Synthesis of Some New Folic Acid-Based Heterocycles of Anticipated Biological Activity.

To date, no fused heterocycles have been formed on folic acid molecules; for this reason, and others, our target is to synthesize new derivatives of folic acid as isolated or fused systems. Folic acid 1 reacted with ethyl pyruvate, triethyl orthoformate, ethyl chloroformate, thioformic acid hydrazide, and aldehydes to give new derivatives of folic acid 2-6a,b. Moreover, It reacted with benzylidene malononitrile, acetylacetone, ninhydrin, ethyl acetoacetate, ethyl cyanoacetate, and ethyl chloroacetate to give the pteridine fused systems 10-15, respectively. Ethoxycarbonylamino derivate 5 reacted with some nucleophiles containing the NH2 group, such as aminoguanidinium hydrocarbonate, hydrazine hydrate, glycine, thioformic acid hydrazide, and sulfa drugs in different conditions to give the urea derivatives 16-20a,b. Compound 4 reacted with the same nucleophiles to give the methylidene amino derivatives 21-24a,b. The fused compound 10 reacted with thioglycolic acid carbon disulfide, malononitrile, and formamide to give the four cyclic fused systems 25-30, respectively. The biological activity of some synthesized showed moderate effect against bacteria, but no effect shown towards fungi.

Activation of GPR119 Stimulates Human ß-Cell Replication and Neogenesis in Humanized Mice with Functional Human Islets.

Using humanized mice with functional human islets, we investigated whether activating GPR119 by PSN632408, a small molecular agonist, can stimulate human ß-cell regeneration in vivo. Human islets were transplanted under the left kidney capsule of immunodeficient mice with streptozotocin- (STZ-) induced diabetes. The recipient mice were treated with PSN632408 or vehicle and BrdU daily. Human islet graft function in the mice was evaluated by nonfasting glucose levels, oral glucose tolerance, and removal of the grafts. Immunostaining for insulin, glucagon, and BrdU or Ki67 was performed in islet grafts to evaluate α- and ß-cell replication. Insulin and CK19 immunostaining was performed to evaluate ß-cell neogenesis. Four weeks after human islet transplantation, 71% of PSN632408-treated mice achieved normoglycaemia compared with 24% of vehicle-treated mice. Also, oral glucose tolerance was significantly improved in the PSN632408-treated mice. PSN632408 treatment significantly increased both human α- and ß-cell areas in islet grafts and stimulated α- and ß-cell replication. In addition, ß-cell neogenesis was induced from pancreatic duct cells in the islet grafts. Our results demonstrated that activation of GPR119 increases ß-cell mass by stimulating human ß-cell replication and neogenesis. Therefore, GPR119 activators may qualify as therapeutic agents to increase human ß-cell mass in patients with diabetes.

Sulforaphane Analogues with Heterocyclic Moieties: Syntheses and Inhibitory Activities against Cancer Cell Lines.

Recent studies have shown that sulforaphane (SFN) selectively inhibits the growth of ALDH⁺ breast cancer stem-like cells.Herein, a series of SFN analogues were synthesized and evaluated against breast cancer cell lines MCF-7 and SUM-159, and the leukemia stem cell-like cell line KG-1a. These SFN analogues were characterized by the replacement of the methyl group with heterocyclic moieties, and the replacement of the sulfoxide group with sulfide or sulfone. A growth inhibitory assay indicated that the tetrazole analogs 3d, 8d and 9d were significantly more potent than SFN against the three cancer cell lines. Compound 14c, the water soluble derivative of tetrazole sulfide 3d, demonstrated higher potency against KG-1a cell line than 3d. SFN, 3d and 14c significantly induced the activation of caspase-3, and reduced the ALDH⁺ subpopulation in the SUM159 cell line, while the marketed drug doxrubicin(DOX) increased the ALDH⁺ subpopulation.

SYNTHESIS AND STUDY OF HALOGENATED BENZYLAMIDES OF SOME ISOCYCLIC AND HETEROCYCLIC ACIDS AS POTENTIAL ANTICONVULSANTS.

A series of potential anticonvulsants have been synthesized. There are eight fluorobenzylamides and three chlorobenzylamides of isocyclic or heterocyclic acids. Two not halogenated benzylamides were also synthesized to compare the effect of halogenation. The aim of the research performed was to evaluate whether halogenation of the mother structure is able to improve its anticonvulsant activity. The compounds were tested in Anticonvulsant Screening Project (ASP) of Antiepileptic Drug Development Program (ADDP) of NIH. Compound 1 showed MES ED50 = 80.32 mg/kg, PI = 3.16. Compound 7 showed CKM ED50 = 56.72 mg/kg. Compound 8 showed MES ED50 = 34.23 mg/kg and scPTZ ED50 > 300 mg/kg, PI = 8.53.Compound 13 showed 6Hz ED50 = 78.96, PI = 3.37. The results indicate that fluorination does not improve activity, whereas chlorination in our experiment even reduces it.

Direct activation of the proposed anti-diabetic receptor, GPR119 in cardiomyoblasts decreases markers of muscle metabolic activity.

GPR119 agonists are emerging rapidly as a pharmaceutical treatment of diabetes. Diabetes is a known risk factor for cardiovascular disease yet the cardiac-specific consequences of GPR119 activation are unknown. This study demonstrated that GPR119 agonism in cardiac myoblasts reduces metabolic activity in high and low concentrations of fatty acids, with high concentrations of palmitate largely attenuating the effects of the GPR119 agonist, PSN632408. The effects of GPR119 activation on gene and protein markers of metabolism were dependent on fatty acid exposure. Activating GPR119 did not affect cell hypertrophy of lipid accumulation regardless of lipid exposure. These results suggest that the pathways activated in response to GPR119 modulation in cardiac muscle cells differ between healthy and metabolically dysregulated states. However regardless of the pathway activated by GPR119, these effects may cause detrimental reductions to oxidative/metabolic capacity under both conditions. Thus further development of GPR119 agonists for treating metabolic diseases is warranted.

5-membered cyclic hydroxamic acids as HDAC inhibitors.

The new histone deacylases inhibitors (HDACi) were synthesized in the class of 5-membered cyclic hydroxamic acids (5-CHA), showing medium size CHA as a new Zn-binding group. New reaction sequence was proposed for the synthesis of 5-membered alkylidene-cyclic-hydroxamic acids starting from butyrolactone. Compound 10c showed low µM activity on HeLa cell extracts. From these results, cyclic hydroxamic acids will be further investigated to find more potent compounds.

The melanocortin-4 receptor is expressed in enteroendocrine L cells and regulates the release of peptide YY and glucagon-like peptide 1 in vivo.

The melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly enriched GPCR in peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent antisecretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal (i.p.) administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions.

GPR119 regulates genetic markers of fatty acid oxidation in cultured skeletal muscle myotubes.

Gene knockout and agonist studies indicate that activation of the G protein-coupled receptor, GPR119, protects against diet-induced obesity and insulin resistance. It is not known if GPR119 activation in skeletal muscle mediates these effects. To address this uncertainty, we measured GPR119 expression in skeletal muscle and determined the effects of PSN632408, a GPR119 agonist, on the expression of genes and proteins required for fatty acid and glucose oxidation in cultured myotubes. GPR119 expression was readily detected in rat skeletal muscle and mRNAs were induced by 12 weeks of high-fat feeding. Treatment of cultured mouse C2C12 myotubes with 5 µM PSN632408 or 0.5 mM palmitate reduced expression of mRNAs encoding fatty acid oxidation genes to similar extents. More so, treatment with PSN632408 decreased AMPKα (Thr172 phosphorylation) activity in the absence of palmitate and ACC (Ser79 phosphorylation) activity in the presence of palmitate. In human primary myotubes PSN632408 decreased expression of PDK4 and AMPKα2 mRNA in myotubes derived from obese donors. These data suggest GPR119 activation in skeletal muscle may impair fatty acid and glucose oxidation.

N-heterocyclic dronic acids: applications and synthesis.

Substituted hydroxymethylenebisphosphonic acid derivatives--either as dronic acids or their dronate sodium salts, are important pharmaceuticals in the treatment of diseases arising from excessive bone-resorption. Potential has also been identified in areas ranging from parasite-growth inhibition to immunological and cancer therapeutics. Representative clinically relevant N-heterocyclic derivatives include zoledronic and risedronic acids. The biochemical background and mechanism of action of these drugs are discussed, along with trends in structural development and future prospects. Synthetic routes to dronates are then summarized. The most popular route to valuable dronic acids involves the 3- component condensation of a substituted acetic acid, phosphorous acid, and phosphorus trichloride. However, the protocols recorded in the literature are very diverse. This review gives a critical account of reported methods, explores the contradictions and suggests a practical synthetic procedure after clarifying the inconsistencies described. Possible mechanisms of the reaction are also discussed.

Stimulating beta cell replication and improving islet graft function by GPR119 agonists.

G protein-coupled receptor 119 (GPR119) is predominantly expressed in ß cells and intestinal L cells. In this study, we investigated whether oleoylethanolamide (OEA), a GPR119 endogenous ligand, and PSN632408, a GPR119 synthetic agonist, can stimulate ß-cell replication in vitro and in vivo and improve islet graft function in diabetic mice. We found that OEA and PSN632408 significantly increased numbers of insulin(+)/5-bromo-2'-deoxyuridine (BrdU)(+) ß cells in cultured mouse islets in a dose-dependent manner. All diabetic recipient mice, given marginal syngeneic islet transplants with OEA or PSN632408 or vehicle, achieved normoglycemia at 4 weeks after transplantation. However, normoglycemia was achieved significantly faster in OEA- or PSN632408-treated diabetic mice than in vehicle-treated diabetic mice (P < 0.05). The percentage of insulin(+)/BrdU(+) ß cells in islet grafts in OEA- and PSN632408-treated mice was significantly higher than in vehicle-treated mice (P < 0.01). Our data demonstrated that OEA and PSN632408 can stimulate ß-cell replication in vitro and in vivo and improve islet graft function. Targeting GPR119 is a novel therapeutic approach to increase ß-cell mass and to improve islet graft function by stimulating ß-cell replication.

Dinuclear copper complexes of organic claw: potent inhibition of protein tyrosine phosphatases.

Three dinuclear copper complexes of organic claw ligands (2,2',2″,2'''-(5-R-2-hydroxy-1,3-phenylene)bis(methylene)bis(azanetriyl)tetraacetic acid, R=methyl (H(5)L1), chloro (H(5)L2) and bromo (H(5)L3)): [Cu(2)NaL1(H(2)O)(2)] (1), [Cu(2)HL2(H(2)O)(2)] (2), [Cu(2)NaL3(H(2)O)(2)] (3), have been synthesized and characterized by elemental analyses, infrared spectra, thermo-gravimetric analyses, X-ray diffraction analysis, electrospray ionization mass spectra, pH-potentiometric titration, molar conductivity. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T cell protein tyrosine phosphatase (TCPTP), Megakaryocyte protein tyrosinephosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) are evaluated in vitro. The three copper complexes exhibit potent and almost same inhibition against PTP1B and SHP-1 with IC(50) values ranging from 0.15 to 0.31µM, about 2-fold stronger inhibition than against PTP-MEG2, 10-fold stronger inhibition than against TCPTP, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Molecular docking analyses confirm the inhibition model. Fluorescence titration studies suggest that the complexes bond to PTP1B with the formation of a 1:1 complex. The results demonstrate that copper complexes that are potent PTPs inhibitors but have different inhibitory effects over different PTPs, may be explored as new practical inhibitors towards individual PTP with some specificity.

Peptide YY is critical for acylethanolamine receptor Gpr119-induced activation of gastrointestinal mucosal responses.

Peptide YY (PYY) is released following food intake and regulates intestinal function and glucose homeostasis, but the mechanisms underpinning these processes are unclear. Enteroendocrine L cells contain PYY and express the acylethanolamine receptor, Gpr119. Here, we show that Gpr119 activation inhibited epithelial electrolyte secretion in human and mouse colon in a glucose-sensitive manner. Endogenous PYY selectively mediated these effects, since PYY(-/-) mice showed no Gpr119 response, but responses were observed in NPY(-/-) mice. Importantly, Gpr119 responses in wild-type (WT) mouse tissue and human colon were abolished by Y(1) receptor antagonism, but were not enhanced by dipeptidylpeptidase IV blockade, indicating that PYY processing to PYY(3-36) was not important. In addition, Gpr119 agonism reduced glycemic excursions after oral glucose delivery to WT mice but not PYY(-/-) mice. Taken together, these data demonstrate a previously unrecognized role of PYY in mediating intestinal Gpr119 activity and an associated function in controlling glucose tolerance.

Endogenous and synthetic agonists of GPR119 differ in signalling pathways and their effects on insulin secretion in MIN6c4 insulinoma cells.

BACKGROUND AND PURPOSE: GPR119 is a G protein-coupled receptor that is preferentially expressed in islet cells and mediates insulin secretion. Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA) act as endogenous ligands for this receptor, whereas PSN375963 and PSN632408 are two recently reported synthetic agonists. In this study, we explored mechanisms underlying GPR119-induced insulin secretion. In addition, we assessed the potential utility of the synthetic agonists as tools for exploring GPR119 biology. EXPERIMENTAL APPROACH: We examined natural and synthetic GPR119 agonist activity at GPR119 in MIN6c4 and RINm5f insulinoma cells. We evaluated insulin secretion, intracellular calcium [Ca(2+)](i), ion channel involvement and levels of cAMP. KEY RESULTS: We report that increases in insulin secretion induced by OEA were associated with increased cAMP and a potentiation of glucose-stimulated increases in [Ca(2+)](i). We also demonstrate that ATP-sensitive K(+) and voltage-dependent calcium channels were required for GPR119-mediated increases in glucose-stimulated insulin secretion. In contrast to OEA, the synthetic GPR119 agonist PSN375963 and PSN632408 have divergent effects on insulin secretion, cAMP and intracellular calcium in MIN6c4 cells. CONCLUSIONS AND IMPLICATIONS: The endogenous ligand OEA signals through GPR119 in a manner similar to glucagon-like peptide-1 (GLP-1) and its receptor with respect to insulin secretion, [Ca(2+)](i) and cAMP. In addition, PSN375963 and PSN632408 substantially differ from OEA and from one another. These studies suggest that the commercially available synthetic agonists, although they do activate GPR119, may also activate GPR119-independent pathways and are thus unsuitable as GPR119-specific pharmacological tools.

Synthesis and growth inhibition activity of alpha-bromoacrylic heterocyclic and benzoheterocyclic derivatives of distamycin A modified on the amidino moiety.

The design, synthesis and in vitro activities of novel alpha-bromoacryloyl pyrazole, imidazole and benzoheterocyclic derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the distamycin frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of alpha-bromoacrylic derivatives of distamycin.

A novel class of endothelin-A receptor antagonists, (R)-2-(benzo[1,3]dioxol-5-yl)-6-isopropyloxy-2H-chromene-3-carboxylic acids (S-1255). Conformational analysis of basic structure, crucial for ET(A) antagonism, in solution and solid states.

Conformational studies of potent and selective endothelin-A (ET(A)) receptor antagonists, 4-substituted (R)-2-(benzo[1,3]-dioxol-5-yl)-6-isopropoxy-2H-chromene-3-carboxylic acids, are reported. X-ray crystallography and NMR studies of the 4-anisyl derivative 2 (S-1255), the stable atropisomers 3 and the 4-n-butyl derivative 4 reveal that the A-, B- and C-rings in these compounds adopt a L-like conformation in both solution and solid states. Molecular mechanics calculation shows that this L-like conformation is an inevitable conformation as determined by intramolecular steric repulsions. These 2H-chromene derivatives bound to an ET(A) receptor with IC(50) values of less than 1 nM, whereas the dihydro compounds 7 and 9 not having the L-like conformation showed weaker affinities. These results suggest that the L-like conformation is specifically recognized by the active site of the ET(A) receptor. The roles of the L-like conformation in the receptor binding are discussed.

Synthesis and biological activity of potent heterocyclic thiol-based inhibitors of endothelin-converting enzyme-1.

Directed screening of metalloprotease inhibitors identified CGS 30084 (1) as a potent inhibitor of endothelin-converting enzyme-1 (ECE-1) in vitro (IC(50)=77 nM). Herein we report the syntheses and biological activities of analogues containing modified biphenyl moieties, bearing heterocyclic proximal rings. Compound 20, the thioacetate ethyl ester prodrug derivative of compound 19a, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.