Design,synthesis and biological evaluation of novel substituted benzoylguanidine derivatives as potent Na+/H+ exchanger inhibitors

A novel series of substituted benzoylguanidine derivatives were designed and synthesized as potent NHE1 inhibitors. Most compounds can significantly inhibit NHE1-mediated platelet swelling in a concentration-dependent manner, among which compound 5f (IC₅₀ = 3.60 nM) and 5l (IC₅₀ = 4.48 nM) are 18 and 14 times respectively more potent than cariporide (IC₅₀ = 65.0 nM). Furthermore, when tested in vivo and in vitro, compound 5f showed superior cardioprotective effects against SD rat myocardial ischemic-reperfusion injury over cariporide, representing a promising lead compound for further exploration.     

Synthesis,molecular modeling and SAR study of novel pyrazolo[5,1-f][1,6]naphthyridines as CB2 receptor antagonists/inverse agonists

Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affinity at CB₁ and CB₂ receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology, the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel compounds feature a p-tolyl (8a–i) or a 2,4-dichlorophenyl (8j) motif at the C₅-position of the tricyclic pyrazolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C₂-position includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, compound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the highest CB₂ receptor affinity (K_i = 33 nM) and a high degree of selectivity (K_iCB₁/K_iCB₂ = 173:1), whereas a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, K_i = 53 nM) and the myrtanyl derivative 8j (K_i = 67 nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were determined, showing antagonist/inverse agonist properties for such compounds. Docking studies conducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) disclosed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB₂ inverse agonists.     

Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of K_d using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 μM–46 nM) to JNK3. Among them, compound 16f exhibited potent activities (K_d = 46 nM). Especially, 16f was also found to present a potent cell protective effect (IC₅₀ = 1.09 μM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.     

Design,synthesis and anticholinesterase activity of a novel series of 1-benzyl-4-((6-alkoxy-3-oxobenzofuran-2(3H)-ylidene) methyl) pyridinium derivatives

A novel series of benzofuranone-ylidene-methyl benzylpyridinium derivatives (6a–u) were synthesized as acetylcholinesterase inhibitors. The anticholinesterase activity of synthesized compounds was measured using colorimetric Ellman’s method. It was revealed that some synthesized compounds exhibited high anticholinesterase activity, among them compound 6b was the most active compound (IC₅₀ = 10 ± 6.87 nM).     

Fragment-based discovery of novel and selective mPGES-1 inhibitors Part 1: Identification of sulfonamido-1,2,3-triazole-4,5-dicarboxylic acid

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase that catalyzes the conversion of prostaglandin PGH₂ to PGE₂ and represents a novel target for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human mPGES-1 (IC₅₀ of 1.1 μM) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a cell-free assay. In addition, the activity of compound 6f was again tested at 10 μM concentration in presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent, therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more potent mPGES-1 inhibitors.     

Syntheses of coumarin–tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase,Aβ aggregation,and β-secretase

Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer’s disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine–coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (K_i = 16.7 nM) against human AChE and about 2-fold lower potency (K_i = 16.1 nM) against BChE than tacrine (K_i = 35.7 nM for AChE, K_i = 8.7 nM for BChE), respectively. In addition, some of the tacrine–coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine–coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.     

Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic anhydrase isoforms hCA IX and hCA XII

Three series of novel heterocyclic compounds (3a–3g, 4a–4g and 5a–5g) containing benzenesulfonamide moiety and incorporating a 1,2,4-triazole ring, have been synthesized and investigated as inhibitors against four isomers of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozymes hCA I and II, compounds of two series (3a–3g and 4a–4g) showed K_i values in the range of 84–868 nM and 5.6–390 nM, respectively whereas compounds of series 5a–5g were found to be poor inhibitors (K_i values exceeding 10,000 nM in some cases). Against hCA IX and XII, all the tested compounds exhibited excellent to moderate inhibitory potential with K_i values in the range of 2.8–431 nM and 1.3–63 nM, respectively. Compounds 3d, 3f and 4f exhibited excellent inhibitory potential against all of the four isozymes hCA I, II, IX and XII, even better than the standard drug acetazolamide (AZA) whereas compound of the series 5a–5g were comparatively less potent but more selective towards hCA IX and XII.     

Synthesis and structure–activity relationship of 3β-(4-alkylthio, -methylsulfinyl,and -methylsulfonylphenyl)tropane and 3β-(4-alkylthiophenyl)nortropane derivatives for monoamine transporters

Early studies led to the identification of 3β-(4-methoxyphenyl)tropane-2β-carboxylic acid methyl ester (5) with high affinity at the DAT (IC₅₀ = 6.5 nM) and 5-HTT (K_i = 4.3 nM), while having much less affinity at the NET (K_i = 1110 nM). In the present study, we replaced the 4′-methoxy group of the 3β-phenyl ring with a bioisosteric 4′-methylthio group to give 7a. We also synthesized a number of 3β-(4-alkylthiophenyl)tropanes 7b–e, 3β-(4-methylsulfinylphenyl) and 3β-(4-methylsulfonylphenyl)tropane analogues 7f–h as well as the 3β-(4-alkylthiophenyl)nortropane derivatives 8–11 to further characterize the structure–activity relationship of this type of compound for binding at monoamine transporters. With exception of the 4′-methylsulfonyl analogue 7h, all the tested compounds possessed high binding affinities at the 5-HTT. The K_i values ranged from 0.19 nM to 49 nM. The 3β-(4-methylthiophenyl)tropane 7a and its N-(3-fluoropropyl) analogue 9a and N-allyl analogue 10a are the most selective compounds for the 5-HTT over the NET (NET/5-HTT = 314–364) in the series. However, none of the compounds showed selectivity similar to 5 for both the DAT and 5-HTT relative to the NET. This study provided useful SAR information for rational design of potent and selective monoamine transporter inhibitors.     

Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.     

Discovery of new [1,4]dioxino[2,3-f]quinazoline-based inhibitors of EGFR including the T790M/L858R mutant

A novel series of 2,3-dihydro-[1,4]dioxino[2,3-f]quinazoline derivatives were designed, synthesized and evaluated as reversible and noncovalent epidermal growth factor receptor (EGFR) inhibitors. Most of the compounds exhibited good potency against EGFR^wt and some showed moderate to excellent potency against EGFR^T790M/L858R mutant. The half-maximal inhibitory concentration (IC₅₀) values of twenty-one compounds against EGFR^wt were less than 50 nM, and those of six compounds were less than 10 nM. The IC₅₀ values of eleven compounds against EGFR^T790M/L858R were less than 100 nM. Among these, compound b1 displayed the most potent inhibitory activity against EGFR^wt (IC₅₀ = 2.0 nM) and EGFR^T790M/L858R (IC₅₀ = 6.9 nM). Compounds with excellent inhibitory activities against EGFR^wt and EGFR^T790M/L858R kinase inhibitory activities showed good antiproliferative activities against H358 and A549 cells. Docking study was performed to position compound b1 into the EGFR active pocket to determine the probable binding conformation.     

Toward discovery of mutant EGFR inhibitors; Design,synthesis and in vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives

A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type (WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type (EGFR WT) with IC₅₀ values in the low nanomolar range (<0.495–9.05 nM) and displayed more potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed. Compounds 4d, 6f, 7e showed significant inhibition (IC₅₀ = 1.76–2.38 μM) in these mutant lines and significant Her2 enzyme inhibition (IC₅₀ = 19.2–40.6 nM) compared to lapatinib (60.1 nM). The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested, compounds 6f and 7e showed about eight and three folds respectively greater potency than gefitinib. Our structure–activity relationships (SAR) studies suggested that presence of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for developing inhibitors against wild type and mutant EGFR.     

Synthesis,biological evaluation and molecular modeling of 1,3,4-thiadiazol-2-amide derivatives as novel antitubulin agents

A series of 1,3,4-thiadiazol-2-amide derivatives (6a–w) were designed and synthesized as potential inhibitors of tubulin polymerization and as anticancer agents. The in vitro anticancer activities of these compounds were evaluated against three cancer cell lines by the MTT method. Among all the designed compounds, compound 6f exhibited the most potent anticancer activity against A549, MCF-7 and HepG2 cancer cell lines with IC₅₀ values of 0.03 μM, 0.06 μM and 0.05 μM, respectively. Compound 6f also exhibited significant tubulin polymerization inhibitory activity (IC₅₀ = 1.73 μM), which was superior to the positive control. The obtained results, along with a 3D-QSAR study and molecular docking that were used for investigating the probable binding mode, could provide an important basis for further optimization of compound 6f as a novel anticancer agent.     

4-Functionalized 1,3-diarylpyrazoles bearing 6-aminosulfonylbenzothiazole moiety as potent inhibitors of carbonic anhydrase isoforms hCA I,II, IX and XII

A series of 24 novel heterocyclic compounds—functionalized at position 4 with aldehyde (5a–5f), carboxylic acid (6a–6f), nitrile (7a–7f) and oxime (8a–8f) functional groups—bearing 6-aminosulfonybenzothiazole moiety at position 1 of pyrazole has been synthesized and investigated for the inhibition of four isoforms of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozyme hCA I, compounds 6a–6f showed medium-weak inhibitory potential with K_i values in the range of 157–690 nM with 6a showing better potential than the standard drug acetazolamide (AZA). Against hCA II, all the compounds showed excellent to moderate inhibition with K_i values of compounds 5a, 5d, 5f, 6a–6f, 8d and 8f lower than 12 nM (K_i of AZA). Against hCA IX, all the compounds showed moderate inhibition with the exception of 6e which showed nearly 9 fold a better profile compared to AZA, whereas against hCA XII, four compounds 6e, 7a, 7b and 7d showed K_i in the same order as that of AZA. Carboxylic acid 6e was found to be an excellent inhibitor of both hCA IX and XII, with K_i values of 2.8 nM and 5.5 nM, respectively.     

Synthesis and anti-proliferative activity of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs against human tumor cell lines

Based on previous SAR studies on N-benzylindole and barbituric acid hybrid molecules, we have synthesized a series of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs (3a–i) and evaluated them for their in vitro growth inhibition and cytotoxicity against a panel of 60 human tumor cell lines. Compounds 3c, 3d, 3f and 3g were identified as highly potent anti-proliferative compounds against ovarian, renal and breast cancer cell lines with GI₅₀ values in low the nanomolar range. The 4-methoxy-N-benzyl analog (3d) was the most active compound with GI₅₀ values of 20 nM and 40 nM against OVCAR-5 ovarian cancer cells and MDA-MB-468 breast cancer cells, respectively. Two other analogs, 3c (the 4-methyl-N-benzyl analog) and 3g (the 4-fluoro-N-benzyl analog) exhibited equimolar potency against MDA-MB-468 cells GI₅₀ = 30 nM). Analog 3f (the 4-chloro-N-benzyl analog) exhibited a GI₅₀ value of 40 nM against renal cancer cell line A498. These results suggest that aromatic substituted N-benzylindole dimethylbarbituric acid hybrids may have potential for development as clinical candidates to treat a variety of solid tumors.     

Synthesis and evaluation of new N6-substituted adenosine-5′-N-methylcarboxamides as A3 adenosine receptor agonists

A number of N⁶-substituted adenosine-5′-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a–4f and 5a–5f, showed modest receptor affinity for the A₃AR with K_i values in the low to mid μM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N⁶-position (in compounds 4g and 5g) significantly improved the affinity with K_i values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalised linker was introduced. The N⁶-phenyl series, compounds 7a–7d, demonstrated low to mid nanomolar receptor affinities (K_i = 2.3–45.0 nM), with 7b displaying 109-fold selectivity for the A₃AR (vs A₁). The N⁶-benzyl series 9a–9c also proved to be potent and selective A₃AR agonists and the longer chain length linker 13 was tolerated at the A₃AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A₃AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For example, compound 9c reduced cell death by 68.0 ± 3.6%.     

Bioisosterism of urea-based GCPII inhibitors: Synthesis and structure–activity relationship studies

We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure–activity relationship studies of the P1′ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with K_i values below 20 nM. Among them, compound 32d (K_i = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1′ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d.     

The discovery of potent glycine transporter type-2 inhibitors: Design and synthesis of phenoxymethylbenzamide derivatives

We describe the discovery of phenoxymethylbenzamide derivatives as a novel class of glycine transporter type-2 (GlyT-2) inhibitors. We found hit compound 1 (human GlyT-2, IC₅₀ = 4040 nM) in our library and converted its 1-(1-(naphthalen-2-ylmethyl)piperidin-4-yl)pyrrolidin-3-yl group to an 1-(N,N-dimethylaminopropyl)piperidyl group and its tert-butyl group to a trifluoromethyl group to obtain N-(1-(3-(dimethylamino)propyl)piperidin-4-yl)-4-((4-(trifluoromethyl)phenoxy)methyl)benzamide (20). Compound 20 showed good inhibitory activity against human GlyT-2 (IC₅₀ = 15.3 nM) and exhibited anti-allodynia effects in a mouse neuropathic pain model.     

8-Substituted 1,3-dimethyltetrahydropyrazino[2,1-f]purinediones: Water-soluble adenosine receptor antagonists and monoamine oxidase B inhibitors

Multitarget approaches, i.e., addressing two or more targets simultaneously with a therapeutic agent, are hypothesized to offer additive therapeutic benefit for the treatment of neurodegenerative diseases. Validated targets for the treatment of Parkinson’s disease are, among others, the A_2A adenosine receptor (AR) and the enzyme monoamine oxidase B (MAO-B). Additional blockade of brain A₁ ARs may also be beneficial. We recently described 8-benzyl-substituted tetrahydropyrazino[2,1-f]purinediones as a new lead structure for the development of such multi-target drugs. We have now designed a new series of tetrahydropyrazino[2,1-f]purinediones to extensively explore their structure–activity-relationships. Several compounds blocked human and rat A₁ and A_2AARs at similar concentrations representing dual A₁/A_2A antagonists with high selectivity versus the other AR subtypes. Among the best dual A₁/A_2AAR antagonists were 8-(3-(4-chlorophenyl)propyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (41, K_i human A₁: 65.5 nM, A_2A: 230 nM; K_i rat A₁: 352 nM, A_2A: 316 nM) and 1,3-dimethyl-8-((2-(thiophen-2-yl)thiazol-4-yl)methyl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (57, K_i human A₁: 642 nM, A_2A: 203 nM; K_i rat A₁: 166 nM, A_2A: 121 nM). Compound 57 was found to be well water-soluble (0.7 mg/mL) at a physiological pH value of 7.4. One of the new compounds showed triple-target inhibition: (R)-1,3-dimethyl-8-(2,1,3,4-tetrahydronaphthalen-1-yl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (49) was about equipotent at A₁ and A_2AARs and at MAO-B (K_i human A₁: 393 nM, human A_2A: 595 nM, IC₅₀ human MAO-B: 210 nM) thus allowing future in vivo explorations of the intended multi-target approach.     

Cyanoguanidine-based lactam derivatives as a novel class of orally bioavailable factor Xa inhibitors

The N,N′-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K_i = 6.5 nM, EC_2xPT = 32 μM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand–protein interactions are discussed.     

Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC₅₀ = 11 nM; PC-3 antiproliferative IC₅₀ = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC₅₀ = 10 nM; A2780 antiproliferative IC₅₀ = 7 nM) in complex with the NAMPT protein was also determined.