Determination of cyclodextrins and their derivatives by capillary electrophoresis with indirect UV and conductivity detection.

A fast and simple capillary electrophoretic method suitable for the determination of native alpha-, beta-, gamma-cyclodextrins, their randomly substituted tert-butyl derivatives (average degree of substitution 3.8-4.4), heptakis (2,6-di-O-methyl)- and heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin was developed. Naphthyl-2-sulfonic acid (2-NSA), 3-iodobenzoic acid (3-IBA) and (1S)-1-phenylethylamine (PHEA) were tested as selective complex forming and UV absorbing background electrolyte additives. The composition of optimized background electrolyte for the separation of uncharged cyclodextrins and their derivatives was: 15 mM 3-iodobenzoic acid titrated with tris[hydroxymethyl]aminomethane to pH 8.0, 5% (v/v) of acetonitrile. A complete resolution of mono-2-O-, mono-3-O- and mono-6-O-carboxymethyl-beta-cyclodextrin regioisomers was achieved in the optimized background electrolyte system: 40 mM PHEA titrated with 2-[N-morpholino]ethanesulfonic acid to pH 5.6. In addition to indirect UV detection a contactless conductometric detector was successfully utilized.

Determination of cyclodextrin content using periodate oxidation by capillary electrophoresis.

A simple and general method suitable for the determination of cyclodextrin content in various matrices is described. The proposed method involves selective cleavage of C-C bonds with vicinal hydroxyl groups by means of periodate (Malaprade's reaction). The amount of produced iodate is monitored by capillary electrophoresis. Optimized electrophoretic conditions (20 mM disodium tetraborate with 1 mM tetradecyltrimethylammonium bromide, direct UV detection lambda = 200 nm) ensure complete separation of periodate and iodate ions and sufficient sensitivity towards iodate. Under optimized reaction conditions (2-fold excess of periodate, temperature 70 degrees C) reproducible quantitative results were obtained for alpha-, beta- and gamma-cyclodextrins as model samples. The proposed method was tested on a real sample of acrylamide--2'-O-allyl-beta-CD copolymer. The values of beta-cyclodextrin content were compared with those obtained by reference NMR measurement and were found to be identical.

Efficacy of the acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against feline immunodeficiency virus.

The acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated for their efficacy and side effects in a double-blind placebo-controlled trial using naturally occurring feline immunodeficiency virus (FIV)-infected cats. This natural retrovirus animal model is considered highly relevant for the pathogenesis and chemotherapy of HIV in humans. Both PMEA and FPMPA proved effective in ameliorating the clinical symptoms of FIV-infected cats, as measured by several clinical parameters including the incidence and severity of stomatitis, Karnofsky's score, immunologic parameters such as relative and absolute CD4+ lymphocyte counts, and virologic parameters including proviral DNA levels in peripheral blood mononuclear cells (PBMC) of drug-treated animals. In contrast with PMEA, FPMPA showed no hematologic side effects at a dose that was 2.5-fold higher than PMEA.

Acyclic nucleotide analogs derived from 8-azapurines: synthesis and antiviral activity.

Reaction of phosphoroorganic synthons with 8-azaadenine, 8-aza-2, 6-diaminopurine, and 8-azaguanine using cesium carbonate yielded regioisomeric 8-azapurine N7-, N8-, and N9-(2-(phosphonomethoxy)alkyl) derivatives. This reaction followed by deprotection afforded isomeric 2-(phosphonomethoxy)ethyl (PME), (S)-(3-hydroxy-2-(phosphonomethoxy)propyl) [(S)-HPMP], (S)-(3-flouro-2-(phosphonomethoxy)propyl) [(S)-FPMP], (S)-(2-(phosphonomethoxy)propyl) [(S)-PMP], and (R)-(2-(phosphonomethoxy)propyl) [(R)-PMP] derivatives. 13C NMR spectra were used for structural assignment of the regioisomers. None of the 8-isomers exhibited any antiviral activity against herpesviruses, Moloney murine sarcoma virus (MSV), and/or HIV. 9-(S)-HPMP-8-azaadenine (23) and PME-8-azaguanine (65) were active against HSV-1, HSV-2, and CMV at 0.2-7 micrograms/mL, VZV at 0.04-0.4 microgram/mL, and MSV (at 0.3-0.6 microgram/mL). PME-8-azaguanine (65) and (R)-PMP-8-azaguanine (71a) protected MT-4 and CEM cells against HIV-1- and HIV-2-induced cytopathicity at a concentration of approximately 2 micrograms/mL.

Separation of cyclodextrins and their derivatives by thin-layer and preparative column chromatography.

Cyclodextrins, their derivatives and products of their partial hydrolysis are clearly separated by chromatography on silica gel by mobile phases containing aqueous ammonia and organic solvent (acetonitrile or 1-propanol). This system allows separation of compounds with different numbers of substituents on cyclodextrin, but does not allow the separation of isomers; the system can be used both for thin-layer chromatography and for preparative column chromatography. The isomers in the fraction thus obtained can be in some cases separated, after peracetylation, by chromatography on silica gel using a mobile phase consisting of methanol and dichloromethane. This chromatographic sequence potentially yields derivatives of cyclodextrins in quantities which enable evaluation of their solubilization and biological properties.

Regioselectivity of alkylation of cyclomaltoheptaose (beta-cyclodextrin) and synthesis of its mono-2-O-methyl, -ethyl, -allyl, and -propyl derivatives.

Mono-2-O-methyl-, -2-O-ethyl-, and -2-O-allyl-cyclomaltoheptaose were prepared by alkylations of cyclomaltoheptaose in dilute aqueous alkali, and mono-2-O-propylcyclomaltoheptaose was obtained by hydrogenation of the allyl derivative. All the 2-O-alkyl derivatives were less soluble in water than was cyclomaltoheptaose. All formed inclusion complexes with toluene in aqueous solution, but only the methyl ether was less soluble in the water-toluene system than in water. The solubilities of the other ethers in water were enhanced by the addition of toluene. Partial methylation of cyclomaltoheptase with 13C-enriched dimethyl sulfate in dilute aqueous alkali yielded mixtures of products. The substitution patterns were analyzed by GLC-MS of the alditol acetates, prepared by hydrolysis, reduction, and acetylation, and by 13C NMR after complete permethylation with nonenriched reagent. The results showed that methylation at O-2 is a predominant but not an exclusive reaction; as expected, the regioselectivity decreases with increasing degree of methylation.

Inhibition of visna virus replication by 2',3'-dideoxynucleosides and acyclic nucleoside phosphonate analogs.

A series of acyclic nucleoside phosphonate (ANP) and 2',3'-dideoxynucleoside (ddN) derivatives were evaluated for their inhibitory effects on visna virus replication and maedi/visna virus-induced syncytium formation in sheep choroid plexus cells. Most ANP derivatives inhibited virus replication and syncytium formation within a concentration range of 0.2 to 1.8 microM. Among the most active ANP derivatives ranked (R)-9-(2-phosphonomethoxypropyl)adenine, (R)-9-(2-phosphonomethoxypropyl)-2,6-diaminopurine, and (S)-9-(3-fluoro-2-phosphonomethoxypropyl)adenine. Of the ddN derivatives, 2',3'-dideoxycytidine (ddCyd) proved to be the most inhibitory to visna virus-induced syncytium formation (50% effective concentration, 0.02 microM). The purine ddN analogs (i.e., 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, and 2,6-diaminopurine-2',3'-dideoxyribosine) were 10- to 30-fold less effective, and the thymidine derivatives 2',3'-didehydro-2',3'-dideoxythymidine (D4T) and 3'-azido-2',3'-dideoxythymidine (AZT) were more than 500-fold less inhibitory to visna virus than ddCyd. The 5'-triphosphate forms of AZT and D4T were 100- to 600-fold more inhibitory to visna virus particle-derived reverse transcriptase than was the 5'-triphosphate of ddCyd. The apparent discrepancy between the inhibitory effects of these ddN derivatives on virus replication and viral reverse transcriptase activity most likely reflects differences in the metabolic conversion of ddCyd versus D4T and AZT in sheep choroid plexus cells.

Differential antiherpesvirus and antiretrovirus effects of the (S) and (R) enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo antiretrovirus activities of (R)-9-(2-phosphonomethoxypropyl)-2,6-diaminopurine.

The (S)- and (R)-enantiomers of acyclic purine nucleoside phosphonate analogs (i.e., 3-hydroxy-2-phosphonomethoxypropyl [HPMP] derivatives, 3-fluoro-2-phosphonomethoxypropyl [FPMP] derivatives, and 2-phosphonomethoxypropyl [PMP] derivatives of adenine [A], 2-aminopurine, 2,6-diaminopurine [DAP], and guanine [G]) have been synthesized and evaluated for antiviral activity. As a rule, the HPMP derivatives proved effective against DNA viruses but not RNA viruses or retroviruses. In particular, (S)-HPMPA, (S)-HPMPDAP, and (R)- and (S)-HPMPG were exquisitely inhibitory to herpes simplex virus type 1 (50% effective concentrations, 0.63, 0.22, 0.10, and 0.66 microM, respectively). The FPMP and PMP derivatives showed marked inhibitory activities against retroviruses but not DNA viruses. The (S)-enantiomer of FPMPA and the (R)-enantiomer of PMPA were approximately 30- to 100-fold more effective against human immunodeficiency virus and Moloney murine sarcoma virus (MSV) than their enantiomeric counterparts. In contrast, both (S)- and (R)-enantiomers of the DAP and G derivatives proved equally effective against retroviruses, except for (R)-PMPDAP, which was 15- to 40-fold more inhibitory than (S)-PMPDAP. (R)-PMPDAP emerged as the most potent and selective inhibitor of MSV-induced transformation of murine C3H/3T3 cells and human immunodeficiency virus-induced cytopathicity in MT-4 and CEM cells (50% effective concentration, approximately 0.1 to 0.6 microM). When administered intraperitoneally at a single dose as low as 2 mg/kg, (R)-PMPDAP efficiently decreased MSV-induced tumor formation in newborn NMRI mice and significantly increased the survival time of MSV-infected mice. In addition, upon oral administration to MSV-infected mice, (R)-PMPDAP showed marked antiretroviral efficacy.

Phosphorylation of 9-(2-phosphonomethoxyethyl)adenine and 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine by AMP(dAMP) kinase from L1210 cells.

Acyclic nucleotide analogues 9-(2-phosphonomethoxyethyl)adenine (PMEA) and 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine ((S)-HPMPA) which display potent antiviral activity are transformed in the cells to their mono- and disphosphoryl derivatives. We purified from mouse L1210 cells the enzyme that in two steps phosphorylates PMEA and (S)-HPMPA to their diphosphoryl derivatives and found that it co-purifies with AMP(dAMP) kinase activity; the best substrates of this enzyme were AMP, ADP and dAMP. Other nucleoside 5'-triphosphates or creatine phosphate could not be substituted for ATP as a phosphate donor. Our results also indicated that at least one other enzyme (creatine kinase) is capable of transforming the monophosphoryl derivatives of the studied compounds to their respective diphosphates.

9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] derivatives of purines: a class of highly selective antiretroviral agents in vitro and in vivo.

A new class of compounds, 9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] [(RS)-FPMP] derivatives of purines, is described that has selective activity against a broad spectrum of retroviruses [including human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)] but not other RNA or DNA viruses. This activity spectrum is completely different from that of the parental compounds, 9-[(2S)-3-hydroxy-2-phosphonylmethoxypropyl] [(S)-HPMP] derivatives of purines, which are active against a broad range of DNA viruses. The racemic (RS)-FPMP derivatives of adenine and 2,6-diaminopurine, termed (RS)-FPMPA and (RS)-FPMPDAP, respectively, are markedly more selective as in vitro antiretroviral agents than their 9-(2-phosphonylmethoxyethyl) (PME) counterparts, PMEA and PMEDAP. Also, (RS)-FPMPA and (RS)-FPMPDAP have a substantially higher therapeutic index in mice in inhibiting Moloney murine sarcoma virus-induced tumor formation and associated death and are markedly less inhibitory to human bone marrow cells than PMEA and PMEDAP. The diphosphate derivative of (RS)-FPMPA [(RS)-FPMPApp] is a potent and selective inhibitor of HIV-1 reverse transcriptase but not of HSV-1 DNA polymerase or DNA polymerase alpha. (RS)-FPMPApp, akin to PMEA diphosphate (PMEApp), acts as a DNA chain terminator. The DNA chain-terminating properties of PMEApp and (RS)-FPMPApp seem to be a prerequisite for acyclic nucleoside phosphonates to exhibit antiretrovirus (i.e., anti-HIV) activity.