Methyl analogues of the experimental Alzheimer drug phenserine: synthesis and structure/activity relationships for acetyl- and butyrylcholinesterase inhibitory action.

With the goal of developing potential Alzheimer's pharmacotherapeutics, we have synthesized a series of novel analogues of the potent anticholinesterases phenserine (2) and physostigmine (1). These derivatives contain methyl (3, 4, 6), dimethyl (5, 7, 8, 10, 11) and trimethyl (14) substituents in each position of the phenyl group of the phenylcarbamoyl moieties, and with N-methyl and 6-methyl substituents (12, 13, 31, 33). We also quantified the inhibitory action of these compounds against human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). An analysis of the structure/anticholinesterase activity relationship of the described compounds, together with molecular modeling, confirmed the catalytic triad mechanism of the binding of this class of carabamate analogues within AChE and BChE and defined structural requirements for their differential inhibition.

Phenserine regulates translation of beta -amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development.

The reduction in levels of the potentially toxic amyloid-beta peptide (Abeta) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Abeta precursor protein (betaAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces betaAPP levels in vivo. Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in betaAPP processing. Phenserine treatment resulted in decreased secretion of soluble betaAPP and Abeta into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of betaAPP protein expression by phenserine was posttranscriptional as it suppressed betaAPP protein expression without altering betaAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5'-mRNA leader sequence of betaAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Abeta levels by regulating betaAPP translation via the recently described iron regulatory element in the 5'-untranslated region of betaAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of betaAPP expression that can result in a substantial reduction in the level of Abeta.

Mapping the binding site of colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354.

2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with beta-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of beta-tubulin in protofilaments (Nogales, E. , Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203), we found two potential binding sites. At one, entirely encompassed within beta-tubulin, the C2- and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the alpha/beta interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.

Anti-AIDS agents part 41: synthesis and anti-HIV activity of 3',4'-di-o-(-)-camphanoyl-(+)-cis-khellactone (DCK) lactam analogues.

DCK lactam analogues were synthesized and evaluated for anti-HIV activity against HIV-1 replication in H9 lymphocyte cells. 4-Methyl-DCK lactam (11a) exhibited potent anti-HIV activity with EC50 and therapeutic index values of 0.00024 microM and 119,333, respectively.

Antitumor agents. 199. Three-dimensional quantitative structure-activity relationship study of the colchicine binding site ligands using comparative molecular field analysis.

Inhibitors of tubulin polymerization interacting at the colchicine binding site are potential anticancer agents. We have been involved in the synthesis of a number of colchicine site agents, such as thiocolchicinoids and allocolchicinoids, which are colchicine analogues, and 2-phenyl-quinolones and 2-aryl-naphthyridinones, which are the amino analogues of cytotoxic antimitotic flavonoids. The most cytotoxic of the latter compounds strongly inhibit binding of radiolabeled colchicine to tubulin, and these agents therefore probably bind in the colchicine site of tubulin. We have applied conventional CoMFA and q(2)-GRS CoMFA to identify the essential structural requirements for increasing the ability of these compounds to form tubulin complexes. The CoMFA model for the training set of 51 compounds yielded cross-validated R(2) (q(2)) values of 0.637 for conventional CoMFA and 0.692 for q(2)-GRS CoMFA. The predictive power of this model was confirmed by successful activity prediction for a test set of 53 compounds with known potencies as inhibitors of tubulin polymerization. The activities of 88% of the compounds were predicted with absolute value of residuals of less than 0.5. The predictive q(2) values were 0.546 for conventional CoMFA and 0.426 for q(2)-GRS CoMFA. The conventional CoMFA model with the highest predictive q(2) (0.546) was analyzed in detail in terms of underlying structure-activity relationships.

The experimental Alzheimer drug phenserine: preclinical pharmacokinetics and pharmacodynamics.

Phenserine, a phenylcarbamate of physostigmine, is a new potent and highly selective acetylcholinesterase (AChE) inhibitor, with a > 50-fold activity versus butyrylcholinesterase (BChE), in clinical trials for the treatment of Alzheimer's disease (AD). Compared to physostigmine and tacrine, it is less toxic and robustly enhances cognition in animal models. To determine the time-dependent effects of phenserine on cholinergic function, AChE activity, brain and plasma drug levels and brain extracellular acetylcholine (ACh) concentrations were measured in rats before and after phenserine administration. Additionally, its maximum tolerated dose, compared to physostigmine and tacrine, was determined. Following i.v. dosing, brain drug levels were 10-fold higher than those achieved in plasma, peaked within 5 min and rapidly declined with half-lives of 8.5 and 12.6 min, respectively. In contrast, a high (> 70%) and long-lasting inhibition of AChE was achieved (half-life > 8.25 h). A comparison between the time-dependent plasma AChE inhibition achieved after similar oral and i.v. doses provided an estimate of oral bioavailability of 100%. Striatal, in vivo microdialysis in conscious, freely-moving phenserine-treated rats demonstrated > 3-fold rise in brain ACh levels. Phenserine thus is rapidly absorbed and cleared from the body, but produces a long-lasting stimulation of brain cholinergic function at well tolerated doses and hence has superior properties as a drug candidate for AD. It selectively inhibits AChE, minimizing potential BChE side effects. Its long duration of action, coupled with its short pharmacokinetic half-life, reduces dosing frequency, decreases body drug exposure and minimizes the dependence of drug action on the individual variations of drug metabolism commonly found in the elderly.

Synthesis of novel phenserine-based-selective inhibitors of butyrylcholinesterase for Alzheimer's disease.

Four novel analogues (8-11) of cymserine (2) were synthesized by methods similar to those recently developed for the total syntheses of N8-norphenserine (Yu, Q. S.; et al. J. Med. Chem. 1997, 40, 2895-2901) and N1,N8-bisnorphenserine (Yu, Q. S.; et al. J. Med. Chem. 1998, 41, 2371-2379). As our structure-activity studies predicted, these compounds are highly potent and selective inhibitors of human butyrylcholinesterase (BChE) and will test the novel hypothesis that BChE inhibitors are useful in the treatment of Alzheimer's disease. In a similar manner, the same modifications that provided BChE selectivity were applied to the acetylcholinesterase (AChE)-selective inhibitor, tolserine (5), to provide the novel tolserine analogues 12-15. As predicted, these modifications altered the AChE-selective action of tolserine (5) to favor a lack of cholinesterase enzyme subtype selectivity.

Asymmetric solid-phase synthesis of (3'R,4'R)-di-O-cis-acyl 3-carboxyl khellactones.

[formula: see text] We describe a practical parallel synthesis of (3'R,4'R)-di-O-cis-acyl 3-carboxyl khellactones on a solid phase in high yield. The highlights of this synthesis include a Knoevenagel condensation, asymmetric dihydroxylation, catalyzed acylation, and product cleavage from the solid support.

Antitumor agents. 185. Synthesis and biological evaluation of tridemethylthiocolchicine analogues as novel topoisomerase II inhibitors.

Several 1,2,3-tridemethyldeacetylthiocolchicine derivatives have been synthesized and evaluated for cytotoxic activity against various human tumor cell lines and for their inhibitory effects on DNA topoisomerases in vitro. Exhaustive demethylation of thiocolchicine analogues completely changes their biological profiles. Instead of displaying antitubulin activity, most target compounds inhibited topoisomerase II activity. Only compounds with a larger side chain, such as 15a, 23a, and 24a, did not interfere with topoisomerase II enzymatic functions. The cytotoxicity of target compounds was reduced by 3 orders of magnitude compared to that of colchicine in most cell lines. The hydrophilicity of phenolic compounds might prevent drug passage through the cell plasma membrane and, thus, be responsible for the relatively weak cytotoxicity. To test this hypothesis, 27-30 were prepared from 16a by protecting all hydroxy groups with esters with an aim to facilitate drug transportation. In vitro cytotoxicity assays indicated that 27 was more potent than its parent compound in all tested tumor cell lines and showed tissue selective cytotoxicity with a significant inhibitory effect against KB cells (IC50 = 2.7 microg/mL). Therefore, we propose that 27 acts as a prodrug, liberating 16a to exert its antitopoisomerase activity and, finally, to cause cell death.

Syntheses and anticholinesterase activities of (3aS)-N1, N8-bisnorphenserine, (3aS)-N1,N8-bisnorphysostigmine, their antipodal isomers, and other potential metabolites of phenserine.

Hydrolysis of the carbamate side chains in phenserine [(-)1] and physostigmine [(-)2] yields the metabolite (-)-eseroline (3), and the red dye rubreserine (4) on air oxidation of the former compound. Both compounds lacked anticholinesterase activity in concentrations up to 30 mM, which would be unachievable in vivo. A second group of potential metabolites of 1 and 2 are the N1,N8-bisnorcarbamates (-)9 and (-)10, prepared from (3aS)-N8-benzylnoresermethole (-)12 by the carbinolamine route. These entirely novel compounds proved to be highly potent inhibitors of acetylcholinesterase [(-)9] and of acetyl- and butyrylcholinesterase (AChE and BChE) [(-)10], respectively. To elucidate further the structure/anticholinesterase activity relationship of the described compounds, the antipodal isomers (3aR)-N1,N8-bisnorcarbamates (+)9 and (+)10 were likewise synthesized from (3aR)-N8-benzylnoresermethole (+)12 and assessed. The compounds possessed moderate but less potent anticholinesterase activity, with the same selectivity as their 3aS enantiomers. Finally, the anticholinesterase activities of intermediates N1, N8-bisnorbenzylcarbamates (-)18, (-) 19, (+)18, and (+)19, also novel compounds, were additionally measured. The 3aS enantiomers proved to be potent and selective inhibitors of BChE, particularly (-)19, whereas the antipodal isomers lacked activity.

Antitumor agents. Part 186: Synthesis and biological evaluation of demethylcolchiceinamide analogues as cytotoxic DNA topoisomerase II inhibitors.

Demethylation of colchiceinamide (2) and its analogues (3-10) afforded a novel class of mammalian DNA topoisomerase II inhibitors (2a-10a) without displaying tubulin inhibitory activity. All target compounds inhibited the catalytic activity of topoisomerase II at drug concentrations at 100 microM. An in vitro cytotoxicity assay indicated that compounds 3a and 8a were strong and tissue-selective cytotoxic agents against the MCF-7 breast cancer cell line (IC50 = 0.36 and 0.48 microgram/mL, respectively) and the CAKI-1 renal cancer cell line (IC50 = 0.72 and 0.96 microgram/mL, respectively).

Total syntheses and anticholinesterase activities of (3aS)-N(8)-norphysostigmine, (3aS)-N(8)-norphenserine, their antipodal isomers, and other N(8)-substituted analogues.

N(8)-Benzylesermethole (6) was prepared from 5-methoxytryptamine (1) in five steps. Resolution of compound 6 by dibenzoyl- and ditoluyltartaric acid provided enantiomers (-)- and (+)-7. After demethylation, reaction with isocyanates and catalytic debenzylation over hydrogen, the total syntheses of (-)- and (+)-N(8)-norphysostigmine [(-)- and (+)-11] and (-)- and (+)-N(8)-norphenserine [(-)- and (+)-12] were accomplished, (-)-N(8)-Norphysostigmine [(-)-11] and (-)-N(8)-norphenserine [(-)-12] were also obtained by transformations of natural physostigmine [(-)-13] and phenserine [(-)-14] prepared from (-)-13. The absolute configurations and optical purity of compounds (-)-11, (-)-12, (+)-11, and (+)-12 were confirmed by a comparison of their optical rotations with those of the compounds synthesized from physostigmine [(-)-13]. The anticholinesterase activities of N(8)-nor- and N(8)-substituted analogues, (-)- and (+)-9, -10, -11, -12, 15, and 16, were compared with those of physostigmine [(-)- and (+)-13] and phenserine [(-)- and (+)-14] and are reported.

Antitumor agents. 172. Synthesis and biological evaluation of novel deacetamidothiocolchicin-7-ols and ester analogs as antitubulin agents.

A series of novel 7-O-substituted deacetamidothiocolchicine derivatives has been synthesized and evaluated for their inhibitory activity against tubulin polymerization, the binding of [3H]-colchicine to tubulin, and the growth of human Burkitt lymphoma cells. Of these new derivatives, thiocolchicone (8), wherein an acetamido group in thiocolchicine is replaced by a carbonyl oxygen at C(7), was obtained from deacetythiocolchicine (6) by Schiffs base equilibration and acid hydrolysis. Reduction of thiocolchiocone with sodium borohydride yielded the racemic alcohol 9, the structure of which was verified by X-ray crystallographic analysis. Optically pure alcohols 9a,b were obtained by treatment of 9 with the optically pure reagent (1S)-(-)-camphanic chloride followed by chromatographic separation of the camphanate esters and hydrolysis of the diastereomers. X-ray crystallographic analysis established the aS,7S-configuration of 9a. Racemic and optically active esters 11-15, 11a,b, 12a, 14a, and 15a were obtained by esterification of the corresponding alcohols. The compounds showing activity equivalent to or greater than (-)-thiocolchicione (2a) in all the biological assays were three (-)-aS,7S optically pure enantiomers: the alcohol 9a, the acetate 11a (an oxygen isostere of thiocolchicine), and the isonicotinoate 15a. In addition, the ketone 8 and two (-)-aS,7S enantiomers (12a, 14a) had high activity in the biochemical assays with tubulin but reduced antiproliferative activity. In all cases, optically pure isomers with the (-)-aS,7S configuration exhibited greater biological activity than racemic mixtures or isomers or isomers with the (+)-aR,7R configuration.

Antitumor agents--CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis.

(+)-Thiocolchicine (2b) was prepared from (+/-)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (-)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (-)-camphanylated diastereomer, had potent activity in all assays comparable to that of (-)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.

Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine.

The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 A. using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[14C] Carbonyl)-3- demethylthiocolchicine ([14C]3CTC). This beta-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)- 3-demethythiocolchicine ([14C]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radio-labeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [14C]3CTC and beta-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 A of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 A distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.

Maze learning in aged rats is enhanced by phenserine, a novel anticholinesterase.

A new generation of cholinesterase inhibitors is expected to overcome some limitations of the therapeutic use of anticholinesterases. Phenserine is a long-acting and selective inhibitor of acetylcholinesterase with a preferential brain uptake. We have assessed the effects of chronic phenserine tartrate treatment on performance of aged Fischer-344 rats in the 14-unit T-maze. Phenserine (1-3 mg kg-1, i.p.) treatment for 5 days significantly reduced the number of errors made in the Stone maze. Other performance variables were also improved. No side effects were noted across 5 days treatment at doses of 1-2 mg kg-1. Phenserine can therefore improve the performance of aged rats in this complex maze task without producing obvious side effects.

The nitrogen of the acetamido group of colchicine modulates P-glycoprotein-mediated multidrug resistance.

The substituents of drug molecules and the specific amino acid residues of P-glycoprotein (P-gp) implicated in drug/protein interactions are largely unknown. We have used a series of colchicine analogs modified on the A, B, and C rings to identify the discrete chemical groups on the colchicine molecule that are required for recognition by P-gp. For this, the toxicity of these analogs was tested on independent cell clones expressing either of the two mouse mdr genes, mdr1 and mdr3, known to confer multidrug resistance. Modifications of the methoxy groups on the A and C rings modulated cellular toxicity but had no effect on P-gp recognition; however, modifications at the C7 position of the B ring, in particular the removal of the nitrogen atom of the acetamido group, had a dramatic effect. Analogs bearing a hydrogen at that position were not substrates for P-gp. The importance of the nitrogen at C7 was independently verified in thiocolchicine and allocolchicine analogs similarly modified, although overall levels of resistance to these compounds were somewhat reduced compared to their colchicine counterparts. The study of allocolchicine congeners bearing a six-carbon C ring and of two other analogs completely lacking a B ring suggested that intact B and C rings were important for interaction with P-gp. These results suggest that the structural determinants for cytotoxicity (tubulin binding) and P-gp recognition map to nonoverlapping sites in the colchicine analogs analyzed. Examination of calculated molar refractivities (CMR) revealed that only compounds showing CMR values greater than 9.7 were P-gp substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mescaline and some of its analogs on cholinergic neuromuscular transmission.

Mescaline (3,4,5-trimethoxyphenylethylamine; MES) and its analogs, anhalinine (ANH) and methylenemescaline trimer (MMT) were investigated, using sciatic-sartorius preparations of the frog and cortical tissue from the rat. The effects of MES and its analogs were examined with respect to muscle twitch, resting membrane potential and nicotinic receptor binding. Mescaline and its analogs (10-100 microM) blocked both directly and neurally evoked twitches but their effects on neurally evoked twitches were greater than those on directly evoked twitches. Mescaline, ANH and MMT decreased amplitude of the miniature endplate and endplate potentials, decreased acetylcholine (ACh) quantal content, hyperpolarized the resting membrane potential and prolonged duration of the action potential. They did not significantly displace the binding of [125I]-alpha-bungarotoxin (alpha-BTX) to nicotinic receptors, at concentrations which blocked neuromuscular transmission. These results suggest that MES and its analogs inhibit cholinergic neuromuscular transmission by blocking release of ACh; they also affect K+ conductance.