Synthesis, structure analysis, antitumor evaluation and 3D-QSAR studies of 3,6-disubstituted-dihydro-1,2,4,5-tetrazine derivatives.

3,6-Diaryl-dihydro-1,2,4,5-tetrazine derivatives were synthesized and their structures were confirmed by single-crystal X-ray diffraction. Monosubstituted dihydrotetrazines are the 1,4-dihydro structure, but disubstituted dihydrotetrazines are the 1,2-dihydro structure. The results of further research indicated there may be a rearrangement during the synthesis process of disubstituted dihydrotetrazines. Their antitumor activities were evaluated against A-549 and P388 cells in vitro. The results showed several compounds to be endowed with cytotoxicity in the low micromolar range. Two compounds were highly effective against A-549 cell and IC50 values were 0.575 and 2.08 µM, respectively. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were carried out on 37 1,2,4,5-tetrazine derivatives with antitumor activity against A-549 cell. Models with good predictive abilities were generated with the cross validated q(2) values for CoMFA and CoMSIA being 0.744 and 0.757, respectively. Conventional r(2) values were 0.978 and 0.988, respectively, the predicted R(2) values were 0.916 and 0.898, respectively. The results provide the tool for guiding the design and synthesis of novel and more potent tetrazine derivatives.

Correlation between Spectral CT Parameters and Ki67 Expression in Hepatocellular Carcinoma.

OBJECTIVE: The objective of this study was to analyze the relationship between quantitative parameters of spectral CT and the Ki67 expression index of tumor cells in hepatocellular carcinoma (HCC). METHODS: A total of 19 patients who underwent preoperative spectral CT dual-phase enhancement and who were diagnosed with HCC by postoperative pathology were prospectively selected. Patients with ≥10% Ki67-positive tumor cells formed a high-Ki67 group, and those with <10% Ki67- positive cells formed a low-Ki67 group. The iodine concentrations (ICs) of the lesion and the descending aorta were measured during the arterial and venous phases. Relative iodine concentration (RIC) was calculated thus: RIC=IClesion/ICdescending aorta. CT values of the lesions at 40 and 70 keV were measured during the enhanced arterial and venous phases. The slope of the spectral curve (λ) was calculated thus: λ = (40 keV-70 keV) /(70-30). To compare the differences in quantitative parameters between the high- and low-Ki67 groups, either an independent samples t-test (normal distribution) or a Mann-Whitney U test (non-normal distribution) was used. Receiver operating characteristic curves were used to evaluate the effectiveness of spectral CT parameters in distinguishing between high-Ki67 and low-Ki67 groups. Pearson correlation analysis was used to evaluate the correlation between spectral CT quantitative parameters and Ki67 expression. RESULTS: IC, RIC and λ values for the high-Ki67 group in arterial and venous phases were higher than those for the low-Ki67 group, P < 0.05. IC, RIC, and λ values in the arterial phase were 0.83, 0.89, and 0.75, respectively; in the venous phase, the values of these three parameters were 0.76, 0.77, and 0.69, respectively. IC, RIC, and λ were positively correlated with Ki67 expression in both arterial and venous phases, with a highest correlation of 0.82 for arterial-phase RIC. CONCLUSION: The quantitative parameters of spectral CT in HCC were correlated with Ki67 expression. This finding may make it easier for clinicians to determine whether a tumor is high or low in Ki67 before surgery.

Synthesis of novel thiazolyl-pyrimidines and their anticancer activity in vitro.

A series of novel compounds 7-43 were prepared via the condensation of enaminones 4a-h and the guanidines carbonate 6a-f. The structures of these newly synthesized compounds were confirmed by (1) H-NMR, MS, EA and IR. All the compounds were tested for their cytotoxic activity in vitro against human cancer cell lines including Ishikawa, A549, BEL-7404, SPC-A-01 and SGC-7901. Most of them showed moderate cytotoxic against the tested cell lines. Among them, the most potent compounds 9 and 30 exhibited more efficient activity against Ishikawa, A549.

4-{4-Methyl-2-[(meth-yl)(2-methyl-phen-yl)amino]-1,3-thia-zol-5-yl}-N-(3-methyl-phen-yl)pyrimidin-2-amine.

In the title compound, C(23)H(23)N(5)S, the thia-zole ring and pyrimidine ring are almost coplanar, making a dihedral angle of 4.02 (9)°. in the crystal, weak inter-molecular N-H⋯N inter-actions link pairs of molecules into centrosymmetric dimers.

4-{2-[(3,4-Dichloro-phen-yl)(meth-yl)amino]-4-methyl-1,3-thia-zol-5-yl}-N-(3-methyl-phen-yl)pyrimidin-2-amine.

In the title compound, C(22)H(19)Cl(2)N(5)S, the thia-zole and pyrimidine rings are almost co-planar, making a dihedral angle of 6.48 (7)°. In the crystal, intermolecular N-H⋯N hydrogen bonds link pairs of molecules into centrosymmetric dimers..

N-(3-Meth-oxy-phen-yl)-4-{4-methyl-2-[(meth-yl)(4-methyl-phen-yl)amino]-1,3-thia-zol-5-yl}pyrimidin-2-amine.

The asymmetric unit of the title compound, C(23)H(23)N(5)OS, contains two independent mol-ecules. In one mol-ecule, the thia-zole and pyrimidine rings are almost co-planar, making a dihedral angle of 2.48 (8)°. In the other mol-ecule, the corresponding dihedral angle is 12.82 (8)°. The crystal structure is stabilized by weak inter-molecular N-H⋯N and C-H⋯O inter-actions that extend along the b axis.

Synthesis and anticancer evaluation of alpha-lipoic acid derivatives.

alpha-Lipoic acid derivatives were synthesized and evaluated for their in vitro anticancer activities against NCI-460, HO-8910, KB, BEL-7402, and PC-3 cell lines. The results, for most compounds exhibited dose-dependent inhibitory property and several compounds had good inhibitions at the dose of 100 microg/mL. Compound 17 m was further selected for in vivo evaluation against S180 xenograft in ICR mice, which had 24.7% tumor-weight inhibition through intragastric administration of 200mg/kg of body weight. Moreover, the LD(50) in mice for 17 m through ig exceeded 1000 mg/kg of body weight.

Synthesis and anticancer evaluation of thiazolyl-chalcones.

Thirty-seven (E)-1-(4-methyl-2-arylaminothiazol-5-yl)-3-arylprop-2-en-1-ones were synthesized via Claisen-Schmidt condensation of 1-(4-methyl-2-(arylamino)thiazol-5-yl)ethanone with the corresponding arylaldehydes. All these thiazolyl-chalcones were characterized and evaluated by MTT assay on human cancer cell lines BGC-823, PC-3, NCI-H460, BEL-7402 in vitro. Compounds 5, 8, 26, 37 and 41 are effective against cancer cell lines with IC(50)s below 10 µM. The antitumor activity in ICR mice bearing sarcoma 180 tumors indicates compounds 10 and 41 have moderate in vivo activity with 22-25% tumor-weight inhibition.

[Apoptosis of human lung carcinoma cell line EBC-1 induced by N, N'-di-(m-methylphenyl)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide and its molecular mechanism].

OBJECTIVE: To study whether N, N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) inhibits proliferation and induces apoptosis in human lung carcinoma cell line EBC-1 cells and its molecular mechanism. METHODS: Different concentrations of ZGDHu-1 and different times of culture were used to treat EBC-1 cells in vitro. The inhibition of proliferation was measured by BrdU-ELISA. Cell apoptosis was detected by Annexin V/PI staining and cellular DNA fragmentation ELISA. Phosphorylated p38MAPK and STAT3 were examined by flow cytometry. The protein expressions of bcl-2, bax, p53, Fas, and caspase-3 were detected by Western blot analysis. RESULTS: ZGDHu-1 inhibited EBC-1 cell proliferation within a certain range of treating times and does, with a 24 h IC(50) of (295 ± 25) ng/ml, 48 h of (112 ± 8) ng/ml and 72 h of (23 ± 2) ng/ml. The EBC-1 cell apoptosis was confirmed by Annexin V/PI labeling and cellular DNA fragmentation ELISA in a dose-related manner. When EBC-1 cells were treated with 50, 200, and 500 ng/ml ZGDHu-1 for 48 h, the expression rates of phosphor-p38MAPK protein were 67.4%, 88.2%, 91.1%, respectively, and that of the control was 10.6%. That of STAT3 protein were 56.5%, 43.6% and 34.6%, respectively, and that of the control was 89.1%. The expression of bax, p53 and Fas protein was significantly increased, that of bcl-2 was not changed, and that of caspase-3 was significantly decreased by the ZGDHu-1 treatment. CONCLUSION: ZGDHu-1 can inhibit proliferation and induce apoptosis in EBC-1 cells. The mitochondrial pathway mediated by Fas may be one of its mechanisms. The apoptosis of EBC-1 cells may associate with up-regulation of phosphor-p38MAPK and down-regulation of phosphor-STAT3 in the cells.

Vadimezan: 2-(5,6-dimethyl-9-oxo-9H-xanthen-4-yl)acetic acid.

In the title mol-ecule, C(17)H(14)O(4), the C atom of the carboxyl group deviates by 1.221 (3) Šfrom the plane [maximum deviation = 0.0122(2) Å] of the tricycic ring system. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link the mol-ecules into centrosymmetric dimers, and π-π inter-actions [centroid-centroid distances = 3.491 (3), 3.591 (3), 3.639 (3) and 3.735 (3) Å] link these dimers into layers parallel to the ac plane. Weak inter-molecular C-H⋯O inter-actions further consolidate the crystal packing.

The synthesis and biological evaluation of some caffeic acid amide derivatives: E-2-cyano-(3-substituted phenyl)acrylamides.

A series of caffeic acid amide derivatives 2-cyano-(3-substituted phenyl)acrylamides were synthesized via Knoevenogal condensation of substituted benzaldehydes with cyanoacetamides. The structure of compound 1f was determined as E-isomer by X-ray diffractive analysis. The biological screening tests in vitro showed that compound 1b has obvious inhibitory activities against human gastric carcinoma cell line BGC-823, human nasopharyngeal carcinoma cell line KB and human hepatoma cell line BEL-7402 with IC(50) values of 5.6 microg/mL, 13.1 microg/mL and 12.5 microg/mL, respectively. Some preliminary structure-activity relationships (SAR) were also proposed which may provide a direction for further study.

Synthesis and biological evaluation of amide derivatives of diflunisal as potential anti-inflammatory agents.

To improve the medicinal activity, the structure of diflunisal has been modified. Twenty-one amide derivatives of diflunisal were synthesized starting from diflunisal in three steps with total yields from 72% to 89%. All compounds were identified by (1)H NMR, MS, and elemental analysis. The anti-inflammatory and analgesic activities for 19 compounds were evaluated. It was found that 5m possesses an excellent anti-inflammatory activity and a good analgesic activity, maybe a potential anti-inflammatory agent.

Synthesis and biological evaluation of amide derivatives of diflunisal as potential anti-tumor agents.

To discover the new medicinal activity, the structure of diflunisal has been modified. Forty amide derivatives of diflunisal were synthesized starting from diflunisal in three steps. Their inhibition growth rate of human lung cancer cell (A549) and human endometrial adenocarcinoma cell (Ishikawa) in vitro was evaluated. The preliminary assay results showed that compounds 6j, 7o and 8c exhibited good anti-tumor activities and excellent selectivity for the Ishikawa cell, may be potential anti-tumor agents.

(E)-Ethyl 2-cyano-3-(3,4-dihydr-oxy-5-nitro-phen-yl)acrylate.

The title compound, C(12)H(10)N(2)O(6), was synthesized via a Knoevenagel condensation and crystallized from ethanol. In the crystal, strong classical inter-molecular O-H⋯O hydrogen bonds and weak C-H⋯N contacts link the mol-ecules into ribbons extending along [010]. Intra-molecular O-H⋯O and C-H⋯N contacts support the planar conformation of the mol-ecules (mean deivation 0.0270 Å).

N-(3,4-Dichloro-phen-yl)thio-urea.

In the title compound, C(7)H(6)Cl(2)N(2)S, the benzene ring and the mean plane of the thio-urea fragment [-N-C(=S)-N] make a dihedral angle of 66.77 (3)°. Inter-molecular N-H⋯S and N-H⋯Cl hydrogen bonds link the mol-ecules into a three-dimensional network.

Synthesis of trans-caffeate analogues and their bioactivities against HIV-1 integrase and cancer cell lines.

Forty caffeate analogues were synthesized via a convenient method starting from vanillin with moderate to good yields. The testing of biological activity of these compounds against HIV-1 integrase indicates that four compounds: bornyl caffeate, bornyl 2-nitrocaffeate, 5-nitrocaffeic acid and 5-nitrocaffeic acid phenethyl ester (5-nitroCAPE) possess a good HIV integrase inhibitory activity, IC(50) 19.9, 26.8, 25.0 and 13.5 microM , respectively. Twelve caffeate analogues were tested by MTT assay on growth of human hepatocellular carcinoma BEL-7404, human breast MCF-7 adenocarcinoma, human lung A549 adenocarcinoma and human gastric cancer BCG823 cell lines, respectively. And the best result is IC(50) 5.5 microM for CAPE against BEL-7404.

[Effect of ZGDHu-1 on proliferation and apoptosis of A549 cells in vitro and antitumor activity in vivo].

This study is to explore the mechanism and effect of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation and apoptosis of A549 cells in vitro and on A549 xenograft tumor in nude mice. With different concentrations of ZGDHu-1 at different times were used to treat A549 cells in vitro. The proliferation was determined by living cell count, SRB assay and Brdu-ELISA. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin V/PI and Hoechst 33258 labeling method. The nude mice model of A549 xenograft tumor was established by subcutaneous inoculation. The suppression activity of ZGDHu-1 by intraperitoneal injection on xenograft mice model was detected. The expressions of bcl-2, bax and p53 gene and protein were analyzed by RT-PCR and flow cytometry. ZGDHu-1 can inhibit A549 cell proliferation viability within a certain range of treating time and does, and a majority of A549 cells were arrested in G2-M phase. The A549 cells apoptosis was confirmed by typical cell morphology, DNA fragment, Sub G1 phase, Hoechst 33258 and Annexin V/PI labeling method with a time and dose related manner. When the xenograft tumor mice model were treated with 10, 20 and 40 mg x kg(-1) ZGDHu-1 for 14 days, the tumor growth inhibition rate were 43.7%, 56.9% and 60.0%, respectively. The expression of bax, bax/bcl-2 and p53 gene and protein increased significantly and bcl-2 decreased slightly by the treatment of ZGDHu-1. ZGDHu-1 can significantly suppress the growth of A549 xenograft tumor in vivo and inhibited proliferation by inducing tumor cell apoptosis in vitro. The mechanism may associate with its up-regulation of bax and p53 during the apoptosis process.

ZGDHu-1 induces G2/M phase arrest and apoptosis in Kasumi-1 cells.

The present study examined the effects of N,N'­di­(m­methylphenyi)­3, 6­dimethyl­1, 4­dihydro­1,2,4,5­tetrazine­1,4­dicarboamide (ZGDHu­1), a novel oxazine derivative, in Kasumi­1 cells. Following incubation with various concentrations of ZGDHu­1, fluorescence­activated cell sorting (FACS) was used in order to detect changes in mitochondrial membrane permeability in Kasumi­1 cells. Western blot analysis was performed in order to analyze the expression of nuclear factor­κB, inhibitor of κB and AML1/ETO. In addition FACS was used to analyze leukemia cell cycles and the expression levels of cyclin, cyclin­dependent kinases and cyclin­dependent kinase inhibitors in G2/M phase were determined using FACS and western blot analysis. The upregulation of reactive oxygen species production and mitochondrial membrane permeability was ascribed to apoptosis. The growth of Kasumi­1 cells was inhibited through the downregulation of nuclear factor­κB, degradation of AML1/ETO fusion protein and cell cycle arrest at the G2/M phase. This study documented that G2/M regulatory molecules, including cyclin B1, cell division control (cdc)2 and cdc25c were downregulated and checkpoint kinase 1 (CHK1), p53, p27, phospho­cdc25c, phospho­CHK1 and phospho­p53 were upregulated following treatment with ZGDHu­1. In the present study, pretreatment with CHIR­124, a selective CHK1 inhibitor, abrogated G2/M arrest via ZGDHu­1. These results demonstrated the anti­tumor activity of ZGDHu­1, which may therefore a potential target for further investigation and may be useful for the treatment of patients with t(8;21) acute myeloid leukemia.

N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) suppresses the proliferation of PANC-1 pancreatic cancer cells via apoptosis and G2/M cell cycle arrest.

Pancreatic cancer is one of the human gastrointestinal malignancies with a high mortality and poor prognosis. Approximately eighty percent of patients are diagnosed with unresectable or metastatic disease. Thus, development of novel chemicals in the treatment of pancreatic cancer is imperative. This study aimed to investigate the anticancer effects of N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a new tetrazine derivative, on the PANC-1 pancreatic cancer cell line and clarify the underlying molecular mechanism. Using an MTT assay, we found that ZGDHu-1 significantly suppressed the proliferation of PANC-1 cells in a time- and dose-dependent manner. Moreover, according to the morphological and flow cytometric analysis, the results indicated that ZGDHu-1 induced PANC-1 cell apoptosis and G2/M cell cycle arrest in a dose-dependent manner. In the western blot analysis, expression of the pro-apoptotic Bax gene was upregulated while the anti-apoptotic Bcl-2 gene was downregulated following treatment with ZGDHu-1. ZGDHu-1 also activated pro-caspase-3 and PARP and increased the expression of NF-κB inhibitor IκB. Furthermore, the expression levels of G2/M regulatory molecules such as cyclin B1 and cdc2 were decreased while that of Chk1 was increased. These results suggested that ZGDHu-1 suppressed the proliferation of pancreatic cancer cells, rendering it a potential therapeutic drug for the treatment of pancreatic cancer.

How much water is made "non-free" by 36% native hemoglobin?

At equilibrium, the concentration ratio of poly(ethylene gycol) (PEG-4000) in a dialysis sac containing a 35.1% solution of native bovine hemoglobin over that in the external solution is 0.196 +/- 0.028 (mean +/- SD). This apparent equilibrium distribution constant or rho-value of 0.196, when viewed side-by-side with the near-equal distribution of sucrose and raffinose in similar native-hemoglobin dominated water suggests all (rather than 80%) of the water in this solution has been altered by the native hemoglobin and is no longer free liquid water. Based on Ling's equation for solute exclusion, we found that an excess of water-to-water interaction energy of a mere 4.25 cal/mole could account for both the observed exclusion of PEG-4000 and non-exclusion of sucrose and raffinose. Finally, the long-range action of (even this relatively inactive) native hemoglobin on the dynamic water structure was compared with the exclusion of coated latex microspheres from the altered water 100 microm from the surface of polyvinylalcohol gel (Zheng and Pollack) --in the light of Ling's new theory of ad infinitum water polarization-orientation (under idealized conditions) first publicized at the Gordon Research Conference on "Interfacial Water in Cell Biology" on the campus of the Mount Holyoke College in June 2004.