Discovery of quinolone derivatives as antimycobacterial agents

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is an important public health issue. Current first-line drugs administered to TB patients have been in use for over 40 years, whereas second-line drugs display strong side effects and poor compliance. Additionally, designing effective regimens to treat patients infected with multi- and extremely-drug-resistant (MDR and XDR) strains of TB is challenging. In this report, we screened our compound library and identified compound 1 with antituberculosis activity and a minimal inhibitory concentration (MIC) against M. tuberculosis of 20 μg mL⁻¹. Structure optimization and the structure–activity relationship of 1 as the lead compound enabled the design and synthesis of a series of quinolone derivatives, 6a1–6a2, 6b1–6b36, 6c1, 6d1–6d14, 7a1–7a2, 7b1–7b2, 7c1, 8a1–8a5, 9a1–9a4 and 10a1–10a6. These compounds were evaluated in vitro for anti-tubercular activity against the M. tuberculosis H₃₇Rv strain. Among them, compounds 6b6, 6b12 and 6b21 exhibited MIC values in the range of 1.2–3 μg mL⁻¹ and showed excellent activity against the tested MDR-TB strain (MIC: 3, 2.9 and 0.9 μg mL⁻¹, respectively). All three compounds were non-toxic toward A549 and Vero cells (>100 and >50 μg mL⁻¹, respectively). In addition, an antibacterial spectrum test carried out using compound 6b21 showed that this compound specifically inhibits M. tuberculosis. These can serve as a new starting point for the development of anti-TB agents with therapeutic potential.

6b21: MIC against M. tb H₃₇Rv = 1.2 μg mL⁻¹, MIC against drug-resistant strains = 0.9 μg mL⁻¹, solubility = 132 μg mL⁻¹, non-cytotoxicity.     

Peptide deformylase inhibitors as antibacterial agents: identification of VRC3375, a proline-3-alkylsuccinyl hydroxamate derivative, by using an integrated combinatorial and medicinal chemistry approach

Peptide deformylase (PDF), a metallohydrolase essential for bacterial growth, is an attractive target for use in the discovery of novel antibiotics. Focused chelator-based chemical libraries were constructed and screened for inhibition of enzymatic activity, inhibition of Staphylococcus aureus growth, and cytotoxicity. Positive compounds were selected based on the results of all three assays. VRC3375 [N-hydroxy-3-R-butyl-3-(2-S-(tert-butoxycarbonyl)-pyrrolidin-1-ylcarbonyl)propionamide] was identified as having the most favorable properties through an integrated combinatorial and medicinal chemistry effort. This compound is a potent PDF inhibitor with a K(i) of 0.24 nM against the Escherichia coli Ni(2+) enzyme, possesses activity against gram-positive and gram-negative bacterial pathogens, and has a low cytotoxicity. Mechanistic experiments demonstrate that the compound inhibits bacterial growth through PDF inhibition. Pharmacokinetic studies of this drug in mice indicate that VRC3375 is orally bioavailable and rapidly distributed among various tissues. VRC3375 has in vivo activity against S. aureus in a murine septicemia model, with 50% effective doses of 32, 17, and 21 mg/kg of body weight after dosing by intravenous (i.v.), subcutaneous (s.c.), and oral (p.o.) administration, respectively. In murine single-dose toxicity studies, no adverse effects were observed after dosing with more than 400 mg of VRC3375 per kg by i.v., p.o., or s.c. administration. The in vivo efficacy and low toxicity of VRC3375 suggest the potential for developing this class of compounds to be used in future antibacterial drugs.     

Peptide deformylase as an antibacterial drug target: target validation and resistance development

New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10(-7)). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.     

High-throughput screen for detecting antimycobacterial agents.

A simple, robust assay system which can be used to screen for inhibitors of mycobacterial growth has been developed. A strain of the rapidly growing saprophyte Mycobacterium aurum is used as the test organism. Inhibition of its growth is highly predictive of activity against Mycobacterium tuberculosis, which cannot itself be used in screening because of its growth characteristics and highly infectious nature. The viability of M. aurum in the presence of a test sample is monitored by measuring the uptake of radiolabelled uracil into the cells. In a microtiter plate format, the screen has the potential for testing several thousand samples per day.     

Halophenyl furanopyrimidines as potent and selective anti-VZV agents

Bicyclic furano pyrimidines have been previously reported by us to be highly potent and selective inhibitors of varicella zoster virus (VZV). p-Alkyl phenyl analogues are particularly potent with EC50 values below 1 nM. In this article we report the synthesis and anti-VZV activity of a series of halophenyl analogues, with variation in the nature (F, Cl, Br) and location (o, m, p) of the halogen substituent. The compounds show a range of activities from ca. 10 nM to > 50 microM. In most cases, ortho substitution leads to greatest activity, meta substitution is in general poor, and the effect of p-substitution shows a marked dependence on the halogen atom. The p-fluorophenyl compound is unique amongst compounds of this class in being inactive as an antiviral. The possible origins of these marked SARs are discussed.     

N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.     

Neuraminidase inhibitors as anti-influenza virus agents.

Influenza virus neuraminidase (NA) catalyses the cleavage of sialic acid residues terminally linked to glycoproteins and glycolipids and plays an important role in the replication of the virus. Recently, several potent NA inhibitors have been synthesized based on the rational design of mimicking the transition state of the sialic acid cleavage. Zanamivir and oseltamivir (GS 4104, the prodrug of GS 4071) have emerged as promising influenza NA inhibitors for the treatment and prophylaxis of human influenza virus infection. This review describes the recent work toward the discovery and development of influenza NA inhibitors.     

Design of translactam HCMV protease inhibitors as potent antivirals

Human cytomegalovirus (HCMV) is an important pathogen for which there is a significant unmet medical need. New HCMV antivirals, active against novel molecular targets, are undoubtedly needed as the currently available drugs ganciclovir, cidofovir, and foscarnet, which are all viral DNA inhibitors, suffer from limited effectiveness, mainly due to the development of drug resistance, poor bioavailability, and toxicity. One of the newer molecular targets that has been exploited in the search for better drug candidates is HCMV protease. Our deltaAla HCMV protease (wild type variant with the internal cleavage site deleted) was cloned and expressed in E. coli. This viral enzyme was used to develop HCMV protease assays to evaluate potential inhibitors. The chirally pure (SRS)-alpha-methyl pyrrolidine-5,5-trans-lactam template was synthesized, which together with the natural substrate requirements of HCMV protease and detailed SAR, was used to design potent and selective mechanism based inhibitors of HCMV protease. The mechanism of action of these inhibitors of HCMV protease was investigated by ESI/MS, and the X-ray crystal structure of the HCMV protease was used to refine our selective viral enzyme inhibitors to obtain plasma stable antivirals. A novel ELISA antiviral assay was developed which, together with a cytotoxicity assay, enabled us to discover anti-HCMV drug candidates equivalent in potency to ganciclovir that had good pharmacokinetics in the dog and good brain and ocular penetration in the guinea pig.     

Furano pyrimidines as novel potent and selective anti-VZV agents.

Bicyclic furano pyrimidine nucleosides have been found to be highly potent and selective inhibitors of varicella zoster virus (VZV). They are inactive against herpes simplex virus and have been known for several decades as (unwanted) synthetic by-products in the Pd-catalysed coupling of acetylenes to 5-iodo nucleosides. These fluorescent bicyclic nucleosides are now established as a new family of potent antivirals. They are unusual in that they exhibit complete specificity for VZV and require an alkyl (or alkylaryl) side-chain for biological activity. The latter requirement confers extremely high lipophilicities on these compounds, unknown amongst chemotherapeutic nucleosides, which may be of considerable importance in formulation, dosing and tissue distribution. The most potent compounds reported are p-alkylaryl compounds, with EC50 values below 1 nM versus VZV and selectivity index values of around 1,000,000. Here, we review the discovery, synthesis, characterization, antiviral profile, SAR, mechanism of action and development prospects for this new family of antivirals.     

In vitro activities of peptide deformylase inhibitors against gram-positive pathogens

The activities of six peptide deformylase (PDF) inhibitors against 107 respiratory tract pathogens were studied and compared to those of ciprofloxacin and amoxicillin-clavulanate. Against Streptococcus pneumoniae, BB-83698 and BB-83815 were the most active PDF inhibitors (MIC at which 90% of the organisms tested were inhibited [MIC(90)], 0.25 microg/ml). Five of the agents showed similar activity against Moraxella catarrhalis (MIC(90), 0.12 microg/ml). All PDF inhibitors were less active against Haemophilus influenzae; BB-3497 was the most active agent (MIC(90), 2 microg/ml). Five agents were studied against Chlamydia spp. and showed activity similar to that of ciprofloxacin (MIC, 0.5 to 4 microg/ml). This study demonstrates that PDF inhibitors have the potential to be developed for the treatment of respiratory tract infections.     

Flavonoids as RTK inhibitors and potential anticancer agents

Tyrosine kinase receptors (RTKs) play a crucial role in the regulation of the cell division cycle. Currently more than 50 RTKs divided into several subfamilies have been described. The inhibition of these enzymes has emerged as an important research-area. Compounds able to inhibit the activity of these enzymes are expected to display antiproliferative properties. Flavonoids are representative of various small molecules acting as RTK inhibitors. These naturally occurring compounds are able to bind to the ATP-binding site of several kinases. The most plausible current hypothesis explaining the action of these substances on kinases is that the chromenone moiety of the flavonoid acts as a mimetic of the adenine moiety of ATP, the receptor co-factor. In this review, we report recent results on the activity of natural and synthetic derivatives of flavonoids as inhibitors of RTKs. Mechanistic aspects, the therapeutic usefulness, and the potential clinical use are discussed.     

Serine protease inhibitors as anti-hepatitis C virus agents

Approximately 3% of the worldwide population (i.e., more than 170 million people) are chronically infected with the hepatitis C virus (HCV). An estimated 20% of these patients will develop liver cirrhosis within a mean of 20 years, and 2–5% of cirrhotic patients will die of end-stage liver disease or hepatocellular carcinoma. The currently approved antiviral therapy with pegylated interferon (pegIFN) and ribavirin induces a sustained virological response (SVR) in 40–50% of patients infected with genotype 1, the most prevalent HCV type. In this review, we focus on the development and clinical application of serine protease inhibitors as anti-HCV agents. Although highly active in inducing a significant decline of serum HCV RNA, the rapid development of resistance must be counteracted in combination with other antiviral agents, currently pegIFN-α and ribavirin. Two serine protease inhibitors have reached clinical Phase III trials, increasing SVR rates and shortening treatment duration when combined with pegIFN and ribavirin. Trials of interferon-free targeted combination therapies are currently underway.     

New pyridine and chromene scaffolds as potent vasorelaxant and anticancer agents

Based on studies that have reported the association between cancer and cardiovascular diseases, new series of pyridine- (3a–o) and/or chromene- (4a–e) carbonitrile analogous were designed, synthesized and screened for their vasodilation and cytotoxic properties. The majority of the new chemical entities demonstrated significant vasodilation efficacies, compounds 3a, 3h, 3j, 3m, 3o, 4d and 4e exhibited the most promising potency with IC₅₀ = 437.9, 481.0, 484.5, 444.8, 312.1, 427.6 and 417.2 μM, respectively, exceeding prazosin hydrochloride (IC₅₀ = 487.3 μM). Compounds 3b–e, 3k and 3l also, revealed moderate vasodilation activity with IC₅₀ values ranging from 489.7 to 584.5 μM. In addition, the anti-proliferative activity evaluation of the experimental compounds at 10 μM on the MCF-7 and MDA-MB 231 breast cancer cell lines illustrated the excellent anti-proliferative properties of derivatives 3d, 3g and 3i. Compound 3d was the most potent analogue with IC₅₀ = 4.55 ± 0.88 and 9.87 ± 0.89 μM against MCF-7 and MDA-MB 231, respectively. Moreover, compound 3d stimulated apoptosis and cell cycle arrest at the S phase in MCF-7 cells in addition to its capability in accumulation of cells in pre-G1 phase and activating caspase-3. Furthermore, the molecular docking of 3d was performed to discover the binding modes within the active site of caspase-3. 3d, as the only common bi-functional agent among the tested hits, demonstrated that new pyridine-3-carbonitrile derivatives bearing cycloheptyl ring systems offer potential as new therapeutic candidates with combined vasodilation and anticancer properties.

Series of pyridine- (3a–o) and/or chromene- (4a–e) carbonitrile scaffolds have been designed, synthesized and evaluated for their bi-function activities, 3d was the only common derivative having combined vasodilation and anticancer properties.     

Protein tyrosine kinase inhibitors as novel therapeutic agents.

Protein tyrosine kinases (PTKs) play a key role in normal cell and tissue development. Enhanced PTK activity is intimately correlated with proliferative diseases, such as cancers, leukemias, psoriasis, and restenosis. This realization prompted us to systematically synthesize tyrosine phosphorylation inhibitors (tyrphostins) as potential drugs. Over the years, we have demonstrated the ability to synthesize selective tyrphostins aimed at different receptor, as well as at nonreceptor, tyrosine kinases. Some of these tyrphostins have shown efficacy in vivo as antileukemic agents and antirestenosis agents. AG 490, a Jak-2 inhibitor, is potent against recurrent pre-B acute lymphoblastic leukemia. AG 1295, a selective platelet-derived growth factor receptor kinase inhibitor, inhibits 50% of balloon injury-induced stenosis in the phemoral arteries of pigs. AG 1517 (SU 5271), a potent epiderminal growth factor receptor kinase inhibitor, is currently in clinical trials for psoriasis. Similarly, SU 5416, a potent kinase inhibitor of the vascular endothelial growth factor receptor/kinase domain receptor/Flk-1, is currently in clinical trials as an anticancer agent by virtue of its strong anti-angiogenic activity. These findings demonstrate that the identification of PTKs that play a key role in a defined disease state can lead to a selective drug. Tyrphostins also show efficacy in vivo in inflammatory diseases such as sepsis, cirrhosis, and experimental autoimmune encephalitis.     

SGLT inhibitors as antidiabetic agents: a comprehensive review

Diabetes is one of the most common disorders that substantially contributes to an increase in global health burden. As a metabolic disorder, diabetes is associated with various medical conditions and diseases such as obesity, hypertension, cardiovascular diseases, and atherosclerosis. In this review, we cover the scientific studies on sodium/glucose cotransporter (SGLT) inhibitors published during the last decade. Our focus on providing an exhaustive overview of SGLT inhibitors enabled us to present their chemical classification for the first time.

Diabetes is one of the most common disorders that substantially contributes to an increase in global health burden.