Modifications on the heterocyclic base of ganciclovir, penciclovir, acyclovir - syntheses and antiviral properties.

АBSTRACTEsters of the antiherpetic drugs ganciclovir, penciclovir with the bile acids (cholic, chenodeoxycholic and deoxycholic) and amino acid esters of acyclovir were generated and evaluated for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The antiviral assays demonstrated that modified analogs of ACV and PCV are less active compared to the initial substances against HSV-1and HSV-2. CC50 for ganciclovir-deoxycholate corresponded to the CC50 of the other analogs and its activity is lower than ganciclovir. Obtained results show that tested modification do not improve bioavailability of nucleoside analogs in cells.

C-5 hydroxyethyl and hydroxypropyl acyclonucleosides as substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK): syntheses and biological evaluation.

The efficient syntheses of 5-(2-hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains are reported. A synthetic approach that included the alkylation of an N-anionic-5-substituted pyrimidine intermediate (method A) provided the target acyclonucleosides in significantly higher overall yields in comparison to those obtained by method B using sylilation reaction. The phosphorylation assays of novel compounds as potential substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK) showed that solely pyrimidine 5-substituted acyclonucleosides with a penciclovir-like side chain acted as a fraudulent substrates of HSV-1 TK. Moreover, the uracil derivative with penciclovir-like side chain with less bulky 2-hydroxyethyl substituent at C-5 proved to be a better substrate than the corresponding one with a 3-hydroxypropyl substituent. Therefore, this acyclonucleoside was selected as a lead compound for the development of a positron emission tomography HSV-1 TK activity imaging agent.

Studies on the synthesis of N'-acetyl aza-analogues of ganciclovir-unexpected liability of N'-(2-hydroxyethyl)-azanucleosides under basic conditions.

The O'-pivaloyl diesters of N'-acetyl-azanucleosides were obtained from N-[1,3-di(pivaloyloxy)prop-2-yl]-N-(pivaloyloxymethyl)acetamide and a silylated nucleobase under Vorbruggen's conditions. Unexpectedly, de-pivaloylation of the diesters under basic conditions afforded the corresponding nucleobase and N-acetylserinol. Mechanistic investigations showed that these products result from the following cascade of spontaneous transformations initiated by the mono de-pivaloylation of the starting diesters. N'-Deacetylation of the resultant mono-esters via the intramolecular N-O acetyl migration is the key step of the cascade; the corresponding NH-azanucleosides in the form of O-acetyl-O'-pivaloyl diesters are formed. Fragmentation of these diester intermediates gives the nucleobase and O-acetyl-O'-pivaloylserinol. Conversion of the latter to N-acetylserinol involves the selective O-N acetyl migration followed by de-pivaloylation of the resulting N-acetyl-O-pivaloylserinol.

A high yield robotic synthesis of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) and 9-[3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F] FHPG) for gene expression imaging.

The aim of this study was to develop an automated synthesis of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) and 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) using a Scanditronix Anatech RB III robotic system. [(18)F]HF was produced via (18)O(p, n)(18)F using a Scanditronix MC17F cyclotron. On average, a typical run produced [(18)F]FHBG and [(18)F]FHPG with an uncorrected radiochemical yield of 19% and 16%, respectively, at end of synthesis (EOS) from irradiation of 95% enriched [(18)O]water. The total synthesis time was 80 min. The retention time of [(18)F]FHBG and [(18)F]FHPG (the radio-peak) was 3.9 and 4.0 min, respectively, which was consistent with the [(19)F]FHBG and [(19)F]FHPG ultraviolet peak. The radiochemical purity was greater than 97%. A robotic, automated method for [(18)F]FHBG and [(18)F]FHPG radiosynthesis is therefore feasible. The radiation burden for the operator can be reduced as much as possible. Sufficient radioactivities of [(18)F]FHBG and [(18)F]FHPG could be obtained for non-invasive monitoring the expression of transfected gene in vivo with positron emission tomography (PET).

Synthesis of a technetium-99m labeled tricyclic ganciclovir analog for non-invasive reporter gene expression imaging.

A potential radiopharmaceutical and HSV1-TK substrate, 3-((1,3-dihydroxypropan-2-yloxy)-methyl)-6-(4-(3-((2-mercaptoethyl)(2-(2-mercaptoethyl-amino)-ethyl)amino)propoxy)phenyl)-3H-imidazopurin-9(5H)-one-oxo-technetium(V), was synthesized via a converging approach and its chemical structure was comparatively characterized with a non-radioactive analog. The final radiochemical purity and yield were 97 and 73%, respectively.

Synthesis, physicochemical properties and antiviral activities of ester prodrugs of ganciclovir.

The purpose of this study was to synthesize a series of diester prodrugs of ganciclovir (GCV), for improving ocular and oral bioavailability and therapeutic activity. Solubility, logP, pH stability profile, in vitro antiviral activity, cytotoxicity, inhibition profile and ocular tissue hydrolysis of the GCV prodrugs were measured. Val-Val-GCV and Val-Gly-GCV diesters were found to exhibit greater aqueous stability compared to Val-GCV and Gly-Val-GCV while ocular tissue hydrolysis demonstrated Val-Gly-GCV and Gly-Val-GCV to be more stable. Val-Val-GCV and Val-GCV diesters were the most lipophilic compounds and were predicted to possess a partition coefficient 295- and 12-fold greater than that of GCV, respectively. All the prodrugs possess much higher aqueous solubility than the parent drug GCV. Ex vivo uptake in the rabbit eye indicates that the prodrugs have high uptake potential. The prodrugs showed no increase in cytotoxicity compared to GCV, instead there was a marked increase in their potency against human cytomegalovirus (HCMV) as well as HSV-1 and HSV-2. This should allow therapeutic response to be seen at a lower concentration that can be achieved more easily, than the drugs currently being used. In conclusion, the diester GCV prodrugs demonstrated excellent chemical stability, high aqueous solubility and markedly enhanced antiviral potency against the herpes viruses without any increase in cytotoxicity.

Fluorosubstitution and 7-alkylation as prospective modifications of biologically active 6-aryl derivatives of tricyclic acyclovir and ganciclovir analogues.

A series of fluorine containing tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) were synthesized and evaluated for their activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumour cells. It was found that fluorine substitution reduced the antiviral activity, but most of the new compounds were pronounced cytostatic agents with potency and selectivity similar to those of parental ACV and GCV. Compounds 12, 13 and 16 seem to be promising as labeled substrates for (19)F NMR studies of the HSV TK-ligand interaction and/or monitoring of their metabolites in cells expressing HSV TK.

Synthesis and fluorescent properties of 6-(4-biphenylyl)-3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine analogues of acyclovir and ganciclovir.

Tricyclic (T) analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) carrying the 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine system [i.e., 6-(4-BrPh)TACV, 5 and 6-(4-BrPh)TGCV, 6] were transformed into 6-[(4'-R2)-4-biphenylyl] derivatives of TACV (7-9) and TGCV (10-12) by Suzuki cross coupling with 4-substituted phenylboronic acids. Compound 11 (R2 = CH2OH) showed a high (approximately 1000) selectivity index against herpes simplex virus type 1 (HSV-1) together with advantageous fluorescence properties (emission in visible region, little overlap with absorption and moderate intensity).

Synthesis and biological activity of strongly fluorescent tricyclic analogues of acyclovir and ganciclovir.

In search of strongly fluorescent tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2), derivatives of the 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine system, several 6-[4-(acyloxy)phenyl], 6-[4-(acylamino)phenyl], and 6-[4-(phenoxycarbonyloxy)phenyl]-substituted TACV and TGCV analogues were synthesized and evaluated for their activity against herpes simplex virus types 1 and 2 in cell culture. All TACV and TGCV analogues showed strong fluorescence (quantum yield of 30-65% vs 2-aminopurine 100%). The 6-[4-(phenoxycarbonyloxy)phenyl]-substituted compounds 11 and 19 displayed the best combination of the fluorescence and antiviral potency.

Fluorescent tricyclic analogues of acyclovir and ganciclovir. A structure-antiviral activity study.

Of a series of new guanine base modified tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2), derivatives of the 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine system, evaluated for activity against herpes simplex virus type 1 and 2, several fluorescent analogues, 6-(4-MeOPh)-TACV (8), 7-Me-6-Ph-TACV (17), 6-(4-MeOPh)-TGCV (27), and 7-Me-6-Ph-TGCV (28), were obtained that showed similar potency and selectivity as the parent compounds. The activity was found to be strongly dependent on the nature and steric demands of the substituents in the 6 and/or 7 position.

A simplified one-pot synthesis of 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy.

9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG, 2) has been synthesized by nucleophilic substitution of N(2)-(p-anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethoxy)-3-toluenesulfonyloxy-2-propoxy]methyl]guanine (1) with potassium [18F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 degrees C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 degrees C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 degrees C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 degrees C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126 +/- 0.022, n=5 and 0.165 +/- 0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ( approximately 5-30 microg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains approximately 10-25 microg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is readily amenable for automation.

In vitro and in vivo activity of 1-O-hexadecylpropanediol-3-phospho-ganciclovir and 1-O-hexadecylpropanediol-3-phospho-penciclovir in cytomegalovirus and herpes simplex virus infections.

Human cytomegalovirus (HCMV) and herpes simplex virus (HSV) can cause a wide variety of clinical manifestations in man. Ganciclovir (GCV) is effective against HCMV infection when administered by the intravenous route and may be used orally in large doses for prophylaxis of HCMV infections in organ transplantation patients and in AIDS patients. In previous studies with acyclovir (ACV), we found that covalent attachment of an alkyl glycerol phosphate moiety greatly increased oral bioavailability and increased antiviral activity against hepatitis B virus. Adducts of ACV with alkyl propanediol phosphate were more active than the alkyl glycerol phosphate analogue in vitro in 2.2.15 cells, which constitutively produce hepatitis B virus. To see if this strategy would work for two other poorly absorbed nucleoside analogues, we synthesized 1-O-hexadecylpropanediol-3-phospho-GCV (HDP-P-GCV) and 1-O-hexadecyl-propanediol-3-phospho-penciclovir (HDP-P-PCV), and evaluated the in vitro antiviral activity, selectivity and oral antiviral activity of both compounds versus GCV or PCV in mice infected with HSV-1 or HDP-P-GCV versus murine cytomegalovirus (MCMV). HDP-P-GCV is orally active in both MCMV and HSV-1 infection in mice with antiviral activity equivalent to (HSV-1) or greater than oral GCV (MCMV). Oral HDP-P-PCV was more active than PCV orally versus intranasal HSV-1 infection in mice.

Intravitreal toxicology and duration of efficacy of a novel antiviral lipid prodrug of ganciclovir in liposome formulation.

PURPOSE: To evaluate the intraocular safety and antiviral treatment efficacy of the sustained lipid prodrug of ganciclovir, 1-O-hexadecylpropanediol-3-phospho-ganciclovir (HDP-P-GCV), as an intravitreal injectable drug system for viral retinitis. METHODS: HDP-P-GCV was synthesized by coupling 1-O-hexadecyl-propanediol-3-phosphate to either free hydroxyl of ganciclovir in pyridine with dicyclohexylcarbodiimide as catalyst. The compound was formulated into liposomes. The antiviral activity was assessed by DNA reduction in vitro, and intraocular safety was assessed by ophthalmoscopy, electrophysiology, and histology after intravitreal injections, with resultant intravitreal concentrations of 0.2, 0.632, 1.12, and 2 mM. The treatment efficacy was evaluated by simultaneous intravitreal injection of HDP-P-GCV and herpes simplex virus type 1 (HSV-1) or by intravitreal injection of HDP-P-GCV at various times before HSV-1 intravitreal inoculation. Retinitis was scored with ophthalmoscopy and compared with controls. RESULTS: In vitro, the IC50 of HDP-P-GCV against HSV-1 and human cytomegalovirus (HCMV) infected cells was 0.02 and 0.6 microM, respectively. In rabbits in vivo, HDP-P-GCV dispersed evenly and maintained a good vitreous clarity at all doses except 2 mM final intravitreal concentration. Although cataracts were observed in some eyes at the higher doses, they were not observed in eyes with 0.2 mM final intravitreal concentration. No other indications of ocular toxicity were observed. Intravitreal injection of HDP-P-GCV with resultant 0.2 mM intravitreal concentration in the HSV-1 retinitis rabbit model demonstrated a complete protection of the retina with the simultaneous treatment strategy and a 4 (P = 0.03) to 6-(P = 0.058) week significant protection of retina with the pretreatment strategies when compared with ganciclovir or blank liposome controls. CONCLUSIONS: In the rabbit model of HSV-1 retinitis HDP-P-GCV acts as a long-lasting intravitreal injectable anti-CMV or anti-HSV compound. This self-assembling liposome system could be applicable for many compounds available for intraocular diseases.

9-[(3-[18F]-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]-FHPG): a potential imaging agent of viral infection and gene therapy using PET.

A no-carrier-added synthesis of 9-[(3-[18F]-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) is reported. The 9-[(1,3-dihydroxy-2-propoxy)methyl)guanine (DHPG) was converted to 9-[N2,O-bis(methoxytrityl)-3-(tosyl)-2-propoxy-methyl]guanine by treatment with methoxytrityl chloride followed by tosylation. The tosylate was reacted with [18F]-KF in the presence of kryptofix 2.2.2. to produce the 3-fluoro-N2-O-bis-(methoxytrityl) derivative. Removal of the methoxytrityl protecting groups by acid hydrolysis produced [18F]-FHPG. The labeled product was purified by HPLC on a reverse-phase C18 column, and eluted in 9 min with a mobile phase of 5% acetonitrile in water. The radiochemical yield was 7-17%, with an average of 10% in 10 runs (corrected for decay to EOB). The radiochemical purity was > 99%, and specific activities with an average of 526 mCi/mumol were obtained. The synthesis time was 70-80 min, including HPLC purification and determination of radiochemical purity and specific activity.

Metabolism and pharmacokinetics of a double prodrug of ganciclovir in the rat and monkey.

Ganciclovir (GCV), which is used in the treatment of human cytomegalovirus infections, is poorly absorbed orally. A double prodrug of GCV, the dipivalate ester of 6-deoxy-GCV (6-dGCV) (called 6-dGCV-DPiv), was given orally to rats (25 mg/kg) and resulted in a nearly 7-fold enhancement of GCV bioavailability compared with administration of GCV alone and a 2-fold increase compared with administration of 6-dGCV. The prodrug was rapidly hydrolyzed and extensively oxidized by first-pass metabolism in such a way that only GCV, 6-dGCV, and a small amount of the monopivalate ester of 6-dGCV were observed in rat plasma. In cynomolgus monkey was given the prodrug orally (22.5 mg/kg), two additional metabolites were observed--the 8-hydroxy analogs of GCV and dGCV. The double prodrug approach demonstrated the potential for enhanced oral delivery of GCV in humans.

An enantiospecific synthesis of the human cytomegalovirus antiviral agent [(R)-3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid.

The racemic isosteric phosphonate of ganciclovir monophosphate (BW2482U89, SR3745, [3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid, 1) has potent and selective in vitro activity against human cytomegalovirus. An enantiospecific synthesis of the R-enantiomer of compound 1 starting from L-arabinose was developed. The synthesis involved (1) the preparation of a chiral acyclic moiety, (2) the coupling of the chiral acyclic moiety to diacetylguanine, (3) the introduction of phosphorus, and (4) the final deprotection. The R-enantiomer, which has stereochemistry analogous to the natural compound GMP, was tested against human cytomegalovirus and had an IC50 of 1.7 microM, which was approximately 2-fold more active than the racemic material. Both racemic and chiral compounds were less toxic than ganciclovir to bone marrow progenitor cells in an in vitro assay.

Synthesis and antiviral activity of 3-substituted derivatives of 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purines, tricyclic analogues of acyclovir and ganciclovir.

9-[(2-Hydroxyethoxy)methyl]guanine (acyclovir, 1a) and 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, ganciclovir, 1b) were transformed to their respective tricyclic derivatives, 3-substituted 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purines 2b, 3a, and 3b. The 6-methyl-substituted compound 2b was obtained following reaction of 1b with bromoacetone. A two-step approach via 1-(2,2-diethoxyethyl) intermediates 4a,b was the most effective for the preparation of the derivatives unsubstituted in the appended ring (3a,b). The novel acyclonucleosides, in particular ganciclovir derivative 2b, proved markedly active against herpes simplex virus type 1 and 2, varicella-zoster virus, and cytomegalovirus.

Synthesis and antiviral activity of 9-alkoxypurines. 1. 9-(3-Hydroxypropoxy)- and 9-[3-hydroxymethyl)propoxy]purines.

Reaction of hydroxyl-protected derivatives of hydroxyalkoxyamines (3a,b,c) with either 4,6-dichloro-2,5-diformamidopyrimidine (5) or 4,6-dichloro-5-formamidopyrimidine (31) and subsequent cyclization of the resultant 6-(alkoxyamino)pyrimidines (6, 17, 32, 35) by heating with diethoxymethyl acetate afforded 9-alkoxy-6-chloropurines (7, 18, 33, 36), which were converted subsequently to 9-(3-hydroxypropoxy)- and 9-[3-hydroxy-2-(hydroxymethyl)propoxy] derivatives of guanine, 2-amino-6-chloropurine, 2-amino-6-alkoxypurines, 2-aminopurine, 2,6-diaminopurine, adenine, hypoxanthine, and 6-methoxypurine (8, 12, 13, 19-21, 23-26, 34, 37-39). Carboxylic acid esters (9-11, 14-16, 27-29) and a cyclic phosphate derivative (22) of the 9-(hydroxyalkoxy)guanines (8, 21) and 2-amino-9-(hydroxyalkoxy)purines (13, 26) were also prepared. The guanine derivatives (8, 21) showed potent and selective activity against herpes simplex virus types 1 and 2 and varicella zoster virus in cell cultures and 8 is more active than acyclovir. Although without significant antiviral activity in cell cultures, the 2-aminopurines (13, 14-16, 26-29) and 2-amino-6-alkoxypurines (12, 23-25) are well absorbed after oral administration to mice and are converted efficiently to the antiviral guanine derivatives (8, 21) in vivo.