Memory of chirality (MOC) is an emerging strategy in asymmetric synthesis. It has been applied to enolate chemistry, reactions involving carbocation intermediates, and to radical systems. In this strategy the chirality of an enantiopure reactant is transferred to the dynamic chirality of a reactive intermediate to produce stereospecific product. 1,4-Benzodiazepin-2-ones have been described as a "privileged" structure in medicinal chemistry. In addition to their uses as anxiolytics (Valium ®) and anti-epileptic agents (Clonopin ®), they have shown activity as HIV Tat antagonist, ras farnesyltransferase inhibitors in cancer cells, and antiarrhythmic agents. Because of the utility of this scaffold in the area of medicinal chemistry, it has served as a template in libraries for tens of thousands of compounds. Despite the vast diversity of 1,4-benzodiazepin-2-ones, there are few routes to enantiomerically enriched 3,3-disubstituted benzodiazepines containing a "quaternary" stereogenic center. This research will discuss the stereochemical properties of 1,4-benzodiazepin-2-ones, and provide a novel approach to synthesize enantiomerically enriched "quaternary" benzodiazepines with stereogenic centers through MOC, without the use of external chiral sources. / Ph. D.

O transtorno do pânico (TP) é um transtorno de ansiedade caracterizado por ataques de pânico recorrentes e inesperados, com um prognóstico crônico. Entre as drogas utilizadas no tratamento do TP, os benzodiazepínicos (BZs) de alta potência, como o alprazolam e o clonazepam, apresentam a vantagem de serem eficazes logo no início do tratamento. Assim como outras drogas BZs, tais como o diazepam e o flurazepam, estes compostos também são empregados como ansiolíticos no tratamento de pacientes com transtorno de ansiedade generalizada. O mecanismo da ação primária dessas drogas ocorre pela interação com os sítios de ligação BZs presentes nos receptores do ácido gama-aminobutírico do tipo A (GABAA), facilitando a neutotransmissão GABAérgica. Entretanto, ainda permanecem desconhecidos os substratos neurais envolvidos no efeito panicolítico causado pelos BZs. Dentre os substratos em potencial, a substância cinzenta periaquedutal dorsal (SCPD), uma estrutura mesencefálica criticamente relacionada à fisiopatogênica do TP, apresenta alta densidade de receptores GABAA e de sítios de ligação BZs. Neste trabalho avaliamos o envolvimento do complexo receptor GABAA/BZ presente na SCPD no efeito panicolítico promovido pela administração sistêmica de alprazolam em ratos Wistar. Para isso, empregamos o labirinto em T elevado (LTE), que além da resposta de fuga, que é associada ao pânico, também permite avaliar a resposta de esquiva inibitória, associada à ansiedade. Neste modelo, o alprazolam inibe a expressão da resposta de fuga, indicando efeito panicolítico e inibe a aquisição da esquiva inibitória, sugestivo de efeito ansiolítico. Além do LTE, também empregamos os modelos experimentais da hipóxia e o de Vogel, associados ao pânico e a ansiedade, respectivamente. Os resultados obtidos mostraram que o efeito panicolítico promovido pela administração sistêmica de alprazolam, observado na resposta de fuga do LTE, foi bloqueado pela administração intra-SCPD de flumazenil, antagonista dos sítios de ligação BZs, ou de bicuculina, antagonista dos receptores 10 GABAA. No teste da hipóxia, o efeito panicolítico causado pela administração sistêmica de alprazolam foi inibido, porém não significativamente bloqueado, pela administração intra-SCPD de bicuculina. Já o efeito ansiolítico, observado na resposta de esquiva do LTE e no teste do beber punido de Vogel, não foi bloqueado pela administração intra-SCPD de flumazenil ou de bicuculina. No conjunto, nossos resultados sugerem que o complexo receptor GABAA/BZ da SCPD está envolvido no efeito panicolítico, mas não ansiolítico, promovido pela administração sistêmica de alprazolam. / Panic Disorder (PD) is an anxiety disorder characterized by recurrent and unexpected panic attacks with a chronic prognosis. Among the drugs used to treat PD, highpotency benzodiazepines (BZs), such as alprazolam and clonazepam, have the advantage of causing significant effects early in the treatment. Like others BZs, such as diazepam and flurazepam, these compounds are also used as anxiolytics in the treatment of patients with generalized anxiety disorder. The primary mechanism of action of these drugs is the interaction with BZs binding sites present at gammaaminobutyric acid type A receptors (GABAA), facilitating GABAergic neurotransmission. However, it remains yet unknown the neural substrates involved in the panicolytic-like action caused by BZs. Among the potential substrates, the dorsal periaqueductal gray matter (DPAG), a mesencephalic structure critically associated with the physiopathology of PD, presents a high density of GABAA receptors and of BZs binding sites. In this work, we evaluated the participation of the GABAA/BZ receptor complex present in the DPAG in the panicolytic-like effect caused by systemic administration of alprazolam in Wistar rats. For this, we use the elevated T-maze (ETM), that besides the escape response which is associated with panic, also allows the measurement of inhibitory avoidance acquisition, which has been related to anxiety. In this model, alprazolam inhibits the expression of escape, indicating a panicolytic-like effect and inhibits the acquisition of inhibitory avoidance, suggestive of an anxiolytic effect. In addition to the ETM, animals were also tested in the hypoxia and Vogel\'s conflict tests, which have been associated with panic and anxiety, respectively. The results showed that the panicolytic-like effect caused by alprazolam in ETM\'s escape response was blocked by intra-DPAG injection of flumazenil, a BZs binding site antagonist, or bicuculline, a GABAA receptor antagonist. In the hypoxia test, the panicolytic-like effect caused by alprazolam was inhibited, but not significantly blocked, by intra-DPAG injection of bicuculline. The anxiolytic effect observed in the 12 ETM\'s avoidance task or in the Vogel\'s conflict test was not blocked by intra-DPAG injection of flumazenil or bicuculline. Taken together, our results suggest that the GABAA/BZ receptor complex located in the DPAG is involved in the panicolytic, but not anxiolytic, effect caused by systemic administration of alprazolam.

<p>Important information on the bioactive conformation of biologically active peptides may be obtained by studies of rigid peptides or well-defined secondary structure mimetics incorporated into pseudopeptides. The structural requirements for the interaction of angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) with its AT₁ and AT₂ receptors were the subject of this study.</p><p>The main objectives of this work were to synthesize secondary structure mimetics and incorporate these into Ang II. Ang II has been suggested to adopt a turn conformation around Tyr⁴ when interacting with its AT₁ receptor. Therefore, two <i>γ</i>- and one <i>β</i>-turn mimetic scaffolds based on the benzodiazepine structure were synthesized and decorated with side chains. The scaffolds replaced the turn region around Tyr⁴. Most of the pseudopeptides obtained after incorporation into Ang II exhibited high AT₂/AT₁ selectivity and nanomolar affinity to the AT₂ receptor. One pseudopeptide encompassing a <i>β</i>-turn mimetic also displayed AT₁ receptor affinity.</p><p>We hypothesized that the position of the guanidino group of the arginine residue and the N-terminal end, in relation to the tyrosine side chain, was critical for AT₂ receptor affinity. Conformational evaluation of the pseudopeptides revealed that in all the compounds with AT₂ receptor affinity the arginine side chain and the N-terminal end could reach common regions, not accessible to the inactive compound. It is proposed that Ang II has a more extended bioactive conformation when binding to the AT₂ receptor than when binding to the AT₁ receptor.</p><p>Furthermore, in a Gly scan of Ang II only replacement of the arginine residue reduced the affinity for the AT₂ receptor considerably. Some N-terminal modified Ang II analogues were also synthesized and it was concluded that truncated Ang II analogues interact with the AT₂ receptor differently than Ang II.</p><p>Three of the synthesized pseudopeptides were evaluated in AT₂ receptor functional assays and were found to act as agonists.</p>

Important information on the bioactive conformation of biologically active peptides may be obtained by studies of rigid peptides or well-defined secondary structure mimetics incorporated into pseudopeptides. The structural requirements for the interaction of angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) with its AT1 and AT2 receptors were the subject of this study. The main objectives of this work were to synthesize secondary structure mimetics and incorporate these into Ang II. Ang II has been suggested to adopt a turn conformation around Tyr4 when interacting with its AT1 receptor. Therefore, two γ- and one β-turn mimetic scaffolds based on the benzodiazepine structure were synthesized and decorated with side chains. The scaffolds replaced the turn region around Tyr4. Most of the pseudopeptides obtained after incorporation into Ang II exhibited high AT2/AT1 selectivity and nanomolar affinity to the AT2 receptor. One pseudopeptide encompassing a β-turn mimetic also displayed AT1 receptor affinity. We hypothesized that the position of the guanidino group of the arginine residue and the N-terminal end, in relation to the tyrosine side chain, was critical for AT2 receptor affinity. Conformational evaluation of the pseudopeptides revealed that in all the compounds with AT2 receptor affinity the arginine side chain and the N-terminal end could reach common regions, not accessible to the inactive compound. It is proposed that Ang II has a more extended bioactive conformation when binding to the AT2 receptor than when binding to the AT1 receptor. Furthermore, in a Gly scan of Ang II only replacement of the arginine residue reduced the affinity for the AT2 receptor considerably. Some N-terminal modified Ang II analogues were also synthesized and it was concluded that truncated Ang II analogues interact with the AT2 receptor differently than Ang II. Three of the synthesized pseudopeptides were evaluated in AT2 receptor functional assays and were found to act as agonists.

Benzodiazepines are widely abused by anxious individuals. Consequently, this thesis modeled anxiety in a mouse model in order to investigate benzodiazepine motivation within this sub-population. Using the Tube test of Social Dominance and the Resident/Intruder Paradigm I investigated whether animals identified as dominant or submissive/defeated would differentially display a preference for 0.25 mg/kg midazolam in a conditioned place preference paradigm. Consistent with my hypotheses, benzodiazepine preference was mediated by negative reinforcement as submissive but not dominant mice displayed a preference for midazolam. Furthermore, different neural systems mediated benzodiazepine preference dependent on the stress status of the animal (acute vs. chronic stress) such that, acutely stressed animals experienced benzodiazepine preference through a dopamine-independent pathway whereas chronically stressed animals experienced benzodiazepine preference through a dopamine-dependent pathway. Within chronically stressed mice, blockade of either D1 or D2 receptors attenuated benzodiazepine preference.

Benzodiazepines are widely abused by anxious individuals. Consequently, this thesis modeled anxiety in a mouse model in order to investigate benzodiazepine motivation within this sub-population. Using the Tube test of Social Dominance and the Resident/Intruder Paradigm I investigated whether animals identified as dominant or submissive/defeated would differentially display a preference for 0.25 mg/kg midazolam in a conditioned place preference paradigm. Consistent with my hypotheses, benzodiazepine preference was mediated by negative reinforcement as submissive but not dominant mice displayed a preference for midazolam. Furthermore, different neural systems mediated benzodiazepine preference dependent on the stress status of the animal (acute vs. chronic stress) such that, acutely stressed animals experienced benzodiazepine preference through a dopamine-independent pathway whereas chronically stressed animals experienced benzodiazepine preference through a dopamine-dependent pathway. Within chronically stressed mice, blockade of either D1 or D2 receptors attenuated benzodiazepine preference.

O transtorno do pânico (TP) é um transtorno de ansiedade caracterizado por ataques de pânico recorrentes e inesperados, com um prognóstico crônico. Entre as drogas utilizadas no tratamento do TP, os benzodiazepínicos (BZs) de alta potência, como o alprazolam e o clonazepam, apresentam a vantagem de serem eficazes logo no início do tratamento. Assim como outras drogas BZs, tais como o diazepam e o flurazepam, estes compostos também são empregados como ansiolíticos no tratamento de pacientes com transtorno de ansiedade generalizada. O mecanismo da ação primária dessas drogas ocorre pela interação com os sítios de ligação BZs presentes nos receptores do ácido gama-aminobutírico do tipo A (GABAA), facilitando a neutotransmissão GABAérgica. Entretanto, ainda permanecem desconhecidos os substratos neurais envolvidos no efeito panicolítico causado pelos BZs. Dentre os substratos em potencial, a substância cinzenta periaquedutal dorsal (SCPD), uma estrutura mesencefálica criticamente relacionada à fisiopatogênica do TP, apresenta alta densidade de receptores GABAA e de sítios de ligação BZs. Neste trabalho avaliamos o envolvimento do complexo receptor GABAA/BZ presente na SCPD no efeito panicolítico promovido pela administração sistêmica de alprazolam em ratos Wistar. Para isso, empregamos o labirinto em T elevado (LTE), que além da resposta de fuga, que é associada ao pânico, também permite avaliar a resposta de esquiva inibitória, associada à ansiedade. Neste modelo, o alprazolam inibe a expressão da resposta de fuga, indicando efeito panicolítico e inibe a aquisição da esquiva inibitória, sugestivo de efeito ansiolítico. Além do LTE, também empregamos os modelos experimentais da hipóxia e o de Vogel, associados ao pânico e a ansiedade, respectivamente. Os resultados obtidos mostraram que o efeito panicolítico promovido pela administração sistêmica de alprazolam, observado na resposta de fuga do LTE, foi bloqueado pela administração intra-SCPD de flumazenil, antagonista dos sítios de ligação BZs, ou de bicuculina, antagonista dos receptores 10 GABAA. No teste da hipóxia, o efeito panicolítico causado pela administração sistêmica de alprazolam foi inibido, porém não significativamente bloqueado, pela administração intra-SCPD de bicuculina. Já o efeito ansiolítico, observado na resposta de esquiva do LTE e no teste do beber punido de Vogel, não foi bloqueado pela administração intra-SCPD de flumazenil ou de bicuculina. No conjunto, nossos resultados sugerem que o complexo receptor GABAA/BZ da SCPD está envolvido no efeito panicolítico, mas não ansiolítico, promovido pela administração sistêmica de alprazolam. / Panic Disorder (PD) is an anxiety disorder characterized by recurrent and unexpected panic attacks with a chronic prognosis. Among the drugs used to treat PD, highpotency benzodiazepines (BZs), such as alprazolam and clonazepam, have the advantage of causing significant effects early in the treatment. Like others BZs, such as diazepam and flurazepam, these compounds are also used as anxiolytics in the treatment of patients with generalized anxiety disorder. The primary mechanism of action of these drugs is the interaction with BZs binding sites present at gammaaminobutyric acid type A receptors (GABAA), facilitating GABAergic neurotransmission. However, it remains yet unknown the neural substrates involved in the panicolytic-like action caused by BZs. Among the potential substrates, the dorsal periaqueductal gray matter (DPAG), a mesencephalic structure critically associated with the physiopathology of PD, presents a high density of GABAA receptors and of BZs binding sites. In this work, we evaluated the participation of the GABAA/BZ receptor complex present in the DPAG in the panicolytic-like effect caused by systemic administration of alprazolam in Wistar rats. For this, we use the elevated T-maze (ETM), that besides the escape response which is associated with panic, also allows the measurement of inhibitory avoidance acquisition, which has been related to anxiety. In this model, alprazolam inhibits the expression of escape, indicating a panicolytic-like effect and inhibits the acquisition of inhibitory avoidance, suggestive of an anxiolytic effect. In addition to the ETM, animals were also tested in the hypoxia and Vogel\'s conflict tests, which have been associated with panic and anxiety, respectively. The results showed that the panicolytic-like effect caused by alprazolam in ETM\'s escape response was blocked by intra-DPAG injection of flumazenil, a BZs binding site antagonist, or bicuculline, a GABAA receptor antagonist. In the hypoxia test, the panicolytic-like effect caused by alprazolam was inhibited, but not significantly blocked, by intra-DPAG injection of bicuculline. The anxiolytic effect observed in the 12 ETM\'s avoidance task or in the Vogel\'s conflict test was not blocked by intra-DPAG injection of flumazenil or bicuculline. Taken together, our results suggest that the GABAA/BZ receptor complex located in the DPAG is involved in the panicolytic, but not anxiolytic, effect caused by systemic administration of alprazolam.

<p>The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections.</p><p>An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [¹¹C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [¹¹C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The <i>in vivo</i> visualization of specific binding could not be achieved, though <i>in vitro</i> binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors.</p><p>The use of the glucose analogue [¹⁸F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-¹⁴C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [¹¹C]Cyano-dizocilpine, [¹¹C]4-NMPB, and [¹¹C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABA_A receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [¹¹C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [¹¹C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [¹¹C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.</p>

The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections. An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [11C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [11C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The in vivo visualization of specific binding could not be achieved, though in vitro binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors. The use of the glucose analogue [18F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-14C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [11C]Cyano-dizocilpine, [11C]4-NMPB, and [11C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABAA receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [11C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [11C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [11C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.

No description available.