Over-activation of EFTUD2 correlates with tumor propagation and poor survival outcomes in hepatocellular carcinoma

PurposeElongation factor Tu GTP-binding domain containing 2 (EFTUD2) is an essential constituent of U5 small nuclear ribonucleoproteins (snRNPs) and plays a crucial role in spliceosome activation and cancer. The mechanism of EFTUD2 on carcinogenesis and development of liver cancer still need further study.MethodsBioinformatic analysis was performed to find differential expressed genes and related pathways. Western blotting and quantitative PCR assays were used to verify the EFTUD2 expression in HCC cell lines and tumor tissues of liver cancer patients. Transfection of shRNAs in SKHEP1 and Huh7 cell lines was conducted to explore the mechanisms of EFTUD2 in HCC. CCK-8 method, colony formation, and cell cycle detection kit were used to detect the proliferation. A tumor model in nude mice was used to explore the role of EFTUD2 in liver cancer in vivo.ResultsBased on the tumor tissues and para-tumor tissues in our HCC patients, we identified EFTUD2 as highly expressed in HCC tissues (P < 0.001). Bioinformatic analysis from the TCGA database also supported this biological phenomenon (P = 1.911e−17). Furtherly, the results of clinical specimens and TCGA data suggested that higher EFTUD2 expression levels correlated with high histologic grades, high pathological grades, and poor survival prognoses in HCC patients. And knockdown of EFTUD2 suppressed cell proliferation and colony formation in vitro. In vivo, knockdown of EFTUD2 constrained the tumor growing and expansion derived from SKHEP1 cells and induced a decrease in the tumor volume and tumor weight resected from nude mice. Furthermore, RNA sequencing based on EFTUD2 knockdown revealed that EFTUD2 affected target genes concerned with the cell cycle. Flow cytometric analyses in the SKHEP1 cell model revealed that knockdown significantly suppressed cell cycle course and caused cell cycle arrest in the G1 phase. CyclinD1 proteins were also inhibited by knocking down of EFTUD2.

Over-activation of EFTUD2 correlates with tumor propagation and poor survival outcomes in hepatocellular carcinoma.

PURPOSE: Elongation factor Tu GTP-binding domain containing 2 (EFTUD2) is an essential constituent of U5 small nuclear ribonucleoproteins (snRNPs) and plays a crucial role in spliceosome activation and cancer. The mechanism of EFTUD2 on carcinogenesis and development of liver cancer still need further study. METHODS: Bioinformatic analysis was performed to find differential expressed genes and related pathways. Western blotting and quantitative PCR assays were used to verify the EFTUD2 expression in HCC cell lines and tumor tissues of liver cancer patients. Transfection of shRNAs in SKHEP1 and Huh7 cell lines was conducted to explore the mechanisms of EFTUD2 in HCC. CCK-8 method, colony formation, and cell cycle detection kit were used to detect the proliferation. A tumor model in nude mice was used to explore the role of EFTUD2 in liver cancer in vivo. RESULTS: Based on the tumor tissues and para-tumor tissues in our HCC patients, we identified EFTUD2 as highly expressed in HCC tissues (P < 0.001). Bioinformatic analysis from the TCGA database also supported this biological phenomenon (P = 1.911e-17). Furtherly, the results of clinical specimens and TCGA data suggested that higher EFTUD2 expression levels correlated with high histologic grades, high pathological grades, and poor survival prognoses in HCC patients. And knockdown of EFTUD2 suppressed cell proliferation and colony formation in vitro. In vivo, knockdown of EFTUD2 constrained the tumor growing and expansion derived from SKHEP1 cells and induced a decrease in the tumor volume and tumor weight resected from nude mice. Furthermore, RNA sequencing based on EFTUD2 knockdown revealed that EFTUD2 affected target genes concerned with the cell cycle. Flow cytometric analyses in the SKHEP1 cell model revealed that knockdown significantly suppressed cell cycle course and caused cell cycle arrest in the G1 phase. CyclinD1 proteins were also inhibited by knocking down of EFTUD2. CONCLUSION: EFTUD2 is markedly overexpressed in HCC tumor tissues. High EFTUD2 expression in HCC patients is associated with clinical features. Moreover, we confirmed that EFTUD2 shows a pivotal role in HCC cell proliferation and cell cycle course and could be a possible therapeutic avenue in HCC through disturbing EFTUD2.

[The correlation between findings of whole-body diffusion weighted imaging and clinical result in patients with multiple myeloma].

Objective: To explore the relationship between the findings of whole body diffusion weighted imaging (WBDWI) and the clinical result in patients with multiple myeloma. Methods: A total of 43 cases of multiple myeloma patients were retrospectively collected from May 2015 to May 2017 in Tianjin First Central Hospital.Twenty-nine cases were male and 14 were female. The median age was 54 years old with a range from 36 to 73 years old. The patients were divided into two groups with and without abnormal findings pending on whole body diffusion weighted imaging. The clinical data and the ADC value were compared between the two groups, as well as comparison in patients with abnormal findings between pre-and post-treatment. Results: In 43 patients, normal findings on WBDWI were found in 10 cases, 7 males, 3 females, age (59±9) years old, and abnormal findings in 33 cases, 22 males, 11 females, age (57±10) years old.No statistical differences of age and gender were found between two groups (P>0.05) .The ratio of plasma cells and the proportion of ß(2) microspheres in patients with abnormal WBDWI (50.0% (14.0%, 78.0%) , 4.8 (2.7, 7.7) mg/L)were significantly higher than those in the normal group(5.0% (2.5%, 15.0%) , 2.4 (2.0, 3.7) mg/L) (P<0.05).ADC value in different body parts of abnormal group including costal ((0.66±0.15)×10(-3) mm(2)/s), sternal bone((0.71±0.20)×10(-3) mm(2)/s), clavicles((0.67±0.17)×10(-3) mm(2)/s), thoracic vertebra((0.63±0.17)×10(-3) mm(2)/s), lumber vertebra((0.69±0.20)×10(-3) mm(2)/s), pelvic((0.83±0.36)×10(-3) mm(2)/s), proximal humerus((0.76±0.13)×10(-3) mm(2)/s), proximal femur((0.64±0.17)×10(-3) mm(2)/s), shaft of femur((0.70±0.22)×10(-3) mm(2)/s), proximal tibia((0.97±0.18)×10(-3) mm(2)/s), shaft of tibia((0.83±0.18)×10(-3) mm(2)/s) which were significantly higher than those of normal group (all P<0.05). The albumin concentration of the patients after treatment was significantly higher than those before treatment (P<0.05). Conclusion: Different imaging findings on WBDWI can reflect clinical different result in patients with multiple myeloma, and when WBDWI is normal, the clinical symptoms are mild. When abnormal findings detected on WBDWI, the clinical symptoms are still severe although albumin concentration increased after treatment.

Simultaneous removal of aqueous Zn2+, Cu2+, Cd2+, and Pb2+ by zeolites synthesized from low-calcium and high-calcium fly ash.

In this study, zeolites were synthesized from low-calcium (LCZ) and high-calcium (HCZ) fly ash, respectively. Subsequently, the zeolites were tested for their removal effectiveness for four aqueous cations, namely, Zn2+, Cu2+, Cd2+, and Pb2+, as a function of contact time, pH value, adsorbent dosage, and initial concentration of heavy metals. Both zeolites were characterized by X-ray diffraction, X-ray fluorescence spectrometry, scanning electron microscopy, specific surface area, and cation exchange capacity. The results show that HCZ mainly consists of an unnamed zeolite (Na6[AlSiO4]6·4H2O), whereas LCZ mainly consists of faujasite-type zeolite. The optimum sorption conditions were pH = 6.0; adsorbent dosage = 1.0 g·L-1; temperature = 25 °C; contact time = 100 min; and initial heavy metal concentration = 100 mg·L-1. The sorption kinetics of the four aqueous cations on both LCZ and HCZ followed the pseudo-second-order kinetic model, and the sorption isotherm data fitted well with the Langmuir isotherm model. For LCZ, the maximum adsorption capacities of Zn2+, Cu2+, Cd2+, and Pb2+ were 155.76, 197.86, 123.76, and 186.22 mg·g-1, respectively. For HCZ, the values were 154.08, 183.15, 118.91, and 191.94 mg·g-1, respectively. The zeolites were regenerated by NaCl solution (1 mol·L-1) and showed high removal efficiency. In conclusion, zeolites produced by fly ash are promising materials for removing Zn2+, Cu2+, Cd2+, and Pb2+ from wastewater.

Simultaneous immobilization of ammonium and phosphate from aqueous solution using zeolites synthesized from fly ashes.

Zeolites were synthesized from silica-rich (SF-Z) and calcium-rich (CF-Z) fly ashes, respectively, and their performance in immobilizing ammonium and phosphate was investigated through batch experiments. The cation exchange capacity and phosphate immobilization capacity of SF-Z were identified as 2.79 meq/g and 12.97 mg/g while those of CF-Z were 0.69 meq/g and 87.41 mg/g, respectively. The mixture of SF-Z and CF-Z (MSC-Z) immobilized simultaneously ammonium and phosphate, and the ratio of SF-Z to CF-Z depended on the ammonium and phosphate concentrations in wastewater and the discharge standard. The adsorption processes of ammonium and phosphate on MSC-Z followed Ho's pseudo-second-order model and the intra-particle diffusion was a rate-controlling step. The Langmuir model produced better suitability to the equilibrium data. The thermodynamic study revealed that the adsorption of both ammonium and phosphate on MSC-Z was an endothermic reaction. After treatment by MSC-Z, the ammonium and phosphate concentrations in wastewater from a sewage treatment plant decreased from 7.45 and 1.42 mg/L to 2.06 and 0.51 mg/L, respectively, and met Surface Water Environment Quality Standard in China δ. These results show that the immobilization of ammonium and phosphate in wastewater can be achieved by the combination of zeolites synthesized from silica-rich and calcium-rich fly ashes.

Ribose modified nucleosides and nucleotides as ligands for purine receptors.

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.

Growth suppression of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor.

A mutant epidermal growth factor receptor (variously called DeltaEGFR, de2-7 EGFR, or EGFRvIII) containing a deletion of 267 amino acids of the extracellular domain is frequently highly expressed in human malignant gliomas and has been reported for cancers of the lung, breast, and prostate. We tested the efficacy of a novel monoclonal anti-DeltaEGFR antibody, mAb 806, on the growth of intracranial xenografted gliomas in nude mice. Systemic treatment with mAb 806 significantly reduced the volume of tumors and increased the survival of mice bearing xenografts of U87 MG.DeltaEGFR, LN-Z308.DeltaEGFR, or A1207.DeltaEGFR gliomas, each of which expresses high levels of DeltaEGFR. In contrast, mAb 806 treatment was ineffective with mice bearing the parental U87 MG tumors, which expressed low levels of endogenous wild-type EGFR, or U87 MG.DK tumors, which expressed high levels of kinase-deficient DeltaEGFR. A slight increase of survival of mice xenografted with a wild-type EGFR-overexpressing U87 MG glioma (U87 MG.wtEGFR) was effected by mAb 806 concordant with its weak cross-reactivity with such cells. Treatment of U87 MG.DeltaEGFR tumors in mice with mAb 806 caused decreases in both tumor growth and angiogenesis, as well as increased apoptosis. Mechanistically, in vivo mAb 806 treatment resulted in reduced phosphorylation of the constitutively active DeltaEGFR and caused down-regulated expression of the apoptotic protector, Bcl-XL. These data provide preclinical evidence that mAb 806 treatment may be a useful biotherapeutic agent for those aggressive gliomas that express DeltaEGFR.

Ring-Constrained (N)-methanocarba nucleosides as adenosine receptor agonists: independent 5'-uronamide and 2'-deoxy modifications.

Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation preferred at adenosine receptors (ARs), were synthesized and tested in binding assays. The 5'-uronamide modification preserved [N6-(3-iodobenzyl)] or enhanced (N6-methyl) affinity at A3ARs, while the 2'-deoxy modification reduced affinity and efficacy in a functional assay.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin.

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of A6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. A6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, A6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of A6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or A6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving A6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

The utilization of a unified pharmacophore query in the discovery of new antagonists of the adenosine receptor family.

Pharmacophore queries from previously known potent selective A3 antagonists were generated by Chem-X. These queries were used to search a pharmacophore database of diverse compounds (CNS-Set). In vitro assays of 186 'hits' yielded over 30 active compounds, for four adenosine receptor subtypes. This search strategy may also be applicable to the discovery of new ligands via receptor homology data.

Selective A(3) adenosine receptor antagonists: water-soluble 3, 5-diacyl-1,2,4-trialkylpyridinium salts and their oxidative generation from dihydropyridine precursors.

A(3) adenosine receptor antagonists are sought for their potential antiinflammatory, antiasthmatic, and antiischemic properties. We have found that 3,5-diacyl-1,2,4-trialkyl-6-phenylpyridinium derivatives constitute a novel class of selective A(3) adenosine receptor antagonists. The structure-activity relationships of this class of antagonists, incorporating the 3-thioester, have been explored. The most potent analogue in this group was 2, 4-diethyl-1-methyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl -6-phe nylpyridinium iodide (11), which had an equilibrium inhibition constant (K(i)) value of 219 nM at human A(3) receptors (binding of [(125)I]AB-MECA (N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine)) expressed in Chinese hamster ovary (CHO) cells and >10 microM at rat brain A(1) and A(2A) receptors and at recombinant human A(2B) receptors. Compound 11 could be generated through oxidation of the corresponding 3,5-diacyl-1,2,4-trialkyl-6-phenyl-1,4-dihydropyridine, 24, with iodine or in the presence of rat brain homogenates. A 6-cyclopentyl analogue was shown to increase affinity at human A(3) receptors upon oxidation from the 1-methyl-1,4-dihydropyridine analogue, 25, to the corresponding pyridinium derivative, 23 (K(i) 695 nM), suggesting a prodrug scheme. Homologation of the N-methylpyridinium derivatives to N-ethyl and N-propyl at the 1-position caused a progressive reduction in the affinity at A(3) receptors. Modifications of the alkyl groups at the 2-, 3-, 4-, and 5-positions failed to improve potency in binding at A(3) receptors. The pyridinium antagonists are not as potent as other recently reported, selective A(3) receptor antagonists; however, they display uniquely high water solubility (43 mM for 11). Compound 11 antagonized the inhibition of adenylate cyclase elicited by IB-MECA in CHO cells expressing the human A(3) adenosine receptor, with a K(B) value of 399 nM, and did not act as an agonist, demonstrating that the pyridinium salts are pure antagonists.

Differential expression of peroxiredoxin subtypes in human brain cell types.

The peroxiredoxin (Prx) protein is expressed widely in animal tissues and serves an antioxidant function associated with removal of cellular peroxides. We have cloned two Prx genes and observed differential expression of Prx-I and Prx-II (formerly NKEF-A and NKEF-B) in purified rat brain cell cultures (Sarafian et al. [1998] Mol. Chem. Neuropathol. 34:39-51). We have examined regional and cell-type-specific expression of Prx-I and Prx-II in paraffin sections of human brain using immunohistochemical methods. These studies revealed a clear segregation of expression of these two gene products in different brain cell types. In the cerebral cortex, cerebellum, basal ganglia, substantia nigra, and spinal cord, Prx-I was expressed primarily in astrocytes, while Prx-II was expressed exclusively in neurons. Prx-I was also prominently expressed in ependymal cells and subependymal matrix of substantia nigra and basal ganglia. Prx-II was not expressed at uniform density in all neurons. In general, small neurons such as cerebellar granule neurons displayed little or no staining, while large neurons, such as hippocampal pyramidal and Purkinje neurons were heavily stained. The absence of expression of Prx-I in neurons and the selective expression of Prx-II in large neurons suggest that these antioxidant enzymes serve distinct functional roles that may reflect the different functions and biochemical activities of these cell types. Restricted expression of these genes may also contribute to the selective vulnerability of these cells to a wide variety of neuropathologic conditions.

Functionalized congeners of 1,4-dihydropyridines as antagonist molecular probes for A3 adenosine receptors.

4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure-activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 microM.

Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A3 adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201). In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values at recombinant human and rat A3 adenosine receptors were determined using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine). Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A3 receptor affinity, resulting in Ki values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A3 receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A3 receptors. Several analogues were nearly equipotent at rat and human A3 receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a Ki value of 248 nM at human A3 receptors. Using affinity data and a general pharmacophore model for A3 adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r2pred = 0.873) for compounds in the test set. A rhodopsin-based model of the human A3 receptor was built, and the pyridine reference ligand 2,3,4, 5-tetraethyl-6-phenyl-pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.

Monoclonal anti-androgen receptor antibodies: production, characterization and potential diagnostic applications.

Several monoclonal antibodies (mAbs) and novel mAb-based assays for the androgen receptors (AR) have been developed. Large amounts of the recombinant human AR protein produced by a baculovirus expression system were used as an antigen to produce mAbs. Twenty-nine AR-specific mAbs were first confirmed by Western blot analysis and were then characterized for their immunoglobulin isotypes, epitopes, and epitope localization in AR. Novel assays using flow cytometry and sandwich enzyme-linked immunosorbent assays (ELISA) were established to detect AR-expressing cells and to quantify soluble AR protein, respectively. Using immunostaining, we identified several anti-AR mAbs exclusively recognizing AR within the nuclei of the prostate cancer cell line LNCaP and of prostate tissues in both frozen and paraffin-embedded sections, whereas other mAbs could detect AR in both nuclear and cytoplasmic compartments. Interestingly, certain mAbs, such as G122-25 and G122-77, could distinguish the androgen-bound AR from the unoccupied AR. In sum, many purified AR protein and anti-AR mAbs, together with the assays developed, could be powerful tools for the study of functional AR and for the diagnosis of prostatic cancers.

Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors.

Radiolabeled ZM 241385 (4-(2-[7-amino-2- ¿furyl¿¿1,2,4¿triazolo¿2,3-a¿¿1,3,5¿triazin-5-ylaminoethyl)p henol), has previously been used as a high affinity radioligand for the labeling of A2A adenosine receptors in cell membranes. Another subtype, the A2B receptor, is the least well-defined subtype of adenosine receptors and lacks selective pharmacological probes. In the present study, we have used [3H]ZM 241385 as a radioligand to label recombinant human A2B adenosine receptors in HEK-293 cell membranes, that do not express A2A adenosine receptors, and found that the pharmacological profile is consistent with the SAR of A2B receptors. Saturable, specific binding (Kd 33.6 nM, Bmax 4.48 pmol/mg protein) that was best described by a one-site model was found, and specific binding was approximately 75% of total binding. [3H]ZM 241385 binding was displaceable by a large number of compounds known to interact with A2B receptors; thus, this method has promise as a tool in the search for agonists and antagonists selective for this subtype. Xanthine analogs, which are antagonists, proved to be the most potent displacers. The Ki of XAC, xanthine amine congener, was 12.3 nM, while CPX (8-cyclopentyl-1,3-dipropylxanthine) was less potent. The non-selective triazoloquinazoline antagonist CGS 15943 (Ki 16.4 nM), which is similar in structure to ZM 241385, was slightly less potent than XAC. The non-xanthine A2B-antagonist alloxazine displaced [3H]ZM 241385-binding with a Ki of 462 nM, similar to its affinity in functional assays. Adenosine derivatives known to activate this receptor subtype, such as NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA (N6-phenylisopropyladenosine), were considerably less potent than the 8-substituted xanthines examined.

Synthesis and adenosine receptor affinity of 7-beta-D-ribofuranosylxanthine.

7-beta-D-Ribofuranosylxanthine, a previously unreported isomer of xanthosine, was prepared in four steps from 7-benzylxanthine. The procedure, which involves the use of pivaloyloxymethyl groups to protect the xanthine ring, was also applied to preparation of some 1-N-alkyl derivatives of 7-ribosylxanthine. Adenosine receptor affinity for these compounds was determined. 7-beta-D-Ribofuranosylxanthine was found to have higher affinity and greater selectivity for the A1 receptor than previously reported xanthine nucleosides, and to be a partial agonist.

Synthesis and biological activity of a new series of N6-arylcarbamoyl, 2-(Ar)alkynyl-N6-arylcarbamoyl, and N6-carboxamido derivatives of adenosine-5'-N-ethyluronamide as A1 and A3 adenosine receptor agonists.

A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ +-b earing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors expressed in CHO cells. The derivatives contained the 5' substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ + (NECA). While the carboxamido derivatives (9-13) showed affinity for A1 receptors, the urea derivatives (30-45) showed different degrees of affinity and selectivity for the A3 adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (Ki = 9 nM) and selectivity for the A3 receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-be ta-D-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A3 adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A2A and/or A3 receptors but rather showed a dramatic reduction of A2A affinity, resulting in compounds with good A2A/A3 selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A1 and A3 receptor subtypes, excluding the A2A receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A3 receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [35S]GTP-gamma-S.

Structure-activity relationships and molecular modeling of 3, 5-diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2- and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2, 4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate , 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.