Discovery and Synthesis of Hydroxy-l-Proline Blockers of the Neutral Amino Acid Transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2).

As a conformationally restricted amino acid, hydroxy-l-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened synthetic hydroxy-l-proline derivatives using electrophysiological and radiolabeled uptake methods against amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We have discovered a novel class of alkoxy hydroxy-pyrrolidine carboxylic acids (AHPCs) that act as selective high-affinity inhibitors of the SLC1 family neutral amino acid transporters SLC1A4 and SLC1A5. AHPCs were computationally docked into a homology model and assessed with respect to predicted molecular orientation and functional activity. The series of hydroxyproline analogs identified here represent promising new agents to pharmacologically modulate SLC1A4 and SLC1A5 amino acid exchangers which are implicated in numerous pathophysiological processes such as cancer and neurological diseases.

The Lateral Metalation of Isoxazolo[3,4-d]pyridazinones towards Hit-to-Lead Development of Selective Positive Modulators of Metabotropic Glutamate Receptors.

Isoxazolo[3,4-d] pyridazinones ([3,4-d]s) were previously shown to have selective positive modulation at the metabotropic glutamate receptor (mGluR) Subtypes 2 and 4, with no functional cross-reactivity at mGluR1a, mGluR5, or mGluR8. Additional analogs were prepared to access more of the allosteric pocket and achieve higher binding affinity, as suggested by homology modeling. Two different sets of analogs were generated. One uses the fully formed [3,4-d] with an N6-aryl with and without halogens. These underwent successful selective lateral metalation and electrophilic quenching (LM&EQ) at the C3 of the isoxazole. In a second set of analogs, a phenyl group was introduced at the C4 position of the [3,4-d] ring via a condensation of 4-phenylacetyl-3-ethoxcarbonyl-5-methyl isoxazole with the corresponding hydrazine to generate the 3,4-ds 2b and 2j to 2n.

Synthesis, crystal structures, and Hirshfeld analysis of three hexa-hydro-quinoline derivatives.

Three hexa-hydro-quinoline derivatives were synthesized and crystallized in an effort to study the structure-activity relationships of these calcium-channel antagonists. The derivatives are ethyl 4-(2-meth-oxy-phen-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxyl-ate, C22H27NO4, (I), ethyl 4-(4-meth-oxy-phen-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carb-ox-yl-ate, C22H27NO4, (II), and ethyl 4-(3,4-di-hydroxy-phen-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxyl-ate, C21H24NO5, (III). In these hexa-hydro-quinoline derivatives, common structural features such as a flat-boat conformation of the 1,4-di-hydro-pyridine (1,4-DHP) ring, an envelope conformation of the fused cyclo-hexa-none ring, and a substituted phenyl group at the pseudo-axial position are retained. Hydrogen bonds are the main contributors to the packing of the mol-ecules in these crystals.

10-Alkoxy-anthracenyl-isoxazole analogs have sub-micromolar activity against a Glioblastoma multiforme cell line.

A series of 10-alkoxy-Anthryl-isoxazole-pyrrole-doubletails (RO-AIMs) were synthesized using a crown ether assisted nucleophilic aromatic substitution followed by a modified Schotten-Baumann reaction. The novel RO-AIMs described here exhibit robust growth inhibition for the human SNB19 CNS glioblastoma cell line, and biphenyl analog 8c had activity in the nanomolar regime, which represents the most efficacious compound in the AIM series to date. Computational modeling for RO-AIMs binding in a ternary complex with c-myc quadruplex DNA and its helicase DHX36 is presented which represents our current working hypothesis.

Syntheses and crystal structures of a nitro-anthracene-isoxazole and its oxidation product.

The syntheses and structures of an unexpected by-product from an iodination reaction, namely, ethyl 5-methyl-3-(10-nitro-anthracen-9-yl)isoxazole-4-carb-oxy-l-ate, C21H16N2O5, (I), and its oxidation product, ethyl 3-(9-hy-droxy-10-oxo-9,10-di-hydro-anthracen-9-yl)-5-methyl-isoxazole-4-carboxyl-ate, C21H17NO5 (V) are described. Compound (I) crystallizes with two mol-ecules in the asymmetric unit in which the dihedral angles between the anthracene fused-ring systems and isoxazole ring mean planes are 88.67 (16) and 85.64 (16)°; both mol-ecules feature a disordered nitro group. In (V), which crystallizes with one mol-ecule in the asymmetric unit, the equivalent dihedral angle between the almost planar anthrone ring system (r.m.s. deviation = 0.029 Å) and the pendant isoxazole ring is 89.65 (5)°. In the crystal of (I), the mol-ecules are linked by weak C-H⋯O inter-actions into a three-dimensional network and in the extended structure of (V), inversion dimers linked by pairwise O-H⋯O hydrogen bonds generate R 2 2(14) loops.

Liposomal Encapsulated FSC231, a PICK1 Inhibitor, Prevents the Ischemia/Reperfusion-Induced Degradation of GluA2-Containing AMPA Receptors.

Strokes remain one of the leading causes of disability within the United States. Despite an enormous amount of research effort within the scientific community, very few therapeutics are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity. Calcium-permeable AMPA receptors (AMPARs), lacking the GluA2 subunit, contribute to calcium cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of GluA2 AMPAR subunits following oxygen-glucose deprivation/reperfusion (OGD/R) is, at least in part, mediated by protein-interacting with C kinase-1 (PICK1). The purpose of the present study is to evaluate whether treatment with a PICK1 inhibitor, FSC231, prevents the OGD/R-induced degradation of the GluA2 AMPAR subunit. Utilizing an acute rodent hippocampal slice model system, we determined that pretreatment with FSC231 prevented the OGD/R-induced association of PICK1-GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein levels. This suggests that the interaction between GluA2 and PICK1 serves as an important step in the ischemic/reperfusion-induced reduction in total GluA2 levels.

10-N-heterocylic aryl-isoxazole-amides (AIMs) have robust anti-tumor activity against breast and brain cancer cell lines and useful fluorescence properties.

A novel series of anthracenyl-isoxazole amide (AIM) antitumor agents containing N-heterocycles in the 10 position (N-het) were synthesized using palladium cross-coupling. The unique steric environment of the N-het-AIMs required individual optimization in each case. Lanthanide mediated double activation was used to couple the dimethylamino pyrrole moiety, required for antitumor action. Robust antitumor activity was observed against breast and brain cancer cell lines. The compounds were docked with the c-myc oncogene promoter sequence, which adopts a G4 quadruplex DNA conformation, and represents the working hypothesis for biological action. The N-het-AIMs have useful fluorescence properties, allowing for observation of their distribution within tumor cells.

Synthesis and crystal structures of a bis-(3-hy-droxy-cyclo-hex-2-en-1-one) and two hexa-hydro-quinoline derivatives.

The title compound I, 2,2'-[(2-nitro-phen-yl)methyl-ene]bis-(3-hy-droxy-5,5-di-methyl-cyclo-hex-2-enone), C23H27NO6, features a 1,3-ketone-enol conformation which is stabilized by two intra-molecular hydrogen bonds. The most prominent inter-molecular inter-actions in compound I are C-H⋯O hydrogen bonds, which link mol-ecules into a two-dimensional network parallel to the (001) plane and a chain perpendicular to (11). Both title compounds II, ethyl 4-(4-hy-droxy-3,5-di-meth-oxy-phen-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carb-oxyl-ate, C23H29NO6, and III, ethyl 4-(anthracen-9-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxyl-ate, C29H29NO3, share the same structural features, such as a shallow boat conformation of the di-hydro-pyridine group and an orthogonal aryl group attached to the di-hydro-pyridine. Inter-molecular N-H⋯O bonding is present in the crystal packing of both compound II and III.

Ethyl 5-methyl-3-[11-(pyridin-2-yl)-6,11-di-hydro-6,11-ep-oxy-dibenzo[b,e]oxepin-6-yl]isoxazole-4-carboxylate: a bicyclic acetal from the rearrangement of an anthracenyl isoxazole.

The title compound, C26H20N2O5, is a rearrangement product of an o-pyridinyl anthracenyl isoxazole ester. It features a bicyclic acetal structure, which has two extended almost co-planar ring systems, which subtend a fold angle of 102.17 (5)°. In the crystal, the mol-ecules are closely knitted together through C-H⋯N and C-H⋯O hydrogen bonds and form chains of alternating enanti-omers propagating along the c-axis direction.

Crystal structure of ethyl 4-[4-(di-methyl-amino)-phen-yl]-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxyl-ate.

In the title racemic compound, ethyl 4-(4-di-methyl-amino-phen-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxyl-ate, the common structural features in this type of compound, such as the flat-boat conformation of the 1,4-di-hydro-pyridine (1,4-DHP) ring, the envelope conformation of the fused cyclo-hexa-none, and the substituted phenyl ring at the pseudo-axial position and orthogonal to the 1,4-DHP ring, are present. In the crystal, mol-ecules are linked via N-H⋯O and C-H⋯O hydrogen bonds, forming layers parallel to the (10) plane.

Crystal structure of the major quadruplex formed in the promoter region of the human c-MYC oncogene.

The c-MYC oncogene mediates multiple tumor cell survival pathways and is dysregulated or overexpressed in the majority of human cancers. The NHE III1 region of the c-MYC promoter forms a DNA quadruplex. Stabilization of this structure with small molecules has been shown to reduce expression of c-MYC, and targeting the c-MYC quadruplex has become an emerging strategy for development of antitumor compounds. Previous solution NMR studies of the c-MYC quadruplex have assigned the major conformer and topology of this important target, however, regions outside the G-quartet core were not as well-defined. Here, we report a high-resolution crystal structure (2.35 Å) of the major quadruplex formed in the NHE III1 region of the c-MYC promoter. The crystal structure is in general agreement with the solution NMR structure, however, key differences are observed in the position of nucleotides outside the G-quartet core. The crystal structure provides an alternative model that, along with comparisons to other reported quadruplex crystal structures, will be important to the rational design of selective compounds. This work will aid in development of ligands to target the c-MYC promoter quadruplex with the goal of creating novel anticancer therapies.

AIMing towards improved antitumor efficacy.

Using the structure-activity relationship emerging from previous Letter, and guided by pharmacokinetic properties, new AIMs have been prepared with both improved efficacy against human glioblastoma cells and cell permeability as determined by fluorescent confocal microscopy. We present our first unambiguous evidence for telomeric G4-forming oligonucleotide anisotropy by NMR resulting from direct interaction with AIMs, which is consistent with both our G4 melting studies by CD, and our working hypothesis. Finally, we show that AIMs induce apoptosis in SNB-19 cells.

The effect of bromine scanning around the phenyl group of 4-phenylquinolone derivatives.

Three quinolone compounds were synthesized and crystallized in an effort to study the structure-activity relationship of these calcium-channel antagonists. In all three quinolones, viz. ethyl 4-(4-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, (I), ethyl 4-(3-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, (II), and ethyl 4-(2-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, (III), all C21H24BrNO3, common structural features such as a flat boat conformation of the 1,4-dihydropyridine (1,4-DHP) ring, an envelope conformation of the fused cyclohexanone ring and a bromophenyl ring at the pseudo-axial position and orthogonal to the 1,4-DHP ring are retained. However, due to the different packing interactions in each compound, halogen bonds are observed in (I) and (III). Compound (III) crystallizes with two molecules in the asymmetric unit. All of the prepared derivatives satisfy the basic structural requirements to possess moderate activity as calcium-channel antagonists.

Diethyl 4-(biphenyl-4-yl)-2,6-dimethyl-1,4-di-hydro-pyridine-3,5-di-carboxyl-ate.

The title compound, C25H27NO4, has a flattened di-hydro-pyridine ring. The benzene and phenyl rings are synclinal to one another, forming a dihedral angle of 49.82 (8)°; the axis of the biphenyl rings makes an 81.05 (9)° angle to the plane of the di-hydro-pyridine ring. In the crystal, N-H⋯O hydrogen bonds link the mol-ecules into chain motifs running along the a-axis direction. The chains are cross-linked by C-H⋯O inter-actions, forming sheet motifs running slightly off the (110) plane, together with an intermolecular interaction between head-to tail biphenyl groups, thus making the whole crystal packing a three-dimensional network. Intra-molecular C-H⋯O hydrogen bonds are also observed.

4-isoxazolyl-1,4-dihydropyridines: a tale of two scaffolds.

The association of the isoxazole and dihydropyridine (DHP) ring systems fused at the 4'-isoxazolyl- to the 4-position of the DHP has produced a combination scaffold, the isoxazolyl-DHPs (IDHPs) with unique conformational characteristics. The IDHPs are useful in probing biological activity, as exemplified by our efforts in the fields of voltage gated calcium channel (VGCC) antagonists and inhibitors of the multi-drug resistance (MDR) transporter. A strategically placed methyl group produced a signifcant change at the VGCC, with (R)-(+)-1-phenyl-prop-2-yl (3.7 nM) > phenethyl (22.9 nM) > (S)-(-)-1-phenyl-prop-2-yl (210 nM), a eudismic ratio of 56.7. Branching at the C-5 of the isoxazole produced a 25% increase in MDR binding, and replacing the DHP C-3 ester with a functionalized amide also gave a dramatic increase in binding affinity. Opportunities for combined scaffolds - including examples containing IDHPs - are waiting to be discovered: because new biology is driven by new chemistry.

Ethyl 3-(9-chloro-10-oxo-9,10-di-hydro-anthracen-9-yl)-5-methyl-isoxazole-4-carboxyl-ate.

The asymmetric unit of the title compound, C21H16ClNO4, contains two independent mol-ecules (A and B), each adopting a conformation wherein the isoxazole ring is roughly orthogonal to the anthrone ring. The dihedral angle between the mean plane of the isoxazole (all atoms) and the mean plane of the anthrone (all atoms) is 88.48 (3)° in one mol-ecule and 89.92 (4)° in the other. The ester is almost coplanar with the isoxazole ring, with mean-plane dihedral angles of 2.48 (15) and 8.62 (5)°. In both mol-ecules, the distance between the ester carbonyl O atom and the anthrone ketone C atom is about 3.3 Å. The anthrone ring is virtually planar (r.m.s. deviations of 0.070 and 0.065 Å) and adopts a shallow boat conformation in each mol-ecule, as evidenced by the sum of the six intra-B-ring torsion angles [41.43 (15) and 34.38 (15)° for molecules A and B, respectively]. The closest separation between the benzene moieties of anthrones A and B is 5.1162 (7) Å, with an angle of 57.98 (5)°, consistent with an edge-to-face π-stacking inter-action. In the crystal, weak C-H⋯O and C-H⋯N inter-actions link the mol-ecules, forming a three-dimensional network.

Fluorescent probes of the isoxazole-dihydropyridine scaffold: MDR-1 binding and homology model.

Isoxazole-1,4-dihydropyridines (IDHPs) were tethered to fluorescent moieties using double activation via a lanthanide assisted Weinreb amidation. IDHP-fluorophore conjugate 3c exhibits the highest binding to date for IDHPs at the multidrug-resistance transporter (MDR-1), and IDHP-fluorophore conjugates 3c and 7 distribute selectively in SH-SY5Y cells. A homology model for IDHP binding at MDR-1 is presented which represents our current working hypothesis.

Microwave accelerated synthesis of isoxazole hydrazide inhibitors of the system xc- transporter: Initial homology model.

Microwave accelerated reaction system (MARS) technology provided a good method to obtain selective and open isoxazole ligands that bind to and inhibit the Sxc- antiporter. The MARS provided numerous advantages, including: shorter time, better yield and higher purity of the product. Of the newly synthesized series of isoxazoles the salicyl hydrazide 6 exhibited the highest level of inhibitory activity in the transport assay. A homology model has been developed to summarize the SAR results to date, and provide a working hypothesis for future studies.

3-(1,3-Di-phenyl-propan-2-yl)-4-methyl-6-phenyl-isoxazolo[3,4-d]pyridazin-7(6H)-one.

In the title compound, C27H23N3O2, the geminal benzyl groups branching out from the methine adjacent to the isoxazole group are both syn-oriented to the methyl group of the pyridazinone moiety, as reflected by C-C distances of 3.812 (2) and 4.369 (2) Šbetween the methyl carbon and the nearest ring carbon of each benzyl group. This kind of conformation is retained in CDCl3 solution, as evidenced by distinct phenyl-shielding effects on the (1)H NMR signals of the methyl H atoms. The isoxazolo[3,4-d]pyridazin ring system is virtually planar (r.m.s. deviation from planarity = 0.031 Å), but the N-bonded phenyl group is inclined to the former by an ring-ring angle of 55.05 (3)°. In the crystal, the T-shaped mol-ecules are arranged in an inter-locked fashion, forming rod-like assemblies along [10-1]. The mol-ecules are held together by unremarkable weak C-H⋯N, C-H⋯O and C-H⋯π inter-actions (C-O,N,C > 3.4 A), while significant π-π-stacking inter-actions are absent.