Design and synthesis of novel delta opioid receptor agonists and their pharmacologies

We re-examined the accessory site of the 4,5-epoxymorphinan skeleton by camdas conformational analysis in an effort to deign novel δ opioid receptor antagonists. We synthesized three novel compounds (SN-11, 23 and 28) with a 10-methylene bridge and without a 4,5-epoxy ring. Among them, compounds SN-23 (17-isobutyl derivative) and SN-28 (17-methyl derivative) showed very strong agonist activity (over 10 times more than TAN-67). SN-28 also showed high δ selectivity. The δ agonist activity of SN-23 was weaker than that of SN-28, but in terms of the δ selectivity, SN-23 was higher than that of SN-28. These unexpected results indicated that the 4,5-epoxy ring, but not the 10-methylene bridge, was an accessory site in δ opioid receptor agonists.     

Essential structure of opioid κ receptor agonist nalfurafine for binding to the κ receptor 2: synthesis of decahydro(iminoethano)phenanthrene derivatives and their pharmacologies

To clarify the essential structures of an opioid κ receptor selective agonist, nalfurafine, for binding to the κ receptor, we designed and synthesized some nalfurafine derivatives and the decahydro(iminoethano)phenanthrene derivatives with a cyclohexene moiety as a surrogate for the phenol ring. In addition to the 6-amide side chain and the 17-nitrogen substituted by a cyclopropylmethyl group, the 4,5-epoxy ring, phenolic hydroxy group, and angular hydroxy group played important roles in eliciting the binding properties of nalfurafine but these three moieties were not indispensable for binding to the κ receptor. Moreover, the phenol ring was also not essential for the binding to the κ receptor, and the cyclohexene moiety would play an important role in fixing the conformation of decahydro(iminoethano)phenanthrene derivatives to effectively raise the amide side chain, rendering a conformation that resembled the active one of nalfurafine.     

Essential structure of orexin 1 receptor antagonist YNT-707, Part I: Role of the 4,5-epoxy ring for binding with orexin 1 receptor

The essential structure of the orexin 1 receptor (OX₁R) antagonist YNT-707 (2) was clarified, particularly the roles to OX₁R antagonist activities of the 3-OMe, the 4,5-epoxy ring, the 14-hydroxy group, and the orientation of the 6-amide side chain.The 3-OMe and 17-sulfonamide group were shown to be essential for the OX₁R antagonistic activity. The 4,5-epoxy ring plays an important role for the active orientation of the 6-amide group. The 14-hydroxy group could lower the activity of the 6β-amide isomer by the interaction of the 14-hydroxy group with the 6-amide group, which could orient the 6-amide group toward the upper side of the C-ring.Finally, we proposed the difference in the active conformation between OX₁R and κ opioid receptor (KOR), especially in the orientation of the 6-amide group which is expected to be a useful guide for medicinal chemists to design OX₁R ligands.     

Synthesis and anticancer activities of ageladine A and structural analogs

A series of ageladine A analogs that include 2-aminoimidazo[4,5-c]azepines (seven-membered rings) and 2-amino-3H-imidazo[4,5-c]pyridine (six-membered rings) derivatives were synthesized and evaluated for their anticancer effects against several human cancer cell lines and MMP-2 inhibition in vitro. Only compounds possessing the aromatic azepine (seven-membered ring) core showed anticancer activity with IC₅₀ values in the low micromolar range.     

Synthesis of pyrrolomorphinan derivatives as κ opioid agonists

We synthesized pyrrolomorphinan derivatives 6, 7, and 9 to examine whether the pyrrole ring would be an accessory site in the κ opioid receptor selective antagonist, nor-binaltorphimine. Derivative 6 had an α,β-unsaturated ketone substituent that strongly bound to the κ receptor. The compound with the highest κ receptor selectivity, 6e, produced a dose-dependent antinociceptive effect in the mouse acetic acid writhing test. However, derivatives 7 and 9, which did not have α,β-unsaturated ketone substituents, showed less κ receptor selectivity than compound 6. Based on structure–activity relationships, we proposed that these compounds adopted active structures for κ selective agonist activity. The pyrrole ring would not function as an accessory site, but the ability of the side chain on the pyrrole ring to localize above the C-ring appeared to confer κ selective agonist activity. These results will promote the design of novel κ agonists.     

Essential structure of orexin 1 receptor antagonist YNT-707, Part IV: The role of D-ring in 4,5-epoxymorphinan on the orexin 1 receptor antagonistic activity

The orexin 1 receptor (OX₁R) antagonists carrying a morphinan skeleton such as YNT-707 (2) and YNT-1310 (3) showed potent and extremely high selective antagonistic activity against OX₁R. In the course of our study of the essential structure of YNT-707 for high binding affinity against OX₁R, we prepared derivatives of 2 without the D- and 4,5-epoxy rings to clarify the roles of these structural determinants toward OX₁R antagonistic activity. The D- and 4,5-epoxy rings played important roles for the active orientation of the 17-sulfonamide and 6-amide side chains. Finally, we identified the simple structure required for selective OX₁R antagonistic activity in the complex morphinan skeleton, which is expected to be a useful scaffold for further design of OX₁R ligands.     

2-Aryl-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols as a class of antitumor agents selectively active in securin−/− cells

A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitumor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure–activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was comparable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher metabolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against MCF-7 and MDA-MB-361 lines and was selected for further evaluation.     

The molecular and crystal structures of 4-N-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparagine trihydrate and 4-N-(β-d-glucopyranosyl)-l-asparagine monohydrate. The X-ray analysis of a carbohydrate–peptide linkage

X-ray analyses have shown that the glucopyranose rings of GlcNAc-Asn [4-N-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-l-asparagine] and Glc-Asn [4-N-(beta-d-glucopyranosyl)-l-asparagine] both have the C-1 chair conformation and also that the glucose-asparagine linkage of each molecule is present in the beta-anomeric configuration. The dimensions (the estimated standard deviations of the last digit are in parentheses) of the glycosidic bond in GlcNAc-Asn and Glc-Asn are, respectively, C((1))-N((1)) 0.1441(6)nm, 0.146(2)nm; angle O((5))-C((1))-N((1)) 106.8(3) degrees , 105.7(8) degrees ; angle C((2))-C((1))-N((1)) 111.1(4) degrees , 110.4(9) degrees ; angle C((1))-N((1))-C((9)) 121.4(4) degrees , 120.5(9) degrees . The glycosidic torsion angle C((9))-N((1))-C((1))-C((2)) is 141.0 degrees and 157.6 degrees in GlcNAc-Asn and Glc-Asn respectively. Hydrogen-bonding is extensive in these two crystal structures and does affect one torsion angle in particular. Two very different values of chi(1)(N-C(alpha)-C(beta)-C(gamma)) occur for the asparagine residue of the two different molecules; the values of chi(1), -69.0 degrees in GlcNAc-Asn and 61.9 degrees in Glc-Asn, correspond to two different staggered conformations about the C(alpha)-C(beta) bond as the NH(3) (+) group is adjusted to different hydrogen-bonding patterns. The two trans-peptide groups in GlcNAc-Asn show small distortions in planarity whereas that in Glc-Asn is more non-planar. The mean plane through the atoms of the amide group at C((2)) in GlcNAc-Asn is approximately perpendicular (69 degrees ) to the mean plane through the C((2)), C((3)), C((5)) and O((5)) atoms of the glucose ring and that at C((1)) is less perpendicular (65 degrees ). The mean plane through the atoms of the amide group in Glc-Asn makes an angle of only 55 degrees with the mean plane through these same four atoms of the glucose ring. The N((1))-H bond of the amide at C((1)) is trans to the C((1))-H bond in these two compounds; the N((2))-H bond of the amide at C((2)) is trans to the C((2))-H bond in GlcNAc-Asn. The values of the observed and final calculated structure amplitudes have been deposited as Supplementary Publication SUP 50035 (26 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Synthesis and cytotoxicity of allobetulin derivatives

A variety of oxygen-, nitrogen-, sulfur-, and platinum-containing allobetulin derivatives, including those with different positions of double bonds in rings A and B, the penta- and hexacyclic ring A, and the 21-acetyl-20,28-epoxy-18α,19βH-ursanoisomeric cycle E, have been synthesized, and the screening of their antineoplastic activity in vitro (cytotoxicity) has been carried out. A significant cytotoxic activity was exhibited by (3R,5R)-19β,28-epoxy-4,5-seco-18α-olean-3(5)-ozonide toward MeWo melanoma cells and by 2,3-indolo-21β-acetyl-20β,28-epoxy-18α,19βH-ursane toward SR leukosis cells. The 3S,5S-diastereoisomer of the former compound showed no cytotoxicity.     

An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels

Slow inactivation of Kv1 channels involves conformational changes near the selectivity filter. We examine such changes in Shaker channels lacking fast inactivation by considering the consequences of mutating two residues, T449 just external to the selectivity filter and V438 in the pore helix near the bottom of the selectivity filter. Single mutant T449F channels with the native V438 inactivate very slowly, and the canonical foot-in-the-door effect of extracellular tetraethylammonium (TEA) is not only absent, but the time course of slow inactivation is accelerated by TEA. The V438A mutation dramatically speeds inactivation in T449F channels, and TEA slows inactivation exactly as predicted by the foot-in-the-door model. We propose that TEA has this effect on V438A/T449F channels because the V438A mutation produces allosteric consequences within the selectivity filter and may reorient the aromatic ring at position 449. We investigated the possibility that the blocker promotes the collapse of the outer vestibule (spring-in-the-door) in single mutant T449F channels by an electrostatic attraction between a cationic TEA and the quadrupole moments of the four aromatic rings. To test this idea, we used in vivo nonsense suppression to serially fluorinate the introduced aromatic ring at the 449 position, a manipulation that withdraws electrons from the aromatic face with little effect on the shape, net charge, or hydrophobicity of the aromatic ring. Progressive fluorination causes monotonically enhanced rates of inactivation. In further agreement with our working hypothesis, increasing fluorination of the aromatic gradually transforms the TEA effect from spring-in-the-door to foot-in-the-door. We further substantiate our electrostatic hypothesis by quantum mechanical calculations.     

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing.     

Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3β-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRβ activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50?ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRβ-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.     

Crystal Structure of a Tightly Bound Inhibitor of Adenosine Transport N6-(4-Nitrobenzyl)-β-D-2′-Deoxyadenosine Methanol Solvate,C17H18N6O5.1/2 CH3OH

Abstract

The molecular structure of N⁶-(4-nitrobenzyl)-β-D-2′-deoxyadenosine (I) has been determined by single crystal X-ray diffraction. A potent inhibitor of adenosine permeation in cultured S49 mouse lymphoma cells, I binds tightly (K_D 2.4 nM) to high affinity membrane sites present on the nucleoside transporter elements of these cells. Compound I crystallizes in the trigonal space group P3₂21 with unit cell dimensions a = b = 8.0009(9)Å, c = 49.174(8)Å, and Z = 6. The structure was solved by direct methods and refined by least-squares to a final R = 0.038. The mean plane of the 4-nitrobenzyl group, an important substituent for potent nucleoside transport inhibition in a series of S6-substituted 6-thioinosine derivatives, is inclined at an angle of 120.6° to the plane of the adenine ring. The torsion angles around the methylene carbon atom of this benzyl group are C(6)-N(6)-C(10)-C(11), 96.6° and N(6)-C(10)-C(11)-C(12), 93.6°. The glycosidic torsion angle, X, is 217.1° which corresponds to the common anti nucleoside conformation. The deoxyribose ring, however, has the unusual C(1′)-exo conformation, with C(1′) displaced 0.608Å from the plane of C(2′), C(3′), C(4′) and O(4′). The conformation about the exocyclic C(4′)-C(5′) bond is gauche⁺.     

Synthesis and X-ray structures of sulfate esters of fructose and its isopropylidene derivatives. Part 1: 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose 1-sulfate and 4,5-O-isopropylidene-beta-D-fructopyranose 1-sulfate

2,3:4,5-Di-O-isopropylidene-beta-D-fructopyranose 1-sulfate have been synthesized by treatment of 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose with pyridine-sulfur trioxide complex. Direct hydrolysis of the isopropylidene group at C-4, C-5 gave 2,3-O-isopropylidene-beta-D-fructopyranose 1-sulfate. The crystal and molecular structures of ammonium (1a) and potassium (1b) salts of diisopropylidene derivative and ammonium (2) salt of monoisopropylidene derivative were determined by X-ray crystallography. Data for 1a and 1b were collected in 120 K and in 150 K for 2. All salts crystallized in P2(1)2(1)2(1) space group. There are three independent anions in asymmetric unit in 1b. Pyranose rings in the diisopropylidene derivative salts studied adopt 2S(0) twist boat conformation, whereas in the monoisopropylidene exists in a slightly distorted chair conformation (4C(1)). A staggered conformation is preferred by the sulfate group as indicated by values of C-(ester)-S-O(terminal) torsion angles: -173.2(4) degrees in 1a, 175.1(6) degrees in anion A of 1b, 170.8(6) degrees in anion C of 1b and 177.9(2) degrees in 2. However, strong interactions such as potassium-oxygen and H-bonds may affect the geometry: in anion B of 1b the value of the torsion angle is 139.4(6) degrees.     

Crystal structure of formycin 5'-phosphate: an explanation for its tight binding to AMP nucleosidase

Formycin 5'-monophosphate (FMP) is a strong competitive inhibitor of AMP nucleosidase with Km/Kis from 1200 to 2600 depending on the source of the enzyme. The crystal structure of FMP has been determined in order to understand the basis for its high affinity for AMP nucleosidases and other biological properties. The key structural features of FMP are (1) the base is the N(7)-H tautomer, (2) the N(3) of the base forms an intramolecular hydrogen bond to the phosphate oxygen O(1), (3) the glycosyl torsion angle is syn with O(4')-C(1') relative to C(9)-C(4) being -6.43 degrees, and (4) the furanose ring pucker is C(3')-endo, with a pseudorotation angle of 20.3 degrees. The major difference between the AMP and FMP structures is that the glycosyl torsion angles differ by 190 degrees. The computed conformational energy necessary to distort AMP so that it has the same glycosyl torsion angle as FMP is 4.6 kcal/mol. This corresponds to a 2100-fold difference in binding energy, in good agreement with the observed interaction between AMP nucleosidase and FMP.     

Synthesis of 6,14-epoxymorphinan derivatives and their pharmacologies

A novel 6,14-epoxymorphinan benzamide derivative (NS22) that was previously reported showed opioid κ receptor agonistic activity and analgesic activity. The unsatisfactory κ selectivity of NS22 led us to synthesize its derivatives to improve the opioid κ receptor selectivity and the agonist activity. In the course of SAR of the various derivatives, 17-benzyl-6,14-epoxymorphinan derivatives (KNT-33, 53, 55, 80, 90, 133) were found to show high selectivities and affinities for the opioid κ receptor. In addition, KNT-33, 53, 55 showed dose-dependent analgesic effects in acetic acid writhing tests. Therefore, 17-benzyl substituents may play an important role for developing κ selectivity.     

Changing of the cell division axes drives vulva evolution in nematodes

Vulval epithelial tubes invaginate through concerted cell migration, ring formation, stacking of rings and intra-ring cell fusion in the nematodes Caenorhabditis elegans, Oscheius tipulae and Pristionchus pacificus. The number of rings forming the invaginations is invariantly seven, six, and eight, respectively. We hypothesize that each ring is formed from pairs of symmetrically positioned primordial vulval cells following three premises: If the final cell division is left-right, the daughters will fuse, migrate and form only one ring. If these cells do not divide, one ring will form. If the final division is anterior-posterior, two rings will form. We test the ring hypothesis and found coincidence between the patterns of vulva cell divisions and the number of rings for 12 species. We find heterochronic variations in the timing of division, migration and fusion of the vulval cells between species. We report a unique ring-independent pathway of vulva formation in Panagrellus redivivus. C. elegans lin-11(n389) mutation results in cell fate transformations including changes in the orientation of vulval cell division. lin-11 animals have an additional ring, as predicted by the ring hypothesis. We propose that the genetic pathway determining how vulval cells invaginate evolves through ring-dependent and ring-independent mechanisms.     

The two-conformer hypothesis: 2,3,4,6-tetra-O-methyl-mannopyranosyl and -glucopyranosyl oxacarbenium ions

Computational chemistry can give information about the probable conformations of reactive intermediates that are difficult to determine experimentally. Based on density functional theory (DFT) calculations of tetra-O-methyl-D-mannopyranosyl and -glucopyranosyl oxacarbenium ions, two families of conformations, which we call B0 and B1, were found. For the manno configuration, a 4H3 and 3E almost isoenergetic pair were found, whereas for the gluco-configuration a 4H3 and 5S1 pair favouring 4H3 were calculated. These results corroborate earlier results and suggest that this two or more conformer hypothesis is general. Nucleophilic attack on these pairs of cations was modelled with methanol and led to four cases to consider namely alpha- or beta-attack on B0 or B1. The resulting complexes (G0, G1 and F0, F1) demonstrate facial selectivity. The relative energies of these complexes are dominated by intramolecular hydrogen bonding and the conformational consequences to the pyranose ring of changes in the C-5-O-5-C-1-C-2 torsion angle. Constrained variation of the nucleophilic oxygen (methanol) to C-1 distance shows that these ion dipole complexes are the only minima with this constraint.     

DFT study of the interaction of cytidine and 2′-deoxycytidine with Li, Na, and K: effects of metal cationization on sugar puckering and stability of the N-glycosidic bond

Density functional theory (DFT) calculations were performed at the B3LYP level with a 6-311++G(d,p) basis set to systematically explore the geometrical multiplicity and binding strength for complexes formed by Li⁺, Na⁺, and K⁺ with cytidine and 2′-deoxycytidine. All computational studies indicate that the metal ion affinity (MIA) decreases from Li⁺ to Na⁺ and K⁺ for cytosine nucleosides. For example, for cytidine the affinity for the above metal ions are 79.5, 55.2, and 41.8 and for 2′-deoxycytidine, 82.8, 57.4, and 42.2 kcal/mol, respectively. It is also interesting to mention that linear correlations between calculated MIA values and the atomic numbers (Z) of the above metal ions were found. The influence of metal cationization on the coordination modes and the strength of the N-glycosidic bond in cytosine nucleosides have been studied. In all cases, the N1-C1′ bond distance changes upon introducing a positive charge in the nucleosides. It has been found that metal binding significantly changes the values of the phase angle of pseudorotation P in the sugar unit of these nucleosides. With respect to the sugar ring, metal binding changes the values of the glycosyl torsion angle and sugar ring conformation. The present calculations in the gas phase provide the first clues on the intrinsic chemistry of these systems and may be of value for studies of the influence of metal cations on the conformational behavior and function of nucleic acids.     

Dissection and Modulation of the Four Distinct Activities of Nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation

Nisin A is a pentacyclic antibiotic peptide produced by various Lactococcus lactis strains. Nisin displays four different activities: (i) it autoinduces its own synthesis; (ii) it inhibits the growth of target bacteria by membrane pore formation; (iii) it inhibits bacterial growth by interfering with cell wall synthesis; and, in addition, (iv) it inhibits the outgrowth of spores. Here we investigate the structural requirements and relevance of the N-terminal thioether rings of nisin by randomization of the ring A and B positions. The data demonstrate that: (i) mutation of ring A results in variants with enhanced activity and a modulated spectrum of target cells; (ii) for the cell growth-inhibiting activity of nisin, ring A is rather promiscuous with respect to its amino acid composition, whereas the bulky amino acid residues in ring B abolish antimicrobial activity; (iii) C-terminally truncated nisin A mutants lacking rings D and E retain significant antimicrobial activity but are unable to permeabilize the target membrane; (iv) the dehydroalanine in ring A is not essential for the inhibition of the outgrowth of Bacillus cells; (v) some ring A mutants have significant antimicrobial activities but have decreased autoinducing activities; (vi) the opening of ring B eliminates antimicrobial activity while retaining autoinducing activity; and (vii) some ring A mutants escape the nisin immune system(s) and are toxic to the nisin-producing strain NZ9700. These data demonstrate that the various activities of nisin can be engineered independently and provide a basis for the design and synthesis of tailor-made analogs with desired activities.