N-Haloacetyl-amino-acid amides as active-site-directed inhibitors of papain and cathepsin B

A series of N-haloacetyl-amino-acid amides were synthesized and tested as models of cysteine-protease inhibitors. They irreversibly inactivated papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Apparent second-order rate constants of inactivation ranging from 65 to 16 700 M⁻¹ s⁻¹ were observed. Reactivity against papain, as compared to glutathione, was increased 16 400-fold for N-bromoacetyl-leucine isopentylamide and 25 700-fold for the corresponding iodoacetyl derivative; these increases are probably due to proximity effects. No inhibition of trypsin, chymotrypsin and porcine pancreatic elastase was observed. Haloacetamides represent an interesting class of easily synthesized, efficient, irreversible inhibitors of cysteine proteases, which have low non-specific alkylating properties.     

Peptidyl and azapeptidyl methylketones as substrate analog inhibitors of papain and cathepsin B

Peptidyl methylketones containing Phe, Tyr, Tyr(I), Tyr(I₂), Leu and Ile in P₂ were synthesized and tested as substrate analog reversible inhibitors of papain and bovine spleen cathepsin B. The most effective cathepsin B inhibitor contained Tyr(I₂) and displayed an inhibition constant of 4.7 μM at pH 6.8 and 25°C, while Leu or Ile gave practically inert analogs. Replacement of the amino acids in P₂ with the analogous α-azaamino acids, as well as the glycine in P₁ with α-azaglycine, led to complete loss of inhibiting activity. Introducing alkoxy substituents at the methyl adjacent to the ketone group generally resulted in more effective inhibitors, with inhibition constants in the micromolar range for both papain and cathepsin B.     

Natural biflavones as novel inhibitors of cathepsin B and K

Cathepsin B and K, two important members in lysosomal proteases, involve in many serious human diseases, such as tumor and osteoporosis. In order to find their novel inhibitors, we performed the inhibition assays of cathepsin B and cathepsin K in vitro, randomly screened compounds from plants, and found six biflavones, named AMF1-5 and HIF, can potently inhibit cathepsin B and cathepsin K, especially AMF4 and HIF with IC(50) of 0.62 and 0.58 microM against cathepsin B. They are novel inhibitors for cathepsin B and K. Inhibition and flexible docking studies indicated that these biflavones are reversible inhibitors of cathepsin B, and their binding patterns and interaction modes with cathepsin B made them more specific to cathepsin B endopeptidase.     

Synthesis and evaluation of mansonone F derivatives as topoisomerase inhibitors

A series of mansonone F (MF) derivatives were designed and synthesized. These compounds were found to be strong inhibitors for topoisomerases, with much more significant inhibition for topoisomerase II rather than topoisomerase I. The best inhibitor showed 20 times stronger anti-topoisomerase II activity than a positive control Etoposide. The cytotoxic activity of these MF derivatives was evaluated against human cancer cell lines CNE-2 and Glc-82, which showed that these compounds were potent antitumor agents. The structure-activity relationships (SARs) study revealed that o-quinone group and pyran ring are important for their cytotoxic activity.     

QSAR analysis of 1,3-diaryl-4,5,6,7-tetrahydro-2H-isoindole derivatives as selective COX-2 inhibitors

Quantitative structure-activity relationship (QSAR) analysis was performed on a series of 1,3-diaryl-4,5,6,7-tetrahydro-2H-isoindole for their cyclooxygenase-2 (COX-2) inhibition. QSAR investigations were based on Hansch's extra thermodynamic multi-parameter approach and receptor surface analysis (RSA). QSAR investigations reveal that steric and electrostatic interactions are primarily responsible for COX-2 enzyme-ligand interaction. QSAR model derived from Hansch analysis demonstrated that COX-2 inhibitory activity is correlated with sum of atomic polarizability (Apol), number of hydrogen-bond donor groups (HBD), energy of the highest occupied molecular orbital (HOMO), desolvation free energy for water (F(H(2)O)) and fraction of areas of molecular shadow in the XY and ZX planes over area of enclosing rectangle (Sxyf and Sxzf) with r ranges 0.870-0.904. The best model was obtained from RSA model having r = 0.940 with good predictive ability (predicted compounds in training set and test set within +/- 1.0 unit of pIC(50)) and can be used in designing better selective COX-2 inhibitors among the congeners in future.     

Acetylenic and allenic derivatives of 2-(5-methoxy-1-methylindolyl) alkylamines: Synthesis and evaluation as selective inhibitors of the monoamine oxidases A and B

We report the synthesis and in vitro studies as MAO-A and MAO-B inhibitors of a new series of 2-(5-methoxy-1-methylindolyl)alkylamines, including 2-(5-methoxy-1-methylindolyl)amine 9 and its N-methyl derivative 10, 1-[2-(5-methoxy-1-methylindolyl)]ethylamine 1 and its N-methyl derivative 2 and 3-[2-(5-methoxy-1-methylindolyl)propylamine 11 and its N-methyl derivative 12, so as some of the N-(2-propynyl), N-(2,3-butadienyl) and N-(2-butynyl) derivatives of 1, 2, 11 and 12 (compounds 3–8 and 13–18). All compounds were found to inhibit both MAO-A and MAO-B. Comparison with a previous series of 2-(5-methoxy-1-methylindolyl)methylamine derivatives (A1–A9) shows that in the new series, the inhibitory potency towards MAO-A generally decreases, whereas the inhibitory potency towards MAO-B generally increases, resulting in a loss of the selectivity for MAO-A seen in the reference compounds A1–A9. Structure-activity relationships are discussed.     

Identification of 1-isopropylsulfonyl-2-amine benzimidazoles as a new class of inhibitors of hepatitis B virus

A series of 1-isopropylsulfonyl-2-amine benzimidazole derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. In general, these derivatives are potent HBV inhibitors (IC(50)<4 microM) with high selectivity indices (SIs>40). Compounds 5b-e, g, j, and 9a were among the most prominent compounds, with IC(50)s of 0.70-2.0 microM and SIs of 41-274. The potent anti-HBV activity and safety profiles of the most promising compounds 5d and j (IC(50)s=0.70 microM, SIs>120) demonstrate the potential of this series of benzimidazoles for the development of new anti-HBV drugs.     

Design,synthesis and structure-activity relationships of a series of 9-substituted adenine derivatives as selective phosphodiesterase type-4 inhibitors

Adenine derivatives substituted in position 9 have been demonstrated to have potent cyclic nucleotide phosphodiesterase (PDE) inhibition properties with high selectivity toward PDE-4. Starting from our initial lead compound 9-(2-fluorobenzyl)-N(6)-methyl-2-trifluoromethyladenine (4, NCS613), we designed and synthesized a new series of 9-substituted derivatives for developing structure-activity relationship studies. This new series of derivatives showed increased potencies and better selectivity profiles. Structural modifications were achieved in parallel on three different positions of the adenine ring, and led to the following observations: (i) introduction of a lipophilic substituent such as trifluoromethyl, n-propyl group or iodine in the C-2 position is favourable for both the PDE-4 inhibitory activity and the selectivity towards other isoenzymes; (ii) functionalization of the N9 benzyl group with a 2-methoxy substituent led to remarkably more active compounds; (iii) replacement of the N(6)-methylamino moiety by other amino groups is detrimental to the activity. Among all derivatives prepared, the 9-(2-methoxybenzyl)-N(6)-methyl-2-trifluoromethyladenine (9r), 9-(2-methoxybenzyl)-N(6)-methyl-2-n-propyladenine (9s), and the 2-iodo-9-(2-methoxybenzyl)-N(6)-methyladenine (13b) were found to be the most potent inhibitors within this series (PDE-4-IC(50)=1.4, 7.0, and 0.096 nM, respectively). Compared to our reference compound 4, which showed an IC(50) of 42 nM, the derivative 13b was found 450-fold more potent. Moreover, 2-iodo-9-(2-methoxybenzyl)-N(6)-methyladenine (13b) and 9-(2-methoxybenzyl)-N(6)-methyl-2-trifluoromethyladenine (9r), were at least 50000-150000 times more selective for the PDE-4 than for the other PDE families. Additionally, these new derivatives showed improved efficiency in inhibiting the TNFalpha release from mononuclear cells from healthy subjects (e.g. adenines 7l, 9s and 13b). Thus, compounds 7l, 9r, 9s and 13b are among the most potent and selective PDE-4 inhibitors reported so far and represent very promising pharmacological tools for a better understanding of the signal transduction involving cyclic AMP within the cell: this pathway is implicated in the physiology and the pathophysiology of inflammation, asthma and autoimmune disorders.     

Discovery and kinetic evaluation of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as inhibitors of cathepsin B

Cathepsin B is a lysosomal cysteine protease that has various physiological and pathophysiological functions. We present here the discovery of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as inhibitors of cathepsin B, starting from screening of a library of variously 2,4,6-trisubstituted 1,3,5-triazines and 1,3,5-triazin-2(1H)-ones on three different human cathepsins. The synthesis and enzymatic evaluation of a focused library of new 1,3,5-triazin-2(1H)-ones is also described. The detailed kinetics analyses have shown that these compounds can act as reversible, partial mixed-type inhibitors of cathepsin B, with K(i) and K(i)' values in the low micromolar range. The inhibitory activities of selected compounds were also assessed against two related cysteine proteases, cathepsin H and cathepsin L, to estimate their selectivity; these compounds have a selective profile for catB and catL over catH.     

Synthesis and antibacterial activity of novel 15-membered macrolide derivatives: 4″-Carbamate, 11,12-cyclic carbonate-4″-carbamate and 11,4″-di-O-arylcarbamoyl analogs of azithromycin

4″-Carbamate, 11,12-cyclic carbonate-4″-carbamate and 11,4″-di-O-arylcarbamoyl analogs of azithromycin were designed, synthesized and evaluated. The 4″-carbamate analogs retained excellent activity against erythromycin-susceptible Staphylococcus pneumoniae and showed improved activity against erythromycin-resistant Staphylococcus pneumoniae. Compared with 4″-carbamate analogs, 11,12-cyclic carbonate-4″-carbamate analogs exhibited improved activity against erythromycin-resistant Staphylococcus pneumoniae encoded by the mef gene or the erm and mef genes, and 11,4″-di-O-arylalkylcarbamoyl analogs showed greatly improved activity (0.25–0.5 μg/mL) against erythromycin-resistant Staphylococcus pneumoniae encoded by the erm gene. Among them, the novel series of 11,4″-di-O-arylalkylcarbamoyl analogs 7a–k exhibited potent and balanced activity against susceptible and resistant bacteria. In particular, compounds 7f and 7k were the most effective against susceptible bacteria and resistant bacteria encoded by the erm gene or the mef gene.     

Benzophenone-based derivatives: a novel series of potent and selective dual inhibitors of acetylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation

The leading mechanistic theory of Alzheimer’s disease (AD) is the “amyloid hypothesis” which states that the accumulation of the amyloid β protein (Aβ), and its subsequent aggregation into plaques, is responsible for the initiation of a cascade of events resulting in neurodegeneration and dementia. The anti-amyloid disease-modifying approach, based on the decrease in the production of Aβ, gained thus a paramount importance. The aim of this study was the design and synthesis of a new series of acetylcholinesterase inhibitors (AChEIs) endowed with anti-Aβ aggregating capability. These dual binding inhibitors, being able to interact both with the peripheral anionic site (PAS) of AChE and the catalytic subsite, proved to be able to inhibit the AChE-induced Aβ aggregation. Thus, starting from the lead compound 1, an AChEI composed by a benzophenone scaffold and a N,N′-methylbenzylamino group, a substantial modification aimed at targeting the PAS was performed. To this aim, different amino-terminal side chains were incorporated into this main framework, in order to mimic the diethylmethylammonium alkyl moiety of the pure PAS ligand propidium. The synthesized compounds proved to effectively and selectively inhibit AChE. Moreover, compounds 16a-c and 18a,b, with a propoxy and a hexyloxy tether respectively, showed a good activity against the AChE-induced Aβ aggregation. In particular, molecular modeling studies confirmed that compounds carrying the diethylaminopropoxy and the diethylaminohexyloxy side chains (compounds 16a and 19a, respectively) could suitably contact the PAS pocket of the enzyme.     

Synthesis and bio-activity evaluation of tetraphenyl(phenylamino) methylene bisphosphonates as antioxidant agents and as potent inhibitors of osteoclasts in vitro

A new series of tetraphenyl bisphosphonates have been elegantly synthesized by one-pot method and were characterized by elemental analysis, FTIR, ¹H, ¹³C, ³¹P NMR, mass spectra and evaluated for their in vitro antibone resorptive activity by inhibiting growth of osteoclasts. Two bisphosphonates 3g and 3f showed marked inhibition ratio (8 μM and 10 μM) and emerged as lead compounds. All compounds were tested for their antioxidant (DPPH scavenging, reducing power and inhibition of lipid peroxidation). They exhibited potent in vitro antioxidant activity dose-dependently.     

Synthesis of amide and urea derivatives of benzothiazole as Raf-1 inhibitor

A series of amide and urea derivatives of benzothiazole have been synthesized and evaluated for their antiproliferative profile in human SK-Hep-1 (liver), MDA-MB-231 (breast), and NUGC-3 (gastric) cell lines. Among them, compounds 1-2, 16-18, 23, and 25-26 had potent to moderate inhibitory activities. Further these compounds were investigated for their ability to inhibit Raf-1 activity.     

Synthesis and biological activities of triazole derivatives as inhibitors of InhA and antituberculosis agents

InhA, the enoyl reductase from the mycobacterial type II fatty acid biosynthesis pathway, is a target for the development of novel drugs against tuberculosis. We exploited copper-catalyzed [3+2] cycloaddition between alkynes and different azides to afford 1,4-disubstituted triazole or α-ketotriazole derivatives. Several compounds bearing a lipophilic chain mimicking the substrate were able to inhibit InhA. Among them, 1-dodecyl-4-phenethyl-1H-1,2,3-triazole displayed a minimum inhibitory concentration inferior to 2 μg/mL against Mycobacterium tuberculosis H37Rv.     

Acyclic nucleoside thiophosphonates as potent inhibitors of HIV and HBV replication

9-[2-(Thiophosphonomethoxy)ethyl]adenine 3 and (R)-9-[2-(Thiophosphonomethoxy)propyl]adenine 4 were synthesized as the first thiophosphonate nucleosides bearing a sulfur atom at the α-position of the acyclic nucleoside phosphonates PMEA and PMPA. Thiophosphonates S-PMEA 3 and S-PMPA 4 were evaluated for in vitro activity against HIV-1 (subtypes A to G), HIV-2 and HBV-infected cells, and found to exhibit potent antiretroviral activity. We showed that their diphosphate forms S-PMEApp 5 and S-PMPApp 6 are readily incorporated by wild-type (WT) HIV-1 RT into DNA and act as DNA chain terminators. Compounds 3 and 4 were evaluated for in vitro activity against a broad panel of DNA and RNA viruses and displayed beside HIV a moderate activity against herpes simplex virus and vaccinia viruses. In order to measure enzymatic stabilities of the target derivatives 3 and 4, kinetic data and decomposition pathways were studied at 37 °C in several media.     

Synthesis and biological evaluation of derivatives of 4-deoxypodophyllotoxin as antitumor agents

In an attempt to generate compounds with superior bioactivity and reduced toxicity, a series of derivatives of deoxypodophyllotoxin were synthesized by reacting 4′-demethyl-4-deoxypodophyllotoxin with substituted piperazines or their amino acid amides. The cytotoxic activity of these compounds against three human cancer cell lines was evaluated. We found that p-nitrophenylpiperazine substitution (Compound 8b) led to an increase in the potency of the compound. Compound 8b exhibited the most potent cytotoxicity against A-549, HeLa and SiHa cells (IC₅₀ values were 0.102, 0.180 and 0.0195 μM, respectively). In addition, flow cytometric analysis showed that 8b induced cell cycle arrest in the G1 phase accompanied by apoptosis in A-549 cells.     

Design of benzimidazole- and benzoxazole-2-thione derivatives as inhibitors of bacterial hyaluronan lyase

Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC₅₀ values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch.     

Synthesis, biological activity and HPLC validation of 1,2,3,4-tetrahydroacridine derivatives as acetylcholinesterase inhibitors

The synthesis and biochemical evaluation of new hybrids of tacrine (THA) and 4-fluorobenzoic acid (4-FBA) possessing activity towards acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition are presented. The compounds of interest were obtained from the reaction of activated 4-FBA and diamino derivatives of 1,2,3,4-tetrahydroacridine. The compounds C6-2KW/HCl, C6-4KW/HCl and C6-3KW/HCl have four-fold higher antiacetylcholinesterase activity than THA. All of the acquired compounds present higher selectivity towards AChE than THA and lower selectivity towards BuChE. In addition, a rapid, selective and stability-indicating HPLC method was developed and validated for the determination of C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl. THA and 4-FBA were found to be the main impurities. Chromatographic separation was achieved isocratically on a Waters Symmetry C18 150 × 3.9 mm, 4 μm column with a mobile phase of acetonitrile/buffer (17 mM sodium dodecyl sulphate and 8.3 mM sodium dihydrogen phosphate, 50:50 v/v) (overall pH 4). A 1.5 ml/min flow rate and a 247 nm wavelength were chosen for this method. C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl were subjected to acidic and basic hydrolysis, chemical oxidation, thermal exposition at 60 °C and intense UV light. The limits of detection (LOD) and quantification (LOQ) were less than 2 μg/ml and 6 μg/ml for C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl, 0.04 μg/ml and 0.12 μg/ml for THA, 0.42 μg/ml and 1.41 μg/ml for 4-FBA, respectively.     

Synthesis and evaluation of aliphatic-chain hydroxamates capped with osthole derivatives as histone deacetylase inhibitors

Our previous studies have demonstrated that osthole, a Chinese herbal compound, could be incorporated into the hydroxycinnamide scaffold of LBH-589, a potent HDAC inhibitor, as an effective hydrophobic cap; the resulting compounds showed significant potency against several HDAC isoforms. Here, we presented a series of osthole derivatives fused with the aliphatic-hydroxamate core of suberoylanilide hydroxamic acid (SAHA), a clinically-approved HDAC inhibitor. Several compounds showed potent activity against nuclear HDACs. Further assays against individual HDAC isoforms revealed that some compounds showed not only SAHA-like activity towards HDAC1, -4 and -6, they inhibited HDAC8 by log difference than SAHA and thus exhibited a broader HDAC inhibition spectrum. Among them, compound 6g showed potent antiproliferative effect on several human cancer cell lines.     

Syntheses and characterization of novel oxoisoaporphine derivatives as dual inhibitors for cholinesterases and amyloid beta aggregation

A series of 3-substituted (5c-5f, 6c-6f) and 4-substituted (10a-10g) oxoisoaporphine derivatives were synthesized. It was found that all these synthetic compounds had IC₅₀ values at micro or nano molar range for cholinesterase inhibition, and most of them could inhibit amyloid β (Aβ) self-induced aggregation with inhibition ratio from 31.8% to 57.6%. The structure-activity relationship studies revealed that the derivatives with higher selectivity on AChE also showed better inhibition on Aβ self-induced aggregation. The results from cell toxicity study indicated that most quaternary methiodide salts had higher IC₅₀ values than the corresponding non-quaternary compounds. This study provided potentially important information for further development of oxoisoaporphine derivatives as lead compounds for the treatment of Alzheimer’s disease.