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Lanzhou Alkaline Stretched Noodles (LASN) has introduced by Meng et al. in previous research paper (2007). In order to discover the LASN specialty wheat quality requirement for allelic variations at Glu-1of northwest China spring wheats, 2 northwest China spring wheat cultivars and 39 elite F6 breeding lines were adopted to determine the high-molecular-weight glutenin subunits (HMW-GS) composition by one step one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) based on the protocol of Singh et al. (1991). The results showed that the wheat quality and LASN quality were characterised by Glu-1alleles significantly. Glu-A1/1 correlated to high protein content and better extensibility (L) than other allelic variations. Glu-A1/1, Glu-B1/17+18 and Glu-D1/5+10 were beneficial to dough strength (W), meanwhile Glu-A1/2* and Glu-D1/5+10 were good to dough tenacity (P). Glu-D1/5+10 strongly correlated to high volume of SDS-sediment significantly. Allelic variations at Glu-A1/ (1, 2*), Glu-B1/17+18 and Glu-D1/5+10 were beneficial alleles for wheat quality as well as LASN quality. However, Glu-A1/null and Glu-D1/2+12 were inferior alleles for wheat quality and LASN quality.

We have identified 5-(3-chlorothiophen-2-yl)-3-(4-trifluoromethylphenyl)-1,2,4-oxadiazole (1d) as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 1d has good activity against several breast and colorectal cancer cell lines but is inactive against several other cancer cell lines. In a flow cytometry assay, treatment of T47D cells with 1d resulted in arrest of cells in the G1 phase, followed by induction of apoptosis. SAR studies of 1d showed that the 3-phenyl group can be replaced by a pyridyl group, and a substituted five-member ring in the 5-position is important for activity. 5-(3-Chlorothiophen-2-yl)-3-(5-chloropyridin-2-yl)-1,2,4-oxadiazole (4l) has been found to have in vivo activity in a MX-1 tumor model. Using a photoaffinity agent, the molecular target has been identified as TIP47, an IGF II receptor binding protein. Therefore, our cell-based chemical genetics approach for the discovery of apoptosis inducers can identify potential anticancer agents as well as their molecular targets.

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G2/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure−activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC50 of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI50 values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI50 value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.

By applying a novel cell- and caspase-based HTS assay, a series of N-phenyl nicotinamides has been identified as a new class of potent inducers of apoptosis. Through SAR studies, a 20-fold increase in potency was achieved from a screening hit N-(4-methoxy-2-nitrophenyl)pyridine-3-carboxamide (1) to lead compound 6-methyl-N-(4-ethoxy-2-nitrophenyl)pyridine-3-carboxamide (10), with an EC50 of 0.082 μM in the caspase activation assay in T47D breast cancer cells. The N-phenyl nicotinamides also were found to be active in the growth inhibition assay where compound 10 had a GI50 value of 0.21 μM in T47D cells. More importantly, compound 10 was found to be equipotent in MES-SA cells and paclitaxel-resistant, p-glycoprotein overexpressed MES-SA/DX5 cells. Compounds 1 and 6-chloro-N-(4-ethoxy-2-nitrophenyl)pyridine-3-carboxamide (8), a more potent analogue, were found to arrest T47D cells in G2/M phase of the cell cycle followed by induction of apoptosis as measured by flow cytometry. Compound 8, which was more potent than 1 in the caspase activation assay, also was found to be more potent in G2/M arrest and apoptosis assay. These data confirm that the cell-based caspase activation assay is useful for screening for inducers of apoptosis, as well as for SAR studies and lead optimization. Upon further characterization, N-phenyl nicotinamides were found to be inhibitors of microtubule polymerization in vitro. The identification of N-phenyl nicotinamides as a novel series of inducers of apoptosis demonstrates that our cell- and caspase-based HTS assay is useful for the discovery and optimization of potentially novel anticancer agents.

N-Pentafluorobenzoyl−R110 (1a) and N-(2,3,4,5-tetrafluorobenzoyl)−R110 (1b) with enhanced cell retention properties, were prepared from rhodamine 110 (R-110) and the corresponding polyfluorobenzoyl chloride. N-Ac-DEVD-N‘-pentafluorobenzoyl−R110 (3a) and N-Ac-DEVD-N‘-(2,3,4,5-tetrafluorobenzoyl)−R110 (3b) were prepared as tetrapeptide substrates for caspases. Substrate 3b was efficiently cleaved by human recombinant caspase-3 in an enzyme assay. Substrate 3b also was efficiently cleaved in cell-based apoptosis assays. After cleavage in apoptotic cells by activated caspases, the substrate becomes fluorescent as measured by flow cytometry. These substrates should prove useful in cell-based assays for studying apoptosis inducers and inhibitors.

A molecular electronegativity distance vector based on 13 atomic types, called MEDV-13, is a descriptor for predicting the biological activities of molecules based on the quantitative structure−activity relations (QSAR). The MEDV-13 uses a modified electrotopological state (E-state) index to substitute for the relative eletronegativity (q) of non-hydrogen atoms in the molecule of interest in the MEDV and a topological distance for the relative distance (d) in the MEDV. For an organic molecule containing several chemical elements such as C, H, O, N, S, F, Cl, Br, I, and P, the MEDV-13 includes at best 91 descriptors. Then it is essential to employ a principal component regression (PCR) technique to derive a QSAR model relating the biological activities to the MEDV-13. The MEDV-13 is used to study the QSAR of the corticosteroid-binding globulin (CBG) binding affinity of the steroids and the activity inhibiting angiotensin-converting enzyme (ACE) of dipeptides, and resulting models have a comparable quality to the current three-dimensional (3D) methods such as CoMFA though the MEDV-13 is a descriptor based on two-dimensional topological information.

Antagonists at the 1a/2B subtype of the NMDA receptor (NR1a/2B) are typically small molecules that consist of a 4-benzyl- or a 4-phenylpiperidine with an ω-phenylalkyl substituent on the heterocyclic nitrogen. Many of these antagonists, for example ifenprodil (1), incorporate a 4-hydroxy substituent on the ω-phenyl group. In this study, the position of this 4-hydroxy substituent was transferred from the ω-phenyl group to the benzyl or phenyl group located on the 4-position of the piperidine ring. Analogues incorporating pyrrolidine in lieu of piperidine were also prepared. Electrical recordings using cloned receptors expressed in Xenopus oocytes show that high-potency antagonists at the NR1a/2B subtype are obtained employing N-(ω-phenylalkyl)-substituted 4-(4-hydroxyphenyl)piperidine, 4-(4-hydroxybenzyl)piperidine, and (±)-3-(4-hydroxyphenyl)pyrrolidine as exemplified by 21 (IC50 = 0.022 μM), 33 (IC50 = 0.059 μM), and 40 (IC50 = 0.017 μM), respectively. These high-potency antagonists are >1000 times more potent at the NR1a/2B subtype than at either the NR1a/2A or NR1a/2C subtypes. The binding affinities of 21 at α1-adrenergic receptors ([³H]prazosin, IC50 = 0.54 μM) and dopamine D2 receptors ([³H]raclopride, IC50 = 1.2 μM) are reduced by incorporating a hydroxy group onto the 4-position of the piperidine ring and the β-carbon of the N-alkyl spacer to give (±)-27:  IC50 NR1a/2B, 0.026; α1, 14; D2, 105 μM. The high-potency phenolic antagonist 21 and its low-potency O-methylated analogue 18 are both potent anticonvulsants in a mouse maximal electroshock-induced seizure (MES) study (ED50 (iv) = 0.23 and 0.56 mg/kg, respectively). These data indicate that such compounds penetrate the blood−brain barrier but their MES activity may not be related to NMDA receptor antagonism.

A novel series of N-(phenylalkyl)cinnamides related to N-(4-phenylbutyl)-3,4-dihydroxy-β-cyanocinnamide (6, an EGFR-K inhibitor with high antiproliferative activity) was synthesized and tested for antagonism at N-methyl-d-aspartate (NMDA) receptor subtypes. Potency and subunit selectivity were assayed by electrical recordings in Xenopus oocytes expressing three binary combinations of cloned rat NMDA receptor subunits:  NR1a expressed in combination with either NR2A, NR2B, or NR2C. The N-(phenylalkyl)cinnamides are selective antagonists of NR1a/2B receptors. Assayed under steady-state conditions, N-(4-phenylbutyl)-4-hydroxycinnamide (16) has an IC50 value of 77 nM and >1000-fold selectivity with respect to NR1a/2A and NR1a/2C receptors. Potency at α1 adrenergic receptors is low for the four cinnamides tested. Inhibition of NR1a/2B receptors does not correlate with EGFR and ErbB2/neu tyrosine kinase inhibitor activity. The N-(phenylalkyl)cinnamide series we describe provides a novel and structurally diverse framework for designing new NR2B-selective NMDA antagonists as potential CNS therapeutics.

A structure-based search and screen of our compound library identified N-(2-phenoxyethyl)-4-benzylpiperidine (8) as a novel N-methyl-d-aspartate (NMDA) receptor antagonist that has high selectivity for the NR1/2B subunit combination (IC50 = 0.63 μM). We report on the optimization of this lead compound in terms of potency, side effect liability, and in vivo activity. Potency was assayed by electrical recordings in Xenopus oocytes expressing cloned rat NMDA receptors. Side effect liability was assessed by measuring affinity for α1-adrenergic receptors and inhibition of neuronal K⁺ channels. Central bioavailability was gauged indirectly by determining anticonvulsant activity in a mouse maximal electroshock (MES) assay. Making progressive modifications to 8, a hydroxyl substituent on the phenyl ring para to the oxyethyl tether (10a) resulted in a ~25-fold increase in NR1a/2B potency (IC50 = 0.025 μM). p-Methyl substitution on the benzyl ring (10b) produced a ~3-fold increase in MES activity (ED50 = 0.7 mg/kg iv). Introduction of a second hydroxyl group into the C-4 position on the piperidine ring (10e) resulted in a substantial decrease in affinity for α1 receptors and reduction in inhibition of K⁺ channels with only a modest decrease in NR1a/2B and MES potencies. Among the compounds described, 10e (4-hydroxy-N-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperidine, Co 101244/PD 174494) had the optimum pharmacological profile and was selected for further biological evaluation.

A search of our compound library for compounds with structural similarity to ifenprodil (5) and haloperidol (7) followed by in vitro screening revealed that 4-benzyl-1-(4-phenyl-3-butynyl)piperidine (8) was a moderately potent and selective antagonist of the NR1a/2B subtype of NMDA receptors. Substitution on the benzyl group of 8 did not significantly affect NR1a/2B potency, while addition of hydrogen bond donors in the para position of the phenyl group enhanced NR1a/2B potency. Addition of a hydroxyl moiety to the 4-position of the piperidine group slightly reduced NR1a/2B potency while reducing α-1 adrenergic and dopamine D2 receptor binding affinities substantially, resulting in improved overall selectivity for NR1a/2B receptors. Finally, the butynyl linker was replaced with propynyl or pentynyl. When the phenyl was para substituted with amine or acetamide groups, the NR1a/2B potency order was butynyl > pentynyl ≫ propynyl. For the para methanesulfonamide or hydroxyl groups, the order was butynyl ≈ propynyl > pentynyl. The hydroxyl propyne (48) and butyne (23) were among the most potent NR1a/2B antagonists from this study. They both potentiated the effects of l-DOPA in the 6-hydroxydopamine-lesioned rat, a model of Parkinson's disease, dosed at 10 mg/kg ip, but 48 was not active at 30 mg/kg po.

A group of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones was synthesized and assayed for antagonism of rat brain α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors expressed in Xenopus oocytes. The benzodiazepinones inhibited AMPA-activated membrane current responses in a manner consistent with noncompetitive, allosteric inhibition of the receptor−channel complex. The most potent compound in the series was 1-(4-aminophenyl)-7,8-(methylenedioxy)-3,5-dihydro-4H-2,3-benzodiazepin-4-one (6), which had an IC50 of 2.7 μM. For comparison, the reference compound GYKI 52466 (2) had an IC50 of 6.9 μM. Compound 6 also had potent anticonvulsant activity in a mouse maximum electroshock-induced seizure (MES) assay:  the ED50 was 2.8 mg/kg iv, whereas the ED50 for GYKI 52466 was 4.6 mg/kg iv. In contrast to a previous report, the 7,8-dimethoxy analogue of 6 was a low-potency AMPA antagonist (IC50 >100 μM) and weak anticonvulsant (ED50 >10 mg/kg iv). The benzodiazepinones described herein are potent noncompetitive AMPA receptor antagonists that could have therapeutic potential as anticonvulsants and neuroprotectants.

A group of 5-aza-7-substituted-1,4-dihydroquinoxaline-2,3-diones (QXs) and the corresponding 5-(N-oxyaza)-7-substituted QXs were prepared and evaluated as antagonists of ionotropic glutamate receptors. The in vitro potency of these QXs was determined by inhibition of [³H]-5,7-dichlorokynurenic acid ([³H]DCKA) binding to N-methyl-d-aspartate (NMDA)/glycine receptors, [³H]-(S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) binding to AMPA receptors, and [³H]kainate ([³H]KA) binding to KA receptors in rat brain membranes. 5-(N-Oxyaza)-QXs 12a−e all have low micromolar or submicromolar potency for NMDA/glycine receptors and low micromolar potencies for AMPA and KA receptors. QXs 12a−e display 2−12-fold selectivity for NMDA/glycine receptors compared to AMPA receptors, and ~2-fold difference between AMPA and KA potency. In contrast to other QXs that either show high selectivity for NMDA (such as ACEA 1021) or AMPA (such as NBQX) receptors, these molecules are broad spectrum antagonists of ionotropic glutamate receptors. 7-Nitro-5-(N-oxyaza)-QX (12e) is the most potent inhibitor among 12a−e, having IC50 values of 0.69, 1.3, and 2.4 μM at NMDA, AMPA, and KA receptors, respectively. In functional assays on glutamate receptors expressed in oocytes by rat cerebral cortex poly(A⁺) RNA, 7-chloro-5-(N-oxyaza)-QX (12a) and 7-nitro-5-(N-oxyaza)-QX (12e) have Kb values of 0.63 and 0.31 μM for NMDA/glycine receptors, and are 6- and 4-fold selective for NMDA over AMPA receptors, respectively. 5-(N-Oxyaza)-7-substituted-QXs 12a−e all have surprisingly high in vivo potency as anticonvulsants in a mouse maximal electroshock-induced seizure (MES) model. 7-Chloro-5-(N-oxyaza)-QX (12a), 7-bromo-5-(N-oxyaza)-QX (12b), and 7-methyl-5-(N-oxyaza)-QX (12c) have ED50 values of 0.82, 0.87, and 0.97 mg/kg iv, respectively. The high in vivo potency of QXs 12a−e is particularly surprising given their low log P values (~ −2.7). Separate studies indicate that QXs 12a and 12e are also active in vivo as neuroprotectants and also have antinociceptive activity in animal pain models. In terms of in vivo activity, these 5-(N-oxyaza)-7-substituted-QXs are among the most potent broad spectrum ionotropic glutamate antagonists reported.

A series of 4-hydroxy-3-nitroquinolin-2(1H)-ones (HNQs) was synthesized by nitration of the corresponding 2,4-quinolinediols. The HNQs were evaluated as antagonists at the glycine site of NMDA receptors by inhibition of [³H]DCKA binding to rat brain membranes. Selected HNQs were also tested for functional antagonism by electrophysiological assays in Xenopus oocytes expressing either 1a/2C subunits of NMDA receptors or rat brain AMPA receptors. The structure−activity relationships (SAR) of HNQs showed that substitutions in the 5-, 6-, and 7-positions in general increase potency while substitutions in the 8-position cause a sharp reduction in potency. Among the HNQs tested, 5,6,7-trichloro HNQ (8i) was the most potent antagonist with an IC50 of 220 nM in [³H]DCKA binding assay and a Kb of 79 nM from electrophysiological assays. Measured under steady-state conditions HNQ 8i is 240-fold selective for NMDA over AMPA receptors. The SAR of HNQs was compared with those of 1,4-dihydroquinoxaline-2,3-diones (QXs) and 1,2,3,4-tetrahydroquinoline-2,3,4-trione 3-oximes (QTOs). In general, HNQs have similar potencies to QXs with the same benzene ring substitution pattern but are about 10 times less active than the corresponding QTOs. HNQs are more selective for NMDA receptors than the corresponding QXs and QTOs. The similarity of the SAR of HNQs, QXs, and QTOs suggested that these three classes of antagonists might bind to the glycine site in a similar manner. With appropriate substitutions, HNQs represent a new class of potent and highly selective NMDA receptor glycine site antagonists.

A series of aromatic and azepine ring-modified analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione (HBAD) were synthesized and evaluated as antagonists at NMDA receptor glycine sites. Aromatic ring-modified HBADs were generally prepared via a Schmidt reaction with substituted 2-methoxynaphthalene-1,4-diones followed by demethylation. Electrophilic aromatic substitution of benzazepine 3-methyl ethers gave 7-substituted analogs. The preparation of multiply substituted 2-methoxynaphthalene-1,4-diones was effected via Diels−Alder methodology utilizing substituted butadienes with 2-methoxybenzoquinones followed by aromatization. Structural modifications, such as elimination of the aromatic ring, removal of the 3-hydroxyl group, and transfer of the hydroxyl group from C-3 to C-4, were also studied. An initial evaluation of NMDA antagonism was performed using a [³H]MK801 binding assay. HBADs demonstrating NMDA antagonist activity as indicated by inhibition of [³H]MK801 binding were further evaluated employing a [³H]-5,7-dichlorokynurenic acid (DCKA) glycine site binding assay. Selected HBADs were characterized for functional antagonism of NMDA and AMPA receptors using electrophysiological assays in Xenopus oocytes and cultured rat cortical neurons. Antagonist potency of HBADs showed good correlation between the different assay systems. HBADs substituted at the 8-position possessed the highest potency with the 8-methyl (5), 8-chloro (6), and 8-bromo (7) analogs being the most active. For HBAD 6, the IC50 in [³H]DCKA binding assays was 0.013 μM and the Kb values for antagonism of NMDA receptors in oocytes (NR1a/2C) and cortical neurons were 0.026 and 0.048 μM, respectively. HBADs also antagonized AMPA-preferring non-NMDA receptors expressed in oocytes but at a lower potency than corresponding inhibition of NMDA receptors. HBADs demonstrating a high potency for NMDA glycine sites showed the highest steady-state selectivity index relative to AMPA receptors. Substitution at the 6-, 7-, and 9-positions generally reduced or eliminated glycine site affinity. Moving the hydroxyl group from C-3 to C-4 reduced receptor affinity, and potency was eliminated by the removal of the aromatic ring or the hydroxyl group. These data indicate that the HBAD series has specific structural requirements for high receptor affinity. With the exception of substitution at C-8, modified HBADs generally have a lower affinity at NMDA receptor glycine sites than the parent compound 3. Mouse maximum electroshock-induced seizure studies show that the three HBADs selected for testing have in vivo potency with the 6,8-dimethyl analog (52) being the most potent (ED50 = 3.9 mg/kg, iv).