Effect of spirocyclopiperazinium salt compound LXM-15 on spinal nerve injury in rats.

BACKGROUND: Currently available therapies for neuropathic pain show limited efficacy. This study aimed to investigate the anti-nociceptive effect of the spirocyclopiperazinium salt compound LXM-15 in spinal nerve ligation (SNL) rats and to explore the potential mechanisms. METHODS: Mechanical allodynia and thermal hyperalgesia tests were used to evaluate the effects of LXM-15 in SNL rats. The expression of CaMKIIα, CREB, JAK2, STAT3, c-fos and TNF-α was detected by western blotting, ELISA or qRT-PCR analysis. Receptor blocking test was performed to explore possible target. RESULTS: Administration of LXM-15 (1, 0.5, 0.25 mg/kg, i.g.) dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in rats subjected to SNL (p < 0.01, p < 0.05), and the effects were completely blocked by peripheral α7 nicotinic or M4 muscarinic receptor antagonist (p > 0.05). LXM-15 significantly decreased the overexpression of phosphorylated CaMKIIα, CREB, JAK2 and STAT3 proteins and the mRNA levels of TNF-α and c-fos (p < 0.01, p < 05). All of the effects could be blocked by α7 or M4 receptor antagonist. Furthermore, LXM-15 reduced the protein expression of TNF-α and c-fos (p < 0.01, p < 0.05). No significant acute toxicity or abnormal hepatorenal function was observed. CONCLUSIONS: This is the first study to report that LXM-15 exerts significant anti-nociceptive effect on SNL rats. This effect may occur by activating peripheral α7 nicotinic and M4 muscarinic receptors, further inhibiting the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and finally inhibiting the expression of TNF-α and c-fos. SIGNIFICANCE: Existing treatments for neuropathic pain show limited efficacy with severe adverse reactions. This paper is the first to report that LXM-15, a new spirocyclopiperazinium salt compound, exerts a significant anti-nociception in SNL rats without obvious toxicity. The underlying mechanisms include activating peripheral α7 nicotinic and M4 muscarinic receptors, then inhibiting the signalling pathways of CaMKIIα/CREB and JAK2/STAT3 and the expressions of TNF-α and c-fos. This study sheds new light on the development of novel analgesic drugs with fewer side effects.

Anti-arthritic Effect of the Spirocyclopiperazinium Salt Compound LXM-15 in Rats and Its Underlying Mechanism.

In this study, we aimed to evaluate the effects of the spirocyclopiperazinium salt compound LXM-15 on rheumatoid arthritis induced by complete Freund's adjuvant (CFA) in rats and investigate the underlying mechanism. The results showed that LXM-15 significantly inhibited the paw edema and ankle swelling, and alleviated the mechanical allodynia and thermal hyperalgesia responses in the CFA rats. The histopathological results revealed that LXM-15 ameliorated the infiltration of inflammatory cells and joint destruction. The micro-CT scan showed that LXM-15 alleviated bone erosion and increased BMD in the ankle joints of the CFA rats. Western blot analyses showed that LXM-15 significantly reduced the upregulation of phospho-JAK2, phospho-STAT3, phospho-IκBα, and phospho-NF-κBp65, and the overexpression of BDNF in the dorsal root ganglions. ELISA result showed that the protein level of TNF-α in the paw tissue was decreased upon LXM-15 treatment. RT-PCR analysis showed that the mRNA expression levels of c-fos and BDNF were reduced in the dorsal root ganglions by LXM-15 treatment. The LXM-15-mediated anti-arthritic effects were abolished by treatment with hexamethonium (a peripheral nicotinic receptor antagonist), atropine methylnitrate (a peripheral muscarinic receptor antagonist), methyllycaconitine citrate (a selective α7 nicotinic receptor antagonist), and tropicamide (a selective M4 muscarinic receptor antagonist). Collectively, our results demonstrate that LXM-15 exerts anti-arthritic effects in CFA rats. The underlying mechanism may be related to the activation of the peripheral α7 nicotinic receptor and M4 muscarinic receptor by LXM-15, further suppressing the activation of the JAK2/STAT3 and IκBα/NF-κBp65 signaling pathways and, eventually, inhibiting the expression levels of TNF-α, BDNF, and c-fos.

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.

Urea-containing peptide boronic acids as potent proteasome inhibitors.

A novel class of urea-containing peptide boronic acids as proteasome inhibitors was designed by introducing a urea scaffold to replace an amido bond. Compounds were synthesized and their antitumor activities were evaluated. After two rounds of optimizations, the compound I-14 was found to be a potent proteasome inhibitor. Compared with Bortezomib, I-14 showed higher potency against the chymotrypsin-like activity of human 20S proteasome (IC50 < 1 pM), similar potency against four different cancer cell lines (IC50 < 10 nM), and better pharmacokinetic profile. Furthermore, I-14 significantly inhibited tumor growth in Bel7404 mouse xenograft model. The excellent proteasome inhibition by I-14 was rationalized through docking and molecular dynamics studies.

Proteasome Inhibitor YSY01A Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant Human Ovarian Cancer Cells.

Cisplatin is one of the most common drugs used for treatment of solid tumors such as ovarian cancer. Unfortunately, the development of resistance against this cytotoxic agent limits its clinical use. Here we report that YSY01A, a novel proteasome inhibitor, is capable of suppressing survival of cisplatin-resistant ovarian cancer cells by inducing apoptosis. And YSY01A treatment enhances the cytotoxicity of cisplatin in drug-resistant ovarian cancer cells. Specifically, YSY01A abrogates regulatory proteins important for cell proliferation and anti-apoptosis including NF-κB p65 and STAT3, resulting in down-regulation of Bcl-2. A dramatic increase in cisplatin uptake was also observed by inductively coupled plasma-mass spectrometry following exposure to YSY01A. Taken together, YSY01A serves as a potential candidate for further development as anticancer therapeutics targeting the proteasome.

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.

Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer agents: Synthesis, structure-activity relationship analysis and biological evaluation.

A series of new analogs based on the structure of lead compound 10 were designed, synthesized and evaluated for their in vitro anti-cancer activities against four selected human cancer cell lines (HL-60, Bel-7402, SK-BR-3 and MDA-MB-468). Several synthesized compounds exhibited improved anti-cancer activities comparing with lead compound 10. Among them, 1,3,4-oxadiazole analogs 17o showed highest bioactivity with IC50 values of 1.23, 0.58 and 4.29 µM against Bel-7402, SK-BR-3 and MDA-MB-468 cells, respectively. It is noteworthy that 17o has potent anti-proliferation activity toward a panel of cancer cells with relatively less cytotoxicity to nonmalignant cells. The further mechanistic study showed that it induced apoptosis and cell cycle arrest through disrupting spindle assembly in mitotic progression, indicating these synthesized dithiocarbamates represented a novel series of anti-cancer compounds targeting mitosis.

Anticancer Effect of a Novel Proteasome Inhibitor, YSY01A, via G2/M Arrest in PC-3M Cells in vitro and in vivo.

YSY01A is a new tripeptideboronic acid and an analog of PS341. However, YSY01A's antitumor effects and mechanism have not yet been elucidated. This study demonstrates that YSY01A inhibited proteasome activity by combining with the chymotrypsin-like (CT-L) site (ß5i/ß5), the post-glutamyl peptide hydrolase (PGPH) site (ß1i/ß1) and the trypsin-like (T-L) site (ß2i/ß2) in special fluorgonic substrates and proteasome probe tests. We explored the anticancer effect using methyl thiazolyltetrazolium (MTT) or sulforhodamine B (SRB), and PC-3M cells were sensitive to YSY01A among the four cancer cell types tested. The YSY01A antiproliferative effect was stronger than that of PS341. In vivo, YSY01A (1.25, 2.25, and 3.25 mg/kg) inhibited PC-3M cell xenograft tumor growth, and the tumor volume inhibition rate was approximately 40% to 60%. YSY01A arrested PC-3M cells in the G2/M phase of the cell cycle by flow cytometry (FCM). Many proteins related to the cell cycle were analyzed using western blot, and YSY01A was shown to increase p21, p27, cyclinB1, P-cdc2 (tyr15) and wee1 protein expression in both cells and tumor tissue in a concentration-dependent manner. YSY01A, a proteasome inhibitor, exerts anticancer effects on PC-3M cells in vitro and in vivo. The mechanism of the YSY01A-mediated antitumor effect is that the cell cycle is arrested at the G2/M stage. This study suggests that YSY01A may be a novel therapeutic agent for prostate cancer.

Discovery and optimization of novel dual dithiocarbamates as potent anticancer agents.

A series of dual dithiocarbamates were synthesized and evaluated for their in-vitro anticancer activities on human non-small cell lung cancer cell line H460. Nine compounds exhibited significant antiproliferative activities with IC50 less than 1 µM. Among them, compound 14m showed the highest inhibitory activity against H460 cell and inhibited the growth of nine types of tumor cells with IC50 values less than 1 µM. It also achieved IC50 of 54 nM and 23 nM against HepG2 and MCF-7 cell lines, respectively. Preliminary structure-activity relationship study indicated that: a) when the methyl group (region A) is substituted with benzene rings, ortho substitution on the benzene ring is favored for activity; b) substitution with heterocyclic structures at region A exhibited greater impact on the anti-tumor activity of compounds, in which pyridine ring, thiazole ring, coumarin and benzo[b]thiophene are favored and quinoline ring is the most favored; c) substitution with different amines (region B) also showed marked effect on the activity of compounds and dimethylamine and morpholine are preferred to other tested amines.

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.

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.

IC-4, a new irreversible EGFR inhibitor, exhibits prominent anti-tumor and anti-angiogenesis activities.

Accumulating evidence suggested that the irreversible tyrosine kinase inhibitors (TKIs) have potential to override the acquired resistance to target-based therapies. Herein, we reported IC-4 as a novel irreversible TKI for epidermal growth factor receptor (EGFR). IC-4 potentially suppressed proliferation, induced apoptosis and a G2/M cell cycle arrest in breast cancer cells, correlating with inhibition of EGF-induced EGFR activation, but independent of DNA damage. In addition, IC-4 exhibited anti-angiogenetic activities both in vitro and in vivo. It suppressed cell viability and proliferation induced by various growth factors in human umbilical vein endothelial cells (HUVECs). IC-4 also inhibited HUVECs migration and tube formation. In transgenic zebrafish embryo model, IC-4 was shown to suppress formation of intersegmental vessel and development of subintestinal vessels. Taken together, these results demonstrated that IC-4 is a new irreversible EGFR-TKI, exhibiting potent anti-breast cancer and anti-angiogenetic effects.

An efficient three-component, one-pot synthesis of 2-alkylthio-4-amino-5-cyano-6-aryl(alkyl)pyrimidines in water.

A convenient and practical method for the synthesis of 2-alkylthio-4-amino-5-cyano-6-aryl(alkyl)pyrimidines has been developed via a three-component, one-pot reaction from aldehydes, malononitrile and S-alkylisothiouronium salts in water at room temperature. A series of polysubstituted pyrimidines were prepared by this method in moderate to excellent yields. In addition, two kinds of pyrimidine-fused heterocyclic derivatives with potential pharmacological activity were constructed from our 2-alkylthio-4-amino-5-cyano-6-arylpyrimidines.

Novel cathepsin B-sensitive paclitaxel conjugate: Higher water solubility, better efficacy and lower toxicity.

In order to realize the targeted delivery of paclitaxel (PTX) to tumor through an environment-sensitive mechanism, increase its solubility in water and reformulate without toxic excipients, a novel PTX conjugate, PEG-VC-PABC-PTX was designed and synthesized in this study, using p-aminobenzylcarbonyl (PABC), a spacer, and valine-citrulline (VC), a substrate of cathepsin B (C(B)), to link polyethylene glycol (PEG) and PTX. Pegylated PTX (PEG-PTX) which was synthesized and Taxol formulation were prepared as controls. The conjugates were purified and characterized by melting points, (1)H-NMR, ESI-MS or MALDI-TOF-MS. The two conjugates were similar in particle size, water solubility and their effects on MCF-7 cell line in vitro, and both of them induced no obvious toxicity in vivo. The release of PTX from PEG-PTX was faster due to its ester bond, while PEG-VC-PABC-PTX was proved to be C(B)-sensitive in terms of PTX release and its effect on cell cycle. Additionally, PEG-VC-PABC-PTX exhibited significant effects of antitumor, anti-angiogenesis and anti-proliferation in vivo, while the control conjugate was almost inefficacious at the same in vivo test. On the other hand, PTX conjugates demonstrated a thousand-time or more improvement in water solubility compared to PTX, suggesting a very easy way in the preparation and use of its injection. Without involvement of Cremophore EL and ethanol, the PTX conjugate will guarantee less adverse effects as frequently reported for Taxol formulation. Taxol formulation had a higher cytoxicity in vitro than PEG-VC-PABC-PTX likely because of toxic additives. Importantly, the C(B)-sensitive conjugate indicated a similar in vivo efficacy with the Taxol control, but much lower in vivo toxicity at the same doses evidenced by body weight, animal status, liver toxicity and blood count. Moreover, at the tolerant dose, this novel conjugate exhibited significantly better antitumor effect than that of Taxol formulation. In general, the PEG-VC-PABC-PTX conjugate designed in this study demonstrated significant advantages in terms of high water solubility, no toxic surfactant or organic solvent, tumor environment-sensitivity and high therapeutic index.

Novel EGFR inhibitors prepared by combination of dithiocarbamic acid esters and 4-anilinoquinazolines.

On the basis of combination strategy, a novel series of EGFR inhibitors were designed and synthesized by combination of dithiocarbamic acid esters and 4-anilinoquinazolines. The effect of the synthesized compounds on cell proliferation was evaluated by MTT assay in three human cancer cell lines: MDA-MB-468, SK-BR-3 and HCT-116. Two compounds (11d and 11f) were found more potent against all three cell lines and five compounds (11a, 11d-11g) were found more potent against both MDA-MB-468 and SK-BR-3 than Lapatinib. SAR studies revealed that the substituents on C6 and C7 positions of quinazoline, the amine component of dithiocarbamate moiety and the linker greatly affected the activity. This work provides a promising new strategy for the preparation of potent tyrosine kinase inhibitors.

Synthesis and structure-analgesic activity relationships of a novel series of monospirocyclopiperazinium salts (MSPZ).

A series of monospirocyclopiperazinium salts were designed and synthesized to search for a peripherally-acting analgesic drug with low side effects. Extensive SAR studies revealed that a suitable NR(2)R(3) was critical for the analgesic activity, which might be beneficial to expose the cationic nitrogen to bind to the receptor, and possibly interact with the receptor via π-π interaction. Introduction of substituting group on the N(4)-phenyl ring could improve the activity, and the best position was the 4-position. Compound 14n showed more potent analgesic activity (63%, 20 µM/kg, sc) and holds promise for development as a mechanically new analgesic drug.

Silicon powder: the first nonmetal elemental catalyst for aminobromination of olefins with TsNH(2) and NBS.

Silicon powder was found, for the first time, to be an efficient alternative to transition metal catalysts for aminobromination of alpha,beta-unsaturated carbonyl compounds and simple olefins with p-toluenesulfonamide (4-TsNH(2)) and NBS, affording the aminobrominated products in high yields and regio- and stereoselectivity. The high reactivity of electron-rich substrates reveals that the reaction has the electrophilic addition feature.

Copper powder-catalyzed regio- and stereoselective aminobromination of alpha,beta-unsaturated ketones with TsNH2 and NBS as nitrogen and halogen sources.

The regio- and stereoselective aminobromination of alpha,beta-unsaturated ketones catalyzed by copper powder has been established with 4-TsNH(2) and NBS as the nitrogen/bromine sources, respectively. This method provides an easy access for preparation of vicinal aminohalo derivatives in the presence of 1 mol % catalyst. Electron-rich alpha,beta-unsaturated ketones afforded the corresponding aminobrominated products in excellent yields (up to 99.8%), revealing that the addition has an electrophilic feature.

Effect of TM208 on QGY-7703 xenograft tumor growth.

A newly synthesized dithiocarbamate derivative, 4-methylpiperazine-1-carbodithioc-acid-3-cyano-3,3-diphenylpropyl ester hydrochloride (TM208), has demonstrated anticancer effects with low toxicity in earlier studies; however, the mechanism has yet to be identified. We explored antitumor effects of TM208 and the possible mechanisms by which it inhibited the growth of human hepatocellular carcinoma cell line QGY-7703 xenograft tumors. Cell proliferation was evaluated with the sulforhodamine B assay in vitro. The results suggested that TM208 had slightly antiproliferative activity on QGY-7703 cells. The antitumor effect of TM208 was assessed in nude mice xenografted with QGY-7703 tumors. We found that TM208 significantly inhibited tumor growth but did not cause loss of body weight or leukocytopenia. Western blotting was used to detect the expression of protein kinase C alpha, mitogen-activated protein kinase signal pathways, and cell cycle-related proteins. The results showed that TM208 decreased the expression of protein kinase C alpha, phospho-extracellular signal-regulated kinase-1/2, phospho-p38, cyclin B1, cell division cycle 2 (cdc2), and phospho-cdc2 (Thr161) and increased the expression of phospho-cdc2 (Tyr15). Taken together, our data show that TM208 has little antiproliferative effect on QGY-7703 cells in vitro, whereas it significantly inhibits the growth of QGY-7703 xenograft tumors with low toxicity in vivo. The inhibition of mitogen-activated protein kinase signal pathways and the regulation of the G2/M phase may be responsible for its antitumor effects.

SLXM-2, a derivative of cyclophosphamide: mechanism of growth inhibition on hepatocarcinoma 22 cells.

Restructuring of cyclophosphamide (CPA) is a promising method for the development of antineoplastic therapy. This study investigated the inhibitory effects of a derivative of CPA, SLXM-2, on hepatocarcinoma 22 (H22) transplanted into ICR mice as well as its effects on the survival time of mice transplanted with the ascitic fluid-type H22. We found that SLXM-2 inhibited tumor growth and prolonged survival time. Moreover, the compound had little effect in vivo on leukocytes and body weight and a higher lethal dose 50 than CPA. The cell cycle analysis by flow cytometry revealed that SLXM-2 arrested tumor cells in both the S and G2 phases, and the arrest in the G2 phase increased in a dose-dependent manner. Western blotting and reverse transcription-PCR experiments indicated that the observed G2 arrest was associated with an increase of cyclin B1, whereas cell division cycle protein 2 (Cdc2) remained constant. The results, however, showed an accumulation of tyrosine 15 phosphorylated Cdc2 and a reduction of threonine 161 phosphorylated Cdc2. In addition, SLXM-2 led to a decrease in cyclin-dependent kinase 7 and Cdc25c kinase, which participated in inhibiting the G2/M transition. Our data identified two upstream targets leading to the inactivity of the cyclin B1/Cdc2 complex, which explained the arrest in the G2/M phase following SLXM-2 treatment. These results demonstrated the antitumor activity of SLXM-2 and its potential use as an antineoplastic drug.