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A series of 2′-fluorinated C -nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2′-fluoro-2′- C -methyl adenosine C -nucleoside ( 15 ) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant ( 53 ) with favorable pharmacokinetic properties including efficient liver delivery in animals.

The first synthesis of 1'-C-CN, 2'-F, 2'-C-Me pyrimidines is described. Anti-HCV activity was assessed and compared to the 1'-C-CN, 2'-C-Me as well as the 2'-F, 2'-C-Me pyrimidines. A phosphoramidate prodrug of the cytidine derivative showed activity in the low micromolar range against HCV replicons.

The first synthesis of 1'-cyano-2'-C-methyl pyrimidine nucleosides is described. Anti-HCV activity of these nucleosides and their nucleotide phosphoramidate prodrugs was assessed and compared to the 1'-unsubstituted counterparts and to the related 1'-cyano-2'-C-methyl C-nucleoside parent of GS-6620.

A sulfonamide replacement of the P2–P3 amide bond in the context of macrocyclic HCV NS3 protease inhibitors was investigated. These analogs displayed good inhibitory potency in the absence of any P3 capping group. The synthesis and preliminary SAR are described.

Conversion of diethyl 1-amino-2-vinylcyclopropanephosphonate to ethyl 1-amino-2-vinylcyclopropanephosphinate was accomplished by using either nucleophilic or electrophilic carbon reagents. Hydrolysis of the phosphonate diethyl ester to the mono acid followed by oxalyl chloride treatment provided the phosphonomonochloridate, which was treated with nucleophilic organometallic agents to afford phosphinate ethyl esters. Alternatively, the chloridate was reduced to the phosphonous ethyl ester, and then alkylated with various electrophilic alkylating agents to obtain phosphinate ethyl esters.

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.

A potent and novel class of product-like inhibitors of the HCV NS3 protease was discovered by employing a phosphinic acid as a carboxylate isostere. The replicon activity and pharmacokinetic profile of this series of compounds was optimized by exploring the substitution of the phosphinic acid, as well as conformationally constraining these compounds through macrocyclization. The syntheses and preliminary biological evaluation of these phosphinic acids is described.

A series of N1-alkyl pyrimidinediones were designed, synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Our efforts identified compound 10b, which represents the lead compound in this series with pharmacokinetics and antiviral potency that may support once-daily dosing.

A novel class of phosphonate derivatives was designed to mimic the interaction of product-like carboxylate based inhibitors of HCV NS3 protease. A phosphonic acid (compound 2) was demonstrated to be a potent HCV NS3 protease inhibitor, and a potential candidate for treating HCV infection. The syntheses and preliminary biological evaluation of this phosphonate class of inhibitor are described.

A diphosphate of a novel cyclopentyl based nucleoside phosphonate with potent inhibition of HIV reverse transcriptase (RT) (20, IC(50)=0.13 microM) has been discovered. In cell culture the parent phosphonate diacid 9 demonstrated antiviral activity EC(50)=16 microM, within two-fold of GS-9148, a prodrug of which is currently under clinical investigation, and within 5-fold of tenofovir (PMPA). In vitro cellular metabolism studies using 9 confirmed that the active diphosphate metabolite is produced albeit at a lower efficiency relative to GS-9148.

A series of C3 halobenzyl-substituted tricyclic HIV integrase inhibitors was prepared. Improvement in cell-based inhibitor potency was observed in comparison to previously disclosed tricyclic pyrroloquinolines carrying the ‘halobenzyl tail’ at the lactam nitrogen. Animal PK for several of the C3-substituted inhibitors was examined, with a dihaloaryl analog achieving good balance in protein-shifted EC50 and t1/2 in animal PK studies.

GS-9148 (2'-Fd4AP, 4) has been identified as a nucleoside phosphonate reverse transcriptase (RT) inhibitor with activity against wild-type HIV (EC(50)=12 microM). Unlike many clinical RT inhibitors, relevant reverse transcriptase mutants (M184V, K65R, 6-TAMs) maintain a susceptibility to 2'-Fd4AP that is similar to wild-type virus. The 2'-fluorine group was rationally designed into the molecule to improve the selectivity profile and in preliminary studies using HepG2 cells, compound 4 showed no measurable effect on mitochondrial DNA content indicating a low potential for mitochondrial toxicity.

A series of C5-aza tricyclic HIV integrase inhibitors was prepared. A highly potent and orally bioavailable compound (compound 9) was identified and selected for development.

A series of nucleoside phosphonate reverse transcriptase (RT) inhibitors have been synthesized and their anti-HIV activity and resistance profiles evaluated. The most potent analog [5-(6-amino-purin-9-yl)-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid (d4AP) demonstrated a HIV EC50 = 2.1 μM, and the most favorable resistance profile against HIV-1 variants with K65R, M184V or multiple thymidine analog mutations in RT.

Cyclic phosphonomethoxy pyrimidine nucleosides that are bioisosteres of the monophosphate metabolites of HIV reverse transcriptase (RT) inhibitors AZT, d4T, and ddC have been synthesized. The RT inhibitory activities of the phosphonates were reduced for both dideoxy (dd) and dideoxydidehydro (d4) analogs compared to the nucleosides. Bis-isopropyloxymethylcarbonyl (BisPOC) prodrugs were prepared on selected compounds and provided > 150-fold improvements in antiviral activity.

We report a study of the influence of Lewis acids upon the regioselectivity of ring opening of quinolinic[2,3]anhydrides to provide 2-(isopropoxycarbonyl)-nicotinic acids. In the presence of stoichiometric amounts of indium trifluoromethanesulfonate or lanthanum trifluoromethanesulfonate, the desired 2-position ester was generated with greater than 95:5 regioselectivity. This methodology was also applied to 6-methyl-[2,3]-quinoline to provide similar results.

AN1057A/B has shown potent activity against MRSA. A novel and concise route to the synthesis of its heterocycle core 2,5-diamino-5,6-dihydro-1H-pyrimidine-4-one is described. This methodology allows the synthesis of an array of analogs with different amine substitutions at the 2-position.

Graphical abstract A series of 1′-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside were prepared and evaluated for the potential as antiviral agents. These compounds showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R = CN was attributed to inhibition of HCV NS5B polymerase and intracellular concentration of the corresponding nucleoside triphosphate., A series of 1′-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside were prepared and evaluated for the potential as antiviral agents. These compounds showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R = CN was attributed to inhibition of HCV NS5B polymerase and intracellular concentration of the corresponding nucleoside triphosphate.

Several 1′-substituted analogs of Tubercidin C-nucleosides were prepared using a highly convergent synthesis. Good to high diastereoselectivity was achieved using a variety of nucleophiles targeting the 1′-position. The source for this stereoselectivity is herein proposed. It is thought to be attributed to a temperature-dependent chelation of the incoming nucleophile to either the 2′- or 3′-benzyloxy ether of the ribose core.

Rational drug design utilizing available X-ray crystal structures of sialic acid analogues bound to the active site of influenza virus neuraminidase has led to the discovery of a series of potent carbocyclic influenza neuraminidase inhibitors. From this series, GS 4104 (oseltamivir, TAMIFLU) has emerged as a promising antiviral for the treatment and prophylaxis of human influenza infection. This article will summarize the design, discovery, and development of oseltamivir as an oral therapeutic to treat influenza infection.

We herein describe an efficient synthesis of optically active diethyl 1-amino-2-vinylcyclopropane-1-phosphonate (analogous to 1-amino-2-vinylcyclopropane-1-carboxylate). The racemic phosphonate diethyl ester was obtained from an imine derived from aminomethylphosphonate diester and trans-1,4-dibromo-2-butene. Crystallizations of the dibenzoyl- l -tartaric acid salt allowed for separation of enantiomers. The enantiomerically pure material was used to synthesize an extremely potent tripeptide phosphonate inhibitor of HCV NS3 protease. X-ray crystal structure of the inhibitor bound to the HCV NS3 protease confirmed the absolute stereochemistry of the title compound.

A facile construction of the 3,6-diamino-1,2,3,4-tetrahydropyridine-4-one class of compounds is described. Compound 2, a carbon analog at the N-3 position of TAN-1057A/B, was synthesized using this approach.

The structure-activity relationships (SAR) for a new class of potent inhibitors (1) of influenza neuraminidase are described. Systematic modifications of substituents at the C-3, C-4, and C-5 positions of the carbocyclic ring were performed to establish fundamental SAR to assist in the design of potent inhibitors with activity against both of influenza A and B viruses.

The influence of C2-substitution of GS 4071 on the influenza neuraminidase inhibitory activity was investigated. The introduction of lipophilic substituents (chloro, methyl, and methylthio) at the C2 position resulted in a significant decrease of the activity. This result indicates that at the enzyme active site there is limited hydrophobic pocket for group at the C2 position of GS 4071.

Aromatic analogues of sialic acid ( 2 and 3 ) have been synthesized as potential influenza neuraminidase inhibitors. Whereas compound 2 exhibited good neuraminidase inhibitory activity, compound 3 , possessing the glycerol side chain at the C 5 position, was not a neuraminidase inhibitor.

Among retinoic acid receptors (RARs) alpha, beta, and gamma, the messenger RNA level of RAR-gamma is the most readily detectable by Northern blotting in human and mouse skin. This observation suggests that RAR-gamma may play a critical role in the modulation of the therapeutic benefits and side effects of retinoids in skin. To test this hypothesis, 11 RAR-gamma selective retinoids were synthesized based on three related structures. Each compound was found to prefer RAR-gamma when assessed by retinoid-induced transcriptional activity (RAR-gammaRAR-betaRAR-alpha). The apparent Kd for binding to recombinant receptor protein was found to follow a similar trend. To correlate this receptor selectivity with in vivo activity, the compounds were tested topically in the Rhino mouse utriculi reduction and rabbit irritation models, two assays widely used to screen retinoids for efficacy and side effects, respectively. The results indicated that for these compounds, both efficacy in the utriculi reduction assay and irritation potential in rabbits correlated positively with the RAR-gamma transactivation activity, with r2 of 0.9 and 0.8, respectively. These data suggest that RAR-gamma is an important regulator of retinoic acid efficacy in skin and further, that the irritation associated with the use of retinoids is most likely a receptor-mediated process.

TAN-1057A/B has shown potent activity against MRSA. A new approach to the 2,5-diamino-5,6-dihydro-1 H -pyrimidine-4-one derivatives has been developed. TAN-1057A/B and its analogs were efficiently synthesized using this new approach.

A series of 2′-C-methyl branched purine and pyrimidine C-nucleosides were prepared. Their anti-HCV activity and pharmacological properties were profiled, and compared with known 2′-C-Me N-nucleoside counterparts. In particular, 2′-C-Me 4-aza-7,9-dideazaadenosine C-nucleoside (2) was found to have potent and selective anti-HCV activity in vitro as well as a favorable pharmacokinetic profile and in vivo potential for enhanced potency over the corresponding N-nucleoside.

Regiospecific synthesis of alkylated A ring of arotinoids has been achieved by using Me3Al-TMSOSO2CF3 as a key reagent for conversion of carbonyl to a geminal dimethyl functionality.

A potent and novel class of phosphinic acid derived product-like inhibitors of the HCV NS3/4A protease was discovered previously. Modification of the phosphinic acid and quinoline heterocycle led to GS-9256 with potent cell-based activity and favorable pharmacokinetic parameters. Based on these attributes, GS-9256 was advanced to human clinical trial as a treatment for chronic infection with genotype 1 HCV.

A series of thymidine analogues substituted with alkoxy groups of the C-4 position of the furan ring were synthesized. Among these compounds, the methoxy analogue 9 was the most potent inhibitor of herpes simplex virus type 1 thymidine kinase.

Aromatic analogues of sialic acid ( 2 and 3 ) have been synthesized as potential influenza neuraminidase inhibitors. Whereas compound 2 exhibited good neuraminidase inhibitory activity, compound 3 , possessing the glycerol side chain at the C 5 position, was not a neuraminidase inhibitor.

Regiospecific and highly Stereoselective electrophilic addition reactions to the furanoid glycal 10 have been used as key steps in the synthesis of ddA and d4T antiviral nucleosides.

The synthesis of 9-[(2R,5R)-2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]-2,6-diaminopurine ( 2 ) is described. This compound exhibited good inhibitory activity against HIV replication in MT-4 cells.

Two new phosphonate isosteres of d4T monophosphate have been prepared and tested against HIV.

A series of N1-heterocyclic pyrimidinediones were extensively evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Inhibitor 1 is active against NNRTI-resistant viruses including RT mutant K103N. The co-crystal structure of inhibitor 1 with HIV-1 RT revealed that H-bonds are formed with K101 and K103. Efforts to improve the suboptimal pharmacokinetic profile of 1 resulted in the discovery of compound 13, which represents the lead compound in this series with improved pharmacokinetics and similar potency as inhibitor 1.

Phosphonate analogues of d4T and d4A monophosphates have been synthesized using stereocontrolled addition of dimethyl hydroxymethylphosphonate to furanoid glycals. The new phosphonates exhibited a potent activity against HIV.

SAR studies on the para-fluorobenzyl moiety of tricyclic HIV integrase inhibitors are discussed and lead compounds with potency and PK properties comparable to raltegravir were identified.

The synthesis of 9-[(phosphonomethoxy)methyl]guanine (3) and 9-[2-hydroxy-1-(phosphonomethoxy)ethyl]guanine (4) is described.

A series of 9-(phosphonoalkyl)purines, which are analogues of 9-[2-(phosphonomethoxy)ethyl]purines (guanine, PMEG, 1; adenine, PMEA, 2), were synthesized. The analogues were tested for activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), Rauscher murine leukemia virus (R-MuLV), and human immunodeficiency virus type 1 (HIV-1). With variations in the length of the alkyl chain, the optimal activity was achieved with two carbons between the purine base and the phosphonomethoxy functionality. Despite the structural similarity and the close pKa2 value of 8 to that of PMEG, no phosphorylation of 8 was observed by the bovine brain guanylate kinase. Since all isosteric analogues of PMEG (7-9) were not inhibitory against HSV-1 and HSV-2, the presence of the 3'-oxygen atom in the PME purines proved critical for anti-HSV activity. Introduction of the 1'-methyl group on the PMEG side chain significantly reduced its anti-HSV activity. Analogue 11, which is a mimic of the phosphate by incorporation of the alpha,alpha-difluoro carbon, was ineffective against HSV-1 and HSV-2. These results suggest that the structural requirements of PME purines for anti-HSV activity appear to be very strict.

A novel synthetic methodology for the hydroxymethyl substituted acyclic acetal functionality was developed, The rationally designed phosphonate analogue 3 of ganciclovir monophosphate was highly active against human cytomegalovirus.

Phosphonomethoxy nucleoside analogs of the thymine containing nucleoside reverse transcriptase inhibitors (NRTIs), 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and 2',3'-dideoxythymidine (ddT), were synthesized. The anti-HIV activity against wild-type and several major nucleoside-resistant strains of HIV-1 was evaluated together with the inhibition of wild-type HIV reverse transcriptase (RT). Phosphonomethoxy analog of d4T, 8 (d4TP), demonstrated antiviral activity with an EC(50) value of 26 microM, whereas, phosphonomethoxy analogs of ddT, 7 (ddTP), and AZT, 6 (AZTP), were both inactive at concentrations up to 200 microM. Bis-isopropyloxymethylcarbonyl (bisPOC) prodrugs improved the anti-HIV activity of 7 and 8 by >150-fold and 29-fold, respectively, allowing for antiviral resistance to be determined. The K65R RT mutant virus was more resistant to the bisPOC prodrugs of 7 and 8 than bisPOC PMPA (tenofovir DF) 1. However, bisPOC prodrug of 7 demonstrated superior resistance toward the RT virus containing multiple thymidine analog mutations (6TAMs) indicating that new phosphonate nucleoside analogs may be suitable for targeting clinically relevant nucleoside resistant HIV-1 strains.

A novel class of tri-cyclic HIV integrase inhibitors were designed based on conformational analysis of 1,6-naphthyridine carboxamide compound L-870810 and docking the designed inhibitor into the active site of our integrase enzyme model. The efficient syntheses of pyrroloquinoline tri-cyclic analogs are described. The SAR studies resulted in the identification of a lead compound that is more potent and more soluble than L-870810.

This communication details both the syntheses and biological evaluation of a novel class of HIV-1 integrase inhibitors. When the quinoline moiety is replaced with the quinoxoline moiety, the antiviral activity is significantly compromised. Similarly, introduction of imidazole to replace the pyridine ring is deleterious to the potency of the compound against the enzyme. Substitution at the 3-position of the pyridine has been investigated. The presence of the pyridine ring in the tricyclic core is preferred for antiviral activity against HIV integrase.

A series of novel tricyclic inhibitors of HIV-1 integrase enzyme was prepared. The effect of substitution at C-6 of the 9-hydroxy-6,7-dihydropyrrolo[3,4-g]quinolin-8-one compounds was studied in vitro. Inhibitors with small side chains at C-6 were generally well tolerated by the enzyme, and the physicochemical properties of the inhibitors were improved by substitution of a small alkyl group at this position. A second series of analogs bearing a sulfamate at the C-5 position with various C-6 substituents were prepared to explore the interplay between the two groups. The SAR of the two classes are not parallel; modification at C-5 impacts the effect of substitutions at C-6.

A series of fumagillin analogues targeted at understanding tolerability of MetAP2 toward substitution at C4 and C6 were synthesized. Initially, the C6 side chain was maintained as cinnamoyl ester and C4 was modified. It was concluded that replacing the natural C4 of fumagillin with a benzyl oxime at C4 resulted in moderate loss of activity toward binding to MetAP2. Placement of a primary or secondary carbamate at C6 did not improve the potency of compounds toward inhibition of MetAP2. However, the inhibitory activity against MetAP2 was gained back by placing polar groups such as piperazinyl carbamate at C6. Small alkyl substituents on the amine of piperazinyl carbamate were well tolerated.

Isosteric phosphonate analogues of acyclovir and ganciclovir monophosphates were synthesized. The new phosponates exhibited good anti-herpes activity.

The total synthesis for the determination of the absolute stereochemistry of a GS 4104 metabolite 3 is described. In addition, the influenza neuraminidase inhibitory activity of 3 and related intermediates are reported.