Investigation of some benzoquinazoline and quinazoline derivatives as novel inhibitors of HCV-NS3/4A protease: biological,molecular docking and QSAR studies

Morbidity and mortality due to hepatitis C virus (HCV) is a globe health concern. Hence, there is a persistent demand to design and optimize current HCV therapy and develop novel agents. HCV NS3/A4 protease plays an essential role in HCV life cycle and replication. Thus, HCV NS3/A4 protease inhibitors are one of the best therapeutic targets for the identification of novel candidate drugs. Recent studies have shown some benzoquinazolines as potent antiviral agents and promising HAV-3C protease inhibitors. In the present study, a series of benzo[g]quinazolines (1–13) and their quinazoline analogues (14–17) were evaluated for their HCV-NS3/4A inhibitory activities using in vitro assay. Our results revealed that the target compounds inhibited the activity of the NS3/4A enzyme, (IC₅₀ = 6.41 ± 0.12 to 78.80 ± 1.70 μM) in comparison to telaprevir (IC₅₀ = 1.72 ± 0.03 μM) as a reference drug. Compounds 1, 2, 3, 9, 10 and 13 showed the highest activity (IC₅₀ = 11.02 ± 0.25, 6.41 ± 0.12, 9.35 ± 0.19, 9.08 ± 0.20, 16.03 ± 0.34 and 7.21 ± 0.15 μM, respectively). Molecular docking was performed to study the binding modes of the docked-chosen benzo[g]quinazolines, hydrogen bonding, and amino acid residues at the catalytic triad of the NS3/4A enzyme of HCV. The QSAR was determined to explore the relationships between the molecular structures of the targets and their biological activities by developing prediction models among the known HCV NS3/A4 inhibitors and then to predict the inhibitory activity of the target molecules synthesized.

HCV NS3/A4 protease inhibitors are one of the best therapeutic targets for the identification of novel candidate drugs. A series of benzo[g]quinazolines and their quinazoline analogues were evaluated for their HCV-NS3/4A inhibitory activities.     

Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen

West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen ~32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K_i values of 3.2 ± 0.3 μM and 3.4 ± 0.6 μM, respectively. These compounds inhibited the dengue virus type 2 protease with K_i values of 28.6 ± 5.1 μM and 30.2 ± 8.6 μM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 ± 0.4 μM; selectivity index, 100), presumably by inhibition of polyprotein processing.     

Rapid Ganciclovir Susceptibility Assay Using Flow Cytometry for Human Cytomegalovirus Clinical Isolates

Rapid, quantitative, and objective determination of the susceptibilities of human cytomegalovirus (HCMV) clinical isolates to ganciclovir has been assessed by an assay that uses a fluorochrome-labeled monoclonal antibody to an HCMV immediate-early antigen and flow cytometry. Analysis of the ganciclovir susceptibilities of 25 phenotypically characterized clinical isolates by flow cytometry demonstrated that the 50% inhibitory concentrations (IC₅₀s) of ganciclovir for 19 of the isolates were between 1.14 and 6.66 μM, with a mean of 4.32 μM (±1.93) (sensitive; IC₅₀ less than 7 μM), the IC₅₀s for 2 isolates were 8.48 and 9.79 μM (partially resistant), and the IC₅₀s for 4 isolates were greater than 96 μM (resistant). Comparative analysis of the drug susceptibilities of these clinical isolates by the plaque reduction assay gave IC₅₀s of less than 6 μM, with a mean of 2.88 μM (±1.40) for the 19 drug-sensitive isolates, IC₅₀s of 6 to 8 μM for the partially resistant isolates, and IC₅₀s of greater than 12 μM for the four resistant clinical isolates. Comparison of the IC₅₀s for the drug-susceptible and partially resistant clinical isolates obtained by the flow cytometry assay with the IC₅₀s obtained by the plaque reduction assay showed an acceptable correlation (r² = 0.473; P = 0.001), suggesting that the flow cytometry assay could substitute for the more labor-intensive, subjective, and time-consuming plaque reduction assay.     

Synthesis of 2-ethoxycarbonylthieno[2,3-b]quinolines in biomass-derived solvent γ-valerolactone and their biological evaluation against protein tyrosine phosphatase 1B

A series of 2-ethoxycarbonylthieno[2,3-b]quinolines were synthesized in the bio-derived “green” solvent γ-valerolactone (GVL) and evaluated for their inhibitory activities against PTP1B, the representative compound 6a displayed an IC₅₀ value of 8.04 ± 0.71 μM with 4.34-fold preference over TCPTP. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

A series of 2-ethoxycarbonylthieno[2,3-b]quinolines were synthesized in the bio-derived “green” solvent γ-valerolactone and evaluated for their inhibitory activities against PTP1B, compound 6a displayed an IC₅₀ value of 8.04 ± 0.71 μM with 4.34-fold preference over TCPTP.     

Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding Gene UL89

Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC₅₀] = 0.34 μM) than ganciclovir (EC₅₀ = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC₅₀ = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC₅₀ = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC₅₀ for wild-type HCMV = 0.25 ± 0.04 μM, EC₅₀ for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).     

Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K_m values of 4.70 ± 0.059 (mean ± standard error of the mean) and 9.75 ± 1.64 μM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC₅₀] = 68.6 ± 5.20 nM) than pyrimethamine (IC₅₀ = 55.0 ± 2.08 μM) and trimethoprim (IC₅₀ = 50 ± 12.5 μM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.     

A series of four novel alkaline earth metal–organic frameworks constructed of Ca(ii), Sr(ii), Ba(ii) ions and tetrahedral MTB linker: structural diversity,stability study and low/high-pressure gas adsorption properties

A series of four novel microporous alkaline earth metal–organic frameworks (AE-MOFs) containing methanetetrabenzoate linker (MTB) with composition {[Ca₄(μ₈-MTB)₂]·2DMF·4H₂O}_n (UPJS-6), {[Ca₄(μ₄-O)(μ₈-MTB)_3/2(H₂O)₄]·4DMF·4H₂O}_n (UPJS-7), {[Sr₃(μ₇-MTB)_3/2]·4DMF·7H₂O}_n (UPJS-8) and {[Ba₃(μ₇-MTB)_3/2(H₂O)₆]·2DMF·4H₂O}_n (UPJS-9) (UPJS = University of Pavol Jozef Safarik) have been successfully prepared and characterized. The framework stability and thermal robustness of prepared materials were investigated using thermogravimetric analysis (TGA) and high-energy powder X-ray diffraction (HE-PXRD). MOFs were tested as adsorbents for different gases at various pressures and temperatures. Nitrogen and argon adsorption showed that the activated samples have moderate BET surface areas: 103 m² g⁻¹ (N₂)/126 m² g⁻¹ (Ar) for UPJS-7′′, 320 m² g⁻¹ (N₂)/358 m² g⁻¹ (Ar) for UPJS-9′′ and UPJS-8′′ adsorbs only a limited amount of N₂ and Ar. It should be noted that all prepared compounds adsorb carbon dioxide with storage capacities ranging from 3.9 to 2.4 wt% at 20 °C and 1 atm, and 16.4–13.5 wt% at 30 °C and 20 bar. Methane adsorption isotherms show no adsorption at low pressures and with increasing pressure the storage capacity increases to 4.0–2.9 wt% of CH₄ at 30 °C and 20 bar. Compounds displayed the highest hydrogen uptake of 3.7–1.8 wt% at −196 °C and 800 Torr among MTB containing MOFs.

Four novel microporous alkaline earth metal–organic frameworks (AE-MOFs) containing methanetetrabenzoate linker (MTB): UPJS-6, UPJS-7, UPJS-8 and UPJS-9 have been successfully prepared, characterized and tested as adsorbents for different gases.     

A new synthetic route for the preparation of [Os3(CO)10(μ-OH)(μ-H)] and its reaction with bis(diphenylphosphino)methane (dppm): syntheses and X-ray structures of two isomers of [Os3(CO)8(μ-OH)(μ-H)(μ-dppm)] and [Os3(CO)7(μ3-CO)(μ3-O)(μ-dppm)]

The triosmium cluster [Os₃(CO)₁₀(μ-OH)(μ-H)] containing bridging hydride and hydroxyl groups at a common Os–Os edge was obtained in good yield (ca. 75%) from the hydrolysis of the labile triosmium cluster [Os₃(CO)₁₀(NCMe)₂] in THF at 67 °C. [Os₃(CO)₁₀(μ-OH)(μ-H)] reacts with dppm at 68 °C to afford the isomeric clusters 1 and 2 with the general formula [Os₃(CO)₈(μ-OH)(μ-H)(μ-dppm)] that differ by the disposition of bridging dppm ligand. Cluster 1 is produced exclusively from the reaction of [Os₃(CO)₁₀(μ-OH)(μ-H)] with dppm in CH₂Cl₂ at room temperature in the presence of added Me₃NO. Heating cluster 1 at 81 °C furnishes 2 in a process that likely proceeds by the release of one arm of the dppm ligand, followed by ligand reorganization about the cluster polyhedron and ring closure of the pendent dppm ligand. The oxo-capped [Os₃(CO)₇(μ₃-CO)(μ₃-O)(μ-dppm)] (3) has been isolated starting from the thermolysis of either 1 or 2 at 139 °C. Reactions of [Os₃(CO)₁₀(μ-dppm)] with ROH (R = Me, Et) in the presence of Me₃NO at 80 °C furnish [Os₃(CO)₈(μ-OH)(μ,η¹,κ¹-OCOR)(μ-dppm)] (4, R = Me; 5, R = Et). Clusters 1–5 have been characterized by a combination of analytical and spectroscopic studies, and the molecular structure of each product has been established by X-ray crystallography. The bonding in these products has been examined by electronic structure calculations, and cluster 1 is confirmed as the kinetic product of substitution, while cluster 2 represents the thermodynamically favored isomer.

The cluster [Os₃(CO)₁₀(μ-OH)(μ-H)] was obtained in 75% from the hydrolysis of [Os₃(CO)₁₀(NCMe)₂].     

Nucleoside Inhibitors of Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2′-C-methyladenosine (7-deaza-2′-CMA), 2′-C-methyladenosine (2′-CMA), and 2′-C-methylcytidine (2′-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC₅₀]of 5.1 ± 0.4 μM for 7-deaza-2′-CMA, 7.1 ± 1.2 μM for 2′-CMA, and 14.2 ± 1.9 μM for 2′-CMC) and viral antigen production. Notably, 2′-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC₅₀] of ∼50 μM). The anti-TBEV effect of 2′-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2′-CMA showed no detectable cellular toxicity (CC₅₀ > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2′-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2′-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.     

In vitro and in silico studies on clinically important enzymes inhibitory activities of flavonoids isolated from Euphorbia pulcherrima

Introduction: The genus Euphorbia is known to contain diterpenoids, and several isolated compounds which exhibited biological activities including significant multidrug resistance reversal effects. This work is focused on the isolation, in vitro and in silico studies of two natural bio-active flavonoids (1 & 2) isolated from Euphorbia pulcherrima bark for the very first time.Methods: The phytochemical investigation resulted in the identification of two flavonoids: 3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-methoxy-4H-chromen-4-one (1) and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-6-methoxy-4H-chromen-4-one (2), which were isolated for the first time from Euphorbia pulcherrima.Results: The chemical structures of the two isolated compounds were confirmed by ¹H NMR, ¹³C NMR, and ESI-HRMS spectral data. The Bioactivity activity of these compounds was evaluated; results revealed that compounds 1 & 2 exhibit promising urease inhibitory potential with IC50 values of 15.3 ± 2.13 μM and 19.0 ± 2.43 μM, respectively, whereas the positive control thiourea had an IC₅₀ of 21.0 ± 0.23 μM. Similarly, these compounds were also evaluated against the tyrosinase enzyme; results showed that compound 1 displays significant inhibitory activity with an IC₅₀ value of 48.7 ± 2.19 μM, whereas compound 2 exhibited a moderate effect with an IC50 value of 74.8 ± 1.79 μM, when compared with the standard (alpha-kojic acid, IC₅₀ = 47.6 ± 0.67 μM). Additionally, compounds 1 and 2 also exhibited anti-glycation and phosphodiesterase inhibitory activities.Conclusion: Studies dealing with the drug like properties such as in silico screening (docking study) was also carried out to discover the structural features of both compounds 1 and 2. Results indicated that the docking scores of compounds 1 and 2 are in agreement with their IC₅₀ values.

Key messages

• Isolation and characterization of two bioactive flavonoids (1 and 2) from Euphorbia pulcherrima.
• In silico and in vitro enzyme inhibition studies were conducted to identify the therapeutic potential of flavonoids 1 and 2.
• Drug-like properties were calculated to discover important pharmacophoric features.

     

In vitro and in silico studies of SARS-CoV-2 main protease Mpro inhibitors isolated from Helichrysum bracteatum

Discovering SARS-CoV-2 inhibitors from natural sources is still a target that has captured the interest of many researchers. In this study, the compounds (1–18) present in the methanolic extract of Helichrysum bracteatum were isolated, identified, and their in vitro inhibitory activities against SARS-CoV-2 main protease (M^pro) was evaluated using fluorescence resonance energy transfer assay (FRET-based assay). Based on 1D and 2D spectroscopic techniques, compounds (1–18) were identified as 24-β-ethyl-cholesta-5(6),22(23),25(26)-triene-3-ol (1), α-amyrin (2), linoleic acid (3), 24-β-ethyl-cholesta-5(6),22(23),25(26)-triene-3-O-β-d-glucoside (4), 1,3-propanediol-2-amino-1-(3′,4′-methylenedioxyphenyl) (5), (−)-(7R,8R,8′R)-acuminatolide (6), (+)-piperitol (7), 5,7,4′-trihydroxy-8,3′-dimethoxy flavanone (8), 5,7,4′-trihydroxy-6-methoxy flavanone (9), 4′,5-dihydroxy-3′,7,8-trimethoxyflavone (10), 5,7-dihydroxy-3′,4′,5′,8-tetramethoxy flavone (11), 1,3-propanediol-2-amino-1-(4′-hydroxy-3′-methoxyphenyl) (12), 3′,5′,5,7-tetrahydroxy-6-methoxyflavanone (13), simplexoside (piperitol-O-β-d-glucoside) (14), pinoresinol monomethyl ether-β-d-glucoside (15), orientin (16), luteolin-3′-O-β-d-glucoside (17), and 3,5-dicaffeoylquinic acid (18). Compounds 6, 12, and 14 showed comparable inhibitory activities against SARS-CoV-2 M^pro with IC₅₀ values of 0.917 ± 0.05, 0.476 ± 0.02, and 0.610 ± 0.03 μM, respectively, compared with the control lopinavir with an IC₅₀ value of 0.225 ± 0.01 μM. The other tested compounds showed considerable inhibitory activities. The molecular docking study for the tested compounds was carried out to correlate their binding modes and affinities for the SARS-CoV-2 M^pro enzyme with the in vitro results. Analyzing the results of the in vitro assay together with the obtained in silico results led to the conclusion that phenylpropanoids, lignans, and flavonoids could be considered suitable drug leads for developing anti-COVID-19 therapeutics. Moreover, the phenylpropanoid skeleton oxygenated at C3, C4 of the phenyl moiety and at C1, C3 of the propane parts constitute an essential core of the SARS-CoV-2 M^pro inhibitors, and thus could be proposed as a scaffold for the design of new anti-COVID-19 drugs.

Compounds isolated and identified from Helichrysum bracteatum leaves showed promising in vitro inhibitory activities against SARS-CoV-2 main protease (M^pro). Thus, could be considered suitable drug leads for developing anti-COVID-19 therapeutics.     

Nitro/Nitrosyl-Ruthenium Complexes Are Potent and Selective Anti-Trypanosoma cruzi Agents Causing Autophagy and Necrotic Parasite Death

cis-[RuCl(NO₂)(dppb)(5,5′-mebipy)] (complex 1), cis-[Ru(NO₂)₂(dppb)(5,5′-mebipy)] (complex 2), ct-[RuCl(NO)(dppb)(5,5′-mebipy)](PF₆)₂ (complex 3), and cc-[RuCl(NO)(dppb)(5,5′-mebipy)](PF₆)₂ (complex 4), where 5,5′-mebipy is 5,5′-dimethyl-2,2′-bipyridine and dppb is 1,4-bis(diphenylphosphino)butane, were synthesized and characterized. The structure of complex 2 was determined by X-ray crystallography. These complexes exhibited a higher anti-Trypanosoma cruzi activity than benznidazole, the current antiparasitic drug. Complex 3 was the most potent, displaying a 50% effective concentration (EC₅₀) of 2.1 ± 0.6 μM against trypomastigotes and a 50% inhibitory concentration (IC₅₀) of 1.3 ± 0.2 μM against amastigotes, while it displayed a 50% cytotoxic concentration (CC₅₀) of 51.4 ± 0.2 μM in macrophages. It was observed that the nitrosyl complex 3, but not its analog lacking the nitrosyl group, releases nitric oxide into parasite cells. This release has a diminished effect on the trypanosomal protease cruzain but induces substantial parasite autophagy, which is followed by a series of irreversible morphological impairments to the parasites and finally results in cell death by necrosis. In infected mice, orally administered complex 3 (five times at a dose of 75 μmol/kg of body weight) reduced blood parasitemia and increased the survival rate of the mice. Combination index analysis of complex 3 indicated that its in vitro activity against trypomastigotes is synergic with benznidazole. In addition, drug combination enhanced efficacy in infected mice, suggesting that ruthenium-nitrosyl complexes are potential constituents for drug combinations.     

GRL-04810 and GRL-05010, Difluoride-Containing Nonpeptidic HIV-1 Protease Inhibitors (PIs) That Inhibit the Replication of Multi-PI-Resistant HIV-1 In Vitro and Possess Favorable Lipophilicity That May Allow Blood-Brain Barrier Penetration

We designed, synthesized, and identified two novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-04810 and GRL-05010, containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF), and a difluoride moiety, both of which are active against the laboratory strain HIV-1_LAI (50% effective concentrations [EC₅₀s], 0.0008 and 0.003 μM, respectively) with minimal cytotoxicity (50% cytotoxic concentrations [CC₅₀s], 17.5 and 37.0 μM, respectively, in CD4⁺ MT-2 cells). The two compounds were active against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to various antiviral regimens. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1_NL4-3 variants selected by up to 5 μM lopinavir (EC₅₀s, 0.03 and 0.03 μM, respectively) and atazanavir (EC₅₀s, 0.02 and 0.04 μM, respectively). Moreover, they were active against darunavir (DRV)-resistant variants (EC₅₀ in 0.03 to 0.034 μM range for GRL-04810 and 0.026 to 0.043 μM for GRL-05010), while DRV had EC₅₀s between 0.02 and 0.174 μM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (log P) and distribution (log D) coefficients of −0.14 and −0.29, respectively. The in vitro blood-brain barrier (BBB) permeability assay revealed that GRL-04810 and GRL-05010 may have a greater advantage in terms of crossing the BBB than the currently available PIs, with apparent penetration indexes of 47.8 × 10⁻⁶ and 61.8 × 10⁻⁶ cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert efficient activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moieties may well enhance their penetration across the BBB.     

Structural transformation of methasterone with Cunninghamella blakesleeana and Macrophomina phaseolina

An anabolic-androgenic synthetic steroidal drug, methasterone (1) was transformed by two fungi, Cunninghamella blakesleeana and Macrophimina phaseclina. A total of six transformed products, 6β,7β,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (2), 6β,7α,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (3), 6α,17β-dihydroxy-2α,17α-dimethyl-5α-androstane-3,7-dione (4), 3β,6β,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-7-one (5), 7α,17β-dihydroxy-2α,17α-dimethyl-5α-androstane-3-one (6), and 6β,9α,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (7) were synthesized. Among those, compounds 2–5, and 7 were identified as new transformed products. MS, NMR, and other spectroscopic techniques were performed for the characterization of all compounds. Substrate 1 (IC₅₀ = 23.9 ± 0.2 μg mL⁻¹) showed a remarkable anti-inflammatory activity against nitric oxide (NO) production, in comparison to standard LNMMA (IC₅₀ = 24.2 ± 0.8 μg mL⁻¹). Whereas, its metabolites 2, and 7 showed moderate inhibition with IC₅₀ values of 38.1 ± 0.5 μg mL⁻¹, and 40.2 ± 3.3 μg mL⁻¹, respectively. Moreover, substrate 1 was found to be cytotoxic for the human normal cell line (BJ) with an IC₅₀ of 8.01 ± 0.52 μg mL⁻¹, while metabolites 2–7 were identified as non-cytotoxic. Compounds 1–7 showed no cytotoxicity against MCF-7 (breast cancer), NCI-H460 (lung cancer), and HeLa (cervical cancer) cell lines.

Fungal transformation of methasterone resulted in six products (2–7). 2–5, and 7 were identified as new. Substrate 1 showed remarkable anti-inflammatory activity but was cytotoxic. Products 2 and 7 showed moderate activity but were non-cytotoxic.     

Microbial transformation of mestanolone by Macrophomina phaseolina and Cunninghamella blakesleeana and anticancer activities of the transformed products

The microbial transformation of anabolic androgenic steroid mestanolone (1) with Macrophomina phaseolina and Cunninghamella blakesleeana has afforded seven metabolites. The structures of these metabolites were characterized as 17β-hydroxy-17α-methyl-5α-androsta-1-ene-3,11-dione (2), 14α,17β-dihydroxy-17α-methyl-5α-androstan-3,11-dione (3), 17β-hydroxy-17α-methyl-5α-androstan-1,14-diene-3,11-dione (4), 17β-hydroxy-17α-methyl-5α-androstan-3,11-dione (5), 11β,17β-dihydroxy-17α-methyl-5α-androstan-1-ene-3-one (6), 9α,11β,17β-trihydroxy-17α-methyl-5α-androstan-3-one (7), and 1β,11α,17β-trihydroxy-17α-methyl-5α-androstan-3-one (8). All the metabolites, except 5 and 6, were identified as new compounds. Substrate 1 (IC₅₀ = 27.6 ± 1.1 μM), and its metabolites 2 (IC₅₀ = 19.2 ± 2.9 μM) and 6 (IC₅₀ = 12.8 ± 0.6 μM) exhibited moderate cytotoxicity against the HeLa cancer cell line (human cervical carcinoma). All metabolites were noncytotoxic to 3T3 (mouse fibroblast) and H460 (human lung carcinoma) cell lines. The metabolites were also evaluated for immunomodulatory activity, and all were found to be inactive.

The microbial transformation of anabolic androgenic steroid mestanolone (1) with Macrophomina phaseolina and Cunninghamella blakesleeana has afforded seven metabolites. Some of them have exhibited moderate cytotoxicity against HeLa cancer cell line.     

Novel derivatives of eugenol as potent anti-inflammatory agents via PPARγ agonism: rational design,synthesis, analysis,PPARγ protein binding assay and computational studies

Eugenol is a natural product abundantly found in clove buds known for its pharmacological activities such as anti-inflammatory, antidiabetic, antioxidant, and anticancer activities. It is well known from the literature that peroxisome proliferator-activated receptors (PPARγ) have been reported to regulate inflammatory responses. In this backdrop, we rationally designed semi-synthetic derivatives of eugenol with the aid of computational studies, and synthesized, purified, and analyzed four eugenol derivatives as PPARγ agonists. Compounds were screened for PPARγ protein binding by time-resolved fluorescence (TR-FRET) assay. The biochemical assay results were favorable for 1C which exhibited significant binding affinity with an IC₅₀ value of 10.65 μM as compared to the standard pioglitazone with an IC₅₀ value of 1.052 μM. In addition to the protein binding studies, as a functional assay, the synthesized eugenol derivatives were screened for in vitro anti-inflammatory activity at concentrations ranging from 6.25 μM to 400 μM. Among the four compounds tested 1C shows reasonably good anti-inflammatory activity with an IC₅₀ value of 133.8 μM compared to a standard diclofenac sodium IC₅₀ value of 54.32 μM. Structure–activity relationships are derived based on computational studies. Additionally, molecular dynamics simulations were performed to examine the stability of the protein–ligand complex, the dynamic behavior, and the binding affinity of newly synthesized molecules. Altogether, we identified novel eugenol derivatives as potential anti-inflammatory agents via PPARγ agonism.

Rational design, synthesis, analysis, PPARγ protein binding assay and computational studies of novel eugenol derivatives.     

Structural modification of olibergin A,an isoflavonoid,from Dalbergia stipulacea Roxb. and its cytotoxicity

Fifteen derivatives were synthesized from olibergin A, a major isoflavonoid isolated from the stems of Dalbergia stipulacea Roxb. All compounds were evaluated for cytotoxicity against HCT-116, HT-29, MCF-7 and vero cell lines using MTT assay. Cytotoxicity results showed 5-hydroxy-7,2′,4′,5′-tetramethoxyisoflavone (5) was the most active with IC₅₀ values of 19.03 ± 0.70, 10.83 ± 1.65, 12.53 ± 0.70 and 13.53 ± 0.84 μM against HCT-116, HT-29, MCF-7 and vero cell lines, respectively. It should be noted that 5-hydroxy-7,2′,4′,5′-tetramethoxyisoflavone (5) showed two times less toxicity against vero cells than the cisplatin standard (IC₅₀ = 6.55 ± 0.81 μM) while 5 and cisplatin exhibited nearly equal cytotoxicity against the MCF-7 cell line. 5,7,2′,4′,5′-Pentamethoxyisoflavanone (10) showed an IC₅₀ value of 30.34 ± 1.15 μM against the HCT-116 cell line and exhibited weak cytotoxicity against normal cells, the vero cell line. In addition, 5,7,4′-trihydroxy-2′,5′-dimethoxyisoflavan oxime (13) demonstrated cytotoxicity against HT-29 cells with an IC₅₀ value of 31.41 ± 1.38 μM and displayed weak activity toward the vero cell line. The information revealed that these compounds were suitable for development to anticancer agents against HCT-116, HT-29 and MCF-7 cell lines.

Fifteen derivatives were synthesized from olibergin A, a major isoflavonoid isolated from the stems of Dalbergia stipulacea Roxb.     

Synthesis and biological evaluation of novel artemisone–piperazine–tetronamide hybrids

For the first time, six novel artemisone–piperazine–tetronamide hybrids (12a–f) were efficiently synthesised from dihydroartemisinin (DHA) and investigated for their in vitro cytotoxicity against some human cancer cells and benign cells. All the targets showed good cytotoxic activity in vitro. Hybrid 12a exhibited much better inhibitory activity against human liver cancer cell line SMMC-7721 (IC₅₀ = 0.03 ± 0.04 μM for 24 h) than the parent DHA (IC₅₀ > 0.7 μM), and two references, vincristine (VCR; IC₅₀ = 0.27 ± 0.03 μM) & cytosine arabinoside (ARA; IC₅₀ = 0.63 ± 0.04 μM). Furthermore, hybrid 12a had low toxicity against human benign liver cell line LO2 (IC₅₀ = 0.70 ± 0.02 μM for 24 h) compared with VCR, ARA, and DHA in vitro. Moreover, the inhibitory activity of hybrid 12a was obviously enhanced when human liver cancer cell line MHCC97H absorbed Fe²⁺in vitro.

Six novel artemisone–piperazine–tetronamide hybrids were efficiently synthesised and investigated for their cytotoxicity against some human cancer cells.     

Intracellular time course, pharmacokinetics, and biodistribution of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to mice

Intracellular concentrations of isoniazid and rifabutin resulting from administration of inhalable microparticles of these drugs to phorbol-differentiated THP-1 cells and the pharmacokinetics and biodistribution of these drugs upon inhalation of microparticles or intravenous administration of free drugs to mice were investigated. In cultured cells, both microparticles and dissolved drugs established peak concentrations of isoniazid (~1.4 and 1.1 μg/10⁶ cells) and rifabutin (~2 μg/ml and ~1.4 μg/10⁶ cells) within 10 min. Microparticles maintained the intracellular concentration of isoniazid for 24 h and rifabutin for 96 h, whereas dissolved drugs did not. The following pharmacokinetic parameters were calculated using WinNonlin from samples obtained after inhalation using an in-house apparatus (figures in parentheses refer to parameters obtained after intravenous administration of an equivalent amount, i.e., 100 μg of either drug, to parallel groups): isoniazid, serum half-life (t_1/2) = 18.63 ± 5.89 h (3.91 ± 1.06 h), maximum concentration in serum (C_max) = 2.37 ± 0.23 μg·ml⁻¹ (3.24 ± 0.57 μg·ml⁻¹), area under the concentration-time curve from 0 to 24 h (AUC_0-24) = 55.34 ± 13.72 μg/ml⁻¹ h⁻¹ (16.64 ± 1.80 μg/ml⁻¹ h⁻¹), and clearance (CL) = 63.90 ± 13.32 ml·h⁻¹ (4.43 ± 1.85 ml·h⁻¹); rifabutin, t_1/2 = 119.49 ± 29.62 h (20.18 ± 4.02 h), C_max = 1.59 ± 0.01 μg·ml⁻¹ (3.47 ± 0.33 μg·ml⁻¹), AUC_0-96 = 109.35 ± 14.78 μg/ml⁻¹ h⁻¹ (90.82 ± 7.46 μg/ml⁻¹ h⁻¹), and CL = 11.68 ± 7.00 ml·h⁻¹ (1.03 ± 0.11 ml·h⁻¹). Drug targeting to the lungs in general and alveolar macrophages in particular was observed. It was concluded that inhaled microparticles can reduce dose frequency and improve the pharmacologic index of the drug combination.     

New triterpenes from Cimicifuga yunnanensis down-regulating the mRNA expression of CD147, MMP-2, and MMP-9

Eleven new 9,19-cycloartane triterpenes (1–9, 11–12) and one undescribed lanostane-type aglycone (10) were identified from the aerial parts of Cimicifuga yunnanensis. The new structures were elucidated by analysis of spectroscopic data. Compounds 3–5, 7–9, and 11, without obvious cytotoxicity at 50 μM, were evaluated for inhibiting the mRNA expressions of atherosclerosis-related factors of CD147 (extracellular matrix metalloproteinase inducer, EMMPRIN), matrix metalloproteinase 2 (MMP-2) and MMP-9 in phorbol-12-myristate-13-acetate (PMA) induced Human monocytic THP-1 cells by using a quantitative real-time PCR method (q-PCR). Among them, aglycones 7 and 8 showed potent activities, whereas all tested glycosides were inactive. Compounds 7 and 8 suppressed the mRNA expression of CD147 in a dose-dependent manner, with an IC₅₀ value of 3.38 ± 0.27 μM and 8.25 ± 0.33 μM, respectively. Besides, 7 dose-related down-regulated the mRNA expression of MMP-2, and MMP-9, having an IC₅₀ value of 6.32 ± 0.31 μM and 11.57 ± 0.23 μM, respectively. Meanwhile, 8 at 10 μM reduced the mRNA expression of MMP-2 and MMP-9 by 35% and 25%, respectively. Significantly, the migration ability of the induced THP-1 cells was potently and dose-dependently inhibited by 7, with an IC₅₀ value of 5.87 ± 0.27 μM.

Eleven new 9,19-cycloartane triterpenes (CTs) (1–9, 11–12) and one undescribed lanostane-type aglycone (10) were identified from Cimicifuga yunnanensis. CTs aglycones 7 and 8 potently down-regulated the mRNA expression of CD147, MMP-2, and MMP-9.