Myelosuppression and kinase selectivity of multikinase angiogenesis inhibitors.

BACKGROUND: Myelosuppression has been observed with several multikinase angiogenesis inhibitors in clinical studies, although the frequency and severity varies among the different agents. Inhibitors targeting vascular endothelial growth factor receptor (VEGFR) often inhibit other kinases, which may contribute to their adverse-event profiles. METHODS: Kinase selectivity of pazopanib, sorafenib, and sunitinib was evaluated in a panel of 242 kinases. Cellular potency was measured using autophosphorylation assays. Effect on human bone marrow progenitor growth in the presence of multiple growth factors was evaluated and correlated with the kinase selectivity. RESULTS: Sunitinib inhibited more kinases than pazopanib and sorafenib, at potencies within 10-fold of VEGFR-2. All three compounds potently inhibited VEGFR-2, platelet-derived growth factor receptor-beta and c-Kit, However, pazopanib was less active against Flt-3 in both kinase and cellular assays. The inhibitory properties of pazopanib, sorafenib, and sunitinib were dependent on the growth factor used to initiate bone marrow colony formation. Addition of stem cell factor and/or Flt-3 ligand with granulocyte-macrophage colony stimulating factor resulted in significant shifts in potency for sorafenib and sunitinib but less so for pazopanib. CONCLUSION: Activity against c-kit and Flt-3 by multikinase angiogenesis inhibitors provide a potential explanation for the differences in myelosuppression observed with these agents in patients.

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.

Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

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.

4-hydroxy-5,6-dihydro-2H-pyran-2-ones.3. Bicyclic and hetero-aromatic ring systems as 3-position scaffolds to bind to S1' and S2' of the HIV-1 protease enzyme.

5,6-Dihydro-2H-pyran-2-ones are potent inhibitors of HIV-1 protease, which bind to the S1, S2, S1', and S2' pockets and have a unique binding mode with the catalytic aspartyl groups and the flap region of the enzyme. Efforts to explore 3-position heterocyclic scaffolds that bind to the S1' and S2' pockets have provided a number of selected analogs that display high HIV-1 protease inhibitory activity. reserved.

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.

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori.

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

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.

4-hydroxy-5,6-dihydropyrones. 2. Potent non-peptide inhibitors of HIV protease.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (< 475) have one or no chiral centers and are readily synthesized.

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.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein zinc ejection activity of disulfide benzamides and benzisothiazolones: correlation with anti-HIV and virucidal activities.

It has been shown previously by our group and others that a series of four disulfide benzamides with cellular anti-human immunodeficiency virus (HIV) activity can eject zinc from HIV type 1 nucleocapsid protein (NCp7) in vitro while analogs without antiviral activity do not. We also found that the zinc ejection activity correlates with the loss of the ability of NCp7 to bind to HIV psi RNA in vitro. These observations indicate that the antiviral disulfide benzamides may act at a novel retroviral target of action, i.e., the nucleocapsid protein. The present studies examine the relationship among disulfide benzamide structure, in vitro NCp7 zinc ejection activity, and antiviral activity for a larger series of compounds. All of the antiviral disulfide benzamides were found to eject NCp7 zinc, while some disulfide benzamides with zinc ejection activity are not antiviral. Utilizing the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid), it was determined that the o-amido-phenyl disulfides being studied cyclize in aqueous solution to form benzisothiazolones. A series of benzisothiazolones, which are stable in solution in the absence of dithiothreitol, were found to eject NCp7 zinc at a rate similar to that of their disulfide benzamide analogs and to possess similar antiviral activity. It was also found that the relative rates of HIV inactivation by various disulfide benzamides and benzisothiazolones correlate with their relative kinetic rates of NCp7 zinc ejection, which is consistent with the nucleocapsid protein being the target of action of these compounds.

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.

Inhibitors of HIV protease: unique non-peptide active site templates.

New templates were designed and prepared which straddle the active site of HIV-1 protease. These templates were designed to be "flexible scaffolds' upon which substituents could be appended to fill the pockets of HIV protease. The new templates prepared and analysed were 4-hydroxy-5H-furan-2-ones, 4-hydroxy-5,6-dihydropyrones, 3-hydroxy-cyclohex-2-enones, and 4-hydroxy-2(1H)-pyridinones, of which the 4-hydroxy-5,6-dihydropyrones were found to be the most potent inhibitors of HIV-1 protease.

The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.

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.

Competitive inhibition of HIV-1 protease by biphenyl carboxylic acids.

A novel series of nonpeptidic compounds that contain a biphenyl carboxylic acid group have been shown to inhibit HIV-1 protease. The active compounds, most of which are highly soluble, have IC50 values in the range of 3.4-74 microM. The structure-inhibitory activity relationship demonstrates the necessity of the biphenyl carboxylic acid group for inhibition, which is enhanced by the presence of a sulfone group and by halogenation of an adjacent phenyl group. A double reciprocal plot of inhibition data on two of the compounds clearly shows that the inhibition occurs in a competitive manner, with Ki values of 1.1 and 3.4 microM. Inhibition by several of the compounds was found to be reversible and fast-binding, while one of the biphenyl carboxylic acids inhibits in a reversible slow-binding manner. Time-dependent inhibition studies were conducted on this compound, and it was determined to have the kinetic values of kon = 0.18 microM-1min-1, koff = 9.7 x 10(-2)min-1, and Ki = 0.14 microM. Thus, the slow-binding inhibitor is the most potent in the series. Molecular modeling has provided information on a possible binding mode for two different biphenyl carboxylic acid inhibitors of HIV-1 protease.