Design and synthesis of rosiglitazone-ferulic acid-nitric oxide donor trihybrids for improving glucose tolerance.

Glucose intolerance is associated with metabolic syndrome and type 2 diabetes mellitus (T2DM) while some new therapeutic drugs, such as rosiglitazone (Rosi), for T2DM can cause severe cardiovascular side effects. Herein we report the synthesis of Rosi-ferulic acid (FA)-nitric oxide (NO) donor trihybrids to improve glucose tolerance and minimize the side effects. In comparison with Rosi, the most active compound 21 exhibited better effects on improving glucose tolerance, which was associated with its NO production, antioxidant and anti-inflammatory activities. Furthermore, 21 displayed relatively high stability in the simulated gastrointestinal environments and human liver microsomes, and released Rosi in plasma. More importantly, 21, unlike Rosi, had little stimulatory effect on the membrane translocation of aquaporin-2 (AQP2) in kidney collecting duct epithelial cells. These, together with a better safety profile, suggest that the trihybrids, like 21, may be promising candidates for intervention of glucose intolerance-related metabolic syndrome and T2DM.

Novel Derivative of Bardoxolone Methyl Improves Safety for the Treatment of Diabetic Nephropathy.

Currently, no effective and safe medicines are available to treat diabetic nephropathy (DN). Bardoxolone methyl (CDDO-Me) has displayed promising anti-DN activity as well as serious side effects in clinical trials, probably because the highly reactive α-cyano-α,ß-unsaturated ketone (CUK) in ring A of CDDO-Me can covalently bind to thiol functionalities in many biomacromolecules. In this study, we designed and synthesized a γ-glutamyl transpeptidase (GGT)-based and CUK-modified derivative of CDDO-Me (2) to address this issue. 2 can be specifically cleaved by GGT, which is highly expressed in the kidney, to liberate CDDO-Me in situ. It should be noted that 2 exhibited anti-DN efficacy comparable to that of CDDO-Me with much less toxicity in cells and db/db mice, suggesting that its safety is better than CDDO-Me. Our findings not only reveal the therapeutic potential of 2 but also provide a strategy to optimize other synthetic molecules or natural products bearing a pharmacophore like CUK to achieve safer pharmaceutical drugs.

Synthesis and evaluation of 2-cyano-3, 12-dioxooleana-1, 9(11)-en-28-oate-13ß, 28-olide as a potent anti-inflammatory agent for intervention of LPS-induced acute lung injury.

The present study was designed to synthesize 2-Cyano-3, 12-dioxooleana-1, 9(11)-en-28-oate-13ß, 28-olide (1), a lactone derivative of oleanolic acid (OA) and evaluate its anti-inflammatory activity. Compound 1 significantly diminished nitric oxide (NO) production and down-regulated the mRNA expression of iNOS, COX-2, IL-6, IL-1ß, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further in vivo studies in murine model of LPS-induced acute lung injury (ALI) showed that 1 possessed more potent protective effects than the well-known anti-inflammatory drug dexamethasone by inhibiting myeloperoxidase (MPO) activity, reducing total cells and neutrophils, and suppressing inflammatory cytokines expression, and thus ameliorating the histopathological conditions of the injured lung tissue. In conclusion, compound 1 could be developed as a promising anti-inflammatory agent for intervention of LPS-induced ALI.

Potent Inhibition of Nitric Oxide-Releasing Bifendate Derivatives against Drug-Resistant K562/A02 Cells in Vitro and in Vivo.

Multidrug resistance is a major obstacle to successful chemotherapy for leukemia. In this study, a series of nitric oxide (NO)-releasing bifendate derivatives (7a-n) were synthesized. Biological evaluation indicated that the most active compound (7a) produced relatively high levels of NO and significantly inhibited the proliferation of drug-resistant K562/A02 cells in vitro and in vivo. In addition, 7a induced the mitochondrial tyrosine nitration and the intracellular accumulation of rhodamine 123 by inhibiting P-gp activity in K562/A02 cells. Furthermore, 7a remarkably down-regulated AKT, NF-κB, and ERK activation and HIF-1α expression in K562/A02 cells, which are associated with the tumor cell proliferation and drug resistance. Notably, the antitumor effects were dramatically attenuated by an NO scavenger or elimination of the NO-releasing capability of 7a, indicating that NO produced by 7a contributed to, at least partly, its cytotoxicity against drug-resistant K562/A02 cells. Overall, 7a may be a potential agent against drug-resistant myelogenous leukemia.

Discovery of a ring-opened derivative of 3-n-butylphthalide bearing NO/H2S-donating moieties as a potential anti-ischemic stroke agent.

To search for novel anti-ischemic stroke agents with higher potency than a known drug 3-n-butylphthalide (NBP), a series of ring-opened derivatives of NBP bearing both nitric oxide (NO) and hydrogen sulfide (H2S)-donating moieties (NO/H2S-NBP) (8a-8o) were designed, synthesized, and biologically evaluated. The most active compound 8d was more potent than NBP and the corresponding H2S-NBP 10 or NO-NBP 13 in inhibition of the ADP-induced platelet aggregation in vitro. In addition, 8d produced moderate levels of NO and H2S, which could be beneficial for improving cardiovascular and cerebral circulation. More importantly, in a rat model of transient focal cerebral ischemia, oral treatment with 8d improved neurobehavioral function, reduced the infarct brain size and brain-water content, and enhanced the levels of brain antioxidant SOD, GSH and GSH-Px but diminished the level of oxidant MDA. These protective effects of 8d against the ischemia/reperfusion (I/R)-related brain damage were greater than that of NBP, suggesting that 8d may be a promising agent for further investigation.

Discovery of New Monocarbonyl Ligustrazine-Curcumin Hybrids for Intervention of Drug-Sensitive and Drug-Resistant Lung Cancer.

The elevation of oxidative stress preferentially in cancer cells by inhibiting thioredoxin reductase (TrxR) and/or enhancing reactive oxygen species (ROS) production has emerged as an effective strategy for selectively targeting cancer cells. In this study, we designed and synthesized 21 ligustrazine-curcumin hybrids (10a-u). Biological evaluation indicated that the most active compound 10d significantly inhibited the proliferation of drug-sensitive (A549, SPC-A-1, LTEP-G-2) and drug-resistant (A549/DDP) lung cancer cells but had little effect on nontumor lung epithelial-like cells (HBE). Furthermore, 10d suppressed the TrxR/Trx system and promoted intracellular ROS accumulation and cancer cell apoptosis. Additionally, 10d inhibited the NF-κB, AKT, and ERK signaling, P-gp-mediated efflux of rhodamine 123, P-gp ATPase activity, and P-gp expression in A549/DDP cells. Finally, 10d repressed the growth of implanted human drug-resistant lung cancer in mice. Together, 10d acts a novel TrxR inhibitor and may be a promising candidate for intervention of lung cancer.

Synthesis and Biological Evaluation of Novel Olean-28,13ß-lactams as Potential Antiprostate Cancer Agents.

γ-Lactam is an important structural motif in a large number of biologically active natural products and synthetic small pharmaceutical molecules. However, there is currently no effective approach to construct γ-lactam ring directly from natural rigid polycyclic amides. Herein, we report a facile methodology for synthesis of a new group of olean-28,13ß-lactams (10a-j) from their corresponding amides, promoted by an easily available reagent 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), through an intramolecular dehydrogenative C-N coupling reaction via a radical ion mechanism. Biological evaluation indicated that the most active lactam 10h displayed potent antiproliferative activity against human cancer cells but 13.84- to 16.92-fold less inhibitory activity on noncancer cells in vitro. In addition, 10h significantly inhibited the growth of implanted prostate cancer in vivo. Furthermore, 10h induced cell cycle arrest and apoptosis and down-regulated the AKT/mTOR signaling in DU-145 cells. Finally, 10h was more stable in rat plasma and human liver microsomes than CDDO-Me and had little hERG channel inhibitory activity. Collectively, 10h may be a potential antiprostate cancer agent for further investigation.

Synthesis of CDDO-amino acid-nitric oxide donor trihybrids as potential antitumor agents against both drug-sensitive and drug-resistant colon cancer.

Seventeen CDDO-amino acid-NO donor trihybrids (4a-q) were designed and synthesized. Biological evaluation indicated that the most active compound 4c produced high levels of NO and inhibited the proliferation of drug-sensitive (HCT-8, IC50 = 0.294 µM) and drug-resistant (HCT-8/5-FU, IC50 = 0.232 µM) colon cancer cells, which were attenuated by an NO scavenger or typical substrate of PepT1. Furthermore, 4c triggered HCT-8 and HCT-8/5-FU cell apoptosis more strongly than CDDO-Me, inhibited the HIF-1α, Stat3, AKT, and ERK signaling, and induced the nitration of P-gp, MRP1, and BCRP proteins in HCT-8/5-FU cells. Finally, 4c had 4.36-5.53-fold less inhibitory activity against nontumor colon epithelial-like cells (CCD841, IC50 = 1.282 µM) in vitro and inhibited the growth of implanted human drug-resistant colon cancers in mice more potently than CDDO-Me. Together, 4c is a novel trihybrid with potent antitumor activity and may be a promising candidate for the treatment of drug-resistant colon cancer.

The protective effect of CDDO-Me on lipopolysaccharide-induced acute lung injury in mice.

CDDO-Me, initiated in a phase II clinical trial, is a potential useful therapeutic agent for cancer and inflammatory dysfunctions, whereas the therapeutic efficacy of CDDO-Me on LPS-induced acute lung injury (ALI) has not been reported as yet. The purpose of the present study was to explore the protective effect of CDDO-Me on LPS-induced ALI in mice and to investigate its possible mechanism. BalB/c mice received CDDO-Me (0.5mg/kg, 2mg/kg) or dexamethasone (5mg/kg) intraperitoneally 1h before LPS stimulation and were sacrificed 6h later. W/D ratio, lung MPO activity, number of total cells and neutrophils, pulmonary histopathology, IL-6, IL-1ß, and TNF-α in the BALF were assessed. Furthermore, we estimated iNOS, IL-6, IL-1ß, and TNF-α mRNA expression and NO production as well as the activation of the three main MAPKs, AkT, IκB-α and p65. Pretreatment with CDDO-Me significantly ameliorated W/D ratio, lung MPO activity, inflammatory cell infiltration, and inflammatory cytokine production in BALF from the in vivo study. Additionally, CDDO-Me had beneficial effects on the intervention for pathogenesis process at molecular, protein and transcriptional levels in vitro. These analytical results provided evidence that CDDO-Me could be a potential therapeutic candidate for treating LPS-induced ALI.

Bifendate-chalcone hybrids: a new class of potential dual inhibitors of P-glycoprotein and breast cancer resistance protein.

We previously described bifendate-chalcone hybrids as potent P-glycoprotein inhibitors. In the present work, we determine whether these compounds could reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance using HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. Results indicated that compounds 8d, 8f, 8g and 8h could significantly enhance mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP drug efflux function. The most active compound 8g exhibited little intrinsic cytotoxicity (IC50>100 µM), and could reverse BCRP-mediated drug resistance independent of decreasing BCRP expression level. Notably, 8g had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. The present findings, together with the previous results, suggest that 8g might be act as dual inhibitors of P-gp and BCRP.

Discovery of alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as potential dual inhibitors of P-glycoprotein and breast cancer resistance protein.

We recently reported alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as novel P-glycoprotein (P-gp, ABCB1) inhibitors. In this study, their ability to reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance was tested in HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. It was observed that compounds 4d, 4h, 4i increased mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP efflux function. Notably, the inhibitory activity of 4i was comparable to that of the classical BCRP inhibitor Ko143 at an equimolar concentration. Interestingly, 4i had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. These results, together with the previous findings, suggest that 4i may be a dual inhibitor of P-gp and BCRP to warrant further investigation.

Synthesis and evaluation of 2-anilinopyrimidines bearing 3-aminopropamides as potential epidermal growth factor receptor inhibitors.

Novel compounds 12a-i were synthesized and biologically evaluated. Several ones exhibited stronger inhibitory activity than gefitinib against EGFR L858R/T790M and antiproliferative effects on H1975 and HCC827 cells. The 3-aminopropamide in compounds like 12h could be converted to the active acrylamide in the presence of arginine. Importantly, 12h showed improved stability relative to compound 1 whose structure is same to 12h excepting an acrylamide moiety. Interestingly, 12i, a NO donating compound of 12h, showed more potent and selective inhibition than 12h on H1975 cells. Significantly, 12i produced high levels of NO in H1975 cells but not in non-tumorous 16HBE cells, and its inhibition was diminished by NO scavenger. Furthermore, 12i dose-dependently produced inhibitory effects on EGFR downstream signaling in H1975 cells.

Tacrine-flurbiprofen hybrids as multifunctional drug candidates for the treatment of Alzheimer's disease.

Five tacrine-flurbiprofen hybrid compounds (3a-e) were synthesized as multi-target-directed compounds for the treatment of Alzheimer's disease. Compared to tacrine, two compounds (3d and 3e) showed better acetylcholinesterase (AChE) inhibitory activity and others (3b-e) better or the same butyrylcholinesterase (BuChE) inhibitory activity. Notably, 3d showed a mixed-type inhibitory action for AChE, indicating a "dual-binding site action" of both toward the catalytic active site (CAS) and the peripheral anionic site (PAS), whereas for BuChE, a competitive inhibitory action was observed. Furthermore, a cell-based assay on amyloid-ß inhibition demonstrated that the selected target compound 3d effectively inhibits the formation of amyloid-ß in vitro.

Nitric oxide donating anilinopyrimidines: synthesis and biological evaluation as EGFR inhibitors.

To search for potent nitric oxide (NO) donating epidermal growth factor receptor (EGFR) inhibitors, a series of phenylsulfonylfuroxan-based anilinopyrimidines 10a-h were synthesized and biologically evaluated. Compounds 10f-h exhibited potent inhibitory activity against EGFR L858R/T790M and were as potent as WZ4002 in inhibition of H1975 cells harboring EGFR L858R/T790M. Additionally, 10h produced high levels of NO in H1975 cells but not in normal human cells, and its antiproliferative activity was diminished by hemoglobin, an NO scavenger. Furthermore, 10h inhibited EGFR activation and downstream signaling in H1975 cells. These results suggest that the strong antiproliferative activity of 10h could be attributed to the synergic effects of high levels of NO production and inhibition of EGFR and downstream signaling in the cancer cells.

NO-donating tacrine derivatives as potential butyrylcholinesterase inhibitors with vasorelaxation activity.

To search for potent anti-Alzheimer's disease (AD) agents with multifunctional effects, 12 NO-donating tacrine-flurbiprofen hybrid compounds (2a-l) were synthesized and biologically evaluated. It was found that all the new target compounds showed selective butyrylcholinesterase (BuChE) inhibitory activity in vitro comparable or higher than tacrine and the tacrine-flurbiprofen hybrid compounds 1a-c, and released moderate amount of NO in vitro. The kinetic study suggests that one of the most active and highest BuChE selective compounds 2d may not only compete with the substrate for the same catalytic active site (CAS) but also interact with a second binding site. Furthermore, 2d and 2l exhibited significant vascular relaxation effect, which is beneficial for the treatment of AD. All the results suggest that 2d and 2l might be promising lead compounds for further research.

Novel hybrids of (phenylsulfonyl)furoxan and anilinopyrimidine as potent and selective epidermal growth factor receptor inhibitors for intervention of non-small-cell lung cancer.

A series of hybrids (12a-k) from (phenylsulfonyl)furoxan and anilinopyrimidine were synthesized and biologically evaluated as epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Compound 12k exhibited strong and selective EGFR L858R/T790M inhibitory activity (IC50 = 0.047 µM) and displayed antiproliferative effects on EGFR mutation NSCLC cell lines HCC827 (del E746_A750) and H1975 (L858R/T790M) with IC50 values of 0.007 and 0.029 µM, respectively. Additionally, 12k released high levels of NO in H1975 cells but not in normal human cells, and its activity was diminished by pretreatment with a NO scavenger. Furthermore, 12k induced apoptosis of H1975 and HCC827 cells more strongly than WZ4002 (1), inhibited EGFR downstream signaling in H1975 cells, and suppressed the nuclear factor-κB activation in H1975 cells, while 1 had no significant effects under the same conditions. Finally, 12k substantially inhibited tumor growth in an H1975 xenograft mouse model. Overall, 12k might be a promising candidate for the treatment of NSCLC.

Design, synthesis and evaluation of tacrine-flurbiprofen-nitrate trihybrids as novel anti-Alzheimer's disease agents.

To search for multifunctional anti-Alzheimer's disease (AD) agents with good safety, the previously synthesized tacrine-flurbiprofen hybrids 1a and 1b were modified into tacrine-flurbiprofen-nitrate trihybrids 3a-h. These compounds displayed comparable or higher cholinesterase inhibitory activity relative to the bivalent hybrids. Compound 3a was the most potent, which released moderate NO, exerted blood vessel relaxative activity, and showed significant Aß inhibitory effects whereas tacrine and flurbiprofen did not exhibit any Aß inhibitory activity at the same dose. In addition, 3a was active in improving memory impairment in vivo. More importantly, the hepatotoxicity study showed that 3a was much safer than tacrine, suggesting it might be a promising anti-AD agent for further investigation.

Hybrid molecule from O2-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid: a glutathione S-transferase π-activated nitric oxide prodrug with selective anti-human hepatocellular carcinoma activity and improved stability.

A series of hybrids from O(2)-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid (OA) were designed, synthesized, and biologically evaluated as novel nitric oxide (NO)-releasing prodrugs that could be activated by glutathione S-transferase π (GSTπ) overexpressed in a number of cancer cells. It was discovered that the most active compound, 21, released high levels of NO selectively in HCC cells but not in the normal cells and exhibited potent antiproliferative activity in vitro as well as remarkable tumor-retarding effects in vivo. Compared with the reported GSTπ-activated prodrugs JS-K and PABA/NO, 21 exhibited remarkably improved stability in the absence of GSTπ. Importantly, the decomposition of 21 occurred in the presence of GSTπ and was much more effective than in glutathione S-transferase α. Additionally, 21 induced apoptosis in HepG2 cells by arresting the cell cycle at the G2/M phase, activating both the mitochondrion-mediated pathway and the MAPK pathway and enhancing the intracellular production of ROS.

Studies on the enantiomers of ZJM-289: synthesis and biological evaluation of antiplatelet, antithrombotic and neuroprotective activities.

ZJM-289 is a potent racemic agent which inhibits both platelet aggregation and thrombosis superior to a known anti-ischemic stroke drug 3-n-butylphthalide (NBP). Herein, the enantiomers of ZJM-289, (S)-ZJM-289 and (R)-ZJM-289, were synthesized and evaluated for their biological activities. It was observed that the two enantiomers appeared to be almost as effective as ZJM-289 in inhibiting platelet aggregation in vitro and thrombus formation in vivo. Moreover, like ZJM-289, its enantiomers could regulate the ratio of thromboxane B(2) (TXB(2)) and 6-keto-prostaglandin F(1α), and enhanced levels of nitric oxide (NO), cAMP and cGMP, suggesting that the anti-platelet and antithrombotic activities of the enantiomers and ZJM-289 are associated with both the arachidonic acid cascade and cGMP-NO signal pathway. Furthermore, it was found that oral administration of the enantiomers and ZJM-289 for three days significantly reduced the infarct size, brain water content and neurological deficit in rats after cerebral ischemia reperfusion. Importantly, the two enantiomers equally improved blood flow in the ischemic stroke model and modulated endothelial function through releasing moderate levels of NO, which might, at least partially, contribute to their neuroprotection. Collectively, the present study demonstrates that the two enantiomers are as potent as ZJM-289 in inhibition of platelet aggregation and thrombosis and in neuroprotection, and (S)-ZJM-289 shows somewhat better effects than (R)-ZJM-289 and ZJM-289 in a few cases. These findings may provide new insights into the development of therapeutic agents like ZJM-289 for the intervention of thrombosis-related ischemic stroke.