Integrase Inhibitor Prodrugs: Approaches to Enhancing the Anti-HIV Activity of ß-Diketo Acids.

HIV integrase, encoded at the 3'-end of the HIV pol gene, is essential for HIV replication. This enzyme catalyzes the incorporation of HIV DNA into human DNA, which represents the point of "no-return" in HIV infection. Integrase is a significant target in anti-HIV drug discovery. This review article focuses largely on the design of integrase inhibitors that are ß-diketo acids constructed on pyridinone scaffolds. Methodologies for synthesis of these compounds are discussed. Integrase inhibition data for the strand transfer (ST) step are compared with in vitro anti-HIV data. The review also examines the issue of the lack of correlation between the ST enzymology data and anti-HIV assay results. Because this disconnect appeared to be a problem associated with permeability, prodrugs of these inhibitors were designed and synthesized. Prodrugs dramatically improved the anti-HIV activity data. For example, for compound, 96, the anti-HIV activity (EC50) improved from 500 nM for this diketo acid to 9 nM for its prodrug 116. In addition, there was excellent correlation between the IC50 and IC90 ST enzymology data for 96 (6 nM and 97 nM, respectively) and the EC50 and EC90 anti-HIV data for its prodrug 116 (9 nM and 94 nM, respectively). Finally, it was confirmed that the prodrug 116 was rapidly hydrolyzed in cells to the active compound 96.

A novel molecule with notable activity against multi-drug resistant tuberculosis.

Multi-drug resistant tuberculosis (MDR-TB) is emerging as a serious global health problem, which has been elevated through co-infection involving HIV and MDR-Mtb. The discovery of new compounds with anti-MDR TB efficacy and favorable metabolism profiles is an important scientific challenge. Using computational biology and ligand docking data, we have conceived a multifunctional molecule, 2, as a potential anti-MDR TB agent. This compound was produced through a multi-step synthesis. It exhibited significant in vitro activity against MDR-TB (MIC 1.56µg/mL) and its half-life (t1/2) in human liver microsomes was 14.4h. The metabolic profiles of compound 2 with respect to human cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes were favorable. Compound 2 also had relatively low in vitro cytotoxicity in uninfected macrophages. It displayed synergistic behavior against MDR-TB in combination with PA-824. Interestingly, compound 2 also displayed in vitro anti-HIV activity.

Pharmacokinetics and dose-range finding toxicity of a novel anti-HIV active integrase inhibitor.

Integration of viral DNA into human chromosomal DNA catalyzed by HIV integrase represents the "point of no return" in HIV infection. For this reason, HIV integrase is considered a crucial target in the development of new anti-HIV therapeutic agents. We have discovered a novel HIV integrase inhibitor 1, that exhibits potent antiviral activity and a favorable metabolism profile. This paper reports on the pharmacokinetics and toxicokinetics of compound 1 and the relevance of these findings with respect to further development of this integrase-targeted antiviral agent. Oral administration of compound 1 in Sprague Dawley rats revealed rapid absorption. Drug exposure increased with increasing drug concentration, indicative of appropriate dose-dependence correlation. Compound 1 exhibited suitable plasma half-life, extensive extravascular distribution and acceptable bioavailability. Toxicity studies revealed no compound-related clinical pathology findings. There were no changes in erythropoietic, white blood cell or platelet parameters in male and female rats. There was no test-article related change in other clinical chemistry parameters. In addition, there were no detectable levels of bilirubin in the urine and there were no treatment-related effects on urobilinogen or other urinalysis parameters. The preclinical studies also revealed that the no observed adverse effect level and the maximum tolerated dose were both high (>500mg/kg/day). The broad and significant antiviral activity and favorable metabolism profile of this integrase inhibitor, when combined with the in vivo pharmacokinetic and toxicokinetic data and their pharmacological relevance, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

Approaches to the synthesis of a novel, anti-HIV active integrase inhibitor.

The novel HIV-1 integrase inhibitor 1, discovered in our laboratory, exhibits potent anti-HIV activity against a diverse set of HIV-1 isolates and also against HIV-2 and SIV. In addition, this compound displays low cellular cytotoxicity and possesses a favorable in vitro drug interaction profile with respect to isozymes of cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT). However, the total synthesis of this significant HIV integrase inhibitor has not been reported. This contribution describes an optimized, reproducible, multi-step, synthetic route to inhibitor 1. The yield for the separate steps averaged about 80%. The methodologies utilized in the synthesis were, among others, a palladium-catalyzed cross-coupling reaction, a crossed-Claisen condensation, and a hydrazino amide synthesis step. Successful alternative synthetic methodologies for some of the steps are also described.

Synthesis of a novel carbocyclic analog of bredinin.

The natural nucleoside antibiotic, bredinin, exhibits antiviral and other biological activities. While various nucleosides related to bredinin have been synthesized, its carbocyclic analog has remained unknown. Synthesis of this heretofore unknown analog of bredinin is described. The key precursor, (3aS,4R,6R,6aR)-6-((methoxy-methoxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-amine (5), was prepared from the commercially available compound, (1R,4S)-2-azabicyclo[2.2.1] hept-5-en-3-one (4). Our initial approach used intermediate 6, derived in three transformations from 5, for the key photolytic step to produce the desired ring-opened precursor to the target compound. This photochemical transformation was unsuccessful. However, an appropriately protected and related precursor was synthesized from 5 through the following side-chain functional group transformations: elaboration of the amino group through malonyl ester formation, oximation at the central carbon, conversion of ester to amide and catalytic reduction of the oxime group. This precursor, on treatment with triethylorthoformate and catalytic acetic acid in ethanol, underwent cyclization to produce the desired 4-carbamoyl-imidazolium-5-olate ring. Deprotection of the latter product proceeded smoothly to give the carbocyclic analog of bredinin. This target molecule exhibits antiviral activity, albeit low, against a number of RNA viruses. Further biological evaluations are in progress.

Notable difference in anti-HIV activity of integrase inhibitors as a consequence of geometric and enantiomeric configurations.

While some examples are known of integrase inhibitors that exhibit potent anti-HIV activity, there are very few cases reported of integrase inhibitors that show significant differences in anti-HIV activity that result from distinctions in cis- and trans-configurations as well as enantiomeric stereostructure. We describe here the design and synthesis of two enantiomeric trans-hydroxycyclopentyl carboxamides which exhibit notable difference in anti-HIV activity. This difference is explained through their binding interactions within the active site of the HIV-1 integrase intasome. The more active enantiomer 3 (EC50 25nM) was relatively stable in human liver microsomes. Kinetic data revealed that its impact on key cytochrome P450 isozymes, as either an inhibitor or an activator, was minor, suggesting a favorable CYP profile.

A novel anti-HIV active integrase inhibitor with a favorable in vitro cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase metabolism profile.

Research efforts on the human immunodeficiency virus (HIV) integrase have resulted in two approved drugs. However, co-infection of HIV with Mycobacterium tuberculosis and other microbial and viral agents has introduced added complications to this pandemic, requiring favorable drug-drug interaction profiles for antiviral therapeutics targeting HIV. Cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are pivotal determining factors in the occurrence of adverse drug-drug interactions. For this reason, it is important that anti-HIV agents, such as integrase inhibitors, possess favorable profiles with respect to CYP and UGT. We have discovered a novel HIV integrase inhibitor (compound 1) that exhibits low nM antiviral activity against a diverse set of HIV-1 isolates, and against HIV-2 and the simian immunodeficiency virus (SIV). Compound 1 displays low in vitro cytotoxicity and its resistance and related drug susceptibility profiles are favorable. Data from in vitro studies revealed that compound 1 was not a substrate for UGT isoforms and that it was not an inhibitor or activator of key CYP isozymes.

Solid-state tautomeric structure and invariom refinement of a novel and potent HIV integrase inhibitor.

The conformation and tautomeric structure of (Z)-4-[5-(2,6-difluorobenzyl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridin-3-yl]-4-hydroxy-2-oxo-N-(2-oxopyrrolidin-1-yl)but-3-enamide, C27H22F3N3O5, in the solid state has been resolved by single-crystal X-ray crystallography. The electron distribution in the molecule was evaluated by refinements with invarioms, aspherical scattering factors by the method of Dittrich et al. [Acta Cryst. (2005), A61, 314-320] that are based on the Hansen-Coppens multipole model [Hansen & Coppens (1978). Acta Cryst. A34, 909-921]. The ß-diketo portion of the molecule exists in the enol form. The enol -OH hydrogen forms a strong asymmetric hydrogen bond with the carbonyl O atom on the ß-C atom of the chain. Weak intramolecular hydrogen bonds exist between the weakly acidic α-CH hydrogen of the keto-enol group and the pyridinone carbonyl O atom, and also between the hydrazine N-H group and the carbonyl group in the ß-position from the hydrazine N-H group. The electrostatic properties of the molecule were derived from the molecular charge density. The molecule is in a lengthened conformation and the rings of the two benzyl groups are nearly orthogonal. Results from a high-field (1)H and (13)C NMR correlation spectroscopy study confirm that the same tautomer exists in solution as in the solid state.

Discovery of a Potent HIV Integrase Inhibitor that Leads to a Prodrug with Significant anti-HIV Activity.

Worldwide research efforts in drug discovery involving HIV integrase have produced only one compound, raltegravir, that has been approved for clinical use in HIV/AIDS. As resistance, toxicity and drug-drug interactions are recurring issues with all classes of anti-HIV drugs, the discovery of novel integrase inhibitors remains a significant scientific challenge. We have designed a lead HIV-1 strand transfer (ST) inhibitor (IC(50) 70 nM), strategically assembled on a pyridinone scaffold. A focused structure-activity investigation of this parent compound led to a significantly more potent ST inhibitor, 2 (IC(50) 6 ± 3 nM). Compound 2 exhibits good stability in pooled human liver microsomes. It also displays a notably favorable profile with respect to key human cytochrome P450 (CYP) isozymes and human UDP glucuronosyl transferases (UGTs). The prodrug of inhibitor 2, i.e., compound 10, was found to possess remarkable anti-HIV-1 activity in cell culture (EC(50) 9 ± 4 nM, CC(50) 135 ± 7 µM, therapeutic index = 15,000).

A heterocyclic molecule with significant activity against dengue virus.

There are no specific approved drugs or vaccines for the treatment or prevention of infectious dengue virus and there are very few compounds known that inhibit the replication of this virus. This letter describes the concise synthesis of two uracil-based multifunctional compounds. One of these compounds (1) has strong activity against dengue virus. It also exhibits low activity against a few other RNA viruses, but is highly active against yellow fever virus, a related flavivirus. It is likely that the mechanism of action of the antiviral activity of this compound is through its inhibition of the enzyme, inosine monophosphate dehydrogenase (IMPDH). Molecular modeling studies reveal that the compound can have specific hydrogen bonding interactions with a number of amino acids in the active site of IMPDH, a stacking interaction with the bound natural substrate, IMP, and the ability to interfere with the binding of NAD(+) with IMPDH, prior to the hydration step.

Base-functionalized carbocyclic nucleosides: design, synthesis, and mechanism of antiviral activity.

New carbocyclic ribonucleosides with unsaturated groups at the C-2 position of the nucleobase were designed as potential RNA antiviral compounds. The design was based on the expectation that the monophosphates of these compounds would be inhibitors of the enzyme, IMPDH. Appropriate methodologies were developed to achieve the target molecules. Results from the initial in vitro antiviral studies are mentioned. The IMPDH-associated mechanism of the antiviral activity of the most active compound is supported by enzyme inhibition studies.

Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery.

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme of de novo purine nucleotide biosynthesis and is viewed as an important target in the quest for discovery of drugs in the antiviral, antibacterial and anticancer therapeutic areas. This review focuses on the medicinal chemistry, drug discovery and chemical biology of IMPDH. Examples of IMP and cofactor site-directed inhibitors, allosteric inhibitors and isoform-selective inhibitors are presented. Comparison of IMPDHs from different organisms is also made to facilitate the design of species-selective IMPDH inhibitors for drug discovery. Special emphasis in the review is placed on IMPDH from Mycobacterium tuberculosis.

Inosine monophosphate dehydrogenase as a probe in antiviral drug discovery.

Inosine monophosphate (IMP) dehydrogenase (IMPDH) is a significant enzyme in the purine nucleotide biosynthetic pathway. IMPDH is viewed as an important biological target in the quest for drugs in the antiviral therapeutic area. This review article is focused on the chemistry and biology of IMPDH inhibitors and the use of IMPDH inhibition data as a probe in antiviral drug discovery. Examples of both inosine 5' monophosphate and NAD+ site-directed inhibitors are presented. Correlation of antiviral activities with IMPDH inhibition is discussed.

IMPDH as a biological probe for RNA antiviral drug discovery: synthesis, enzymology, molecular docking, and antiviral activity of new ribonucleosides with surrogate bases.

Our interest in the discovery of molecules with antiviral activity against RNA viruses led us to the design of ribonucleosides with surrogate bases with the intent of using inhibition of inosine monophosphate dehydrogenase (IMPDH) as a probe for antiviral drug discovery. A general methodology for the preparation of these compounds is discussed. Kinetic parameters of the inhibition studies with IMPDH, which were carried out spectrophotometrically by monitoring the formation of NADH, are given. Antiviral information and correlation of activity with IMPDH inhibition are discussed.

Biologically-validated HIV integrase inhibitors with nucleobase scaffolds: structure, synthesis, chemical biology, molecular modeling, and antiviral activity.

Integrase, an enzyme of the pol gene of HIV, is a significant viral target for the discovery of anti-HIV agents. In this presentation, we report on the continuation of our work on the discovery of diketo acids, constructed on nucleobase scaffolds, that are inhibitors of HIV integrase. An example of our synthetic approach to inhibitors with purine nucleobase scaffolds is given. Comparison is made between integrase inhibition data arising from compounds with pyrimidine versus purine nucleobase scaffold. Antiviral results are cited.

Isonucleosides: design and synthesis of new isomeric nucleosides with antiviral potential.

Isonucleosides discovered in our laboratory have been found to have interesting antiviral activity. The design, development of methodology, and stereochemical synthesis of new isonucleosides of anti-HCV interest are described. Antiviral results are cited.

A novel strategy to assemble the beta-diketo acid pharmacophore of HIV integrase inhibitors on purine nucleobase scaffolds.

Claisen condensation, the key step in constructing the pharmacophore of aryl beta-diketo acids (DKA) as integrase inhibitors, fails in certain cases of highly electron-deficient heterocycles such as purines. A general synthetic strategy to assemble the DKA motif on the purine scaffold has been accomplished. The synthetic sequence entails a palladium-catalyzed cross-coupling, a C-acylation involving a tandem addition/elimination reaction, and a novel ferric ion-catalyzed selective hydrolysis of an enolic ether in the presence of a carboxylic acid ester.

HIV integrase inhibitors as therapeutic agents in AIDS.

HIV-1 integrase is a protein of Mr 32 000 encoded at the 3'-end of the pol gene. Integration of HIV DNA into the host cell chromosomal DNA apparently occurs by a carefully defined sequence of DNA tailoring (3'-processing (3'P)) and coupling (integration) reactions. Integration of HIV DNA into human DNA represents the biochemical completion of the invasion of the human cell (e.g., T-cell) by HIV. Unlike major successes seen in the development of clinically approved anti-HIV agents against HIV reverse transcriptase and HIV protease, there are no FDA-approved anti-HIV drugs in clinical use where the mechanism of action is inhibition of HIV integrase. This review summarises some key advances in the area of integrase inhibitors with the major focus being on new generation inhibitors. Special emphasis is placed on diketo acids with aromatic and heteroaromatic moieties, diketo acids with nucleobase scaffolds, bis-diketo acids, functionalised naphthyridines and other isosteres of diketo acids. Data pertaining to integrase inhibition and in vitro anti-HIV activity are discussed. Mention is made of drugs in clinical trials, both past (S-1360, L-870,810 and L-870,812 and present (GS-9137 and MK-0518). Other promising drugs, including those from the authors' laboratory, are referred. Resistant mutants arising from key integrase inhibitors and cross-resistance are indicated.

A novel diketo phosphonic acid that exhibits specific, strand-transfer inhibition of HIV integrase and anti-HIV activity.

We have synthesized novel phosphonic acid analogues of beta-diketo acids. Interestingly, the phosphonic acid isostere, 2, of our anti-HIV compound, 1, was an inhibitor of only the strand transfer step, in stark contrast to 1. Compound 2 had lower anti-HIV activity than 1, but was more active and less toxic than the phosphonic acid analogue of L-708906. These isosteric compounds represent the first examples of beta-diketo phosphonic acids of structural, synthetic, and antiviral interest.