Discovery of 2,4-diarylaminopyrimidine derivatives bearing sulfonamide moiety as novel FAK inhibitors.

Two series of 2,4-diarylaminopyrimidine derivatives containing sulfonamide moiety were designed and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most compounds significantly inhibited the enzymatic activities of FAK, and the best compound was 7b (IC50 = 0.27 nM). A majority of aminoethyl sulfonamide derivatives could effectively inhibit the proliferation of human cancer cell lines (HCT116, A549, MDA-MB-231 and Hela) expressing high levels of FAK. Particularly, compounds 7b, 7c, and 7o exhibited more significant efficacy against all of four cancer cell lines within concentrations of 1.5 µM. Furthermore, these three compounds displayed higher selectivity of cancer cells over normal cells (SI value > 14), compared to the positive control TAE226 (SI value = 1.63). Interestingly, introduction of dithiocarbamate moiety to the aminoethyl sulfonamide derivatives can indeed improve the antiproliferative activities against A549 cells. Especially, compound 8d demonstrated most significant cytotoxicity activity against A549 cells with an IC50 value of 0.08 µM, which is 20-fold superior to parent compound 7k. Additionally, compound 7b, which display the best anti-FAK potency, can inhibit the clone formation and migration of HCT-116 cells, and cause cell cycle arrest at G2/M phase, inducing apoptosis by promoting ROS production. Overall, these results suggest that 7b is a valuable FAK inhibitor that deserves further optimization to improve its druggability.

Synthesis of novel sulfonamide derivatives containing pyridin-3-ylmethyl 4-(benzoyl)piperazine-1-carbodithioate moiety as potent PKM2 activators.

Pyruvate kinase M2 isoform (PKM2) plays a key role in cancer progression through both metabolic and non-metabolic functions, thus it is recognized as a potential target for cancer diagnosis and treatment. In this study, we discovered a sulfonamide-dithiocarbamate compound 8a as a novel PKM2 activator from a random screening of an in-house compound library. Then, a series of lead compound 8a analogs were designed and synthesized for screening as potent PKM2 activators. Among them, compound 8b (AC50 = 0.136 µM) and 8k (AC50 = 0.056 µM) showed higher PKM2 activation activities than positive control NZT (AC50 = 0.228 µM), and they (IC50 < 1 µM) exhibited more significant anti-proliferative activities against human tumor cell lines than NZT (IC50 > 10 µM). Especially, compound 8k inhibited the proliferation of multiple cancer cells, but showed little toxicity on normal cells. In addition, we found that compound 8k inhibit the colony formation of MCF7 cells. Western blot analysis demonstrated that 8k could reduce PKM2 nuclear localization and block the downstream signaling pathway of PKM2, resulting in suppression of tumor cell proliferation. Overall, compound 8k may be a promising candidate for further mechanistic investigation of PKM2 and cancer therapy.

A thienopyridine, CB-20, exerts diuretic activity by inhibiting urea transporters.

Urea transporters (UTs) are transmembrane proteins selectively permeable to urea and play an important role in urine concentration. UT-knockout mice exhibit the urea-selective urine-concentrating defect, without affecting electrolyte balance, suggesting that UT-B inhibitors have the potential to be developed as novel diuretics. In this study, we characterized a novel compound 5-ethyl-2-methyl-3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate (CB-20) with UT inhibitory activity as novel diuretics with excellent pharmacological properties. This compound was discovered based on high-throughput virtual screening combined with the erythrocyte osmotic lysis assay. Selectivity of UT inhibitors was assayed using transwell chambers. Diuretic activity of the compound was examined in rats and mice using metabolic cages. Pharmacokinetic parameters were detected in rats using LC-MS/MS. Molecular docking was employed to predict the potential binding modes for the CB-20 with human UT-B. This compound dose-dependently inhibited UT-facilitated urea transport with IC50 values at low micromolar levels. It exhibited nearly equal inhibitory activity on both UT-A1 and UT-B. After subcutaneous administration of CB-20, the animals showed polyuria, without electrolyte imbalance and abnormal metabolism. CB-20 possessed a good absorption and rapid clearance in rat plasma. Administration of CB-20 for 5 days did not cause significant morphological abnormality in kidney or liver tissues of rats. Molecular docking showed that CB-20 was positioned near several residues in human UT-B, including Leu364, Val367, and so on. This study provides proof of evidence for the prominent diuretic activity of CB-20 by specifically inhibiting UTs. CB-20 or thienopyridine analogs may be developed as novel diuretics.

Antinociceptive Effect of Spirocyclopiperazinium Salt Compound DXL-A-24 and the Underlying Mechanism.

The antinociceptive effects of spirocyclopiperazinium salt compound DXL-A-24 on neuropathic pain and chemical-stimulated pain were investigated in this study. After the administration of DXL-A-24, the paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT) were increased in rats suffering from neuropathic pain (chronic constriction injury, CCI) on days 1, 3, 5, 7 and 14 after surgery, and pain responses were inhibited in mice stimulated with chemicals (formalin or acetic acid). In the analysis of antinociceptive targets, the effect of DXL-A-24 was blocked by a peripheral nicotinic acetylcholine receptor (nAChR) antagonist (hexamethonium, Hex) or α7 nAChR antagonist (methyllycaconitine, MLA) in the formalin test. Meanwhile, the effect of DXL-A-24 was also blocked by a peripheral muscarinic acetylcholine receptor (mAChR) antagonist (atropine methylnitrate, Amn) or M4 mAChR antagonist (tropicamide, TRO). The antinociceptive signalling pathway was explored using molecular biology methods in ipsilateral dorsal root ganglions (DRGs) of CCI rats after the administration of DXL-A-24 for 7 days. Western blot analyses showed that the increased levels of phosphorylation of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and cAMP response element-binding protein (CREB) were eliminated, and the qRT-PCR assay showed that the increase in the expression of Tumor necrosis factor alpha (TNF-α) mRNA was reduced. Meanwhile, immunofluorescence staining revealed that the increase in calcitonin gene related peptide (CGRP) expression was inhibited by the administration of DXL-A-24, and the effect was blocked by MLA or TRO. In conclusion, DXL-A-24 exerts significant antinociceptive effects on neuropathic pain and chemical-stimulated pain. The antinociceptive effect of DXL-A-24 is probably attributed to the activation of peripheral α7 nAChR and M4 mAChR, the subsequent inhibition of the CaMKIIα/CREB signalling pathway, and finally the inhibition of TNF-α and CGRP expression.

Transition Metal-Free Intermolecular C(sp2)-H Direct Amination of Furanones via a Redox Pathway.

A direct C(sp2)-H amination of 2-furanones under metal-free conditions was realized. This unprecedented intermolecular C-H to C-N conversion provides rapid access to 4-amino-furanone derivatives and novel aza-heterocycle fused furanone skeletons. A redox mechanism based on a double-Michael-addition intermediate INT2 is proposed and detected by spectrometry.

Anti-inflammatory effect and mechanism of the spirocyclopiperazinium salt compound LXM-15 in rats and mice.

OBJECTIVE: This study aimed to investigate the anti-inflammatory effects of a novel spirocyclopiperazinium salt compound LXM-15, and explore the underlying mechanisms. METHODS: Xylene-induced mouse ear oedema and carrageenan-induced rat paw oedema tests were used to evaluate the anti-inflammatory effects of LXM-15. The protein levels of TNF-α, IL-6, phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were detected by ELISA or Western blot analysis. Additionally, receptor blocking test was performed to explore the possible target. RESULTS: Intragastric administration with LXM-15 (2, 1, 0.5 mg/kg in mice, and 6, 3, 1.5 mg/kg in rats) produced distinct anti-inflammatory effects in vivo, the highest inhibition percentage was 60 and 52%, respectively (P < 0.01). Following treatment with LXM-15 (6 mg/kg, i.g.), the levels of TNF-α and IL-6 in the rats paws were attenuated by 40 and 41%; and the phosphorylation of JAK2 and STAT3 was restrained by 35 and 45%, respectively (P < 0.01). All effects of LXM-15 were blocked by pretreatment with methyllycaconitine citrate or tropicamide. CONCLUSION: This study provides the first report that the spirocyclopiperazinium salt compound LXM-15 displays considerable anti-inflammatory effects. The underlying mechanism may be through activating the peripheral α7 nicotinic acetylcholine receptor and M4 muscarinic acetylcholine receptor, leading to the inhibition of the JAK2/STAT3 signalling pathway, eventually resulting in the reduction of TNF-α and IL-6.

Synthesis and biological evaluation of curcumin derivatives modified with α-amino boronic acid as proteasome inhibitors.

Curcumin is a well-known pharmacophore and some of its derivatives are shown to target 20S proteasome recently. In this report, we designed and synthesized two series of curcumin derivatives modified with different α-amino boronic acids as potent proteasome inhibitors. The synthesized compounds were evaluated for their cytotoxic activities against HCT116 cells, and the results showed that all of them exhibited excellent cell growth inhibitory activity comparing with curcumin, with the IC50 values varying from 0.17 µM to 1.63 µM. Compound II-2F with free boronic acid was assayed for its proteasome inhibitory activity and the results indicated that II-2F exhibited more potent inhibitory activity against ChT-L with high subunit selectivity than any other reported curcumin derivatives.

Antinociception of the spirocyclopiperazinium salt compound LXM-15 via activating α7 nAChR and M4 mAChR and inhibiting CaMKIIα/cAMP/CREB/CGRP signalling pathway in mice.

The aim of this study was to investigate the analgesic effect of the spirocyclopiperazinium salt compound LXM-15 by intragastric administration in thermal and chemical pain models and further to elucidate the possible molecular mechanisms. The results showed that LXM-15 exerted significant antinociception in hot-plate test, formalin test and acetic acid writhing test. Western blot analysis showed that LXM-15 significantly reduced the upregulation of phosphorylation of calcium/calmodulin -dependent protein kinase IIα (CaMKIIα) and cAMP response element-binding protein (CREB), and further decreased the elevation of calcitonin gene related peptide (CGRP) in the dorsal root ganglion (DRG) and spinal cord in mice. ELISA analysis showed the level of cAMP in the spinal cord was decreased by LXM-15. All effects of LXM-15 could be blocked by methyllycaconitine citrate (MLA, a selective α7 nicotinic receptor antagonist) or tropicamide (TRO, a selective M4 muscarinic receptor antagonist). This study first reported that intragastric administration of LXM-15 produced significant analgesic effect, which may be related to the activation of α7 nicotinic acetylcholine receptor and M4 muscarine acetylcholine receptor, and thereby inhibiting CaMKIIα/cAMP/CREB/CGRP signalling pathway.

Synthesis and biological activity of peptide proline-boronic acids as proteasome inhibitors.

On the basis of the application of proline-boronic acid as pharmacophore in the kinase inhibitors and our previous research results, using proline-boronic acid as warhead, two series of peptide proline-boronic acids, dipeptide proline-boronic acids (I) and tripeptide proline-boronic acids (II), were designed and synthesized. All the synthesized compounds were first evaluated for their biological activity against MGC803 cell, and then, the best compound II-7 was selected to test its anti-tumor spectrum on six human tumor cell lines and proteasome inhibition against three subunits. The results indicated that series II have much better biological activities than series I. The compound II-7 exhibited not only excellent biological activities with IC50 values of nM level in both cell and proteasome models, but also much better subunit selectivity. Thus, proline-boronic acid as warhead is reasonable in the design of proteasome inhibitors.

Regioselective Ir(III)-catalyzed C-H alkynylation directed by 7-azaindoles.

Herein we report a novel iridium(iii)-catalyzed ortho-mono-alkynylation of 7-azaindoles under mild conditions. This approach provides a general and straightforward access to form novel 7-azaindole derivatives with ample substrate scope and broad group tolerance.

Pharmacokinetic-pharmacodynamic modeling of the antitumor effect of TM208 and EGFR-TKI resistance in human breast cancer xenograft mice.

AIM: The novel anticancer compound TM208 is an EGFR tyrosine kinase inhibitor (EGFR-TKI). Since the development of resistance to EGFR-TKIs is a major challenge in their clinical usage, we investigated the profiles of resistance following continuous treatment with TM208 in human breast cancer xenograft mice, and identified the relationship between the tumor pEGFR levels and tumor growth inhibition. METHODS: Female BALB/c nude mice were implanted with human breast cancer MCF-7 cells, and the xenograft mice received TM208 (50 or 150 mg·kg(-1)·d(-1), ig) or vehicle for 18 d. The pharmacokinetics (PK) and pharmacodynamics (PD) of TM208 were evaluated. RESULTS: The PK properties of TM208 were described by a one-compartment model with first-order absorption kinetics. Our study showed the inhibitory effects of TM208 on tumor pEGFR levels gradually reached a maximum effect, after which it became weaker over time, which was characterized by a combined tolerance/indirect response PD model with an estimated EC50 (55.9 µg/L), as well as three parameters ('a' of 27.2%, 'b' of 2730%, 'c' of 0.58 h(-1)) denoting the maximum, extent and rate of resistance, respectively. The relationship between the tumor pEGFR levels and tumor growth inhibition was characterized by a combined logistic tumor growth/transit compartment model with estimated parameters associated with tumor growth characteristics kng (0.282 day(-1)), drug potency kTM208 (0.0499 cm(3)/day) and the kinetics of tumor cell death k1 (0.141 day(-1)), which provided insight into drug mechanisms and behaviors. CONCLUSION: The proposed PK/PD model provides a better understanding of the pharmacological properties of TM208 in the treatment of breast cancer. Furthermore, simulation based on a tolerance model allows prediction of the occurrence of resistance.

LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells.

In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr288 and subsequently impaired p53 phosphorylation at Ser315 which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy.

Design, synthesis and biological evaluation of pyridazino[3,4,5-de]quinazolin-3(2H)-one as a new class of PARP-1 inhibitors.

A series of pyridazino[3,4,5-de]quinazolin-3(2H)-one derivatives were designed and synthesized as PARP-1 inhibitors. Most of the synthesized compounds showed good inhibitory activities of PARP-1 and four of them achieved at the IC50 values ranging from 0.0914 µM to 0.244 µM. Two compounds, 1a and 1b, were further tested for their neuroprotective effect in the PC12 cell model injured by H2O2 and both of them exhibited excellent activities.

Asymmetric Michael addition reactions of nitroalkanes to 2-furanones catalyzed by bifunctional thiourea catalysts.

The first bifunctional thiourea catalyzed asymmetric Michael addition reactions of nitroalkanes to 2-furanones are described. The highly functionalized γ-lactones with two or three consecutive stereogenic carbons were obtained in high yields (up to 99%), high diastereoselectivities (up to >20 : 1 dr) and enantioselectivities (up to >99% ee).

New pyridin-3-ylmethyl carbamodithioic esters activate pyruvate kinase M2 and potential anticancer lead compounds.

Pyruvate kinase M2 (PKM2) is a key protein responsible for cancer's Warburg effect. Activation of PKM2 may alter aberrant metabolism in cancer cells, which suggests PKM2 as a tumor selective therapeutic target. In this paper, the lead compound 8 was first discovered as a new kind of PKM2 activator from a random screening of an in-house compound library. Then, a series of lead compound 8 analogs were designed, synthesized and evaluated for their activation of PKM2 and anticancer activities. 7-Azaindole analog 32 was identified as the most potent PKM2 activator. Compounds with potent enzyme activity also exhibited selective anti-proliferation activity on cancer cell lines HCT116, Hela and H1299 compared with non-tumor cell line BEAS-2B. The structure-activity relationships of these compounds were supported by molecular docking results. Preliminary pharmacological studies also showed that compound 32 arrests the cell cycle at the G2/M phase in HCT116 cell line.

CovalentDock Cloud: a web server for automated covalent docking.

Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/.

Thienoquinolins exert diuresis by strongly inhibiting UT-A urea transporters.

Urea transporters (UT) play an important role in the urine concentration mechanism by mediating intrarenal urea recycling, suggesting that UT inhibitors could have therapeutic use as a novel class of diuretic. Recently, we found a thienoquinolin UT inhibitor, PU-14, that exhibited diuretic activity. The purpose of this study was to identify more potent UT inhibitors that strongly inhibit UT-A isoforms in the inner medullary collecting duct (IMCD). Efficient thienoquinolin UT inhibitors were identified by structure-activity relationship analysis. Urea transport inhibition activity was assayed in perfused rat terminal IMCDs. Diuretic activity of the compound was determined in rats and mice using metabolic cages. The results show that the compound PU-48 exhibited potent UT-A inhibition activity. The inhibition was 69.5% with an IC50 of 0.32 µM. PU-48 significantly inhibited urea transport in perfused rat terminal IMCDs. PU-48 caused significant diuresis in UT-B null mice, which indicates that UT-A is the target of PU-48. The diuresis caused by PU-48 did not change blood Na(+), K(+), or Cl(-) levels or nonurea solute excretion in rats and mice. No toxicity was detected in cells or animals treated with PU-48. The results indicate that thienoquinolin UT inhibitors induce a diuresis by inhibiting UT-A in the IMCD. This suggests that they may have the potential to be developed as a novel class of diuretics with fewer side effects than classical diuretics.

Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208.

AIM: To investigate the effects of a novel dithiocarbamate derivative TM208 on human breast cancer cells as well as the pharmacokinetic characteristics of TM208 in human breast cancer xenograft mice. METHODS: Human breast cancer MCF-7 and MDA-MB-231 cells were treated with TM208 or a positive control drug tamoxifen. Cell proliferation was examined using SRB and colony formation assays. Cell apoptosis was analyzed with Annexin V-FITC/PI staining assay. Protein expression was examined with Western blot, ELISA and immunohistochemical analyses. MCF-7 breast cancer xenograft nude mice were orally administered TM208 (50 or 150 mg·kg(-1)·d(-1)) or tamoxifen (50 mg·kg(-1)·d(-1)) for 18 d. On d 19, the tumors were collected for analyses. Blood samples were collected from the mice treated with the high dose of TM208, and plasma concentrations of TM208 were measured using LC-MS/MS. RESULTS: Treatment of MCF-7 and MDA-MB-231 cells with TM208 dose-dependently inhibited the cell proliferation and colony formation in vitro (the IC50 values were 36.38 ± 3.77 and 18.13 ± 0.76 µmol/L, respectively). TM208 (20-150 µmol/L) dose-dependently induced apoptosis of both the breast cancer cells in vitro. In MCF-7 breast cancer xenograft nude mice, TM208 administration dose-dependently reduced the tumor growth, but did not result in the accumulation of TM208 or weight loss. TM208 dose-dependently inhibited the phosphorylation of EGFR and ERK1/2 in both the breast cancer cells in vitro as well as in the MCF-7 xenograft tumor. CONCLUSION: Inhibition of EGFR autophosphorylation plays an important role in the anticancer effect of TM208 against human breast cancer.

A convenient four-component one-pot synthesis of 2-amino-1,3,4-thiadiazoles in water.

A novel four-component one-pot approach for the synthesis of 2-amino-1,3,4-thiadiazoles from primary amines, carbon disulfide, hydrazine, and acyl chlorides has been developed. A series of 5-substituted-2-amino-1,3,4-thiadiazoles were synthesized in medium-to-good yields utilizing this newly developed method.

CovalentDock: automated covalent docking with parameterized covalent linkage energy estimation and molecular geometry constraints.

Covalent linkage formation is a very important mechanism for many covalent drugs to work. However, partly due to the limitations of proper computational tools for covalent docking, most covalent drugs are not discovered systematically. In this article, we present a new covalent docking package, the CovalentDock, built on the top of the source code of Autodock. We developed an empirical model of free energy change estimation for covalent linkage formation, which is compatible with existing scoring functions used in docking, while handling the molecular geometry constrains of the covalent linkage with special atom types and directional grid maps. Integrated preparation scripts are also written for the automation of the whole covalent docking workflow. The result tested on existing crystal structures with covalent linkage shows that CovalentDock can reproduce the native covalent complexes with significant improved accuracy when compared with the default covalent docking method in Autodock. Experiments also suggest that CovalentDock is capable of covalent virtual screening with satisfactory enrichment performance. In addition, the investigation on the results also shows that the chirality and target selectivity along with the molecular geometry constrains are well preserved by CovalentDock, showing great capability of this method in the application for covalent drug discovery.