Ester prodrugs of cyclic 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine: synthesis and antiviral activity

Reaction of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (1) with dicyclohexylcarbodiimide and N,N,-dicyclohexyl-4-morpholinocarboxamidine in dimethylformamide at elevated temperature afforded the corresponding cyclic phosphonate 2, that is, 1-{[(5S)-2-hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-5-azacytosine. Compound 2 exerts strong in vitro activity against DNA viruses, comparable with activity of parent compound 1. Transformation of 2 to its tetrabutylammonium salt followed by reaction with alkyl or acyloxyalkyl halogenides enabled us to prepare a series of structurally diverse ester prodrugs: alkyl (octadecyl), alkenyl (erucyl), alkoxyalkyl (hexadecyloxyethyl), and acyloxyalkyl (pivaloyloxymethyl) (3-6). The introduction of an alkyl, alkoxyalkyl, or acyloxyalkyl ester group to the molecule resulted in an increase of antiviral activity; the most active compound was found to be the hexadecyloxyethyl ester 5. The relative configuration of the diastereoisomer trans-6 was determined using H,H-NOESY NMR.     

Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is an antiviral phosphonate nucleotide analogue that displays activity against a range of herpesviruses. Anion exchange high performance liquid chromatography analysis of the 60% methanol extract from [14C]HPMPC-treated cells reveals the formation of three major metabolites. Two of these were identified as phosphorylated forms of HPMPC, HPMPC phosphate, and HPMPC diphosphate, by liberation of HPMPC upon acid digestion and coelution with synthetic standards on high performance liquid chromatography. The third metabolite, which is resistant to alkaline phosphatase cleavage but sensitive to phosphodiesterase, is proposed to be an HPMPC phosphate adduct. In herpes simplex virus-1-infected cells the same three metabolites are detected, at concentrations comparable to those in uninfected cells. When HPMPC is removed from the medium, the concentrations of the metabolites in cells decrease slowly, with half-lives of approximately 6, 17, and 48 hr for HPMPC phosphate, HPMPC diphosphate, and the HPMPC phosphate adduct, respectively. HPMPC diphosphate inhibits herpes simplex virus-1 and -2 DNA polymerases with a lower Ki than that for DNA polymerase alpha, and enzyme inhibition is competitive in each case. The formation and the persistence of HPMPC phosphates in cells and the selective inhibition of viral DNA polymerases by HPMPC diphosphate can explain why cells pretreated with HPMPC remain refractory to viral infection even long after HPMPC is removed from the medium.     

Synthesis and antiviral activity of the nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine

The acyclic nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl] cytosine (2, HPMPC) was prepared on a multigram scale in 18% overall yield starting from (R)-2,3-O-isopropylideneglycerol. The key step in the nine-step synthetic route is coupling of cytosine with the side-chain derivative 8 which bears a protected phosphonylmethyl ether group. In vitro data showed that HPMPC has good activity against herpes simplex virus types 1 and 2, although it was 10-fold less potent than acyclovir [AVC, 9-[(2-hydroxyethoxy)methyl]guanine]. By comparison, HPMPC exhibited greater activity than ACV against a thymidine kinase deficient strain of HSV 1 and was more potent than ganciclovir [DHPG, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine] against human cytomegalovirus. In vivo, HPMPC showed exceptional potency against HSV 1 systemic infection in mice, having an ED50 of 0.1 mg/kg per day (ip) compared with 50 mg/kg per day for ACV. HPMPC was also more efficacious than ACV in the topical treatment of HSV 1 induced cutaneous lesions in guinea pigs.     

Antiviral activity of triazine analogues of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir) and related compounds

Treatment of 5-azacytosine sodium salt with diisopropyl [(2-chloroethoxy)methyl]phosphonate followed by removal of ester groups with BrSi(CH3)3 afforded 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (3). Reaction of 5-azacytosine with [(trityloxy)methyl]-(2S)-oxirane followed by etherification with diisopropyl (bromomethyl)phosphonate and removal of ester groups gave 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (1). The synthesis of 6-azacytosine congener 2 was analogous using N4-benzoylated intermediates. Compound 1 was shown to exert strong activity against a broad spectrum of DNA viruses including adenoviruses, poxviruses, and herpesviruses (i.e., herpes simplex viruses, varicella zoster virus, and human cytomegalovirus). Decomposition of 1 in alkaline solutions resulted in products 17 and 18. While the N-formylguanidine derivative 17 proved active, the carbamyolguanidine derivative 18 was devoid of antiviral activity.     

Synthesis and antiviral activity of (S)-9-[4-hydroxy-3-(phosphonomethoxy)butyl]guanine

The synthesis of (S)-9-[4-hydroxy-3-(phosphonomethoxy)butyl]guanine (3), starting from (S)-4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane (4), is described. Alkylation of trityl derivative 7 with (diethylphosphono)methyl triflate provided phosphonate 8, which was readily converted to mesylate 12 in three steps. Nucleophilic substitution of the mesylate group of 12 by 2-amino-6-chloropurine sodium salt led to (S)-2-amino-6-chloro-9-[3-[(diethyl-phosphono)methoxy]-4-(tetrahydro- 2H-pyran-2-yloxy)butyl]purine (13). Sequential treatment of 13 with trimethylsilyl bromide and then with 2 N HCl furnished 3. Preliminary in vitro screening indicated that 3 exhibited a potent activity against human cytomegalovirus (HCMV) but was not active against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The adenine and cytosine derivatives (14 and 15) did not exhibit activity against HSV-1 and -2 and HCMV.     

Tyrosine-based 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine and -adenine ((S)-HPMPC and (S)-HPMPA) prodrugs: synthesis, stability, antiviral activity, and in vivo transport studies

Eight novel single amino acid (6-11) and dipeptide (12, 13) tyrosine P-O esters of cyclic cidofovir ((S)-cHPMPC, 4) and its cyclic adenine analogue ((S)-cHPMPA, 3) were synthesized and evaluated as prodrugs. In vitro IC(50) values for the prodrugs (<0.1-50 μM) vs vaccinia, cowpox, human cytomegalovirus, and herpes simplex type 1 virus were compared to those for the parent drugs ((S)-HPMPC, 2; (S)-HPMPA, 1; IC(50) 0.3-35 μM); there was no cytoxicity with KB or HFF cells at ≤100 μM. The prodrugs exhibited a wide range of half-lives in rat intestinal homogenate at pH 6.5 (<30-1732 min) with differences of 3-10× between phostonate diastereomers. The tyrosine alkylamide derivatives of 3 and 4 were the most stable. (l)-Tyr-NH-i-Bu cHPMPA (11) was converted in rat or mouse plasma solely to two active metabolites and had significantly enhanced oral bioavailability vs parent drug 1 in a mouse model (39% vs <5%).     

Antiviral evaluation of octadecyloxyethyl esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against herpesviruses and orthopoxviruses

Our previous studies showed that esterification of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC₅₀ values were observed with ODE–HPMPA and ODE–HPMPC while ODE–HPMPG had intermediate antiviral activity with an EC₅₀ of 40 nM. In HFF cells infected with HCMV, the lowest EC₅₀ values were observed with ODE–HPMPC, 0.9 nM. ODE–HPMPA was highly active with an EC₅₀ of 3 nM, while ODE–HPMPG and ODE–HPMPDAP were also highly active with EC₅₀s of 22 and 77 nM, respectively. Against vaccinia and cowpox viruses, ODE–HPMPG and ODE–HPMPDAP were the most active and selective compounds with EC₅₀ values of 20–60 nM and selectivity index values of 600–3500. ODE–HPMPG was also active against ectromelia virus with an EC₅₀ value of 410 nM and a selectivity index value of 166. ODE–HPMPG and ODE–HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.     

Synthesis, in vitro antiviral evaluation, and stability studies of novel alpha-borano-nucleotide analogues of 9-[2-(phosphonomethoxy)ethyl]adenine and (R)-9-[2-(phosphonomethoxy)propyl]adenine

We describe here the synthesis of 9-[2-(boranophosphonomethoxy)ethyl]adenine (6a) and (R)-9-[2-(boranophosphonomethoxy)propyl]adenine (6b), the first alpha-boranophosphonate nucleosides in which a borane (BH3) group substitutes one nonbridging oxygen atom of the alpha-phosphonate moiety. H-phosphinates 5a and 5b and alpha-boranophosphonates 6a and 6b were evaluated for their in vitro activity against human immunodeficiency virus (HIV) infected cells and against a panel of DNA or RNA viruses. Compounds 5a, 5b, 6a, and 6b exhibited no significant antiviral activity in vitro and cytotoxicity. To measure the chemical and enzymatic stabilities of the target compounds 6a and 6b, kinetic data of decomposition for derivatives 5a, 5b, 6a, 6b, and standard compounds were studied at 37 degrees C in several media. The alpha-boranophosphonates 6a and 6b were metabolized in culture medium into H-phosphinates 5a and 5b, with half-live values of 5.3 h for 6a and 1.3 h for 6b.     

5-Substituted-2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines-acyclic nucleoside phosphonate analogues with antiviral activity

2,4-Diamino-6-hydroxypyrimidines substituted in position 5 by an allyl, benzyl, cyanomethyl, ethoxycarbonylmethyl, phenyl, cyclopropyl, or methyl group were prepared either by C5-alkylation or by formation of the pyrimidine ring by cyclization. Their alkylation with 2-[(diisopropoxyphosphoryl)methoxy]ethyl tosylate afforded N1- and O6-regioisomers that were separated and converted to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis. Reaction of 2,4-diamino-6-[[(diisopropoxyphosphoryl)methoxy]ethoxy]pyrimidine with elemental bromine, N-chloro-, or N-iodosuccinimide gave the corresponding phosphorus-protected 5-bromo-, 5-chloro-, and 5-iodo derivatives, respectively. Their deprotection afforded 2,4-diamino-5-bromo- and -5-chloro-6-[2-(phosphonomethoxy)ethoxy]pyrimidines. 2,4-Diamino-5-methyl-6-[2-(phosphonomethoxy)ethoxy]pyrimidine was synthesized also by cross-coupling of the 5-bromo compound with AlMe(3), followed by deprotection. The compounds showed poor, if any, inhibitory activity against DNA viruses such as herpes simplex virus type 1 and type 2, cytomegalovirus, varicella-zoster virus, and vaccinia virus. In contrast, several 5-substituted 2,4-diaminopyrimidine derivatives markedly inhibited retrovirus replication in cell culture. The 5-methyl derivative was exquisitely inhibitory to human immunodeficiency virus and Moloney murine sarcoma virus-induced cytopathicity in cell culture (EC(50) approximately 0.00018 mumol/mL) but also cytostatic to CEM cell cultures. In contrast, the 5-halogen-substituted derivatives showed pronounced antiretroviral activity (EC(50) = 0.0023-0.0110 mumol/mL), comparable to that of the reference drugs adefovir and tenofovir, but were devoid of measurable toxicity at 0.3 mumol/mL.     

Synthesis and 5-HT2A, 5-HT1A and alpha1-binding affinities of 2-[2-Hydroxy-3-(pyridin-3-yl-methyl)amino]-, 2-[2-hydroxy-3-(2-pyridin-2-yl-ethyl)amino]- and 2-[2-hydroxy-3-(4-N-methyl-piperazin-1-yl)-amino]propoxybenzaldehyde-O-(substituted) benzyl oximes

Some oxime ether-substituted aryloxypropanolamines 3-5, structurally related to the active metabolite 2 of sarpogrelate 1, were synthesized and tested for their affinities at 5-HT2A and 5-HT1A serotoninergic receptors as well as at the alpha1-adrenoceptor. The results show that the compounds possess, at least partially, the ability of the model compounds 1 and 2 to interact with the 5-HT2A-receptors; they have the same selectivity towards 5-HT2A receptors vs alpha1-adrenoceptors.     

Antiarrhythmic and hypotensive activities of 1-[2-hydroxy-3-(4-phenyl-1-piperazinyl)propyl]-pyrrolidin-2-one (MG-1(R,S)) and its enantiomers

The compound MG-1(R,S), (1-[2-hydroxy-3(4-phenyl-1-piperazinyl)propyl]-pyrrolidin-2-one, and its enantiomers were tested for electrocardiographic, antiarrhythmic and hypotensive activities. The racemic mixture (MG-1(R,S)) and its S-enantiomer significantly decreased systolic and diastolic blood pressure and possessed antiarrhythmic activity. The S-enantiomer displayed the greatest effect. The R-enantiomer did not show antiarrhythmic or hypotensive activity. The results suggest that the antiarrhythmic and hypotensive effects of these compounds are related to their adrenolytic properties.     

Evaluation of infrequent dosing regimens with (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]-cytosine (S-HPMPC) on simian varicella infection in monkeys.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (S-HPMPC) was able to prevent simian varicella infection in African green monkeys inoculated intratracheally with virus. A dose of 50 mg/S-HPMPC/kg administered intravenously was shown to prevent the development of rash, reduce viremia and protect the monkeys from death. The 50 mg/kg dose was effective when treatments initiated on day 2 post-infection (p.i.) was given as ten daily doses of 5 mg/kg, as 10 mg/kg administered on five days on an alternate-day schedule, as two 25 mg/kg doses given on day 2 and on day 7 p.i., or as a single injection of 50 mg/kg on day 2. The single 50 mg/kg dose was also effective when treatment was delayed until four days p.i., but was ineffective when treatment was delayed until six days p.i. The 50 mg/kg dose was not effective when given orally by gavage. No evidence of toxicity was noted in daily clinical examinations, or in frequent hematology and clinical chemistry tests performed during the clinical evaluation of the infection.     

Selective inhibition of human cytochrome P450 3A4 by N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1, 1-dimethylethylaminocarbonyl)-4-[(furo[2, 3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethy lpentanamide and P-glycoprotein by valspodar in gene transfectant systems.

Our previous report showed that L754.394 and valspodar (PSC833) are potent inhibitors of midazolam hydroxylation in human jejunum microsomes and vectorial transport of vinblastine in Caco-2 cells, respectively. In the present study, to directly examine the interactions of these compounds as well as other substrates with CYP3A4 and P-glycoprotein (P-gp), we performed in vitro inhibition studies using recombinant CYP3A4-expressed microsomes and an MDR1-transfected cell line, LLC-MDR1, respectively. In CYP3A4-expressed microsomes, both L754.394 and ketoconazole, at a concentration less than 0.5 microM, are the most potent inhibitors of the formation of 1'-hydroxymidazolam, a major metabolite of midazolam formed by CYP3A4. The greatest inhibitory effect on the transcellular transport of digoxin in LLC-MDR1 cells was observed in the presence of valspodar (<0.1 microM), followed by verapamil. From a comparison of the IC(50) values, it was shown that L754.394 and valspodar exhibited the highest selectivity for CYP3A4 and P-gp, respectively. To demonstrate such specificity, both midazolam hydroxylation and digoxin transport were observed in CYP3A4 transfected Caco-2 cells, which coexpress both P-gp and CYP3A4, in the presence or absence of L754.394 (0.5 microM) and valspodar (1.0 microM). L754.394 almost completely inhibited midazolam hydroxylation, but not digoxin transport, whereas almost complete inhibition of digoxin transport was observed in the presence of valspodar, but inhibition of the hydroxylation was minimal. Thus, the present study has demonstrated that L754.394 has a specific inhibitory effect on CYP3A4, whereas valspodar is specific for P-gp.     

Synthesis and antiviral activity of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine.

A number of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and tested in vitro for anti-herpes and anti-human immunodeficiency virus (HIV) activity. Among these analogues, (R)-2'-methyl-PMEG [(R)-3] and 2',2'-dimethyl-PMEG (7) demonstrated potent anti-HIV activity in the XTT assay with EC50 values of 1.0 and 2.6 microM, respectively. The corresponding (S)-2'-methyl-PMEG [(S)-3] was found to be less potent against HIV. In addition, the (R) and (S) enantiomers of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG, 8) were prepared for comparison of biological activity, and shown to be active and equipotent against herpesviruses, but inactive against HIV.     

Microbial metabolism of bornaprine, 3-(diethylamino)propyl 2-phenylbicyclo[2.2.1]heptane-2-carboxylate

Metabolism studies of the anticholinergic drug, bornaprine [3-(diethylamino)propyl 2-phenylbicyclo[2.2.1]heptane-2-carboxylate, an epimeric mixture], in rats, dogs, and humans have been conducted previously, but the identities of the metabolites were not established. Using an in vitro microbial system to study the metabolism of bornaprine resulted in the isolation of four metabolites whose structures were rigorously established using spectroscopic techniques, especially 13C NMR. The four metabolites found were hydroxylated at C-5 or C-6 in the bicyclic ring.     

Investigations on the synthesis and pharmacological properties of 4-alkoxy-2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl]-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones

Synthesis of 2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl] derivatives of 4-alkoxy-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones (8-12) is described. The chlorides used in the above synthesis can exist in two isomeric forms: chain (18-20) and cyclic (19a, 20a). The compounds 8-12 exhibited potent analgesic activity which was superior than that of acetylsalicylic acid in two different tests. Most of the investigated imides suppressed significantly spontaneous locomotor activity in mice.     

Synthesis and antiviral activity of 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purines

Alkylation of 2-amino-6-chloropurine with 5-(2-bromoethyl)-2,2-dimethyl-1,3-dioxane (5) provided 2-amino-6-chloro-9-[2,(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]purine (6) in high yield. This aminochloropurine 6 was readily converted to the antiviral acyclonucleoside 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (1) and to its 6-chloro (10), 6-thio (11), 6-alkoxy (12-17), 6-amino (20), and 6-deoxy (21) purine analogues. The guanine derivative 1 was converted to its xanthine analogue 9. Similarly, alkylation of 6-chloropurine with 5 provided a route to 8, the hypoxanthine analogue of 1. Of these 9-substituted purines, the guanine derivative 1 showed the highest activity against herpes simplex virus types 1 and 2 in cell cultures, and in some tests it was more active than acyclovir, with no evidence of toxicity for the cells. A series of monoesters (30-33) and diesters (24-27, 29) of 1 were prepared, and some of these also showed antiherpes virus activity in cell cultures, the most active ester being the dihexanoate 27.     

Investigations on the synthesis and pharmacological properties of 4-alkoxy-2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl]-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones

Synthesis of 2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl] derivatives of 4-alkoxy-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones (8-12) is described. The chlorides used in the above synthesis can exist in two isomeric forms: chain (18-20) and cyclic (19a, 20a). The compounds 8-12 exhibited potent analgesic activity which was superior than that of acetylsalicylic acid in two different tests. Most of the investigated imides suppressed significantly spontaneous locomotor activity in mice.