5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: potent nonpeptidic inhibitors of HIV protease.

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S(3)' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S(3)' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.

Nonpeptidic HIV protease inhibitors: 6-alkyl-5,6-dihydropyran-2-ones possessing a novel and achiral 3-(2-t-butyl-5-methyl-4-sulfamate)phenylthio moiety.

Dihydropyran-2-ones possessing a sulfamate moiety at the 4-position of the thiophenyl ring were designed to reach S3' pocket of the HIV protease. Synthetic routes for the preparation of thiotosylates possessing 3-(2-t-butyl-5-methyl-4-sulfamate) phenylthio moiety were established. SAR of various sulfamate analogs including HIV protease binding affinities, antiviral activities and therapeutic indices will be described.

Nonpeptidic HIV protease inhibitors: 6-alkyl-5,6-dihydropyran-2-ones possessing achiral 3-(4-amino/carboxamide-2-t-butyl,5-methylphenyl thio) moiety: antiviral activities and pharmacokinetic properties.

Dihydropyran-2-ones possessing amino and carboxamide functionalities on 3-SPh (2-tert-butyl, 5-methyl) ring were synthesized and evaluated for their antiviral activities. Both the enantiomers of inhibitor 15 were synthesized. The in vitro resistance profile, inhibitory activities against cytochrome P450 isozymes and pharmacokinetic properties of inhibitor 15S will be discussed.

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

2,2'-Dithiobisbenzamides derived from alpha-, beta- and gamma-amino acids possessing anti-HIV activities: synthesis and structure-activity relationship.

Nucleocapsid protein (NCp7), which contains highly conserved retroviral zinc fingers, is essential in the early as well as the late phase of human immunodeficiency virus (HIV) life cycle and constitutes a novel target for AIDS therapy. HIV-1 NCp7 is a basic 55 amino acid protein containing two C(X)2C(X)4H(X)4C motif zinc fingers flanked by basic amino acids on each side. 2,2'-dithiobisbenzamides have previously been reported to release zinc from these NCp7 zinc fingers and also to inhibit HIV replication. Specifically, 2,2'-dithiobisbenzamides derived from simple amino acids showed good antiviral activities. The benzisothiazolone 3, the cyclic derivative of 2, was selected for clinical trials as an agent for AIDS therapy. Herein we report the syntheses and antiviral activities, including therapeutic indices, of 2,2'-dithiobisbenzamides derived from alpha-, beta- and gamma-amino acids. Electrospray ionization mass spectrometry was used to study the zinc-ejection activity of these compounds. Among the alpha-amino acid derived 2,2'-dithiobisbenzamides, analogues containing alkyl side chains were found to be antivirally active with good therapeutic indices. 2,2'-Dithiobisbenzamides, derived from beta- and gamma-amino acids, were found to possess better antiviral and therapeutic efficacies than the alpha-amino acid analogues. Thus compound 59 was found to possess an EC50 of 1.9 microM with a therapeutic index of > 50. Interestingly, 2,2'-dithiobisbenzamides derived from alpha-amino acids containing a protected acid function and polar side chains also exhibited very good antiviral activity.

2,2'-Dithiobisbenzamides and 2-benzisothiazolones, two new classes of antiretroviral agents: SAR and mechanistic considerations.

Substituted 2,2'-dithiobisbenzamides and 2-benzisothiazolones were prepared and shown to possess low microM activity with high therapeutic indices against HIV-1, HIV-2 and SIV in cell culture. The mechanism of antiviral action was determined to be directed toward the nucleocapsid protein (NCp7), which contains two zinc fingers and plays vital roles in the viral life cycle. The "active sulfides" of this study cause the extrusion of zinc from these zinc fingers. Structure-activity relationships of the 2,2'-dithiobisbenzamides reveal that the disulfide bond and the ortho benzamide functional groups are essential for activity, with the best compounds having a carboxylic acid, carboxamide, or sulfonamide substituent. The 2-benzisothiazolones are formed from the disulfides both chemically and in vivo and their SAR mimics that of the 2,2'-dithiobisbenzamides. The antiviral activity of the disulfides may require cyclization to the isothiazolones. Two agents, PD 159206 and PD 161374, which showed good antiviral activity, physical properties, and excellent pharmacokinetics in mice, were selected for advanced studies.

A new class of anti-HIV-1 agents targeted toward the nucleocapsid protein NCp7: the 2,2'-dithiobisbenzamides.

As part of the National Cancer Institute's Drug Screening Program, a new class of antiretrovirals active against the human immunodeficiency virus HIV-1 has been identified, and the HIV-1 nucleocapsid protein NCp7 was proposed as the target of antiviral action. The 2,2'-dithiobis-[4'-(sulfamoyl)benzanilide] (3x) and the 2,2'-dithiobis(5-acetylamino)benzamide (10) represented the prototypic lead structures. A wide variety of 2,2'-dithiobisbenzamides were prepared and tested for anti-HIV-1 activity, cytotoxicity, and their ability to extrude zinc from the zinc fingers for NCp7. The structure-activity relationships demonstrated that the ability to extrude zinc from NCp7 resided in the 2,2'-dithiobisbenzamide core structure. The 3,3' and the 4,4' isomers were inactive. While many analogs based upon the core structure retained the zinc extrusion activity, the best overall anti-HIV-1 activity was only found in a narrow set of derivatives possessing carboxylic acid, carboxamide, or phenylsulfonamide functional groups. These functional groups were more important for reducing cytotoxicity than improving antiviral potency or activity vs NCp7. All of the compounds with antiviral activity also extruded zinc from NCp7. From this study several classes of low microM anti-HIV agents with simple chemical structures were identified as possible chemotherapeutic agents for the treatment of AIDS.

Structure-activity relationships of quinolone agents against mycobacteria: effect of structural modifications at the 8 position.

A series of quinolones with substitutions at the 8 position has been prepared as part of a study to examine the relationship between structural modifications at this position and activity against mycobacteria. The compounds were prepared by procedures described in the literature and were evaluated for their activities against Mycobacterium fortuitum and Mycobacterium smegmatis. The activities of the compounds against these two organisms were used as a measure of Mycobacterium tuberculosis activity. The results demonstrate that the contribution of the 8 position to antimycobacterial activity was dependent on the substituent at N-1 and was in the order (i) COMe approximately CBr > CCI > CH approximately CF approximately COEt > N > CCF3 when N-1 was cyclopropyl; (ii) N approximately CH > CF > COMe when N-1 was 2,4-difluorophenyl; (iii) N > or = CH when N-1 was tert-butyl; and (iv) N > CH when N-1 was ethyl. In general, derivatives with piperazine substitutions at C-7 were slightly less active against mycobacteria than the analogs with pyrrolidine substitutions, regardless of the pattern of substitution at the 8 position. Several of the best compounds were evaluated for their potential side effects as well as their activities against Mycobacterium aurum, Mycobacterium avium-M. intracellulare, and M. tuberculosis. These agents exhibited biological profiles similar to or better than those of the positive controls ciprofloxacin and sparfloxacin.

Discovery and optimization of nonpeptide HIV-1 protease inhibitors.

Several small, achiral nonpeptide inhibitors of HIV-1 protease with low micromolar activity were identified by mass screening of the Parke-Davis compound library. Two of the compounds, structurally similar, were both found to be competitive and reversible inhibitors [compound 1, 4-hydroxy-3-(3-phenoxypropyl)-1-benzopyran-2-one: Ki = 1.0 microM; compound 2, 4-hydroxy-6-phenyl-3-(phenylthio)-pyran-2-one: Ki = 1.1 microM]. These inhibitors were chosen as initial leads for optimization of in vitro inhibitory activity based on molecular modeling and X-ray crystallographic structural data. While improvements in inhibitory potency were small with analogues of compound 1, important X-ray crystallographic structural information of the enzyme-inhibitor complex was gained. When bound, 1 was found to displace H2O301 in the active site while hydrogen bonding to the catalytic Asps and Ile50 and Ile150. The pyranone group of compound 2 was found to bind at the active site in the same manner, with the 6-phenyl and the 3-phenylthio occupying P1 and P1', respectively. The structural information was used to develop design strategies to reach three or four of the internal pockets, P2-P2'. This work led to analogues of diverse structure with high potency (IC50 < 10 nM) that contain either one or no chiral centers and remain nonpeptide. The highly potent compounds possess less anti-HIV activity in cellular assays than expected, and current optimization now focuses on increasing cellular activity. The value of the HIV-1 protease inhibitors described is their potential as better pharmacological agents with a different pattern of viral resistance development, relative to the peptide inhibitors in human clinical trials.

Nonpeptidic potent HIV-1 protease inhibitors.

From an initial mass screening lead, (IC50: 3 microM) and information derived from the X-ray crystallographic structure of a related analog, complexed with HIV protease (PR), the design of more potent inhibitors has been advanced. Various structure-guided approaches to fill P1' and P2' pockets using this pyran-2-one template, molecular modeling and X-ray crystallographic studies led to potent compounds. Of particular significance to the design of this series of inhibitors is the displacement of key structural waters. The binding modes of a series of pyran-2-one analogs and comparison of binding modes with different pyran-2-ones, are highlighted. Noteworthy was the discovery of a highly potent (IC50: 0.007 microM) pyran-2-one derivative, containing novel P1' and P2' functionalization and possessing no chiral centers and having low molecular weight. Pyran-2-ones possessing appended groups to reach to the S3 pocket of the enzyme via tethering on the 6-phenyl ring of pyran-2-one ring is also discussed.

Nonpeptidic HIV protease inhibitors: 4-hydroxy-pyran-2-one inhibitors with functional tethers to P1 phenyl ring to reach S3 pocket of the enzyme.

A systematic study of tethering various groups on 6-phenyl ring of 4-hydroxy-6-phenyl-3-[(2-isopropylphenyl)thio]pyran-2-one was performed to increase the binding affinity with HIV protease. This tethering approach was aimed to fill S3 pocket of the enzyme. Thus, tethering hydrophilic groups resulted in more potent inhibitors. Similarly, various aromatic hydrophobic rings as well as heterocyclic rings were explored as tethering substituents to alter the physical properties as well as to enhance the binding affinity with HIV protease. Inhibitor 24, 4-hydroxy-3-[(2-isopropylphenyl)thio]-6-[4-(3-pyridinylmethoxy+ ++ ) phenyl]-2H-pyran-2-one, was evaluated as a prototypic lead structure to study various physical as well as pharmacological properties of this class of HIV protease inhibitors.

Structure-activity relationships of the quinolone antibacterials against mycobacteria: effect of structural changes at N-1 and C-7.

The re-emergence of tuberculosis infections which are resistant to conventional drug therapy has demonstrated the need for alternative chemotherapy against Mycobacterium tuberculosis. As part of a study to optimize the quinolone antibacterials against M. tuberculosis, we have prepared a series of N-1- and C-7-substituted quinolones to examine specific structure-activity relationships between modifications of the quinolone at these two positions and activity against mycobacteria. The compounds, synthesized by literature procedures, were evaluated for activity against Mycobacterium fortuitum and Mycobacterium smegmatis as well as Gram-negative and Gram-positive bacteria. The activity of the compounds against M. fortuitum was used as a barometer of M. tuberculosis activity. The results demonstrate that (i) the activity against mycobacteria was related more to antibacterial activity than to changes in the lipophilicity of the compounds, (ii) the antimycobacterial activity imparted by the N-1 substituent was in the order tert-butyl > or = cyclopropyl > 2,4-difluorophenyl > ethyl approximately cyclobutyl > isopropyl, and (iii) substitution with either piperazine or pyrrolidine heterocycles at C-7 afforded similar activity against mycobacteria.

The synthesis, structure-activity, and structure-side effect relationships of a series of 8-alkoxy- and 5-amino-8-alkoxyquinolone antibacterial agents.

A series of 1-cyclopropyl-6-fluoro-8-alkoxy (8-methyoxy and 8-ethoxy)-quionoline-3-carboxylic acids and 1-cyclopropyl-5-amino-6-fluoro-8-alkoxyquinoline-3-carboxylic acids has been prepared and evaluated for antibacterial activity. In addition, they were also compared to quinolones with classic substitution at C8 (H, F, Cl) and the naphthyridine nucleus in a phototoxicity and mammalian cell cytotoxicity assay. The series of 8-methoxyquinolones had antibacterial activity against Gram-positive, Gram-negative, and anaerobic bacteria equivalent to the most active 8-substituted compounds (8-F and 8-Cl). There was also a concomitant reduction in several of the potential side effects (i.e., phototoxicity and clonogenicity) compared to the most active quinolones with classic substitution at C-8. The 8-ethoxy derivatives had an even better safety profile but were significantly less active (2-3 dilutions) in the antibacterial assay.

Effect of lipophilicity at N-1 on activity of fluoroquinolones against mycobacteria.

The dramatic increase in drug resistant Mycobacterium tuberculosis has caused a resurgence in research targeted toward these organisms. As part of a systematic study to optimize the quinolone antibacterials against mycobacteria, we have prepared a series of N-1-phenyl-substituted derivatives to explore the effect of increasing lipophilicity on potency at this position. The compounds, synthesized by the modification of a literature procedure, were evaluated for activity against Gram-negative and Gram-positive bacteria, Mycobacterium fortuitum and Mycobacterium smegmatis, and the results correlated with log P, pKa, and other attributes. The activity of the compounds against the rapidly growing, less hazardous organism M. fortuitum was used as a measure of M. tuberculosis activity. The results demonstrate that increasing lipophilic character by itself does not correlate with increased potency against mycobacteria. Rather, intrinsic activity against Gram-negative and/or Gram-positive bacteria is the governing factor for corresponding activity against mycobacteria.

Nonpeptidic potent HIV-1 protease inhibitors: (4-hydroxy-6-phenyl-2-oxo-2H- pyran-3-yl)thiomethanes that span P1-P2' subsites in a unique mode of active site binding.

Using molecular modeling and the information derived from the X-ray crystal structure of HIV-1 protease (HIV PR) complexed with the pyran-2-one 1, a series of (4-hydroxy-6-phenyl-2-oxo-2H-pyran-3-yl)thiomethanes was designed and analyzed as novel, nonpeptidic inhibitors of HIV PR. Structure-activity studies led to the discovery of inhibitor 19 having (RS)-1-(cyclopentylthio)-3-methylbutyl functionalization at the C-3 position, which exhibited a Kc of 33 nM. A X-ray crystallographic structure of 19 bound to HIV PR showed that structural water-301 (inhibitor-flap-bridging water) was displaced by the inhibitor. Interestingly, the enol moiety of the pyran-2-one formed a hydrogen bond directly with Asp125 and with Asp25 via a bridging water molecule, thus illustrating a unique mode of active site binding by an HIV PR inhibitor. The pendant cyclopentyl and isobutyl groups of 19 occupied the S1' and S2' binding sites, respectively, whereas the 6-phenyl group occupied a region in between the S1 and S3 pockets of HIV PR. Selected compounds were tested for antiviral activity on H9 cells infected with HIV-1IIIb. A correlation between enzymatic activity and antiviral activity was not found in this series. The best antiviral compound in this series, 18, contained (RS)-3-[cyclopentyl(cyclopentylthio)methyl] functionalization at the C-3 position of the pyran-2-one ring and exhibited a CIC50 of 14 microM and TC50 of 70 microM. These studies demonstrate that potent enzyme inhibition can be achieved by inhibitors that span only three subsites.

A novel nonpeptide HIV-1 protease inhibitor: elucidation of the binding mode and its application in the design of related analogs.

HIV-1 protease has been identified as a significant target enzyme in AIDS research. While numerous peptide-derived inhibitors have been described, the identification of a nonpeptide inhibitor remains an important goal. Using an HIV-1 protease mass screening technique, 4-hydroxy-3-(3-phenoxypropyl)-2H-1-benzopyran-2-one (1) was identified as a nonpeptide competitive inhibitor of the enzyme. Employing a Monte Carlo-based docking procedure, the coumarin was docked in the active site of the enzyme, revealing a binding mode that was later confirmed by the X-ray crystal analysis. Several analogs were prepared to test the binding interactions and improve the overall binding affinity. The most active compound in the study was 4,7-dihydroxy-3-[4-(2-methoxyphenyl)butyl]-2H-1-benzopyran-2-one (31).

Structure-activity and structure-side-effect relationships for the quinolone antibacterials.

The fluoroquinolones represent a major class of antibacterials with great therapeutic potential. Over the years, several structure-activity and side-effect relationships have been developed, covering thousands of analogues, in an effort to improve overall antimicrobial efficacy while reducing undesirable side-effects. In this review, the various structural features of the quinolones which govern antibacterial efficacy and influence the side-effect profile are delineated and summarized at the molecular level. Those features which most remarkably enhance antimicrobial effectiveness are: a halogen (F or Cl) at the 8-position which improves oral absorption and activity against anaerobes; an alkylated pyrrolidine or piperazine at C7 which increases serum half-life and potency vs Gram-positive bacteria; and a cyclopropyl group at N1 and an amino substituent at C5, both of which improve overall potency. Some side-effects of the quinolones are class effects, and cannot be modulated by molecular variation. These include gastrointestinal irritation and arthropathy. Several other potential side-effects are directly influenced by structural modification. For example, CNS effects and drug interactions with theophylline and NSAIDs are strongly influenced by the C7 substituent with simple pyrrolidines and piperazines the worst actors. Increasing steric bulk through alkylation ameliorates these effects. Phototoxicity is determined by the nature of the 8-position substituent with halogen causing the greatest photo reaction while hydrogen and methoxy show little light induced toxicity. Genetic toxicity is controlled in additive fashion by the choice of groups at the 1, 7 and 8 positions. From the analysis, those groups which mutually improve efficacy while reducing side-effects are identified. In addition, preclinical models for determining potential side-effects are discussed.

Synthesis and antibacterial activity of new quinolones containing a 7-[3-(1-amino-1-methylethyl)-1-pyrrolidinyl] moiety. Gram-positive agents with excellent oral activity and low side-effect potential.

A series of the R and S isomers of 7-[3-(1-amino-1-methylethyl)-1-pyrrolidinyl]-1,4-dihydro-4-oxoquinoline- and 1,8-naphthyridine-3-carboxylic acids was prepared to determine the effect on potency of the two methyl groups adjacent to the distal nitrogen in the pyrrolidinyl moiety. The antibacterial efficacy of these dimethylated derivatives was compared to the relevant 7-[3-(aminomethyl)-1-pyrrolidinyl] parent compounds and, to a lesser extent, the 7-[3-(1-aminoethyl)-1-pyrrolidinyl] analogues. The activity of the title and reference compounds was assayed in vitro using an array of Gram-negative and Gram-positive organisms and in vivo using a mouse infection model. Selected derivatives were then screened for potential side effects in a phototoxicity mouse model and an in vitro mammalian cell cytotoxicity protocol. The results showed that the R isomer displayed a 2-20-fold advantage in activity in vitro and a 2-15-fold advantage in vivo over the S isomer. Although equipotent to the 7-[3-(aminomethyl)-1-pyrrolidinyl] parent compounds in vitro, the R isomers of the 7-[3-(1-amino-1-methylethyl)-1-pyrrolidinyl] analogues showed a dramatic increase in in vivo potency, especially via the oral route of administration. These same R isomers also appeared to possess a reduced risk of phototoxicity and cytotoxicity. This combination of superior in vivo performance with a low degree of phototoxicity and mammalian cell cytotoxicity recommends these agents for further study. Of these agents, naphthyridine 16-R represents the optimal blend of potency and safety.