Exploration of the activity of 7-pyrrolidino-8-methoxyisothiazoloquinolones against methicillin-resistant Staphylococcus aureus (MRSA).

A series of 7-(3'-substituted)pyrrolidino-8-methoxyisothiazoloquinolone (ITQ) analogues were prepared, and their antibacterial potency against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were compared. Many of these analogues had MIC ≤ 0.25 µg/mL against quinolone-resistant MRSA strains. The stereochemical preference was explored for a series of 1''-methyl-3'-aminomethylpyrrolidine analogues. Antibacterial activity was generally more favorable with 3'-R, 1''-S configuration. Substitution on the 3'-aminomethyl nitrogen tended to decrease activity, while potency was maintained with disubstitution or aryl substitution at the 1''-carbon. The 7-[(R)-3-((S)-1-aminoethyl)pyrrolidin-1-yl] analogue (6a(R,S)) and the (R)-7-[3-(2-aminopropan-2-yl)pyrrolidin-1-yl] analogue (7a(R)) were found to be the ITQs with the most promising antibacterial profiles. The MICs of these select ITQs versus a panel of clinical MRSA strains were determined, and the ITQs were found to have 8- to 16-fold greater potency than linezolid. These analogues were also evaluated for inhibition of the target enzymes, topoisomerase IV and DNA gyrase, from both wild-type and multidrug resistant strains. The ITQs were up to >30 times more inhibitory against these targets than the fluoroquinolone moxifloxacin.

Isothiazoloquinolones with enhanced antistaphylococcal activities against multidrug-resistant strains: effects of structural modifications at the 6-, 7-, and 8-positions.

We describe the biological evaluation of isothiazoloquinolones (ITQs) having structural modifications at the 6-, 7-, and 8-positions. Addition of a methoxy substituent to C-8 effected an increase in antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and a decrease in cytotoxic activity against Hep2 cells. Removal of fluorine from C-6 or replacement of the C-8 carbon with a nitrogen compromised anti-MRSA activity. When the groups attached at C-7 were compared, the anti-MRSA activity decreased in the order 6-isoquinolinyl > 4-pyridinyl > 5-dihydroisoindolyl > 6-tetrahydroisoquinolinyl. The compound with the most desirable in vitro biological profile was 9-cyclopropyl-6-fluoro-8-methoxy-7-(2-methylpyridin-4-yl)-9H-isothiazolo[5,4-b]quinoline-3,4-dione (7g). This ITQ demonstrated (i) strong in vitro anti-MRSA activity (MIC90 = 0.5 microg/mL), (ii) strong inhibitory activities against S. aureus DNA gyrase and topoisomerase IV, with weak activity against human topoisomerase II, (iii) weak cytotoxic activities against three cell lines, and (iv) efficacy in an in vivo murine thigh model of infection employing MRSA.

Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop.

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3'-end terminal dinucleotide 5'-GT. The exposed 3'-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5'-end of the long terminal repeats (5'-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5'-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5'-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3'-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.

Azido-containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions.

We previously found that azido-containing beta-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 micro M) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 micro M). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3' processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3'-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

Metal-dependent inhibition of HIV-1 integrase by beta-diketo acids and resistance of the soluble double-mutant (F185K/C280S).

The beta-diketo acids (DKAs) represent a major advance for anti-HIV-1 integrase drug development. We compared the inhibition of HIV-1 integrase by six DKA derivatives using the wild-type enzyme or the double-mutant F185K/C280S, which has been previously used for crystal structure determinations. With the wild-type enzyme, we found that DKAs could be classified into two groups: those similarly potent in the presence of magnesium and manganese and those potent in manganese and relatively ineffective in the presence of magnesium. Both the aromatic and the carboxylic or tetrazole functions of DKAs determined their metal selectivity. The F185K/C280S enzyme was markedly more active in the presence of manganese than magnesium. The F185K/C280S integrase was also relatively resistant to the same group of DKAs that were potent in the presence of magnesium with the wild-type enzyme. Resistance was caused by a synergistic effect from both the F185K and C280S mutations. Molecular modeling and docking suggested metal-dependent differences for binding of DKAs. Molecular modeling also indicated that the tetrazole or the azido groups of some derivatives could directly chelate magnesium or manganese in the integrase catalytic site. Together, these experiments suggest that DKAs recognize conformational differences between wild-type and the double-mutant HIV-1 integrase, because they chelate the magnesium or manganese in the enzyme active site and compete for DNA binding.

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors.

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.

Metal-dependent inhibition of HIV-1 integrase.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202-3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN-DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg(2+) at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg(2+)-based assays, while IN inhibition by salicylhydrazides is strictly Mn(2+)-dependent. Additionally, in target and cell-based assays, the MSHs have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerases I and II at concentrations that effectively inhibit IN. These data suggest that MSHs are selective inhibitors of HIV-1 IN and may serve as leads for antiviral therapeutics.

Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Structural determinants for HIV-1 integrase inhibition by beta-diketo acids.

Among all the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3'-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3'-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3'-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.