In an effect to develop for more selective neuronal nicotinic acetylcholine receptor analgesics that have less toxicity and adverse side effects relative to epibatidine, three new classes of epibatidine analogues were synthesized and evaluated in vitro as potential potent selective nAChR ligands. Specifically, three analogues of epibatidine were synthesized to explore the structure-activity relationships of epibatidine relative to neuromuscular blocking activity as well as nAChRs. Both quaternary epibatidine analogues 2 and bis-epibatidine derivative 3 exhibited high binding affinity relative to nicotine. In addition, a new series of 2-(hydroxyalkylpyridyl)-7-azabicyclo[2.2.1]heptane derivatives were synthesized and evaluated as potential ligands for nicotinic acetylcholine receptors. Moreover, two rigid 2-acetoxy-7-azabicyclo[2.2.1]heptane analogues have been prepared to study the binding conformation of acetylcholine at the active sites of the nicotinic acetylcholine receptors.

In an effort to search for a more selective, less toxic neuronal nicotinic acetylcholine receptor analgesic agent in comparison to epibatidine, a series of analogs with hybrid structures of epibatidine and ABT-594 were designed and synthesized. The 1-(pyridyloxymethyl)-7-azabicyclo[2.2.1]heptane ring systems were furnished via an intramolecular cyclization from a trans 1, 4 disubstitituted amino-cyclohexane derivative. The functionalized cyclohexane ring was formed via a [4+2] Diels-Alder cyclization reaction between the acetamidoacrylate and Danishefsky's diene. These 1- (pyridyloxymethyl)-7-azabicyclo[2.2.1]heptane ring systems were then tested in vitro as potential á4â2 nicotinic acetylcholine receptor ligands with high potency and selectivity. In addition, a series of rigid acetylcholine analogs were synthesized from cocaine to study the conformation of acetylcholine, the endogenous neurotransmitter at the nicotinic acetylcholine receptor. A stereoselective reduction of 2-tropinone led to the enantioselective synthesis of the desired acetoxytropane systems. These compounds were also tested in in vivo models for binding affinity and efficacy responses. Anabasamine, an alkaloid isolated from the Central Asian shrub, Anabasis aphylla, was synthesized for the first time. It was targeted due to interesting preliminary biological activity such as exhibiting anticholinesterase activity, anti-inflammatory activity, and facilitated an increase in hepatic alcohol dehydrogenase levels. Only preliminary studies were performed as anabasamine is limited in quantity due to its difficult isolation. A versatile synthetic methodology was developed for the synthesis of anabasamine and related nicotine analogs. This new methodology employed a pyridyl anion addition to valerolactone, for anabasamine, or butyrolactone for the nicotine analog, to afford 5-hydroxy-1-(6-methoxy-pyridin-3-yl)-pentan-1-one or 4-hydroxy-1-(6- methoxy-pyridin-3-yl)-butan-1-one, respectively. A reductive amination provided the piperidine ring moiety and a Suzuki coupling reaction introduced the bipyridyl moiety to anabasamine in five steps and 23% overall yield. In addition, this methodology was applied successfully to the synthesis of nicotine and other related analogs. In particular the synthesis of 6-methoxynicotine, a useful drug intermediate, was generated improving the yield from 16% over five steps to 54% over three steps.

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Sulfoxide-ligand exchange (SLE) and asymmetric halogen-metal exchange (AHME) processes were separately examined for the enantioselective synthesis of functionalized alpha-haloalkylmetal (carbenoid) reagents. Carbenoids derived from SLE were used to effect stereospecific reagent-controlled homologation (StReCH) of boronic esters and those generated via AHME were engaged in Darzens-type chemistry with aldehydes. Abstract for Part 1. Scalemic syn alpha-chloroalkylsulfoxides p-TolS(O)CHClR [R = allyl, (1,3-dioxolan-2-yl)methyl, proparygyl, and 2-(benzyloxy)ethyl] were prepared from the corresponding thioethers by Jackson-Ellman-Bolm catalytic enantioselective sulfoxidation [cat. VO(acac)₂, tert-leucinol derived chiral Schiff base ligand, aq. H₂O₂, CHCl₃; 76-80% yield, >98% ee] followed by non-racemizing chlorination mediated by N-chlorosuccinimide in the presence of potassium carbonate (84-86% yield, syn:anti ≥ 20:1). The corresponding anti diastereoisomers were accessed from their syn epimers by sodium hexamethyldisilazide mediated deprotonation (THF, –78 °C) followed by treatment with either CH₃OH or CD₃OD to yield alpha-[¹H] or alpha-[²H] isotopomers, respectively (88% yield, anti:syn ≥ 17:1). Allyl and (1,3- dioxan-2-yl)methyl substituted chlorosulfoxides reacted with R'Li (t-BuLi or PhLi, THF, –78 °C) to give the expected products of SLE [p-TolS(O)R' and LiCHClR or LiCDClR]; however, neither the benzylether nor propargyl substituted substrates gave wholly satisfactory results under the same reaction conditions. The functionalized carbenoid reagents so obtained, 1-chloro-3-butenyllithium and 1-chloro-2-(1,3- dioxolan-2-yl)ethyllithium, were applied to the StReCH of B-(2-chloropyrid-5-yl) pinacol boronate but only the latter gave acceptable yields of chain extended products. The anti alpha-[²H]-chlorosulfoxide dioxolanyl bearing carbenoid precursor gave superior results to the analogous syn or anti alpha-[¹H]-chlorosulfoxides for StReCH of the B-pyridyl boronate [79% conversion, ≥ 89% ee (99% stereofidelity), vs. ≤ 68% conversion for non-deuterated chlorosulfoxides]. The origin of this isotope effect was traced to a deleterious proton transfer pathway between the alpha- chloroalkyllithium reagent and its chlorosulfoxide precursor. Sequential double iterative StReCH of B-(2-chloropyrid-5-yl) pinacol boronate with two separate portions of (S)-1-[²H]-1-chloro-2-(1,3-dioxolan-2-yl)ethyllithium (generated via SLE with phenyllithium) followed by oxidative work-up (with KOOH) gave (1R,2R)-1,2- [²H]₂-2-(2-chloropyrid-5-yl)-1,2-bis[(1,3-dioxolan-2-yl)methyl]ethanol (40% yield, ≥ 98% ee, dr = 85:15). Substitution of the (R)-configured carbenoid for its antipode in the second StReCH stage above gave the unlike (1S,2R)-isomer of the same pyridylethanol derivative (49% yield, ≥ 98% ee, dr = 79:21). The unlike diastereoisomer was advanced to the trifluoroacetamide of (1R,2R)-1,2-[2H]2-1- amino-2-(2-chloropyrid-5-yl)cyclohex-4-ene (6 steps, 5% overall yield); the non- deuterated isotopomer of this compound was previously advanced to the analgesic alkaloid (–)-epibatidine by Corey and co-workers. Abstract for Part 2. Scalemic planar chiral N,N-dialkyl 2-iodoferrocene carboxamides envisioned as recyclable precursors to ferrocenyl metal reagents for AHME, were prepared from ferrocene carboxylic acid by a three step sequence of: acid chloride formation [(COCl)₂ and cat. DMF)], aminolysis (with R₂NH, R = Me, Et, i-Pr; 65- 80% yield over 2 steps), and sec-butyllithium/(–)-sparteine mediated enantioselective directed ortho-metallation (DoM) followed by iodinolysis (87% yield, ≥ 96% ee). Attempts to access more elaborate 5-substituted 2-iodoferrocene carboxamides via DoM/iodinolysis of ortho-substituted ferrocene carboxamides (Me, Ph, or SiMe₃ substituents) mostly failed; however, analogous trisubstituted ferrocene oxazolines could be synthesized. Treatment of N,N-diisopropyl 2-iodoferrocene carboxamide (298, ≥ 96% ee) with n-BuLi (THF, –78 °C) resulted in complete conversion to the corresponding lithioferrocene (327) via I/Li interchange; subsequent iodinolysis initiated reverse Li/I exchange and returned iodoferrocene 298 without diminished enantiomeric excess, establishing configurational stability for the lithiated ferrocene intermediate. Prochiral (RCHI₂) and racemic (RCHICl) geminal dihalide substrates for AHME studies were prepared by electrophilic quench of dihalomethylsodiums with either Ph(CH₂)₃I or Me₃SiCl (50-78% yield). Of the four dihalides so produced, only prochiral substrate Me₃SiCHI₂ engaged in I/Li exchange with scalemic lithioferrocene 327 resulting in regeneration of its precursor iodoferrocene 298 and the formation of a putative chiral carbenoid Me₃SiCHLiI. Trapping of the carbenoid with aldehydes RCHO (R = Ph, 4-MeOC₆H₄, Ph(CH₂)₂, c-C₆H₁₁) in the presence of Me₂AlCl gave the expected epoxysilane products (35-40% yield, cis:trans ≥ 2:1) but without discernable enantiomeric excess. Hypotheses to account for the apparent lack of stereoinduction in this AHME cycle are presented. Comparable experiments using analogous magnesiated ferrocenes failed to produce putative carbenoid species from the same set of geminal dihalide substrates. / Graduation date: 2012

Os receptores nicotínicos são encontrados no Sistema Nervoso Central e exercem papel na modulação sináptica, na plasticidade neuronal e em diversas funções cognitivas. A diversidade dos receptores nicotínicos associada ao seu possível envolvimento em diferentes processos fisiológicos e patológicos vem sendo amplamente estudado. Vários estudos demonstraram que a administração de agonistas nicotínicos por via sistêmica, intratecal ou intracerebroventricular causa antinocicepção, sendo que este efeito parece depender da ativação de centros supraespinais. O presente estudo examinou o efeito da injeção sistêmica, da microinjção no núcleo pretectal anterior (NPtA) e na substância cinzenta periaquedutal ventrolateral (SCPvl) da epibatidina, um agonista de receptores nicotínicos neuronais, sobre a alodínia mecânica induzida por ligadura dos troncos dos nervos espinais L5 e L6. Também se avaliou o efeito da microinjeção de atropina ou mecamilamina sobre o efeito antinociceptivo induzido pela microinjeção de epibatidina na SCPvl e o efeito da mecamilamina sobre o efeito antialodínico observado após injeção sistêmica da epibatidina. Inicialmente se observou que a ligadura dos troncos L5 e L6 dos nervos espinais induz alodínia mecânica já no 2º dia após cirurgia, mantendo-se constante até pelo menos o 28º dia após a ligadura. Nenhuma alteração no limiar de resposta a estímulos mecânicos foi observada nos animais sham ou na pata contralateral. A injeção sistêmica (3,0 g/kg, i.p.) de epibatinida induz efeito antialodínico, porém essa mesma dose não altera o desempenho dos animais no teste do rota-rod. Além disso, a microinjeção na SCPvl, mas não no NPtA (0,3 g/0,3 L) de epibatidina induziu efeito antialodínico. Mecamilamina microinjetada na SCPvl aboliu o efeito antinociceptivo induzido pela epibatidina microinjetada neste mesmo núcleo e o efeito da epibatidina administrada por via sistêmica. Estes resultados permitem concluir que a injeção sistêmica e central de epibatidina induz efeito antialodínico no modelo de dor neuropática e esse efeito depende da interação dessa substância com receptores nicotínicos localizados na SCPvl. / Nicotinic receptors are found in the central nervous system (CNS) and have an important function in modulating neural synaptic, CNS plasticity and many others cognite functions. A variety of subtypes nicotinic receptores are related with phisiological and pathological process. Recentely, studies have been demostrating that nicotinic agonists injected either systemically ou centrally have an antinociceptive effetc. The present study examined; 1) the effect of epibatidene injected systemically and centrally into the anterior pretectal nucleus (APtN) and into the ventro lateral periaqueductal gray (vlPAG) on the mechanical allodinia induzed by ligation of trunk lombar (L5-L6) spinal nerve; 2) the effect of microinjection of nicotinic antagonist (atropine and mecamylamine) on the antinocicptive effect induzed by epibetidine microinjection into the vlPAG. 3) the effect of mecamylamine on the antiallodinic effect, observed after sistemic administration of epibatadine. It was demostrated that the ligation of trunk lombar (L5-L6) spinal nerve produce mechanical allodinia after two days pos-operative and it was maintained for 28 days. Shan group and the contralataeral paw had no change in the threshold response. Systemic injection of epibatidine (3,0 g/kg, i.p.) induzed antiallodinic effect, without affect motor function. Central injection of epibatidine into the vlPAG (0,3 g/0,3 L) also had an antiallodinic effect, but no effect on APtN was observed. Mecamylamine injected into the vlPAG abolish the antiallodinic effect of epibatidine injected systemic and centrally (vlPAG). These results shows that the epibatidine injected either systemically or into the vlPAG produced antiallodinic effect on the neuropatic pain model and also infer that this antiallodinic effect produced by epibatidine requires the interation of epibatidae with vlPAG nicotinic receptors.

Os receptores nicotínicos são encontrados no Sistema Nervoso Central e exercem papel na modulação sináptica, na plasticidade neuronal e em diversas funções cognitivas. A diversidade dos receptores nicotínicos associada ao seu possível envolvimento em diferentes processos fisiológicos e patológicos vem sendo amplamente estudado. Vários estudos demonstraram que a administração de agonistas nicotínicos por via sistêmica, intratecal ou intracerebroventricular causa antinocicepção, sendo que este efeito parece depender da ativação de centros supraespinais. O presente estudo examinou o efeito da injeção sistêmica, da microinjção no núcleo pretectal anterior (NPtA) e na substância cinzenta periaquedutal ventrolateral (SCPvl) da epibatidina, um agonista de receptores nicotínicos neuronais, sobre a alodínia mecânica induzida por ligadura dos troncos dos nervos espinais L5 e L6. Também se avaliou o efeito da microinjeção de atropina ou mecamilamina sobre o efeito antinociceptivo induzido pela microinjeção de epibatidina na SCPvl e o efeito da mecamilamina sobre o efeito antialodínico observado após injeção sistêmica da epibatidina. Inicialmente se observou que a ligadura dos troncos L5 e L6 dos nervos espinais induz alodínia mecânica já no 2º dia após cirurgia, mantendo-se constante até pelo menos o 28º dia após a ligadura. Nenhuma alteração no limiar de resposta a estímulos mecânicos foi observada nos animais sham ou na pata contralateral. A injeção sistêmica (3,0 g/kg, i.p.) de epibatinida induz efeito antialodínico, porém essa mesma dose não altera o desempenho dos animais no teste do rota-rod. Além disso, a microinjeção na SCPvl, mas não no NPtA (0,3 g/0,3 L) de epibatidina induziu efeito antialodínico. Mecamilamina microinjetada na SCPvl aboliu o efeito antinociceptivo induzido pela epibatidina microinjetada neste mesmo núcleo e o efeito da epibatidina administrada por via sistêmica. Estes resultados permitem concluir que a injeção sistêmica e central de epibatidina induz efeito antialodínico no modelo de dor neuropática e esse efeito depende da interação dessa substância com receptores nicotínicos localizados na SCPvl. / Nicotinic receptors are found in the central nervous system (CNS) and have an important function in modulating neural synaptic, CNS plasticity and many others cognite functions. A variety of subtypes nicotinic receptores are related with phisiological and pathological process. Recentely, studies have been demostrating that nicotinic agonists injected either systemically ou centrally have an antinociceptive effetc. The present study examined; 1) the effect of epibatidene injected systemically and centrally into the anterior pretectal nucleus (APtN) and into the ventro lateral periaqueductal gray (vlPAG) on the mechanical allodinia induzed by ligation of trunk lombar (L5-L6) spinal nerve; 2) the effect of microinjection of nicotinic antagonist (atropine and mecamylamine) on the antinocicptive effect induzed by epibetidine microinjection into the vlPAG. 3) the effect of mecamylamine on the antiallodinic effect, observed after sistemic administration of epibatadine. It was demostrated that the ligation of trunk lombar (L5-L6) spinal nerve produce mechanical allodinia after two days pos-operative and it was maintained for 28 days. Shan group and the contralataeral paw had no change in the threshold response. Systemic injection of epibatidine (3,0 g/kg, i.p.) induzed antiallodinic effect, without affect motor function. Central injection of epibatidine into the vlPAG (0,3 g/0,3 L) also had an antiallodinic effect, but no effect on APtN was observed. Mecamylamine injected into the vlPAG abolish the antiallodinic effect of epibatidine injected systemic and centrally (vlPAG). These results shows that the epibatidine injected either systemically or into the vlPAG produced antiallodinic effect on the neuropatic pain model and also infer that this antiallodinic effect produced by epibatidine requires the interation of epibatidae with vlPAG nicotinic receptors.

Nicotine exerts a therapeutic effect in ulcerative colitis (UC) but the mechanism underlying this effect, is not clear. However, this effect may imply that nicotine has some, as yet to be discovered, effect on the immune system. The aim of the work described in this thesis was to characterise the nicotinic acetylcholine receptors (nAChRs) on human peripheral blood lymphocytes in term of receptor subtype. To achieve this, a combination of radioligand binding assays, pharmacological and molecular biological techniques were used. The data obtained from the binding studies suggested that the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes with a Kd 15 ± 5.759 nM (1.5 ± 5.759 x 10-8 M) and Bmax 2253 ± 409 sites/cell. The competition studies showed that ligands competing with [3H]-(-)-nicotine were (-)-nicotine, epibatidine and α-bungarotoxin, while others ligands for nAChRs displaced radiolabelled nicotine in insignificant quantities. Thus, radioligand-binding experiments suggest that the binding site for nicotine on human peripheral blood lymphocytes is a nAChR containing α7 and possibly α4 or/and b2 containing nAChR subunits. No evidence was obtained to suggest the presence of a non-cholinergic nicotine receptor. Furthermore, considerable subject to subject variation in the specific binding of radiolabelled nicotine was observed. Because of this only tentative conclusions could be drawn from radioligand binding data. Polymerase chain reaction (RT-PCR) was then used to demonstrate mRNA for the subunits of nAChRs suggested by radioligand binding studies. Data obtained show that the human peripheral blood lymphocytes tested, expressed mRNAs for α4, α5, α7, β2 neuronal nAChRs subunits and β1 muscle nAChR subunit. Expression of the α5 mRNA subunit of nAChR was observed in the lymphocytes in each sample of lymphocytes tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between individuals. Finally, Western blot analysis was used to confirm that mRNA expression resulted in the expression of protein for nAChR subunits in human peripheral lymphocytes using monoclonal antibodies against α4, α5, α7, and β2 nAChR subunits, which had been detected by RT-PCR. The results obtained from the Western blot analysis show that protein for α4, α5, and α7 nAChR subunits was expressed in most, but not all of the human peripheral blood lymphocyte samples tested and some of the bands obtained were faint. In contrast, protein for the β2 nAChR subunit was observed in a few samples tested and the bands were faint. From the results obtained in this study, it is possible to conclude that human peripheral blood lymphocytes may contain nAChRs with subunit compositions of α4β2, α4β2α5, and/or α7. However, further studies are necessary to show whether or not the single binding site for nicotine demonstrated by radioligand binding experiments is due to one or all of these nAChRs. Thus, the findings of the present study suggest the presence of nAChR on human peripheral blood lymphocytes. Nicotine and its effect may occur through these non- neuronal nAChRs mechanisms. Such a mechanism of action could account for the beneficial of nicotine in ulcerative colitis. Furthermore, a compound that acts on these receptors, but not on nAChRs found on other cells may have therapeutic utility in the treatment of inflammation.