4d HNC4H8N-C(0)(AZT) 20 >6000 >300 4e NH2-C(0)(AZT) 3.2 >6000 >1875 AZT 3'-azido-3'-deoxythymidine 0.037 80 2300 Nikavir* (H-P(0)(AZT)(0H)) 0.29 648 2240 Compound HIV-1 (in MT-4) HSV-1 (Lj* and Lj/R*) (in Vero E6) HCV (replicon in Huh7) EC5tv pM CC50, pM *ECS0, pM #EC5tv pM CCso, pM ECgo, pM CCgo, pM 5a 9-{2-[(phosphonomethyl)oxlmlno|ethyl}adenlne 57 >1000 354 354 >708 52 173 5b 9-{2-[(phosphonomethyl)-oximino]ethyl}guanine 85 >1000 >300 n/a >800 >300 480 5c 9-{2-[(phosphonomethyl)-oximino]propyl}adenine 100 >1000 >300 n/a >800 >300 973 PMEA 9-(2-phosphonomethoxyethyl)-adenine 2 >227 35 35 >1133 52 n/a Acyclovir 9-(2-hydroxyethylmethyl)-guanine n/a n/a 1.7 >1778 >1778 n/a n/a 1 ЕСЯ - concentration required to Inhibit 5096 of viral replication;1ССХ - concentration required to Inhibit 5096 of cell growth; 3SI-selectivity Index, CC^/ EC^ 4 *іл - half-life In human blood serum. We thank our colleagues from D.I. Ivanovsky Institute of Virology fer testing antiviral activity of the compounds: Ludmila B. Kotnlna, Dmitry N. Nosik, Eduard V. Koramov, Valeria L Andronova and Alla A. Kusch. Conclusions • We assume, the leading anti-HIV activity of 2d may be explained by its fast hydrolysis to the parent nucleoside. • New strategy for synthesis of phosphonates was applied for acyclic nucleotide analogues which demonstrated activity against several type of human viruses. • Depot forms can significantly decrease the toxicity of the parent nucleoside and provide prolonged drug release in vivo. 118

Pavel N Solyev

Engelhardt Institute of Molecular Biology

32 Vavilov St., 119991 Moscow, Russia

Maxim V Yasko

Engelhardt Institute of Molecular Biology

32 Vavilov St., 119991 Moscow, Russia

Inna L Karpenko

Engelhardt Institute of Molecular Biology

32 Vavilov St., 119991 Moscow, Russia

Marina K Kukhanova

Engelhardt Institute of Molecular Biology

32 Vavilov St., 119991 Moscow, Russia

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Copyright (c) 2016 Solyev P.N., Yasko M.V., Karpenko I.L., Kukhanova M.K.

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