Design, synthesis, and structure-activity relationship studies of himbacine derived muscarinic receptor antagonists

A parallel synthesis of racemic himbacine analogs was carried out by N-alkylation of various commercially available cyclic amine derivatives with the alkylating agent 4 which bears the tricyclic unit of himbacine. Several of these analogs have potency comparable to that of himbacine, albeit lacking the desired selectivity. Structure-activity relationship studies support the existence of a hydrophobic pocket in the receptor where the piperidine ring of dihydrohimbacine binds.     

Himbacine derived thrombin receptor (PAR-1) antagonists: structure-activity relationship of the lactone ring

The structure-activity relationship (SAR) of the lactone ring of himbacine derived thrombin receptor (PAR-1) antagonists (e.g., 2-5) is described. The effect of the lactone carbonyl group on binding to PAR-1 is dependent on the substitution pattern of the pyridine ring. A stereoselective intramolecular Michael addition reaction to the vinyl pyridine group was observed for these pyridine analogs of himbacine in basic conditions at elevated temperature.     

Discovery of nor-seco himbacine analogs as thrombin receptor antagonists

Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in c. monkey.     

Inhibitory effects of Mannich bases of heterocyclic chalcones on NO production by activated RAW 264.7 macrophages and superoxide anion generation and elastase release by activated human neutrophils

Some chalcones exert potent anti-inflammatory activities. Mannich bases of heterocyclic chalcones inhibited nitric oxide (NO) production in lipopolysaccharide and interferon-γ stimulated RAW 264.7 macrophages. Also Formyl-Met-Leu-Phe and cytochalasin B induced superoxide anion generation (O2·-) and elastase release in human neutrophils. Mannich bases of heterocyclic chalcone analogs exhibited potent inhibitory effects on NO production with IC(50) values ranges between 10.5 and 0.018 μM, O2·- generation (IC(50) 39.87-0.68 μM) and elastase release (IC(50) 39.74-0.95 μM). Compound 29 (IC(50) 0.055 μM) and 34 (IC(50) 0.018 μM) were showed excellent inhibition on NO production. On the other hand, compounds 2 and 8 showed potent inhibition on O2·- generation and elastase release. Therefore, these four compounds may be new leads for development of anti-inflammatory activities. The structure-activity relationships are also discussed.     

On the "activation" of cytokinins.

A number of cytokinin analogs containing modifications in the heterocyclic moiety were prepared. These compounds were tested for activity as cytokinins and anticytokinins in the tabacco bioassay and the results were used to determine whether any position(s) of the heterocyclic nucleus of cytokinins may require derivatization as part of an over-all "activation" process. 3-substituted 4-alkylaminopyrazolo [3,4-d]pyrimidines and 4-alkylaminopyrrolo[2,3-d]pyrimidines, for example, have (substituted) carbon rather than nitrogen atoms at positions 3 and 5, respectively (analogous to position 7 in purines) and would be predicted to be metabolically stable at these positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable position, and by extension at position 7 in cytokinin analogues which are purines, is not a prerequisite for the expression of cytokinin activity. Similar consideration of other heterocyclic analogs which have cytokinin activity suggests that the active form of a cytokinin can be the exogenous compound itself. Certain structural analogs of cytokinins were found to inhibit the growth of tobacco callus promoted by 6-(3-methyl-2-butenylamino)purine. These compounds were studied as potential cytokinin antagonists, i.e. having activity analogous to the 7-alkylamino-3-methylpyrazolo[4,3-d]pyrimidines (Hecht, S. M., 2068-2610; Skoog, F., Schmitz, R.Y., Hecht, S.M., and Bock, R. M. (1973) Phytochemistry 12, 25-37). The activity of these compounds is discussed and criteria are proposed to distinguish between those species which are specific anticytokinins and those which otherwise inhibit growth.     

Synthetic studies on himbacine,a potent antagonist of the muscarinic M2 subtype receptor. Part 2: synthesis and muscarinic M2 subtype antagonistic activity of the novel himbacine congeners modified at the C-3 position of lactone moiety

With an aim to disclose the convergency and flexibility of our previously explored synthetic route to natural himbacine 1, and moreover, to clarify some novel aspects of the structure-activity relationships of 1, we prepared various structural types of novel himbacine congeners, 3-demethylhimbacine (3-norhimbacine) 2 and 4-epi-3-demethylhimbacine (4-epi-3-norhimbacine) 4-epi-2 and their enantiomers (ent-2 and ent-4-epi-2), 11-methylhimbacine 3, and 3-epihimbacine 4 in optically pure forms by employing our methodology. All of the synthesized congeners correspond to the compounds modified at the C-3 position of gamma-lactone moiety involved in 1. Among these congeners, 3-demethylhimbacine (3-norhimbacine) 2 was found to exhibit more potent muscarinic M(2) receptor binding affinity than natural 1.     

Himbacine derived thrombin receptor (PAR-1) antagonists: SAR of the pyridine ring

The structure-activity relationship (SAR) of the vinyl pyridine region of himbacine derived thrombin receptor (PAR-1) antagonists is described. A 2-vinylpyridyl ring substituted with an aryl or a heteroaryl group at the 5-position showed the best overall PAR-1 affinity and pharmacokinetic properties. One of the newly discovered analogs bearing a 5-(3-pyridyl) substituent showed excellent PAR-1 affinity (Ki = 22 nM) and oral activity with reduced ClogP and improved off-target selectivity compared to an earlier development candidate.     

Pharmacological parameters of muscarinic cholinoreceptors in skeletal muscles

It has been shown that bath application of muscarine delayed the early post-denervation depolarization in the muscle fibers incubated for 3 h in culture medium. The greatest reduction of the post-devervation depolarization was observed with 50 nmol/l muscarine. Atropine, a muscarinic antagonist, clozapine, a specific inhibitor of M1/M5-cholinergic receptors, and nitrocaramiphen, a M1-antagonist, completely removed the hyperpolarizing effect of muscarine. 4-DAMP, a specific inhibitor of M3-cholinergic receptors, himbacine, an antagonist of M2-cholinergic receptors, and tropicamide, a specific inhibitor of M2/M4-cholinergic receptors, failed to prevent the effect of muscarine. A M1/M2 muscarine agonists propargyl and but-2-ynyl esters of arecaidine had apparent muscarine-like effect. Nitrocaramiphen, and not himbacine, prevented the hyperpolarizing effect of these cholinomimetics. It is concluded that muscarine and esters of arecaidine delay the development of early postdenervation depolarization in M1-cholinergic receptors of skeletal muscle.     

Synthesis and muscarinic M(2) subtype antagonistic activity of enantiomeric pairs of 3-demethylhimbacine (3-norhimbacine) and its C(4)-epimer

In the course of our studies of the structure-activity relationships of himbacine 1, a potent antagonist of the M(2) subtype of muscarinic receptor, the four title compounds, 2, ent-2, 3, and ent-3, were synthesized with a highly stereoselective intermolecular Diels-Alder reaction of tetrahydroisobenzofuran 4 with achiral furan-2(5H)-one 5 as a key step, followed by simultaneous optical resolution and epimer separation of the racemic intermediates. Among these compounds, 3-demethylhimbacine (3-norhimbacine) 2, bearing an absolute configuration corresponding to that of 1, was found to show more potent muscarinic M(2) subtype receptor binding activity than natural 1.     

Prejunctional muscarinic receptors regulating neurotransmitter release in airways

Prejunctional pA2 values of five muscarinic antagonists were determined in the guinea-pig trachea under stimulation conditions in which the antagonists alone did not enhance acetylcholine release. The antagonists were partly selective at M1 (pirenzepine), M2 (AQ-RA 741, himbacine) and M3 receptors (hexahydrosiladifenidol, dicyclomine). The profile of the antagonist affinities was different from that obtained at cardiac M2 receptors but resembled the profile reported in the literature for the cloned m4 receptor. This suggests that autoinhibition of acetylcholine release in the trachea is mediated via M4 receptors.     

2-Benzoyl-6-benzylidenecyclohexanone analogs as potent dual inhibitors of acetylcholinesterase and butyrylcholinesterase

In the present study, a series of 2-benzoyl-6-benzylidenecyclohexanone analogs have been synthesized and evaluated for their anti-cholinesterase activity. Among the forty-one analogs, four compounds (38, 39, 40 and 41) have been identified as lead compounds due to their highest inhibition on both AChE and BChE activities. Compounds 39 and 40 in particular exhibited highest inhibition on both AChE and BChE with IC₅₀ values of 1.6 μM and 0.6 μM, respectively. Further structure–activity relationship study suggested that presence of a long-chain heterocyclic in one of the rings played a critical role in the dual enzymes’ inhibition. The Lineweaver–Burk plots and docking results suggest that both compounds could simultaneously bind to the PAS and CAS regions of the enzyme. ADMET analysis further confirmed the therapeutic potential of both compounds based upon their high BBB-penetrating. Thus, 2-benzoyl-6-benzylidenecyclohexanone containing long-chain heterocyclic amine analogs represent a new class of cholinesterase inhibitor, which deserve further investigation for their development into therapeutic agents for cognitive diseases such as Alzheimer.     

Probing of contacts between EcoRII DNA methyltransferase and DNA using substrate analogs and molecular modeling

To determine the molecular mechanism of DNA recognition and catalysis by EcoRII DNA-methyltransferase (M.EcoRII) binding and methylation by the enzyme of 14-mer substrate analogs containing 2-aminopurine or 1',2'-dideoxy-D-ribofuranose in the M.EcoRII recognition site have been studied. Efficiencies of methylation and DNA binding affinities depend on the location of modified nucleoside residues within the M.EcoRII recognition site. A structural model of M.EcoRII in complex with substrate DNA and cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was built using the previously solved structures of Hhal and HaeIII DNA-methyltransferases as templates. The model was constructed according to the recently developed "Frankenstein's monster" approach. Based on the model, amino acid residues taking part in interactions with DNA were predicted. Besides, based on both theoretical and experimental data obtained the groups of atoms of the heterocyclic bases within the M.EcoRII recognition site presumably involved in interaction with the enzyme were proposed.     

Synthesis of a series of residue 1 (pyroglutamic acid) analogs of thyrotrophin releasing hormone.

This report describes the synthesis of 23 analogs of thyrotrophin releasing hormone, L-pyroglutamic acid-L-histidyl-L-prolineamide, where only the pyroglutamic acid moiety is modified. Twelve of the analogs contain different heterocyclic rings or are derivatized pyrrolidone rings. The syntheses of these pyroglutamic acid analogs are also described. Peptide bond formation was generally achieved by catalyzing carbodiimide coupling with 1-hydroxybenzotriazole. The histidine side chain was protected with the 2,4-dinitrophenyl group during di- and tripeptide synthesis and was removed with benzenethiol Final purification of tripeptides involved passage of synthetic material over a Dowex 1--X4 (bicarbonate) column. The route for synthesis of thyrotrophin releasing hormone and its analogs is highly efficient, since the native hormone may be obtained with 90% efficiency.     

Antimuscarinic potency and functional selectivity of oxotremorine analogs at muscarinic receptor subtypes in the rat.

The potency of six antimuscarinic oxotremorine analogs at sympathetic ganglionic M1 and brainstem M2 muscarinic receptors was compared in the rat. Inhibition of the pressor effects of McNA343 or physostigmine was used as functional indicators of M1 and M2 blockade, respectively. 50% inhibitory doses (ID50) were calculated against both cholinomimetics. Of the analogs, PCA-10 was the most potent, with ID50 values of 0.16 and 0.11 mumol/kg versus McNA343 and physostigmine, respectively. A correlation analysis indicated that these analogs did not functionally discriminate between responses mediated by neuronal M1 or M2 muscarinic receptors in vivo. In contrast, these analogs antagonized M1 ganglionic responses at doses which produced negligible antagonism at cardiac and vascular M2 receptors.     

Synthesis and affinity studies of himbacine derived muscarinic receptor antagonists

A series of himbacine (1)-related analogues has been prepared featuring three different isomeric configurations with respect to the B-ring (a, b and natural c) and three different interconnecting two-carbon unsaturated units [natural (E)-ene, (Z)-ene, and yne]. The study of the binding affinities of the nine resulting compounds, including synthetic (+)-himbacine (3c), towards the M(1)-M(4) muscarine receptor subtypes revealed that analogues 3a and 5c display a promising 10-fold selectivity for the M(2) receptor as compared to the M(1) receptor.     

Substrate properties of C'-methylnucleoside triphosphates in a reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase

2 theta-C-methyl substituted and phosphonate analogs of UTP were prepared and together with the synthesized earlier 3'-C-methyl-UTP were investigated in the RNA synthesis reaction catalysed by Escherichia coli RNA-polymerase. Substrate properties of UTP analogs were studied in the presence of all natural triphosphates, in the absence of UTP and under conditions of soil substrate reaction. It was shown that UTP(3'CH3) is incorporated into the RNA chain and terminates further RNA elongation. Another analog UTP (2'CH3) may substitute natural UTP in RNA synthesis, but the effectivity of its incorporation is diminished. Phosphonate analog UTP(5'CH2) is a pseudoterminator of RNA synthesis. The conformational analysis of 2'- and 3'C-methylnucleosides by force-field method of calculation was carried out in order to find energetically forbidden conformations of these analogs due to the collision of bulky methyl group and a heterocyclic base. An attempt was made to fix the conformation of the substrate during its enzymatic transformation.     

Design and structure-activity relationship of heterocyclic analogs of 4-amino-3-benzimidazol-2-ylhydroquinolin-2-ones as inhibitors of receptor tyrosine kinases

Herein are described a series of novel heterocyclic analogs of the 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one scaffold. These compounds are potent inhibitors of receptor tyrosine kinases and exhibit favorable pharmacokinetic profiles. The synthesis and SAR of these compounds are described.     

Synthesis and SAR of novel conformationally restricted oxazolidinones possessing gram-positive and fastidious gram-negative antibacterial activity. Part 2: Amino substitutions on heterocyclic D-ring system

A novel series of conformationally restricted oxazolidinones was synthesized, in which the heterocyclic D ring was substituted with various amino groups. Several analogs exhibited potent activity against both gram-positive and fastidious gram-negative organisms. Certain amino-substituted analogs also exhibited improved aqueous solubility compared to the corresponding un-substituted heterocyclic D-ring analogs.     

Synthesis,characterization, and anti-melanoma activity of tetra-O-substituted analogs of nordihydroguaiaretic acid

Synthesis of seven semi-synthetic analogs of NDGA is described. An approach to NDGA derivatization is described in which the ortho-phenolic groups are tethered together by one atom, forming a 5-membered heterocyclic ring. The analogs were evaluated for cytotoxicity in four cancer cell lines and compared to NDGA and tetra-O-methyl-NDGA (M4N) (1a). NDGA bis-cyclic sulfate (2a), NDGA bis-cyclic carbonate (2b), and methylenedioxyphenyl-NDGA (2d) and NDGA tetra acetate (1b) showed anti-cancer activity in vitro. Two compounds, (1b) and (2b), were evaluated for anticancer activity in a mouse xenograft model of human melanoma and showed dose-dependent activity.     

Studies on adenosine A2a receptor antagonists: comparison of three core heterocycles

Piperazine and (R)-2-(aminomethyl)pyrrolidine derivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine have recently been shown to be potent and selective adenosine A(2a) receptor antagonists. We have replaced the triazolotriazine core structure with two different heterocyclic cores. One of these, the one deriving from [1,2,4]triazolo[1,5-c]pyrimidine, appears to be particularly effective and selected analogs from this series have been shown to be orally active in a mouse catalepsy model of Parkinson's disease.