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Optimization of 4-(N-Cycloamino)phenylquinazolines as a Novel Class of Tubulin-Polymerization Inhibitors Targeting the Colchicine Site

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was modified to produce 4-(N-cycloamino)quinazolines (4a–c and 5a–m). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin- 2(1H)-one (5f), the most potent compound, exhibited high in vitro cytotoxic activity (GI50 1.9–3.2 nM), significant potency against tubulin assembly (IC50 0.77 μM), and substantial inhibition of colchicine binding (99% at 5 μM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death at the same dose.

Micheliolide Derivative DMAMCL Inhibits Glioma Cell Growth In Vitro and In Vivo

Background There is no highly effective chemotherapy for malignant gliomas to date. We found that dimethylaminomicheliolide (DMAMCL), a selective inhibitor of acute myeloid leukemia (AML) stem/progenitor cells, inhibited the growth of glioma cells. Methods The distribution of DMAMCL in brain was analyzed by an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) system. The anti-tumor evaluations of DMAMCL in vitro were performed by MTT, FACS and RT-PCR. In vivo, the mixture of C6 cells and matrigel was injected into caudatum, and the anti-tumor activity of DMAMCL was evaluated by tumor growth and rat survival. The toxicity of DMAMCL was evaluated by body weight, daily food intake, hematological or serum biochemical analyses, and histological appearance of tissues. Results The IC50 values of DMAMCL against the C6 and U-87MG cell lines in vitro were 27.18 ± 1.89 μM and 20.58 ± 1.61 μM, respectively. DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner. In a C6 rat tumor model, daily administration of DMAMCL for 21 days reduced the burden of C6 tumors by 60% to 88% compared to controls, and more than doubled the mean lifespan of tumor-bearing rats. Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma. Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity. Conclusions These results suggest that DMAMCL is highly promising for the treatment of glioma.

Anticancer activity of tuftsin-derived T peptide in postoperative residual tumors.

Immune adjuvants have been used in cancer biotherapies to stimulate immune response to tumor cells. Despite their potential as anticancer reagents, there are several impediments to their use in clinical applications. In this study, we aim to modify the existing tuftsin structure and evaluate its antitumor activity in preclinical models. We synthesized a novel tuftsin derivative, namely, the T peptide (TP), by linking four tuftsin peptides, which showed enhanced stability in vivo. We then evaluated its anticancer activity in a postoperative residual tumor model in mice, where we surgically removed most of the primary tumor from the host, a procedure mimicking clinically postoperative patients. Despite the limited effect in intact solid tumors, TP strongly inhibited relapsed growth of residual tumors in postsurgical mice. Surgical resection of tumors accelerated residual tumor growth, but TP slowed down this process significantly. Interestingly, TP showed similar effects in human xenograft residual models. As an immunomodulator, TP could synergize the functions of macrophages, thus inhibiting the growth of cocultured tumor cells in vitro. Furthermore, TP could shift the macrophages to the tumor-suppressive M1 type and mobilize them to produce elevated cytotoxic TNF-&agr; and NO. As a result, TP effectively prolonged the survival time of tumor-resected mice. Using the postoperative residual tumor models, we provide a body of evidence showing the antitumor activity of TP, which causes no obvious toxicity. Our study highlights the potential of TP as a postoperative adjuvant in cancer therapies.

Preclinical pharmacodynamic evaluation of antibiotic nitroxoline for anticancer drug repurposing

The established urinary antibiotic nitroxoline has recently regained considerable attention, due to its potent activities in inhibiting angiogenesis, inducing apoptosis and blocking cancer cell invasion. These features make nitroxoline an excellent candidate for anticancer drug repurposing. To rapidly advance nitroxoline repurposing into clinical trials, the present study performed systemic preclinical pharmacodynamic evaluation of its anticancer activity, including a methyl thiazolyl tetrazolium assay in vitro and an orthotopic urological tumor assay in vivo. The current study determined that nitroxoline exhibits dose-dependent anti-cancer activity in vitro and in urological tumor orthotopic mouse models. In addition, it was demonstrated that the routine nitroxoline administration regimen used for urinary tract infections was effective and sufficient for urological cancer treatment, and 2 to 4-fold higher doses resulted in obvious enhancement of anticancer efficacy without corresponding increases in toxicity. Furthermore, nitroxoline sulfate, one of the most common metabolites of nitroxoline in the urine, effectively inhibited cancer cell proliferation. This finding increases the feasibility of nitroxoline repurposing for urological cancer treatment. Due to the excellent anticancer activity demonstrated in the present study, and its well-known safety profile and pharmacokinetic properties, nitroxoline has been approved to enter into a phase II clinical trial in China for non-muscle invasive bladder cancer treatment (registration no. CTR20131716).

WX-132-18B, a novel microtubule inhibitor, exhibits promising anti-tumor effects

Cancer drug researchers have been seeking microtubule-inhibiting agents (MIAs) with higher bioactivity and lower toxicity than currently marketed drugs. WX-132-18B, a novel structural compound synthesized at our institute, specifically bound to the colchicine-binding site on tubulin rather than the vinblastine site, and concentration-dependently reduced microtubule content via depolymerization. It exhibited the same cellular phenotypic profiles as the classic MIAs (colchicine, vincristine, and taxol), including inducing cell cycle arrest at the G2/M phase, triggering tumor cell apoptosis, promoting nuclear membrane permeability, reducing mitochondrial membrane potential, and disrupting the redox system balance. Importantly, WX-132-18B displayed more potent in vitro bioactivity (IC50 0.45–0.99 nM) than did the classic MIAs; it inhibited the proliferation of human umbilical vein endothelial cells and seven types of human tumor cells, especially the taxol-resistant breast cancer cells MX-1/T. WX-132-18B also dose-dependently inhibited mice sarcoma, human lung, and gastric cancer xenograft tumors and the formation of tumor blood vessels in mice. In conclusion, WX-132-18B is a novel microtubule-depolymerizing agent that selectively acts on the colchicine-binding site of tubulin and exerts potent in vitro and in vivo anti-tumor effects. These characteristics, along with its anti-angiogenesis and anti-drug resistance properties, make WX-132-18B a promising anti-tumor drug candidate.Cancer drug researchers have been seeking microtubule-inhibiting agents (MIAs) with higher bioactivity and lower toxicity than currently marketed drugs. WX-132-18B, a novel structural compound synthesized at our institute, specifically bound to the colchicine-binding site on tubulin rather than the vinblastine site, and concentration-dependently reduced microtubule content via depolymerization. It exhibited the same cellular phenotypic profiles as the classic MIAs (colchicine, vincristine, and taxol), including inducing cell cycle arrest at the G2/M phase, triggering tumor cell apoptosis, promoting nuclear membrane permeability, reducing mitochondrial membrane potential, and disrupting the redox system balance. Importantly, WX-132-18B displayed more potent in vitro bioactivity (IC50 0.45-0.99 nM) than did the classic MIAs; it inhibited the proliferation of human umbilical vein endothelial cells and seven types of human tumor cells, especially the taxol-resistant breast cancer cells MX-1/T. WX-132-18B also dose-dependently inhibited mice sarcoma, human lung, and gastric cancer xenograft tumors and the formation of tumor blood vessels in mice. In conclusion, WX-132-18B is a novel microtubule-depolymerizing agent that selectively acts on the colchicine-binding site of tubulin and exerts potent in vitro and in vivo anti-tumor effects. These characteristics, along with its anti-angiogenesis and anti-drug resistance properties, make WX-132-18B a promising anti-tumor drug candidate.

In Vivo and Mechanistic Studies on Antitumor Lead 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one and Its Modification as a Novel Class of Tubulin-Binding Tumor-Vascular Disrupting Agents

7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (2), a promising anticancer lead previously identified by us, inhibited tumor growth by 62% in mice at 1.0 mg/kg without obvious signs of toxicity. Moreover, compound 2 exhibited extremely high antiproliferative activity in the NIH-NCI 60 human tumor cell line panel, with low to sub-nanomolar GI50 values (10-10 M level). It also showed a suitable balance between aqueous solubility and lipophilicity, as well as moderate metabolic stability in vivo. Mechanistic studies using Mayers hematoxylin and eosin and immunohistochemistry protocols on xenograft tumor tissues showed that 2 inhibited tumor cell proliferation, induced apoptosis, and disrupted tumor vasculature. Moreover, evaluation of new synthetic analogues (6a-6t) of 2 indicated that appropriate 2-substitution on the quinazoline ring could enhance antitumor activity and improve druglike properties. Compound 2 and its analogues with a 4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one scaffold thus represent a novel class of tubulin-binding tumor-vascular disrupting agents (tumor-VDAs) that target established blood vessels in tumors.

Novel proapoptotic agent SM-1 enhances the inhibitory effect of 5-fluorouracil on colorectal cancer cells in vitro and in vivo

5-Fluorouracil (5-FU) is one of the most important agents used to treat colorectal cancer. However, the therapeutic effect of 5-FU on colon cancer is limited. SM-1 is a novel type of proapoptotic agent that directly activates procaspase-3 to caspase-3, leading to apoptosis in human cancer cells. The aim of the present study was to evaluate the antitumor effects of 5-FU in combination with SM-1. The human colorectal cancer cell lines HCT116 and LoVo were cultured in the presence of SM-1 and 5-FU. The combination of SM-1 and 5-FU treatment exhibited increased proliferation inhibitory effects compared with 5-FU treatment alone in HCT116 and LoVo cells, as determined using an MTT assay. SM-1 significantly decreased the half-maximal inhibitory concentration of 5-FU from 8.07±0.49 to 2.55±0.41 µmol/l in HCT116 cells, and from 7.90±0.98 to 3.14±0.81 µmol/l in LoVo cells. Similarly, the apoptotic activity was increased to 47.95 and 35.19% in HCT116 and LoVo cells, respectively, as determined using Annexin V/propidium iodide staining and flow cytometry. The combination of SM-1 and 5-FU treatment led to significantly increased caspase-3 activity compared with either compound alone. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis revealed the downregulation of B-cell lymphoma 2 and Survivin, and the upregulation of apoptosis regulator Bcl-2-associated X protein and cleaved poly (ADP-ribose) polymerase in HCT116 and LoVo cells. In addition, RT-qPCR identified downregulation of X-linked inhibitor of apoptosis protein mRNA. 5-FU and SM-1 treatment in combination increased tumor proliferation inhibition in HCT116 and LoVo xenograft mouse models of colorectal cancer, compared with SM-1 or 5-FU treatment alone. SM-1 significantly enhanced the antitumor activity of 5-FU in colorectal cancer. These improved effects were due to increased activity of the apoptotic signaling pathway.

Synthesis, biological evaluation, and physicochemical property assessment of 4-substituted 2-phenylaminoquinazolines as Mer tyrosine kinase inhibitors.

Current results identified 4-substituted 2-phenylaminoquinazoline compounds as novel Mer tyrosine kinase (Mer TK) inhibitors with a new scaffold. Twenty-one 2,4-disubstituted quinazolines (series 4-7) were designed, synthesized, and evaluated against Mer TK and a panel of human tumor cell lines aimed at exploring new Mer TK inhibitors as novel potential antitumor agents. A new lead, 4b, was discovered with a good balance between high potency (IC50 0.68μM) in the Mer TK assay and antiproliferative activity against MV4-11 (GI50 8.54μM), as well as other human tumor cell lines (GI50<20μM), and a desirable druglike property profile with low logP value (2.54) and high aqueous solubility (95.6μg/mL). Molecular modeling elucidated an expected binding mode of 4b with Mer TK and necessary interactions between them, thus supporting the hypothesis that Mer TK might be a biologic target of this kind of new active compound.