Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Jakub Jeřábek Supervisors: Prof. PharmDr. Martin Doležal, Ph.D. Prof. Maria Laura Bolognesi Title of Thesis: Design and Synthesis of Hybrid Compounds Based on Tacrine/Resveratrol Derivatives Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder, in which a progressive dementia appears. The cause of AD is currently unknown, however, scientific research has revealed several pathological hallmarks - β-amyloid plaques and neurofibrillary tangles. These changes cause gradual disintegration of nerve cells and they change the metabolism in the brain. The current drugs are not able to treat the cause of the disease, being able only to delay the onset of severe symptoms. The basic drugs for AD treatment are acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitors and, more recently approved, N-methyl- D-aspartate (NMDA) receptor antagonist memantine. These drugs are able to increase cholinergic activity or preventing glutamate excitotoxicity in the patient's brain, thus improving cognitive functions and delaying severe stages of the disease. One of the emerging approaches in drug synthesis represents multi-target-directed ligands (MTDLs). Apart from the ability...

Nous avons repris et étendu les méthodologies bien connues au laboratoire. A partir des b-chloroacrylonitriles, nous avons pu synthétiser grâce une réaction séquentielle de nouveaux 3-amino-2-cyano thiophènes et sélénophènes. Appliquée à d'autres réactifs, cette méthode donne accès à de nouvelles 7-hydroxythiéno [3,2-b] pyridin-5 (4H)-ones ainsi que des 3 amino-2-nitro thiophènes et sélénophènes. la voie de synthèse consistant à préparer des 3-aminothiophènes à partir de cétènes dithioacétals a été modifiée afin d'obtenir de nouveaux 3-amino-4cyanothiophènes. Cette nouvelle méthode a pu être utilisée pour préparer des 4_amino-5-cyano [1,3] thiazoles en deux étapes à partir de la cyanamide. La Tacrine a été le premier médicament utilisé aux Etats-Unis pour lutter contre la maladie d'Alzheimer. Sa forme commerciale (Cognex) a été retiré du marché en 2004. nous avons comme objectif de préparer de nouveaux analogues de la Tacrine à partir des analogues hétérocycliques de l'anthanilonitrile et avoir la même activité que cette dernière tout en évitant les effets secondaires / We have continued and extended a previous research about 3-aminothiophenes. From b-chloroacrylonitriles, we prepared new 3-amino-2-cyano thiophenes et selenophenes thanks a one-pot-threee-steps procedure. Used with other reagents, this method allows the access to new 7-hydroxythieno [3,2-b] pyridin-5 (4H)-ones and 3-amino-2-nitrothiophenes and selenophenes. By an other hand, we modified thr synthesis of 3-aminothiophenes from ketene dithioacetals to obtain new 3-amino-4-cyanothiophenes. from cyanamide, we used this new method to prepare 4-amino-5cyano [1,3] thiazoles in two steps. Tarcine, sold under the name Cognex, was the first drug used in United States fo fight Alzheimer Disease. Our objective is to prepare new heterocyclic analogues of tarcine from the heterocyclic analogues of anthranilonitrile. We hope to have the same activity of Tarcine avoiding side effects.

No description available.

Alzheimer's Disease (AD) is an irreversible, age-related neurodegenerative disorder which causes cognitive impairment and a wide variety of neuropsychiatric and behavioral disturbances. Acetylcholinesterase inhibitors (AChEI) are the mainstay for the treatment of AD. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acylcholinesters with a relative specificity for acetylcholine (ACh). Observation of a deficiency of cholinergic neurotransmission in AD led to the development of AChEI as the first approved treatment for dementia symptoms. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a reversible inhibitor of AChE. It was the first drug approved by the FDA for the treatment of cognitive symptoms of AD. Tacrine is now rarely prescribed as a drug for the treatment of AD due to its high hepatotoxicity in almost 50% of the patients. However, tacrine derivatives have considerable potential for the palliative treatment of AD. Synthesis of various bivalent tacrines led to the improvement in inhibitory potency and selectivity towards inhibition of AChE. Heptylene-linked bis-tacrine has especially shown immense promise to be an ideal AChEI. Thus dimerization of a lead compound seemed to be an ideal strategy where the compound can bind to both catalytic anionic site (CAS) and peripheral anionic site (PAS) on the AChE enzyme. However synthesis of N-alkyl derivatives of expanded tacrines like 12-chloro-2-methyl-6,7,8,9,10,11-hexahydrocycloocta[b]quinoline by the standard SNAr methods was unsuccessful and thus alternatives needed to be developed to synthesize N-alkylated and bivalent 12-chloro-2-methyl-6,7,8,9,10,11-hexahydrocycloocta[b]quinoline. Upon exploring the alternatives, N-arylation by Pd-catalysis seemed to be the most mild and convenient alternative over the standard SNAr procedures. / Master of Science

A doença de Alzheimer (DA) é uma doença neurodegenerativa que causa perda progressiva e irreversível das funções cerebrais, atualmente não tem cura e não existe um tratamento específico eficaz. Uma estratégia para o tratamento paliativo é restaurar o neurotransmissor acetilcolina utilizando fármacos inibidores das enzimas colinesterase (ChEI), nesse contexto a tacrina foi o primeiro fármaco aprovado para o tratamento da DA. Há mais de uma década os análogos dímeros da tacrina, conhecidos como bis-tacrina, mostraram maior eficiência na inibição da enzima acetilcolinesterase (AChE) comparativamente ao fármaco tacrina e seus análogos, devido à ação simultânea em dois sítios da enzima, catalítico e periférico. Desde então, vários compostos dímeros e híbridos contendo o núcleo tacrina tem sido sintetizados e testados como ChEI. Neste trabalho realizou-se a síntese de dímeros quirais do tipo bis-tacrina, onde dois núcleos da tacrina com substituintes quirais estão conectados por uma cadeia espaçadora de carbonos metilênicos. A reação de condensação de Friedlander foi a estratégia adotada para a obtenção do núcleo tacrina, onde uma ciclocetona quiral de origem terpênica foi condensada com um ácido o-aminobenzóico na presença de POCl3, formando os intermediários do tipo 9-cloroacridinas quirais. As ciclocetonas quirais foram sintetizadas a partir da reação de retro-aldol do monoterpeno natural pulegona, comercialmente disponível nas formas (R)-(+)- e (S)-(-). A preparação dos homodímeros envolveu a reação de substituição nucleofílica aromática (SNAr) entre as 9-cloroacridinas e a 1,7-heptanodiamina, que contém a cadeia alquílica espaçadora. A síntese dos heterodímeros necessitou a preparação dos precursores 9-(1,7-diaminoeptil)-1,2,3,4-tetraidroacridina, contendo o núcleo tacrina e o grupo amino separados pela cadeia espaçadora, para posterior reação de SNAr com as 9-cloroacridinas. Os produtos obtidos neste trabalho foram purificados por cromatografia em coluna e caracterizados por espectroscopia de ressonância magnética nuclear (RMN) de 1H e 13C, no infravermelho (IV), atividade óptica e medidas de ponto de fusão. As análises de atividade óptica mostraram que a quiralidade foi mantida nos intermediários e nos produtos finais sintetizados. Foram realizados ensaios biológicos de inibição das enzimas AChE e BuChE com os compostos quirais disponíveis, e os dímeros da série (R) mostraram ser ativos como inibidores das enzimas. / Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which causes progressive and irreversible loss of brain functions and currently has no cure and no effective specific treatment. A strategy for palliative treatment of AD is the restoration of neurotransmitter acetylcholine using drugs cholinesterase inhibitors (ChEI) and tacrine was the first drug approved for the treatment of AD. About fifteen years ago, bis(n)-tacrine analogues linked by an alkylene chain were prepared, and it was proved that these dimeric molecules of tacrine offered a much stronger potency and selectivity toward AChE. Bis(7)-tacrine simultaneously binds at both the CAS and the PAS sites and provides a higher selectivity towards AChE over BuChE.. Since then, several dimmers and hybrid compounds containing the nucleus tacrine, have been synthesized and tested as cholinesterase inhibitors. In this work were carried out the syntheses of chiral homodimers and heterodimers of bis-tacrine type, where two nucleus of tacrine with chiral substituents were connected by an alkyl chain as spacer. The Friedländer condensation reaction was performed to obtain the tacrine nucleus, the cycloketone from a chiral terpene source was condensed with an o-aminobenzoic acid in the presence of POCl3, forming 9-chloroacridine intermediates. The chiral cycloketone were obtained from natural monoterpene pulegone, commercially available in (R)-(+)- and (S)-(-) enantiomers. The preparation of homodimers involved the nucleophilic aromatic substitution (SNAr) reaction between 9-chloroacridines and 1,7-diaminoheptane, which contains the spacer alkyl chain. The synthesis of heterodimers required the preparation of precursor 9-(1,7-diaminoheptyl)-1,2,3,4-tetraydroacridine, containing tacrine nucleus and the amino group separated by spacer chain, for subsequent SNAr reaction with the 9-chloroacridines. The products obtained in this work were purified by column chromatography and characterized by nuclear magnetic resonance (NMR) spectroscopy of 1H and 13C, infrared spectroscopy (IR), optical activity and melting point measurements. The analysis of optical activity showed that the chirality was maintained in the intermediate and final products synthesized. Biological assays of inhibition of AChE and BuChE enzymes were performed with chiral compounds. The dimers of (R)-series were active as cholinesterase inhibitors.

With the incidence of Alzheimer’s disease (AD) growing worldwide and in Canada along with the growing economic and social burdens, the need for more effective therapies becomes of great importance. Since the discovery of AD, a number of proposed theories have arisen to explain the pathophysiology including the i) cholinergic theory, ii) oxidative stress pathways, and iii) metal ion imbalance. The major class of drug therapies to treat AD are cholinesterase inhibitors; however, the “one drug, one target” approach has not proven fruitful and generally becomes ineffective in later stages of disease progression. In this project, we synthesized a library of 1,2,3,4-tetrahydroacridine derivatives (10a-d, 11a-e, 12a-e, and 13a-f) as potential agents to target the cholinergic and oxidative stress pathways of AD. Chapter I provides background information on the role of AChE and BuChE enzymes in AD. Furthermore, this chapter describes the neurotoxicity of reactive oxygen species (ROS) and metals in AD. Chapter II provides a summary of project hypothesis and rationale. Chapter III describes the synthetic details regarding the synthesis of target small molecules. It further describes the principles involved in carrying out biological evaluation such as AChE and BuChE inhibition, antioxidant properties via DPPH stable radical scavenging, iron chelation capacity using ferrozine and in vitro cell viability data in neuroblastoma cells. Chapter IV describes the SAR details on ChE inhibition, antioxidant activities, iron chelation and cell viability profiles and molecular modeling details. A brief conclusion and future directions are included in Chapter V and the final section, Chapter VI provides experimental details for synthetic chemistry including analytical data of synthesized compounds and protocols for biological evaluations. This study identified novel tetrahydroacridine derivatives with nanomolar inhibition of both human AChE and human BuChE enzymes that were more potent relative to the reference agent tacrine. Compound 10d[N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified as a potent inhibitor of BuChE (IC50 = 24.0 nM) and compound 13c [6-chloro-N-(pyridine- 2-ylmethyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified as a potent inhibitor of AChE (IC50 = 95.0 nM) with good inhibition of BuChE (IC50 = 1.61 μM) whereas compound 11e [6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified with an optimum combination of dual AChE and BuChE inhibition (AChE IC50 = 0.9 μM; BuChE IC50= 1.4 μM). In conclusion, our studies provide new insight into the design and development of novel tetrahydroacridine derivatives to target multiple pathological routes of AD.

A doença de Alzheimer (DA) é uma doença neurodegenerativa que causa perda progressiva e irreversível das funções cerebrais, atualmente não tem cura e não existe um tratamento específico eficaz. Uma estratégia para o tratamento paliativo é restaurar o neurotransmissor acetilcolina utilizando fármacos inibidores das enzimas colinesterase (ChEI), nesse contexto a tacrina foi o primeiro fármaco aprovado para o tratamento da DA. Há mais de uma década os análogos dímeros da tacrina, conhecidos como bis-tacrina, mostraram maior eficiência na inibição da enzima acetilcolinesterase (AChE) comparativamente ao fármaco tacrina e seus análogos, devido à ação simultânea em dois sítios da enzima, catalítico e periférico. Desde então, vários compostos dímeros e híbridos contendo o núcleo tacrina tem sido sintetizados e testados como ChEI. Neste trabalho realizou-se a síntese de dímeros quirais do tipo bis-tacrina, onde dois núcleos da tacrina com substituintes quirais estão conectados por uma cadeia espaçadora de carbonos metilênicos. A reação de condensação de Friedlander foi a estratégia adotada para a obtenção do núcleo tacrina, onde uma ciclocetona quiral de origem terpênica foi condensada com um ácido o-aminobenzóico na presença de POCl3, formando os intermediários do tipo 9-cloroacridinas quirais. As ciclocetonas quirais foram sintetizadas a partir da reação de retro-aldol do monoterpeno natural pulegona, comercialmente disponível nas formas (R)-(+)- e (S)-(-). A preparação dos homodímeros envolveu a reação de substituição nucleofílica aromática (SNAr) entre as 9-cloroacridinas e a 1,7-heptanodiamina, que contém a cadeia alquílica espaçadora. A síntese dos heterodímeros necessitou a preparação dos precursores 9-(1,7-diaminoeptil)-1,2,3,4-tetraidroacridina, contendo o núcleo tacrina e o grupo amino separados pela cadeia espaçadora, para posterior reação de SNAr com as 9-cloroacridinas. Os produtos obtidos neste trabalho foram purificados por cromatografia em coluna e caracterizados por espectroscopia de ressonância magnética nuclear (RMN) de 1H e 13C, no infravermelho (IV), atividade óptica e medidas de ponto de fusão. As análises de atividade óptica mostraram que a quiralidade foi mantida nos intermediários e nos produtos finais sintetizados. Foram realizados ensaios biológicos de inibição das enzimas AChE e BuChE com os compostos quirais disponíveis, e os dímeros da série (R) mostraram ser ativos como inibidores das enzimas. / Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which causes progressive and irreversible loss of brain functions and currently has no cure and no effective specific treatment. A strategy for palliative treatment of AD is the restoration of neurotransmitter acetylcholine using drugs cholinesterase inhibitors (ChEI) and tacrine was the first drug approved for the treatment of AD. About fifteen years ago, bis(n)-tacrine analogues linked by an alkylene chain were prepared, and it was proved that these dimeric molecules of tacrine offered a much stronger potency and selectivity toward AChE. Bis(7)-tacrine simultaneously binds at both the CAS and the PAS sites and provides a higher selectivity towards AChE over BuChE.. Since then, several dimmers and hybrid compounds containing the nucleus tacrine, have been synthesized and tested as cholinesterase inhibitors. In this work were carried out the syntheses of chiral homodimers and heterodimers of bis-tacrine type, where two nucleus of tacrine with chiral substituents were connected by an alkyl chain as spacer. The Friedländer condensation reaction was performed to obtain the tacrine nucleus, the cycloketone from a chiral terpene source was condensed with an o-aminobenzoic acid in the presence of POCl3, forming 9-chloroacridine intermediates. The chiral cycloketone were obtained from natural monoterpene pulegone, commercially available in (R)-(+)- and (S)-(-) enantiomers. The preparation of homodimers involved the nucleophilic aromatic substitution (SNAr) reaction between 9-chloroacridines and 1,7-diaminoheptane, which contains the spacer alkyl chain. The synthesis of heterodimers required the preparation of precursor 9-(1,7-diaminoheptyl)-1,2,3,4-tetraydroacridine, containing tacrine nucleus and the amino group separated by spacer chain, for subsequent SNAr reaction with the 9-chloroacridines. The products obtained in this work were purified by column chromatography and characterized by nuclear magnetic resonance (NMR) spectroscopy of 1H and 13C, infrared spectroscopy (IR), optical activity and melting point measurements. The analysis of optical activity showed that the chirality was maintained in the intermediate and final products synthesized. Biological assays of inhibition of AChE and BuChE enzymes were performed with chiral compounds. The dimers of (R)-series were active as cholinesterase inhibitors.

A doença de Alzheimer (DA) é uma doença neurodegenerativa que causa perda progressiva e irreversível das funções cerebrais, atualmente não tem cura e não existe um tratamento específico eficaz. Uma estratégia para o tratamento paliativo é restaurar o neurotransmissor acetilcolina utilizando fármacos inibidores das enzimas colinesterase (ChEI), nesse contexto a tacrina foi o primeiro fármaco aprovado para o tratamento da DA. Há mais de uma década os análogos dímeros da tacrina, conhecidos como bis-tacrina, mostraram maior eficiência na inibição da enzima acetilcolinesterase (AChE) comparativamente ao fármaco tacrina e seus análogos, devido à ação simultânea em dois sítios da enzima, catalítico e periférico. Desde então, vários compostos dímeros e híbridos contendo o núcleo tacrina tem sido sintetizados e testados como ChEI. Neste trabalho realizou-se a síntese de dímeros quirais do tipo bis-tacrina, onde dois núcleos da tacrina com substituintes quirais estão conectados por uma cadeia espaçadora de carbonos metilênicos. A reação de condensação de Friedlander foi a estratégia adotada para a obtenção do núcleo tacrina, onde uma ciclocetona quiral de origem terpênica foi condensada com um ácido o-aminobenzóico na presença de POCl3, formando os intermediários do tipo 9-cloroacridinas quirais. As ciclocetonas quirais foram sintetizadas a partir da reação de retro-aldol do monoterpeno natural pulegona, comercialmente disponível nas formas (R)-(+)- e (S)-(-). A preparação dos homodímeros envolveu a reação de substituição nucleofílica aromática (SNAr) entre as 9-cloroacridinas e a 1,7-heptanodiamina, que contém a cadeia alquílica espaçadora. A síntese dos heterodímeros necessitou a preparação dos precursores 9-(1,7-diaminoeptil)-1,2,3,4-tetraidroacridina, contendo o núcleo tacrina e o grupo amino separados pela cadeia espaçadora, para posterior reação de SNAr com as 9-cloroacridinas. Os produtos obtidos neste trabalho foram purificados por cromatografia em coluna e caracterizados por espectroscopia de ressonância magnética nuclear (RMN) de 1H e 13C, no infravermelho (IV), atividade óptica e medidas de ponto de fusão. As análises de atividade óptica mostraram que a quiralidade foi mantida nos intermediários e nos produtos finais sintetizados. Foram realizados ensaios biológicos de inibição das enzimas AChE e BuChE com os compostos quirais disponíveis, e os dímeros da série (R) mostraram ser ativos como inibidores das enzimas. / Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which causes progressive and irreversible loss of brain functions and currently has no cure and no effective specific treatment. A strategy for palliative treatment of AD is the restoration of neurotransmitter acetylcholine using drugs cholinesterase inhibitors (ChEI) and tacrine was the first drug approved for the treatment of AD. About fifteen years ago, bis(n)-tacrine analogues linked by an alkylene chain were prepared, and it was proved that these dimeric molecules of tacrine offered a much stronger potency and selectivity toward AChE. Bis(7)-tacrine simultaneously binds at both the CAS and the PAS sites and provides a higher selectivity towards AChE over BuChE.. Since then, several dimmers and hybrid compounds containing the nucleus tacrine, have been synthesized and tested as cholinesterase inhibitors. In this work were carried out the syntheses of chiral homodimers and heterodimers of bis-tacrine type, where two nucleus of tacrine with chiral substituents were connected by an alkyl chain as spacer. The Friedländer condensation reaction was performed to obtain the tacrine nucleus, the cycloketone from a chiral terpene source was condensed with an o-aminobenzoic acid in the presence of POCl3, forming 9-chloroacridine intermediates. The chiral cycloketone were obtained from natural monoterpene pulegone, commercially available in (R)-(+)- and (S)-(-) enantiomers. The preparation of homodimers involved the nucleophilic aromatic substitution (SNAr) reaction between 9-chloroacridines and 1,7-diaminoheptane, which contains the spacer alkyl chain. The synthesis of heterodimers required the preparation of precursor 9-(1,7-diaminoheptyl)-1,2,3,4-tetraydroacridine, containing tacrine nucleus and the amino group separated by spacer chain, for subsequent SNAr reaction with the 9-chloroacridines. The products obtained in this work were purified by column chromatography and characterized by nuclear magnetic resonance (NMR) spectroscopy of 1H and 13C, infrared spectroscopy (IR), optical activity and melting point measurements. The analysis of optical activity showed that the chirality was maintained in the intermediate and final products synthesized. Biological assays of inhibition of AChE and BuChE enzymes were performed with chiral compounds. The dimers of (R)-series were active as cholinesterase inhibitors.

Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh). Inhibition of AChE has proven an effective treatment for the memory loss exhibited by early stage Alzheimer's disease (AD) patients; four AChE inhibitors (AChEI) have been approved by the FDA for this purpose. The first AChEI approved for the palliative treatment of AD-related memory loss was 9-amino-1,2,3,4-tetrahydroacridine (tacrine). Inhibition of AChE may present either therapeutic or toxic effects depending upon the dose administered. With the goal of discovering safe and effective pesticides to control the population of Anopheles gambiae, a malaria-transmitting mosquito indigenous to Sub-Saharan Africa, the reoptimization of the tacrine pharmacophore was undertaken. Because the optimized drug would necessarily be a poor inhibitor for human AChE (hAChE), initial ligand design focused on modification to tacrine known to negatively impact the inhibition potency for hAChE. Ultimately, an AChEI was discovered, which exhibited micromolar inhibition of Anopheles gambiae AChE (AgAChE) and essentially no potency for hAChE. Two units of this lead compound were tethered through an alkyl chain to yield a nanomolar inhibitor of AgAChE that was more than 1,100-fold selective for the mosquito enzyme over hAChE. Dimerization of an active inhibitor is an effective strategy to increase the potency and selectivity of AChEI, and many examples of tacrine hetero- and homodimers complexed to AChE can be found in the RCSB Protein Data Bank (PDB). The bond formed between the exocyclic amine moiety and the heterocyclic ring system of tacrine is analogous to an amide bond when tacrine is protonated. Therefore, the rotational profile of protonated N-alkyltacrine should exhibit a conformational profile in which dihedral angles significantly out of the plane formed by the ring system are associated with high energies relative to those when the dihedral angles are nearly coplanar with the ring system. The barrier of rotation (ΔG^‡) produced by this phenomenon in two tacrine derivatives and two quinoline derivatives was experimentally determined using dynamic 1H NMR. These values were compared to density functional theory (DFT) derived values for the same phenomenon. Furthermore, since the ΔG^‡ proved to be impossible to experimentally determine for the optimal model compound for the active site portion of tacrine dimers, N-methyltacrine, the DFT method employed for modeling the ΔG^‡ of the tacrine and quinoline analogs was used to computationally derive the entire rotational conformation diagram of N-methyltacrine. The calculated values were then used to comment on the relative energies of adopting certain conformations found in the X-ray crystal structures of dimer/AChE complexes. / Ph. D.

Nicotinic acetylcholine receptors are ligand-gated ion channels which are located on neuromuscular junction and in central and perifric nervous system. Activity of nicotinic receptor might be modulated by variety of pharmacological agents. In this work, we have focused on the study of the inhibition effect of tacrine and its derivatives on the nicotinic acetycholine receptors of muscle and neuronal type. These derivatives function as acetylcholinesterase inhibitors and also interact with nicotinic acetylcholine receptors. The majority of current forms of treatment of Alzheimer's disease is based on cholinesterase inhibitors. We have studied the mechanism of tacrine and its derivatives by using patch clamp method in the configuration of whole-cell recording. Powered by TCPDF (www.tcpdf.org)