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A new series of homobivalent Dimebon analogs, bis-γ-carbolines with alkylene, phenylenedialkylene, and triazole-containing spacers, was synthesized. Doubling the γ-carboline pharmacophore increased inhibitory potency against acetylcholinesterase (AChE) compared with Dimebon, while keeping Dimebon’s anti-butyrylcholinesterase activity; therefore, leading to inversion of selectivity. Molecular docking revealed the reasons for the increased anti-AChE activity and ability to block AChE-induced aggregation of β-amyloid for bis-γ-carbolines, which became double-site inhibitors of AChE. Conjugates with ditriazole-containing spacers were the most active antioxidants in both the ABTS-test and prevention of lipid peroxidation in brain homogenates without inhibiting the mitochondrial permeability transition (MPT). Conjugates with alkylene (4a–d), phenylenedialkylene (4e), and monotriazole (8) spacers were less active as antioxidants but prevented induction of the MPT and increased the calcium retention capacity of mitochondria. Lead compound 4e showed neuroprotective potential in a cellular calcium overload model of neurodegeneration. Computational studies showed that all the bis-γ-carbolines were expected to have high values for intestinal absorption and very good blood-brain barrier permeability along with good drug-likeness. Overall, the results showed that new homobivalent Dimebon analogs exhibit an expanded spectrum of biological activity and improved pharmacological properties, making them promising candidates for further research and optimization as multitarget agents for Alzheimer’s disease treatment.

Ester of the tubulin ligand, 2-methoxyestradiol, with two molecules of DNA alkylating agent chlorambucil was proposed as a “triple” mutual prodrug for blocking rapid metabolism of the initial steroid. The target conjugate was synthesized via the Steglich esterification. The biotesting results demonstrate a very low cytotoxicity to cancer and conditionally normal cells (EC50 ~100 μmol L−1), a weak inhibition of the cell growth (A549: 20%/48 h), no stimulation of apoptosis, and no influence on the microtubule morphology of human lung carcinoma A549 cells and the ability to increase the rate of microtubule assembling. The sharp distinction of these properties from those for the initial 2-methoxyestradiol and chlorambucil molecules indicates that the synthesized prodrug is stable in vitro and demonstrates an expediency of its further testing in vivo.

This study presents the synthesis of binary tetrohydro-γ-carbolines with ditriazol spacers of varying length, which exhibit anticholinesterase and antioxidant activity, as compared to the original Dimebon prototype. Anticholinesterase activity suggests the potential ability of the new compounds to block β-amyloid aggregation induced by anticholinesterase, making them promising candidates for further research preparations for the treatment of Alzheimer's disease. Particular attention should be paid to the conjugate with an intertriazol hexamethylene spacer, which can be regarded as the leading compound in this series.

The mitochondria-targeting drugs can be conventionally divided into the following groups: those compensating for the energy deficit involved in neurodegeneration, including stimulants of mitochondrial bioenergetics and activators of mitochondrial biogenesis; and neuroprotectors, that are compounds increasing the resistance of mitochondria to opening of mitochondrial permeability transition (MPT) pores. Although compensating for the energy deficit and inhibition of MPT are obvious targets for drugs used in the very early stages of Alzheimer-like pathology, but their use as the monotherapy for patients with severe symptoms is unlikely to be sufficiently effective. It would be optimal to combine targets that would provide the cognitive-stimulating, the neuroprotective effects and the ability to affect specific disease-forming mechanisms. In the design of such drugs, assessment of their potential mitochondrial-targeted effects is of particular importance. The possibility of targeted drug design for simultaneous action on mitochondrial and neurotransmitter's receptors targets is, in particularly, based on the known interplay of various cellular pathways and the presence of common structural components. Of particular interest is directed search for multitarget drugs that would act simultaneously on mitochondrial calcium-dependent functions, the targets (receptors, enzymes, etc.) facilitating neurotransmission, and the molecular targets related to the action of so-called disease-modifying factors, in particular, the formation and overcoming of the toxicity of β-amyloid or hyperphosphorylated tau protein. The examples of such approaches realized on the level of preclinical and clinical trials are presented below.

The synthesized new binary conjugates of tetrahydro-γ-carbolines, which contained ditriazole spacers of different length, exhibited considerable anticholinesterase and antioxidant activity as well as the potential ability to block the acetylcholinesterase-induced aggregation of β-amyloid in contrast to the original prototype Dimebon. This makes the compounds promising candidates for further investigation as drugs for the treatment of Alzheimer's disease. Special attention should be given to the conjugate containing the hexamethylene intertriazole spacer, which can be considered as a leader in this series of compounds.

A synthetic approach to the vinylation and allylation of tetrahydro-γ-carbolines with alkane dimethyl sulfonates in the presence of sodium hydride was developed. The effect of the synthesized 5-vinyl- and 5-allyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles on acetylcholinesterase, butyrylcholinesterase, and carboxylesterase activity, mitochondrial functions, and tubulin polymerization was investigated, and their antioxidant activity was evaluated.

The paper considers the influence of the structure of aminoadamantane–carbazole conjugates linked by five different spacers on their ability to inhibit acetylcholinesterase, butyrylcholinesterase (BChE), and carboxylesterase, the radical scavenging and mitoprotective activity, and tubulin polymerization. The nature of the spacer was found to be determinant for the appearance of anti-BChE and radical-scavenging activity. Aminoadamantane–carbazole conjugates linked by flexible spacers proved to be most effective in these assays. The influence of the conjugates on mitochondrial functions and tubulin polymerization largely depends on the structure of the carbazole moiety. A series of lead compounds promising for further study was identified as potential drugs for the treatment of neurodegenerative diseases, in particular, Alzheimer’s disease.

Phosphorylation of τ-protein, a component of microtubules in brain neurons, is a key pathomorphological sign of Alzheimer’s disease. The development of this intracellular defect can be promoted by Al3+, Fe3+, and Zn2+ ions. The concentrations of these ions in the brain are considerably elevated in Alzheimer’s disease. We performed a comparative study of phosphorylation of microtubular proteins of rat brain in the presence of Al3+, Fe3+, and Zn2+ ions in concentrations of 10-500 μM and microtubular proteins of brain of patients with Alzheimer’s disease. The most likely candidate for the role of a factor that promotes hyperphosphorylation of τ-protein is Al3+ in concentrations of 250 and 500 μM.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer’s disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure–activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced β-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Alzheimer’s disease has a complex multifactorial nature; therefore, a promising approach for the development of efficient therapeutic agents is the concept of multitarget drugs, which affect several biological targets involved in the pathogenesis of the disease. We developed a synthetic algorithm for conjugating several pharmacophoric ligands acting on the key stages of pathogenesis of several neurodegenerative diseases and synthesized hybrid structures combining the γ-carboline fragment of Dimebon with carbazole and tetrahydrocarbazole moieties. Using the complex primary screening system the structures have been revealed that combine the high inhibitory activity and selectivity towards butyrylcholinesterase with the radical-scavenging activity and the ability to potentiate tubulin polymerization to microtubules with a normal structure and/or prevent mitochondrial permeability transition. The lead compound was identified for future optimization and development of new multi-target drugs against neurodegenerative diseases combining the cognitive-stimulating and neuroprotective potentials.

Novel gold(I) complexes, PPh3AuSR (1) and [(Ph3PAu)2SR]2(BF4)2 (2), with a protective antioxidant group R (R = 3,5-di-tert-butyl-4-hydroxyphenyl) were synthesized and characterized by 1H, 13C, 31P NMR, IR and elemental analyses. The crystal structures of compounds 1 and 2 were determined by X-ray diffraction. Compound 2 has a cluster structure with four Au–Au bonds in the crystal. The tetragold(I) dication is composed of four PPh3Au units with bridging sulfur atoms, where the Au4 moiety has a distorted tetrahedral geometry. The biological impact of gold complexes 1 and 2, and their precursors (RSH and AuPPh3Cl), on lipid peroxidation, mitochondrial functions, the tubulin polymerization, activity of glutathione reductase and cell viability were investigated. A comparative study of the structure–activity relationship was performed. We found that RSH and Au complexes 1 and 2 as opposed to AuPPh3Cl, prevented Fe3+ and tBHP-induced lipid peroxidation in isolated mitochondria. The number of 2,6-di-tert-butylphenol fragments influences the inhibitory effect. AuPPh3Cl induced swelling and depolarization of mitochondria, whereas RSH and the gold complexes 1 and 2, containing 2,6-di-tert-butylphenol groups, showed little to null effect. The thiol ligand RSH and AuPPh3Cl as well as gold complexes 1 and 2 have no impact on tubulin polymerization. The gold complexes were found to inhibit glutathione reductase. The compounds were tested for cytotoxicity against a primary culture of rat cerebellar granule cells. A high toxicity was determined for AuPPh3Cl, while introduction of antioxidant phenol groups decreased the cytotoxicity of the Au complexes. These results open up scope for the design of novel low toxic, gold based pharmacological agents.

Unknown conjugates of carbazoles and tetrahydrocarbazoles were obtained by the alkylation of carbazoles and tetrahydrocarbazoles with 3,6-substituted 9-oxiranylmethylcarbazoles. The influence of synthesized 1-(9Н-carbazol-9-yl)-3-(1,2,3,4-tetrahydro-5Н-pyrido[4,3-b]-indol-5-yl)propan-2-ones on functional characteristics of brain mitochondria has been studied. The potential neuroprotective activity of a several representatives of conjugates has been demonstrated using glutamate toxicity model.

Earlier unknown conjugates of carbazoles and γ-carbolines were obtained by the reaction of 3,6-substituted 9-oxiranylmethylcarbazoles and 2,8-substituted 2,3,4,5-tetrahydro-1H-pyrido-[4,3]indoles. The effects of synthesized 1-(9H-carbazol-9-yl)-3-(1,2,3,4-tetrahydro-5H-pyrido-[4,3-b]indol-5-yl)propan-2-ones and their hydrochlorides on neuronal NMDA receptors, polymerization of tubulin to form microtubules, and functional characteristics of rat liver mitochondria were studied.

Two series of organotin(IV) carboxylates based on phenolic antioxidants RCOOH and R(CH2)2COOH (R = 3,5-di-tert-butyl-4-hydroxyphenyl) and natural bile (cholanic) acids of formulae (RCOO)2SnMe2 (1); (RCOO)2SnBu2 (2); (R(CH2)2COO)2SnMe2 (3); (R(CH2)2COO)2SnBu2 (4); (cholate)SnPh3 (5); (deoxycholate)SnPh3 (6); (lithocholate)SnPh3 (7); (cholate)SnMe3 (8); (deoxycholate)SnMe3 (9), and (lithocholate)SnMe3 (10) were synthesized and characterized by 1H, 13C NMR, IR and elemental analysis to study their antioxidant and cytotoxic potential. Compounds 1 and 2 were found by X-ray diffraction analysis to be monomers in the solid state; moreover, the carboxyl groups are coordinated bidentately through O atoms. The distorted octahedron geometry around the Sn center in the monocrystals of 1, 2 was revealed. The antioxidant activity of compounds as radical scavengers and reducing agents was evaluated in spectrophotometrical tests with stable radical DPPH, reduction of Cu2+ (CUPRAC method) and interaction with superoxide anion radical. Cytotoxicity in vitro of compounds has been estimated on human cancer (colon cancer HCT-116, lung carcinoma A549, breast cancer MCF-7, neuroblastoma SH-SY5Y) cell lines and normal fibroblasts cells. The IC50 values varied in 0.002–100 μM range for compounds 1–4 depend on the alkyl substituents at Sn center and carboxylate ligand structure. The influence on Fe3+-induced lipid peroxidation, mitochondrial potential and mitochondrial permeability and tubulin assembly have been studied for the compounds 1 and 2. It was shown that the complexes 1 and 2 slightly depolarize the mitochondria but don’t influence the calcium-induced mitochondrial permeability transition. Organotin complexes with cholic and deoxycholic acids as ligands were revealed to induce apoptosis in A549 cells. The percentage of apoptotic cells were 27.9 and 27.1% for triphenyltin complexes with cholic and deoxycholic derivatives, respectively.

The article provides an overview of our own results of comparative study of influence of ions of iron, zinc and aluminium on the structure of microtubules from tubulin and microtubules associated proteins of rat brain with data on the structure of microtubules from tubulin and microtubules associated proteins from the brain of patients with Alzheimer′s disease (AD). A significant decrease in the amount of soluble tubulin was found in the postmortem brain of AD patients in comparison with the control group in the hippocampus, frontal cortex and substantia nigra, but not in the inferior olive. In vitro polymerization of tubulin and microtubules associated proteins from the brain of AD patients and electron micrographs of microtubules were obtained. The assembly of microtubules from brains of AD patients is disrupted, resulting in defective structures. On the other hand, the study of the influence of Al3+, Fe3+, Zn2+ on the microtubules from rat brains tubulin and microtubules associated proteins assembly and structure has shown that all studied metals are able to reduce the amount of microtubules and induce the assembly of anomal structures. According to the degree of the destructive effect on the microtubules and, accordingly, the possible significance in the pathogenesis of Alzheimer disease, metal ions can be arranged in the following sequence Al3+ > Zn2+ > Fe3+. Moreover, phosphorylation of tubulin and microtubules associated proteins in the presence of aluminum ions to the greatest extent reflects the phosphorylation of these proteins at AD. Comparison of data on the structure of microtubules after their assembly from brains of AD patients tubulin and microtubules associated proteins from brains of AD patients, and from the brain of rats, but in the presence of metal ions, confirm the conclusion about the possible role of the metals in the AD etiopathogenesis., Обобщены собственные результаты сравнительного исследования эффектов ионов железа, цинка и алюминия на структуру микротрубочек в процессе их сборки из тубулина и микротубуллоассоциированных белков мозга крысы с данными по изменению структуры микротрубочек в процессе их сборки из тубулина и белков, ассоциированных с микротрубочками (МАР от англ. microtubules associated proteins), мозга больных болезнью Альцгеймера (БА). В мозге больных БА выявлено значительное снижение количества растворимого тубулина, по сравнению с контрольной группой, в гиппокампе, лобной коре и чёрной субстанции, но не в нижней оливе. Отработаны условия, позволившие впервые провести in vitro полимеризацию тубулина и MAP мозга больных БА и получить электронные микрофотографии образовавшихся структур. Показано, что при БА нарушен процесс сборки микротрубочек, в результате чего образуются дефектные структуры. С другой стороны, исследование влияния ионов Al3+, Fe3+, Zn2+ на сборку и структуру микротрубочек из тубулина и MAP мозга крысы показало, что любой из исследованных нами металлов способен снижать количество микротрубочек и вызывать образование аномальных структур. По степени деформирующего воздействия на микротубулярную систему клеток мозга и, соответственно, возможной значимости в патогенезе БА, ионы металлов можно расположить в следующей последовательности Al3+ > Zn2+ > Fe3+. Более того, спектр фосфорилирования тубулина и MAP в присутствии ионов алюминия в наибольшей степени отражает фосфорилирование этих белков при болезни Альцгеймера. Сопоставление данных о нарушениях структуры микротрубочек, наблюдаемых при их сборке из препаратов тубулина и MAP из мозга больных БА, и из мозга крыс, но в присутствии ионов металлов, подтверждают вывод о возможной роли металлов в этиопатогенезе болезни Альцгеймера.

A series of cytotoxic organotin compounds with Sn–S bonds of formulae Me2Sn(SR)2 (1); Et2Sn(SR)2 (2); (n-Bu)2Sn(SR)2 (3); Ph2Sn(SR)2 (4); R2Sn(SR)2 (5); Me3SnSR (6) Ph3SnSR (7) (R = 3,5-di-tert-butyl-4-hydroxyphenyl), synthesized recently, have been used to study the possible mechanisms of their cytotoxicity. With this aim we have investigated the influence of organotin compounds 1–7 as well as their precursor R2SnCl2 (8) on tubulin assembly in microtubules. Compounds 1, 6–8 reveal the statistically significant inhibition of tubulin polymerization without notable disturbances of microtubules structure. Docking simulations revealed the possibility of organotin compounds to bind in the paclitaxel or vinblastine sites on the tubulin surface, but the mechanism of action of the compounds could not be explained based solely on these data. The influence on mitochondrial potential and induction of mitochondrial permeability transition also have been studied. Organotin compound 7 depolarises the mitochondria and induces the mitochondrial permeability transition. These properties may be the main reason of its cytotoxicity. Since the organotins possess the protective 2,6-di-tert-butylphenol groups the antioxidant potential of these compounds as inhibitors of mitochondrial lipid peroxidation was studied. All compounds under investigation effectively inhibit the Fe3+-induced mitochondrial lipid peroxidation. The comparative study of structure-activity relationship in the lipid peroxidation inhibition was performed. The influence of the organic groups nature, the number of hindered phenol fragments in organotins on their toxic effect is discussed. The introduction of hindered phenol groups decreases the cytotoxicity of compounds. This result opens up the possibility of designing novel anticancer drugs that might possess lower undesirable toxicity against normal cells.

A new group of compounds, promising for the design of original multitarget therapeutic agents for treating neurodegenerative diseases, based on conjugates of aminoadamantane and carbazole derivatives was synthesized and investigated. Compounds of these series were found to interact with a group of targets that play an important role in the development of this type of diseases. First of all, these compounds selectively inhibit butyrylcholinesterase, block NMDA receptors containing NR2B subunits while maintaining the properties of MK-801 binding site blockers, exert microtubules stabilizing properties, and possess the ability to protect nerve cells from death at the calcium overload conditions. The leading compound C-2h has been shown the most promising effects on all analyzed parameters. Thus, these compounds can be regarded as promising candidates for the design of multi-target disease-modifying drugs for treatment of AD and/or similar neuropathologies.

In in vitro experiments, amiridine in concentration of 100, 200, and 300 microM restored disturbed structure of microtubules assembled from tubulin and microtubule-associated proteins isolated from the brain of patients with Alzheimer disease. Tacrine in a concentration of 100, 250, and 500 microM inhibited phosphorylation of tubulin and microtubule-associated proteins isolated from the brain of patients with Alzheimer disease, while memantine and amiridine in the specified concentrations had no effect on phosphorylation.

Al(3+), Fe(3+), and Zn(2+) ions can disturb microtubule assembly from tubulin and microtubuleassociated proteins in rat brain. The main structural forms of these microtubules are rings and tangled bundles. These structures are formed only in the presence of Al(3+) and Fe(3+) ions. Therefore, Zn(2+) ions can be excluded from possible causes of structural abnormalities in microtubules during Alzheimer's disease. Al(3+) ions are the most probable etiological cause of Alzheimer's disease. The concentration of Al(3+) ions affecting the structure of microtubules is one order of magnitude lower than that of Fe(3+) ions (10 and 100 μM, respectively), which corresponds to their brain concentration reported in Alzheimer's disease.

It is shown for the first time that microtubular proteins isolated from the brain of patients with Alzheimer's disease can in vitro polymerize into microtubules with abnormal structure.

The review and summarizes own and literature data about the role of mitochondria as the important target in the search for drugs for the treatment of neurodegenerative diseases. Aging is a major risk factor for sporadic forms of various neurodegenerative diseases, including Alzheimer′s disease. One of the most argued and currently accepted theories is the Mitochondrial Free Radical Theory of Aging. Mitochondrial hypotheses of the development of sporadic forms of neurodegenerative diseases particularly Alzheimer′s disease, are closely connected with it. Impairments of mitochondrial functions lead to a decrease in their ability to regulate calcium homeostasis in the cell and to a decrease in the threshold for the induction of mitochondrial permeability transition (MPT) pores. MPT inhibitors can be considered as a promising approach to the treatment of neurodegenerative diseases, since these drugs can not only exhibit the properties of neuroprotectors, but also can provide normalization of synaptic activity due to increased calcium capacity of mitochondria. The review presents data on the number of MPT inhibitors, including endogenous compounds melatonin and N-acetylserotonin, their bioisosteric analogue Dimebon and a number of other compounds. The use of mitochondria as a basis for the formation of screening strategy for the search for compounds for the treatment of neurodegenerative diseases is of particular interest – both as a test of their potential toxicity, and as a basis for the creation of metabolic stimulants and drugs with neuroprotective and cognitive-stimulating effect., Обобщены собственные и литературные данные, обосновывающие роль митохондрий как важнейшей мишени при поиске препаратов для лечения нейродегенеративных заболеваний. Старение является основным фактором риска спорадических форм различных нейродегенеративных заболеваний, в том числе и болезни Альцгеймера (БА). Одной из наиболее аргументированных и принятых в настоящее время является свободнорадикальная митохондриальная теория старения. Именно с ней тесно связаны и митохондриальные гипотезы развития спорадических форм нейродегенеративных заболеваний и, в частности, БА. Нарушение митохондриальных функций приводит к снижению их способности регулировать гомеостаз кальция в клетке и снижению порога для индукции поры митохондриальной проницаемости (МРТ). Ингибиторы МРТ можно рассматривать как перспективный подход к терапии нейродегенеративных заболеваний, так как эти препараты могут не только проявлять свойства нейропротекторов, но и обеспечивать нормализацию синаптической активности благодаря увеличенной кальциевой ёмкости митохондрий. В обзоре представлены данные о ряде ингибиторов МРТ, включая эндогенные соединения – мелатонин, N-ацетилсеротонин, их биоизостерный аналог димебон и ряд других соединений. Использование митохондрий как основы для формирования скрининговой стратегии поиска соединений для лечения нейродегенеративных заболеваний представляет особый интерес и как тестирование их потенциальной токсичности, и как основа для создания метаболических стимуляторов и препаратов, обладающих нейропротекторным и когнитивно-стимулирующим действием.

We studied the effects of anti-Alzheimer drugs (tacrine, amiridine, and memantine) on phosphorylation of tubulin and microtubule-associated proteins isolated from rat brain, evaluated the capacity of these proteins to polymerize into microtubules after addition of study pharmacological agents, and analyzed the structure of generated microtubules. It was shown that test substances impair assembly of microtubules to a different extent. Dose-dependent effects of these agents on phosphorylation of tubulin and microtubule-associated proteins were observed. Triazolam (not approved for clinical use as anti-Alzheimer drug) in the same concentrations was used as the reference substance in the same tests. It was observed that this substance even in minimal concentration induced the most pronounced changes in microtubule structure. A direct correlation between the capacity of the test substances to modulate tubulin phosphorylation and to impair microtubule structure was found: the more the substance inhibited tubulin phosphorylation, the more it disordered microtubule structure.

Analysis was made of the composition of four structures of human brain in health (n-12), schizophrenia (n-10), and Alzheimer's senile dementia (n-8). Protein maps obtained by means of two-dimensional electrophoresis were used to analyze tissue samples of the structures under study. In the investigation, the neocortex was represented by the frontal cortex (field 10), the old cortex by the hippocamp, the midbrain by black substance, and medulla oblongata by the inferior olive. Comparative study of the protein maps revealed quantitative differences in certain brain structures in health as well as differences between these structures in health and mental pathology.