Neuromuscular blocking agents and spontaneous sympathetic activity.

The action of neuromuscular blocking agents on the spontaneous sympathetic activity has been quantitated. "On line" spectrum analysis has been applied to the action potential of pre- and post ganglionic nerves of the coeliac plexus. The activity, the frequency spectrum and their changes after the injection of clinical and high doses of decamethonium, D-tubocurarine, succinylcholine, gallamine and pancuronium are determined.     

Neuromuscular blocking agents: structure and activity

Correlation of crystal structure analyses and model building of rigid molecules indicate the structural requirements for curariform activity and the structural differences between depolarizing and non-depolarizing neuromuscular blocking agents.     

Synthesis and evaluation of hapalosin and analogs as MDR-reversing agents.

The marine natural product hapalosin and 22 analogs, which incorporated systematic substituent deletions or variations, were prepared. These compounds were evaluated in a cell-based assay for both MDR-reversing activity and general cytotoxicity. Some substituent modifications resulted in lower cytotoxicities, but most structural changes were either detrimental to or did not seriously alter the MDR-reversing activity.     

Discovery of 13-oxa prostaglandin analogs as antiglaucoma agents: Synthesis and biological activity

FP-Class prostaglandin analogs have demonstrated utility for the treatment of glaucoma and ocular hypertension. A series of novel FP prostaglandin analogs was designed to optimize topical ocular activity and reduce ocular side-effects by replacing 13-carbon with oxygen. A facile synthesis was successfully developed for synthesis of the 13-oxa prostaglandins from the commercially available Corey aldehyde benzoate. Among the compounds synthesized, AL-16082 was the most potent prostaglandin FP agonist in vitro. In a prostaglandin FP receptor-linked second-messenger assay, phosphoinositide (PI) turnover, it exhibited a potency value (EC₅₀) of 1.9 nM (78% max. response relative to fluprostenol). The isopropyl ester of AL-16082, compound AL-16049, significantly lowered intraocular pressure (IOP) in the ocular hypertensive monkey eyes by 30%. In the study of acute ocular irritation response in New Zealand albino rabbits, AL-16049 produced lower incidence of hyperemia, swelling, and discharge than PGF_2α (1 μg), and a similar incidence of hyperemia, swelling, and discharge to latanoprost (1.8 μg). AL-16049 also produced no signs of ocular irritation or discomfort in the cat at the doses evaluated.     

Glycyrrhetinic acid and its analogs: A new class of antifilarial agents

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5 μM; IC₅₀: 1.20 μM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a–7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50 μM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC₅₀ values were found to be 8.8 and 2.2 μM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100 mg/kg, s.c. ×5 days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.     

1-substituted tetrahydroisoquinoline analogs: beta adrenoceptor blocking agents in the isolated perfused rabbit heart.

The 1-benzyl and 1-methyl congeners of trimetoquinol were tested for antagonism of receptors which mediate inotropy and chronotropy in the isolated perfused rabbit heart. 1-Benzyltrimetoquinol was found to be a blocker of resting, isoproterenol- and dobutamine-stimulated inotropy at concentrations (10^?7?10^?5M) which did not significantly affect chronotropy ( > 10^?5M). 1-Methyltrimetoquinol was found to be a partial agonist in the resting myocardium, weakly blocking inotropy and chronotropy at doses of 10^?7?10^?5M. At a concentration of 10^?4M, 1-methyltrimetoquinol was an agonist of both chronotropy and inotropy. These stimulatory properties appear to be direct (not affected by prior reserpinization) and antagonized by propranolol. In the isoproterenol-stimulated heart, 1-methyltrimetoquinol was a specific negative inotropic agent at doses (10^?7?10^?5M) above which agonist properties were manifest. At 10^?4M, 1-methyltrimetoquinol acted synergistically with isoproterenol to produce positive inotropy and chronotropy significantly greater than that of isoproterenol alone. Currently, it is believed that the receptors which mediate inotropy and chronotropy are $\text{beta}$ adrenergic in nature. Thus, it would appear that 1-benzyltrimetoquinol is a specific antagonist of those $\text{beta}$-receptors which mediate inotropy, while 1-methyltrimetoquinol is a partial agonist of both inotropic and chronotropic $\text{beta}$-receptors. Further, the response to these compounds does not appear to be proportionate in various regions of the myocardium.     

The natural product CCR5 antagonist anibamine and its analogs as anti-prostate cancer agents

Prostate cancer is a leading cause of death among males in the United States. As the chemokine receptor CCR5 is over-expressed in more aggressive forms of prostate cancer, and is also a critical receptor in inflammation, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents. Anibamine, a natural product CCR5 antagonist, provides a unique molecular scaffold for the generation of novel analogs with possible anti-prostate cancer activity. A series of analogs of anibamine were designed, synthesized and tested against several prostate cancer cell lines. The analogs all acted as CCR5 antagonists at micromolar range affinity to the receptor while their anti-proliferative activity varied depending on the cell line type and their chemical structural properties. Further basal cytotoxicity characterization on these compounds indicated some of them may be suitable for in vivo studies.     

Structure-activity studies of oxazolidinone analogs as RNA-binding agents

We have synthesized and tested a series of novel 3,4,5-tri- and 4,5-disubstituted oxazolidinones for their ability to bind two structurally related T box antiterminator model RNAs. We have found that optimal binding selectivity is found in a small group of 4,5-disubstituted oxazolidinones.     

Antitumor agents. 271: Total synthesis and evaluation of brazilein and analogs as anti-inflammatory and cytotoxic agents

The first total synthesis of the naturally occurring tetracyclic homoisoflavonoid brazilein (1) and 14 new analogs (1a–n) is reported. Target compounds and intermediates were assayed for anti-inflammatory effects on superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB, and for cytotoxic activity against nasopharyngeal (KB), vincristine-resistant nasopharyngeal (KBvin), lung (A549) and prostate (DU-145) human cancer cell lines. The most active compound 1b showed potent effects on superoxide anion generation and elastase release with IC₅₀ values of 1.2 and 1.9 μM, respectively, and was 65 times more potent than phenylmethylsulfonyl fluoride (PMSF), the positive control, in the latter assay. Additionally, 1b exhibited broad spectrum in vitro anticancer activity with IC₅₀ values of 6–11 μM against the four tested cancer cell lines.     

Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

A DNA sequence cleaved by restriction endonuclease R.EcoRI in only one strand.

Restriction endonuclease EcoRI cuts both strands of the DNA sequence

generating two separate frayed ends (Hedgpeth et al., 1972). Here it is shown that under standard digestion conditions, the enzyme also attacks the sequence

but cuts only one strand. The resulting nick is an efficient initiation point for DNA synthesis by Escherichia coli DNA polymerase I, allowing the selective labelling of one strand of the DNA duplex.In buffers of low molarity and high pH (8.5), EcoRI cleaves sequences with the form

(Polisky et al., 1975). Thus it seems that under both sets of conditions the enzyme recognises the four-base-pair core sequence

and that its ability to cleave different adjacent phosphodiester bonds varies with pH and ionic strength.     

A rapid procedure for purification of EcoRI endonuclease.

A convenient and rapid procedure has been developed to purify restriction endonuclease Eco RI. The method involves sonication of cells at low ionic strength, precipitation of the endonuclease with Polymin P (a polyethyleneimine), elution of the enzyme from the Polymin P precipitate, ammonium sulfate precipitation and chromatography on phosphocellulose. The purified restriction endonuclease is free of exonuclease and other endonucleases.     

Determination of lecithin:cholesterol acyltransfer in mouse plasma and the influence of mercaptoethanol and sulphydryl blocking agents on its activity.

1. The cholesterol esterifying activity in mouse plasma has been identified as lecithin:cholesterol acyltransferase (LCAT) on the basis of stoichiometric data, predominant transfer of polyunsaturated fatty acids, wide pH optimum and inhibition of esterification by phospholipase A2 and sulphydryl blocking agents. The esterifying activity differed from that present in plasma of man, rat and other species since it was partially inhibited by mercaptoethanol and other thiols. 2. Stoichiometric correlations between unesterified cholesterol, lecithin and lysolecithin were not exact, suggesting possible involvement of other enzymes in the overall esterification process during in vitro incubation of mouse plasma. 3. The initial rate of cholesterol esterification was determined by in vitro incubation of mouse plasma, whose cholesterol had been labelled by prior in vivo injection of 3H-mevalonic acid. The mean rate was 281 +/- 74 nmol/ml/hr (mean +/- S.D., n = 12) and correlated with unesterified cholesterol concentration (r = 0.73, P less than 0.01).