1,2,3-Trimethoxy-5,11-dihydroindolo[1,2-b]isoquinoline-5,11-dione.

The title compound, C15H19NO5, crystallizes in the monoclinic space group P2(1)/c with four molecules in the asymmetric unit, which differ from each other in the orientation of their methoxy groups. Of the three methoxy groups in each molecule, one lies close to the plane of the molecule and the other two have an out-of-plane conformation where they point in opposite directions. In the crystal structure, four different types of pi-stacks are observed and the molecules pack in two different types of stacking sheets, with alternating molecules A and B in one ribbon and alternating molecules C and D in the other. The supramolecular structure is supported by C-H...O and pi-pi interactions.

Effects of a novel dual lipid synthesis inhibitor and its potential utility in treating dyslipidemia and metabolic syndrome.

We have identified a novel omega-hydroxy-alkanedicarboxylic acid, ESP 55016, that favorably alters serum lipid variables in obese female Zucker (fa/fa) rats. ESP 55016 reduced serum non-HDL-cholesterol (non-HDL-C), triglyceride, and nonesterified fatty acid levels while increasing serum HDL-C and beta-hydroxybutyrate levels in a dose-dependent manner. ESP 55016 reduced fasting serum insulin and glucose levels while also suppressing weight gain. In primary rat hepatocytes, ESP 55016 increased the oxidation of [(14)C]palmitate in a dose- and carnitine palmitoyl transferase-I (CPT-I)-dependent manner. Furthermore, in primary rat hepatocytes and in vivo, ESP 55016 inhibited fatty acid and sterol synthesis. The "dual inhibitor" activity of ESP 55016 was unlikely attributable to the activation of the AMP-activated protein kinase (AMPK) pathway because AMPK and acetyl-CoA carboxylase (ACC) phosphorylation states as well as ACC activity were not altered by ESP 55016. Further studies indicated the conversion of ESP 55016 to a CoA derivative in vivo. ESP 55016-CoA markedly inhibited the activity of partially purified ACC. The activity of partially purified HMG-CoA reductase was not altered by the xenobiotic-CoA. These data suggest that ESP 55016-CoA favorably alters lipid metabolism in a model of diabetic dyslipidemia in part by initially inhibiting fatty acid and sterol synthesis plus enhancing the oxidation of fatty acids through the ACC/malonyl-CoA/CPT-I regulatory axis.

Exploration of the DTrp-NMeLys motif in the search for potent somatostatin antagonists.

Previous studies from this laboratory demonstrated that N-methylation at Lys(5) residue in somatostatin octapeptide antagonist analogues increased the GH release inhibition potency by as much as 300%. We have now further investigated N-methylation of this Lys(5) residue in conjunction with a number of N- and C-terminal modifications previously found to give highly potent somatostatin receptor antagonists. Synthetic analogues were tested in a functional assay for their ability to inhibit somatostatin-inhibited GH release from rat pituitary cells in culture and to displace 125I-labeled somatostatin from CHO cells transfected with the five known human somatostatin receptors. Several interesting observations resulted from the study. Replacement of liphophilic Nal(8) at the C-terminus with a hydrophilic His(8) resulted in the increased affinity and selectivity for type 2 receptor to give the most potent antagonist analogue yet discovered (K(i), 1.5 nM), although in the rat pituitary cells inhibitory activity on somatostatin inhibited GH release decreased somewhat. A His(3) substitution within the cyclic portion of the analogues retained pituitary cell potency and affinity for type 2 receptor as did substitution with Bip(8) and Fpa(1). Replacement of Cpa(1) with Iph(1) did not effect the affinity for type 2 receptor significantly, but did decrease the effects on rat cell GH release. Iph(3) within-ring substitution increased the selectivity for sst(2) appreciably although the affinity for that receptor was considerably decreased. Substitution of Npa(3) resulted in good selectivity for sst(2) receptor. Replacement of Nal(8) with D-Trp(8) also increased the selectivity for type 2 receptor. Use of a 'bivalent ligand' approach in which two peptides were joined by 4,4'-biphenyldicarbonyl as a spacer destroyed the affinity for all the subtypes, however, the bivalent ligand formed with the Ahp spacer displayed significant affinity and high selectivity for the type 2 receptor.

Design, synthesis, and biological characterization of bivalent 1-methyl-1,2,5,6-tetrahydropyridyl-1,2,5-thiadiazole derivatives as selective muscarinic agonists.

Selective muscarinic agonists could be useful in the treatment of neurological disorders such as Alzheimer's disease, schizophrenia, and chronic pain. Many muscarinic agonists have been developed, yet most exhibit at best limited functional selectivity for a given receptor subtype perhaps because of the high degree of sequence homology within the putative binding site, which appears to be buried within the transmembrane domains. Bivalent compounds containing essentially two agonist pharmacophores within the same molecule were synthesized and tested for receptor binding affinity and muscarinic agonist activity. A series of bis-1,2,5-thiadiazole derivatives of 1,2,5,6-tetrahydropyridine linked by an alkyloxy moiety exhibited very high affinity (K(i) < 1 nM) and strong agonist activity. The degree of activity depended on the length of the linking alkyl group, which could be replaced by a poly(ethylene glycol) moiety, resulting in improved water solubility, binding affinity, and agonist potency.

Highly potent and subtype selective ligands derived by N-methyl scan of a somatostatin antagonist.

The search for synthetic peptide analogues of somatostatin (SRIF) which exhibit selective affinities for the five known receptor subtypes (sst1-5) has generated a large number of potent agonists. Some of these agonists display good subtype selectivities and affinities for the subtypes 1, 2, 3, and 5, including analogues created by N-methyl amino acid substitutions in a standard octapeptide analogue format. We have now extended this peptide backbone N-methylation approach to a potent somatostatin receptor antagonist series using the antagonist Cpa-cyclo(DCys-Pal-DTrp-Lys-Thr-Cys)-Nal-NH2 9 reported from this laboratory as the lead structure. Synthetic analogues were tested for their ability to inhibit somatostatin-stimulated GH release from rat pituitary cells in culture and to displace 125I-labeled somatostatin from CHO cells transfected with the five known human somatostatin receptors. Several interesting observations resulted from the study. N-Methylation at the Lys(9) residue (5) increased the rat GH release inhibitory potency nearly 4-fold to 0.73 nM but resulted in little change in the binding affinity for human type 2 receptor. This analogue also had a high affinity of 5.98 nM for sst5 receptor (compared to 1.4 nM for somatostatin itself) and is the first antagonist analogue to be reported with high affinity for sst5. It also had high potency on in vitro inhibition of sst5 mediated intracellular calcium mobilization. These results were considered surprising, since the Lys(9) residue has long been considered to constitute the active center of somatostatin, important both for receptor binding and activation, and suggests important conformational differences between D-Cys(9) somatostatin antagonists and normal agonist structures. More modifications were carried out on this analogue with the aim of improving antagonist potency and/or specificity. Tyr(7) substitution of 5 resulted in an analogue, which had the highest affinity in the series for hsst2 (K(I) 5.51 nM) and an extraordinarily low IC50 of 0.53 nM in the rat pituitary cell assay. However, this analogue lost considerable affinity for sst5 relative to analogue 5. Analogue 16 with DTrp(12) at C-terminus had the highest affinity for hsst2, however, the IC50 in the rat GH release assay was only 11.6 nM. Replacement of Lys(9) in 9 with Dab(9) gave 11 which displayed high binding affinity for sst3, and it was also quite selective for that receptor. Both the sst3 and sst5 antagonists should be of value in assigning the physiological roles to type 3 and 5 receptor, respectively.

N-Methyl scan of somatostatin octapeptide agonists produces interesting effects on receptor subtype specificity.

The search for synthetic analogues of somatostatin which exhibit selective affinities for the five receptor subtypes is of considerable basic and therapeutic interest and has generated a large number of potent agonist analogues with a wide spectrum of binding profiles. In the past, conformational restriction of side chain groups and the peptide backbone has yielded the most interesting results. Under the latter category and as part of the present study, we were interested in the potential effects of N-methylation of peptide bond NH groups on binding affinity since this approach had not been systematically examined with these peptides. This was aided by new chemistries for introducing an N-Me group during regular solid-phase peptide synthesis using Boc protection. A number of interesting effects were noted on relative binding affinities of the two series of agonist sequences chosen, DPhe(5)(or Tyr(5))-c[Cys(6)-Phe(7)-DTrp(8)-Lys(9)-Thr(10)-Cys(11)]Thr(12)-NH(2) (SRIF numbering), at the five known human somatostatin receptors transfected into and stably expressed by CHO cells. N-Methylation of residues 7 (Phe), 10 (Thr), 11 (Cys), and 12 (Thr) largely destroyed affinities for all five receptors. N-Methylation of DTrp in the DPhe series gave an analogue with extraordinarily high affinity for the type 5 receptor for which it was also quite selective. N-Methylation of Lys in both series resulted in retention of type 2 affinity despite this residue constituting the "active center" of somatostatin peptides. N-Methylation of either the N-terminal Tyr residue or of Cys(6) in the Tyr series resulted in analogues with extraordinarily high affinity for the type 3 receptor, also with a degree of specificity. N-Methylation of the peptide bond constrains the conformational space of the amino acid and eliminates the possibility of donor hydrogen bond formation from the amide linkage. The beta-bend conformation of the agonists around DTrp-Lys is stabilized by a transannular intramolecular hydrogen bond(s) between Phe(7) and Thr(10) so methylation of these residues eliminates this source of stabilization. It is expected that several of these analogues will provide additional tools for determining some of the physiological roles played by type 3 and 5 somatostatin receptors which are still far from being fully elucidated.

Design and development of selective muscarinic agonists for the treatment of Alzheimer's disease: characterization of tetrahydropyrimidine derivatives and development of new approaches for improved affinity and selectivity for M1 receptors.

Cholinergic neurons degenerate in Alzheimer's disease, resulting in cognitive impairments and memory deficits, and drug development efforts have focused on selective M1 muscarinic agonists. 5-(3-Ethyl-1,2,4- oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102) stimulates M1 muscarinic receptors in rat brain [Messer, W.S., Jr., Abuh, Y.F., Liu, Y., Periyasamy, S., Ngur, D.O., Edgar, M.A., El-Assadi, A.A., Sbeih, S., Dunbar, P.G., Roknich, S., Rho, T., Fang, Z., Ojo, B., Zhang, H., Huzl, J.J., III, Nagy, P.I., 1997a. J. Med. Chem. 40, 1230-1246.] and improves memory function in rats with lesions of the basal forebrain cholinergic system. Moreover, CDD-0102 exhibits oral bioavailability, few side effects and low toxicity, and thus represents a viable candidate for clinical studies. Despite the development of functionally selective agonists such as xanomeline and CDD-0102, there is room for improvements in ligand affinity and selectivity. The high degree of amino acid homology within transmembrane domains has hindered the development of truly selective agonists. Site-directed mutagenesis, biochemical and molecular modeling studies have identified key amino acid residues such as Thr192 and Asn382 in the binding of agonist to M1 receptors [Huang, X.P., Nagy, P.I., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1999. Br. J. Pharmacol. 126, 735-745.]. Recent work has implicated residues at the top of transmembrane domain VI in the binding of muscarinic agonists and activation of M1 receptors [Huang, X.P., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1998. J. Pharmacol. Exp. Ther. 286, 1129-1139.]. Thus, residues such as Ser388 represent molecular targets for the further development of agonists with improved M1 receptor affinity, selectivity and activity.