Tracy L. Hartman

Antiviral activity of hop constituents against a series of DNA and RNA viruses

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors.

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.

Development of Hexadecyloxypropyl Tenofovir (CMX157) for Treatment of Infection Caused by Wild-Type and Nucleoside/Nucleotide-Resistant HIV

ABSTRACT CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC50s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC50s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

The structure-activity relationships of 2,4(1H,3H)- pyrimidinedione derivatives as potent HIV type 1 and type 2 inhibitors

Since the discovery of the 2,4 (1H,3H)-pyrimidinediones as potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase (RT) this class of compounds has yielded a number of N-1 acyclic substituted pyrimidinediones with substantial antiviral activity, which is highly dependent upon their molecular fit into the binding pocket common to this inhibitory class. We have specifically examined the structure activity relationships of compounds with chemical modification made by substituting homocyclic rather than acyclic moieties at N-1 of the pyrimidinedione. Seventy-four compounds were synthesized and evaluated for antiviral activity against HIV-1 and HIV-2. The homocyclic modifications resulted in compounds with significant activity against both HIV-1 and HIV-2, suggesting these compounds represent a new class of non-nucleoside RT inhibitors. The structure-activity relationship (SAR) evaluations indicated that cyclopropyl, phenyl and 1- or 3-cyclopenten-1-yl substitutions at the N-1 of the pyrimidinedione, the addition of a methyl linker between the cyclic moiety and the N-1 and the addition of a benzoyl group at the C-6 of the pyrimidinedione had the greatest contribution to antiviral activity. Five pyrimidinedione analogues with therapeutic indexes (TIs)>450,000 and a specific analogue (1-cyclopropylmethyl-5-isopropyl-6-(3,5-dimethylbenzoyl)-2,4(1H,3H)-pyrimidinedione), which exhibited a TI of >2,000,000, were identified. None of the analogues were cytotoxic to target cells at the highest in vitro test concentration, which is the upper limit of compound solubility of the analogues in aqueous solution. Thus, we have identified a series of pyrimidinediones with substantially improved antiviral efficacy and range of action and with significantly reduced cellular cytotoxicity.

Comparative Evaluation of the Inhibitory Activities of a Series of Pyrimidinedione Congeners That Inhibit Human Immunodeficiency Virus Types 1 and 2

ABSTRACT Seventy-three analogs of SJ-3366 (1-(3-cyclopenten-1-ylmethyl)-5-ethyl-6-(3,5-dimethylbenzoyl)-2,4(1H,3H)-pyrimidinedione) were synthesized and comparatively evaluated for their ability to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2 and for their ability to suppress virus entry and reverse transcription. These studies were performed to identify inhibitors with activity greater than that of the current lead molecule (SJ-3366) and to utilize structure-activity relationships (SAR) to define the chemical features of the pyrimidinedione congeners responsible for their efficacy, toxicity, and dual mechanism of action against HIV. The results of our SAR evaluations have demonstrated that the addition of the homocyclic moiety at the N-1 of the pyrimidinedione results in acquisition of the ability to inhibit virus entry and extends the range of action of the compounds to include HIV-2. In addition, the results demonstrate that analogs with a methyl linker between the homocyclic substitution and the N-1 of the pyrimidinedione had a greater number of highly active molecules than those analogs possessing ethyl linkers. Six molecules were identified with activity equivalent to or greater than that of SJ-3366, and five additional molecules with highly potent inhibition of reverse transcriptase and virus entry and possessing high efficacy against both HIV-1 and HIV-2 were identified. Six molecules exhibited significant inhibition of viruses with the highly problematic nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance engendering amino acid change K103N in the reverse transcriptase. These evaluations indicate that a new class of NNRTIs has been identified and that these NNRTIs possess highly potent inhibition of HIV-1 with an extended range of action, which now includes HIV-2.

The Continuing Evolution of HIV-1 Therapy: Identification and Development of Novel Antiretroviral Agents Targeting Viral and Cellular Targets

During the past three decades, over thirty-five anti-HIV-1 therapies have been developed for use in humans and the progression from monotherapeutic treatment regimens to todays highly active combination antiretroviral therapies has had a dramatic impact on disease progression in HIV-1-infected individuals. In spite of the success of AIDS therapies and the existence of inhibitors of HIV-1 reverse transcriptase, protease, entry and fusion, and integrase, HIV-1 therapies still have a variety of problems which require continued development efforts to improve efficacy and reduce toxicity, while making drugs that can be used throughout both the developed and developing world, in pediatric populations, and in pregnant women. Highly active antiretroviral therapies (HAARTs) have significantly delayed the progression to AIDS, and in the developed world HIV-1-infected individuals might be expected to live normal life spans while on lifelong therapies. However, the difficult treatment regimens, the presence of class-specific drug toxicities, and the emergence of drug-resistant virus isolates highlight the fact that improvements in our therapeutic regimens and the identification of new and novel viral and cellular targets for therapy are still necessary. Antiretroviral therapeutic strategies and targets continue to be explored, and the development of increasingly potent molecules within existing classes of drugs and the development of novel strategies are ongoing.

Synthesis of alkenyldiarylmethanes (ADAMs) containing benzo[d]isoxazole and oxazolidin-2-one rings, a new series of potent non-nucleoside HIV-1 reverse transcriptase inhibitors

As a continuation of efforts to replace the metabolically labile methyl esters of lead alkenyldiarylmethanes (ADAMs) with stable bioisosteres, compounds bearing benzo[d]isoxazole and oxazolidine-2-one rings were designed and evaluated as a new series of potent HIV-1 non-nucleoside reverse transcriptase inhibitors with anti-HIV activity. All of the resulting ADAMs were found to inhibit HIV-1 RT with poly(rC) x oligo(dG) as the template primer. The most promising compound in this series was ADAM 3, with EC(50) values of 40 nM (vs HIV-1(RF)) and 20 nM (vs HIV-1(IIIB)). Compound 3 also inhibited HIV-1 reverse transcriptase with an IC(50) of 0.91 microM. ADAM 4 has an antiviral EC(50) of 0.6 microM in CEM-SS cells and a plasma half-life of 51.4 min.

Synthesis and anti-HIV activity of a bile acid analog of cosalane

Abstract Cosalane is a novel anti-HIV agent that inhibits the attachment of gp120 to CD4. The therapeutic potential of cosalane is limited by poor oral absorption. In an attempt to target the ileal bile acid transporter and thus facilitate oral bioavailability, a cosalane analog was synthesized in which the disalicylmethane pharmacophore is attached to a bile acid through a linker chain appended to C-17 of the steroid nucleus. The resulting bile acid analog of cosalane retained antiviral activity vs. HIV-1 IIIB and HIV-2 ROD in MT-4 cells, but was less potent than cosalane.

Synthesis of alkenyldiarylmethane (ADAM) non-Nucleoside HIV-1 reverse transcriptase inhibitors with non-Identical aromatic rings

The existing methods for the synthesis of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors proceed from symmetrical benzophenones and therefore result in products with identical aromatic rings. New methods have therefore been devised for the preparation of stereochemically defined ADAMs with non-identical aromatic rings. The new routes rely on palladium-catalyzed reactions, including Sonogashira, Suzuki, Stille, and hydroarylation methodology. Several of the new ADAMs inhibited the cytopathic effect of HIV-1 in cell culture and HIV-1 reverse transcriptase at submicromolar concentrations.

Crystallographic Study of a Novel Subnanomolar Inhibitor Provides Insight on the Binding Interactions of Alkenyldiarylmethanes with Human Immunodeficiency Virus-1 Reverse Transcriptase

Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) nonnucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the back side of the binding pocket, placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the compound (IC(50) </= 1 nM). ADAM 4 was designed to be hydrolytically stable in blood plasma, and an investigation of its hydrolysis in rat plasma demonstrated it has a significantly prolonged half-life in comparison to ADAM lead compounds 1 and 2.