Therapeutic Potential of Heterocyclic Compounds Targeting Mitochondrial Calcium Homeostasis and Signaling in Alzheimer’s Disease and Parkinson’s Disease

1. ANAVEX2-73

ANAVEX2-73 (Blarcamesine) is a non-selective muscarinic acetylcholine receptor (mAChR) and alfa-1 receptor (S1R) ligand that exhibits an important affinity for its pharmacological targets at micromolar concentration.[186] σ1 Receptors are ubiquitously expressed in the central nervous system (CNS) and are located at mitochondria-associated ER membranes, where they interact with IP3 receptors to regulate Ca²⁺ exchange between the ER and mitochondria, thereby resulting in reduced mitochondrial stress, regulation of ion channels, activation of the nuclear erythroid 2-related factor 2 (NRF2) / antioxidant response element (ARE) pathway, and limited apoptosis.[187,188] IP₃ receptor-mediated Ca²⁺ release correlates with variations in the availability of mitochondrial Ca²⁺ and ATP synthesis.[189] A formal concept analysis (FCA) combined with the Knowledge Extraction and Management (KEM) environment was able to identify ANAVEX2-73 (tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride) as a potential genomic biomarker of disease and therapeutic response in a phase IIa clinical trial.[190] An ongoing randomized, double-blind phase III trial of this compound is evaluating the bioavailability, safety, tolerability, and effectiveness of the treatment in patients with AD (Nct03790709), PD with dementia (NCT03774459) and Rett syndrome (NCT03758924).

Intraperitoneal administration of ANAVEX2-73 (0.3-1 mg/kg) reversed scopolamine- and dizocilpine-induced learning impairments one week after intracerebroventricular injection of the neurotoxic Aβ_25-35 peptide in mice.[191] Using the same mouse model, treatment with ANAVEX2-73 inhibited the phosphatidyl-inositol 3-kinase (PI3K)/Akt pathway, thereby resulting in the activation of GSK-3β, improvement of behavioral deficits, and limitation of Aβ seeding and tau-induced pathology.[192] Moreover, the drug preserved mitochondrial integrity and function in isolated mitochondria from the hippocampal brain region of Aβ_25-35 injected mice by increasing the activity of complex IV and oxygen consumption at all states.[193] Exposure to ANAVEX2-73 also decreased the peroxidation of lipids, Bax/Bcl-2 ratio (that determines the cell susceptibility to apoptosis) and cytochrome c release. In an independent study, ANAVEX2-73 restored the respiratory control ratio and preserved complex IV levels from Aβ toxicity in a Ca²⁺-dependent fashion that regulates several tricarboxylic acid cycle (TCA) enzymes including α-ketoglutarate dehydrogenase complex and isocitrate dehydrogenase, responsible for NADH production, a substrate for complex I.[194] ANAVEX2-73 displayed a synergic effect with donepezil (but not with memantine) and restored spontaneous alternation and passive avoidance response in a non-transgenic mouse model of AD.[195] Incubation with ANAVEX2-73 elicited the accumulation of LC3-II-positive puncta and succeeding autophagic flux in cell cultures. C. elegans expressing GFP-LGG-1 (a marker of autophagy) treated with ANAVEX2-73 exhibit increased autophagic activity. In addition, administration of ANAVEX2-73 to Aβ₄₂-expressing nematodes upregulated proteostasis capacity, ultimately resulting in a dissociation and clearance of Aβ₄₂ aggregates.[196]

In summary, ANAVEX2-73 modulates Ca²⁺ release after its interaction with IP₃ receptors and prevents mitochondrial failure in multiple ways, including through the activation of the NRF2/ARE pathway that directly controls the expression of several antioxidant and anti-inflammatory genes. In addition, the molecule promotes autophagosome biogenesis and autophagic flux to degrade aggregated proteins and damaged organelles (such as mitochondria).

ANAVEX2-73 (1-(2,2-diphenyltetrahydro-3-furanyl)-N,N-dimethylmethanamine hydrochloride) 1 unique total synthesis was reported by Foscolos and co-workers.[197] The key step during the synthesis is the reduction-opening of lactone 7 to provide 1,4-diol 8, which was subsequently converted by acid-catalyzed cyclodehydration to ANAVEX2-73 1 (Scheme 1).

2. Caffeine

Caffeine (Mateine), a purine alkaloid present in several plants (Coffea, Camellia, Cola, Cirus, Ilex, Paullinia, and Theobroma), is the most consumed psychoactive substance in the world.[198] Coffee contains a variety of compounds such as caffeine, chlorogenic acid, cafestol, diterpenes and kahweol.[199] Moreover, coffee is a rich source of bioactive components that contribute to its biological activity, including potassium (K⁺), magnesium (Mg²⁺), niacin and potent antioxidants (chlorogenic acid and tocopherol).[200] Research studies have established a strong relationship between frequent caffeine consumption and reduced risk of developing AD and PD, with no detectable adverse effects in the CNS in an exposed population. A large body of literature has shown that mutations in presenilins are linked with Ca²⁺ signaling dysregulation and elevated Ca²⁺ release from the ER in neuronal cultures expressing mutant PS,[201,202,203] transgenic mice engineered to overexpress mutant PS1,[204,205] and fibroblasts from subjects with AD.[206,207,208] PS induces excessive Ca²⁺ accumulation and release in part via its biochemical interaction with the IP3 receptor Ca²⁺ release channel, thereby resulting in an anomalous regulation of Ca²⁺ signaling pathways and stimulation of APP processing and Aβ synthesis,[209,210,211] even prior the formation of plaques and NFTs.[212] Ca²⁺ storage content is higher in mutant presenilin-1 (PS1) knock-in neurons primary cortical neurons from a triple transgenic mouse model of AD (3xTg-AD) compared to non-transgenic cells.[213] Caffeine exposure altered Ca²⁺ signaling and promoted its release, which was independent of extrusion mechanisms or variations in the steady-state concentration of specific Ca²⁺-binding proteins. However, this effect was attributed to the activation of the ryanodine receptors (RyRs) that become sensitized to so-called process of Ca²⁺-induced release by caffeine.

The CAIDE longitudinal epidemiological study suggested an association between mid-life moderate coffee (but no tea) consumption and late-life reduced risk of dementia and AD.[214] Specifically, caffeine intake (3-5 cups per day) reversed cognitive impairment (assessed by Mini-Mental State Examination) in a gender-specific manner (women age 65 and older). A recent mendelian randomization study found that genetically predicted higher caffeine content in the plasma correlates with diminished risk of AD.[215] Following confounding adjustment, it has been described that long-term coffee consumption (≥ 2 cups/day) was associated with a significant cognitive decline decrease in pathological Aβ deposition. Nevertheless, no changes were observed in cortical thickness, cerebral white matter hyperintensities or cerebral glucose metabolism, which are features also related to the neurodegenerative process.[216] The Honolulu Heart Program provided the first evidence showing a potential beneficial effect of caffeine intake in PD patients. Coffee drinkers (28 oz or more per day) had 5-fold lower incidence of developing PD compared to non-caffeine drinkers following an adjustment for both age and pack-years of cigarette smoking.[217] This study was further supported by Ascherio and colleagues.[218] In addition, consumption of decaffeinated coffee was not associated with decreased risk of PD, suggesting that caffeine, rather than other coffee components, accounts for the inverse association observed. The findings showed a significant negative correlation between caffeine intake and risk of developing PD in men but a U-shaped relationship among women. The risk of PD was similar in women using hormones and women who never used hormones. Interestingly, caffeine diminished the risk of PD in menopausal women that did not take hormone replacement but there is a higher risk (4-fold) among hormone users with high caffeine.[219] The risk of PD was even lower when coffee intake is combined with cigarette smoking and nonsteroidal anti-inflammatory drug use, resulting in a cumulative effect.[220]

A different clinical trial confirmed that caffeine consumption mitigates the risk of PD, but its neuroprotective properties may vary depending on its interaction with other factors, such as obesity and low serum cholesterol levels, which can modify the risk of having PD.[221] A meta-analysis involving a large number of participants found a non-linear relationship between coffee consumption and the incidence of PD that achieves the maximum protective effect at 3 cups per day.[222] However, the authors described a linear dose relationship of reduced risk of PD with both tea and caffeine consumption, especially in men compared to women and in European and Asian population relative to USA residents. Based on an open-label, dose-escalation study, caffeine had potential motor and non-motor benefits in subjects with PD, with the maximum tolerated dose of 100-200 mg/day bis in die (BID) without affecting sleep quality.[223] In a randomized controlled trial, administration of 200 mg caffeine BID for six weeks did not improve daytime somnolence in PD patients but possessed the potential to reverse motor symptoms.[224] Transgenic mice overexpressing the human APP gene carrying the Swedish mutation (APPsw) treated with 0.3 mg/mL caffeine in the drinking water for 5 months exhibited an improvement in multiple behavioral measurements.[225] In addition, enzyme-linked immunosorbent assay showed a significant downregulation of both Aβ₄₀ and Aβ₄₂ levels and decreased PS1 and β-secretase (BACE) protein concentration in the hippocampus of these AD mice. Oral supplementation with caffeine for 4 months restored motor performance, anxiety and memory deficits, prevented neuronal death in the CA1 pyramidal layer of the hippocampus, and promoted neurogenesis in the absence of detectable pathological effects on the Aβ pathology in transgenic Tg4-42 and 5xFAD mouse models of AD.[226] Caffeine did not alter the optical density or mRNA expression levels of A1 or A2A receptors in the mouse cerebral cortex or hippocampus.

Aβ burden was reduced in both brain and plasma of APPsw and APP/PS1 mice (which contain human transgenes for both APP bearing the Swedish mutation and PSEN1 containing a L166P mutation) following either acute or chronic caffeine administration.[227] However, amyloid plasma levels were not correlated with (i) Aβ brain content, (ii) cognitive impairment, and (iii) pro-inflammatory cytokine levels in aged mice. Animals receiving caffeine also display an enhanced memory performance. It has been also demonstrated that long-term administration of caffeine is protective in the THY-Tau22 transgenic mouse model of progressive AD-like tau pathology by limitation of spatial memory abnormalities, tau phosphorylation, oxidative stress, and inflammation.[228] Since persistent (but no acute) administration of caffeine increases cerebrospinal fluid (CSF) secretion in rats in comparison to the control-treated group, it has been proposed that chronic caffeine intake can promote CSF production combined with an elevated cerebral blood flow velocity and Na⁺/K⁺-ATPase levels.[229] Protein kinase A (PKA) is a heterotetrametric enzyme comprised of two regulatory and two catalytic subunits that plays an essential role in cell proliferation with an anti-apoptotic activity. The cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) is a transcription factor that modulates a subset of genes implicated in cognition and neuron survival. c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) are mitogen-activated protein kinases that can trigger apoptotic signaling by the upregulation of pro-apoptotic genes through the activation of specific transcription factors or directly, by catalyzing protein phosphorylation. Administration of caffeine upregulated PKA activity, induced CREB phosphorylation, and reduced the levels of phosphorylated JNK and ERK in the striatum (but no frontal cortex) of APPsw mice, suggesting that caffeine pro-apoptotic signaling.[230] Caffeine intake resulted in memory capacity improvement and increased hippocampal brain neurotrophic derived factor (BNDF) and tyrosine receptor kinase B (TrkB) immunoreactivity in PS1/APP double transgenic mice and rats treated with aluminum chloride.[231,232]

Caffeine also has a neuroprotective role in PD. Exposure to caffeine preserved the degeneration of the nigrostriatal DAergic pathway in neurotoxin-based animal models of PD, such as rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Histopathological and immunohistochemical characterization of brain tissue from rotenone-exposed rats show a significant degeneration of SN DA neurons and their projections into the striatum. However, administration of caffeine provided a dose-dependent therapeutic effect against rotenone-mediated neurotoxicity.[233] Injection of caffeine through a peritoneal route improved the behavioral phenotype, normalized brain enzymatic activities of both acetylcholinesterase (AChE) and Na⁺/K⁺-ATPase (which play an important role in memory and learning) and mitigated neuroinflammation and oxidative damage in rotenone-treated rats.[234] Caffeine modulated striatal protein content and catalytic activity of cytochrome P450 (a membrane-bound hemoprotein that plays a central role in the detoxification of xenobiotics), glutathione-S-transferases (a family of phase II detoxification enzymes involved in the protection of macromolecules from attack by ROS, reactive electrophiles, environmental carcinogens, and chemotherapeutic agents) and vesicular monoamine transporter-2 (VMAT-2, an integral presynaptic protein that controls the packaging and release of DA and other neurotransmitters from synaptic vesicles) in mice receiving MPTP for 4 weeks.[235] Using ex vivo ¹H-[¹³C]-NMR spectroscopy, Bagga et al. found that pretreatment with caffeine increased neuronal glutamatergic and GABAergic metabolic activity, and neurotransmission in MPTP-injected mice.[236] Oral supplementation with the coffee component eicosanoyl-5-hydroxytryptamide attenuated MPTP-induced nigrostriatal DAergic cell loss in mice, displayed both antioxidant and anti-inflammatory properties and normalized phosphoprotein phosphatase 2A (PP2A) methylation and activity.[237]

Intraperitoneal injection of 10-20 mg/kg day of caffeine improved motor dysfunction and increased catecholamine levels in a rat model of 6-OHDA-induced striatal lesion.[238] Striatal injection of 6-OHDA elicited apomorphine-induced rotation and impaired locomotor activity in parallel with a loss of DA immunoreactivity and an inflammatory response, but administration of caffeine ameliorated the behavioral and pathological PD-like phenotype. Moreover, it has been also reported that administration of caffeine in the drinking water can exert neuroprotective effects by decreasing the number of inclusions positive for pS129 α-syn, content of TUNEL-stained apoptotic cells, LC3-mediated macroautophagy, and lysosome-associated membrane protein type 2A (LAMP2A) chaperone-related autophagy in mice that received an intracerebral injection of synthetic α-syn fibrils with the A53T missense mutation.[239] Regarding its potential mechanism of action, caffeine is an antagonist of the adenosine-2A receptor (A2AR), which are predominantly localized in the GABAergic striatopallidal neurons projecting from the caudate nucleus and the putamen to the external segment of the globus pallidus (indirect pathway).[240] A2ARs colocalize with DA2 receptors to form heteromeric complexes that mediate the allosteric antagonism between adenosinergic and DAergic transmission.[241] Noteworthy, a double-blind, randomized, crossover study showed that treatment with caffeine improved the pharmacokinetic profile of levodopa by reducing its plasma concentration-time profile and by prolonging its effect.[242]

In summary, while caffeine has complex biological and pharmacological profiles, experimental evidence indicates that caffeine readily crosses the blood-brain barrier and exerts its action by antagonizing A2ARs and confers neuroprotection by stimulating mitochondrial function and by attenuating excitatory neurotransmitter release, oxidative insult, and neuroinflammation. Clinical trials have shown that caffeine significantly enhances AD-related memory loss and improves motor symptoms in PD patients. In addition, caffeine has a well-established long-term safety profile. Together with its low cost and high availability, caffeine is a promising therapeutic agent for the treatment of neurodegenerative diseases.

The most recent total caffeine (1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione) 2 synthesis was reported by Narayan in 2003 from uracil 9.[243] N-Methylation in the presence of a strong base such as sodium hydride in dimethylsulfoxide (DMSO) produced 1,3-dimethyluracil 10, which was nitrated and subsequently reduced to 5-amino-1,3-dimethyluracil 12, using iron and hydrochloric acid. Compound 13 was obtained following two conventional steps and resulted in the formation of theophylline 14 after a reduction process and an intramolecular heterocyclization reaction with iron and acetic acid. The final methylation at position 7 led to the generation of caffeine 2. Therefore, synthesis of caffeine needed seven reaction steps with an ~10% overall yield (Scheme 2).

In recent years, new and improved methodologies for the N-methylation of theophylline 14 have been published. A novel technique uses the Q-tube apparatus in water at over boiling temperature as a green solvent.[244] In a different study, Schnürch’s group optimized the employment of quaternary ammonium salts as monoselective N-methylation reagents (Scheme 3).[245]

3. Diltiazem

Diltiazem (Cardizem) is a non-dihydropyridine Ca²⁺ channel blocker with antihypertensive, antiarrhythmic and vasodilation properties. The drug selectively targets the VOCCs, which are the primary mediators of Ca²⁺ influx into neurons in response to membrane depolarization. P/Q- and N-type VOCCs regulate neurotransmitter release upon arrival of the action potential to the axon terminal in the presynaptic neuron. Glutamate release promotes postsynaptic Ca²⁺ trafficking by activation of NMDA receptors (NMDAR) or through an indirect pathway involving L-type VOCCs.[246] Reduced Ca²⁺ transient at presynaptic or postsynaptic sites can mitigate glutamate-induced excitotoxicity. Therefore, administration of Ca²⁺ channel blockers has become an interesting approach for the treatment of neurodegenerative diseases. PET and postmortem analyses of different brain regions (such as cerebral cortex and amygdala) from AD subjects revealed a marked inhibition of AChE enzymatic activity, responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into acetate and choline.[247,248,249] In contrast, administration of Aβ_25-35 peptide upregulated the activity of AChE through modulation of the L-type VOCCs (increasing intracellular Ca²⁺ concentration) in embryonal carcinoma P19 cells.[250] When cultures were incubated in the presence of diltiazem, the authors found a 75% loss of AChE enzyme activity.

Epidemiological data obtained from individuals with PD indicate that long-term treatment with Ca²⁺ channel blockers targeting Ca²⁺ channels of DA neurons may represent a potential therapeutic strategy, reducing the risk for developing the disease.[251,252,253] A larger study involving 65,001 patients, reinforced the connection between Ca²⁺ channel blockers and diminished incidence of PD.[254] In addition, centrally acting Ca²⁺ channel blockers prevent nigrostriatal DAergic degeneration in parkinsonian mice and non-human primates and improve survival rate in vitro.[178,255,256] There is evidence supporting a beneficial effect of diltiazem against DA toxicity in human neuroblastoma cells.[257] Cav1.2 and Cav1.3 are L-type VOCCs regulate DAergic neuron spontaneous firing activity in the SN region of the brain. DA neurons fire either in a low frequency single-spike pattern or transiently, in a high-frequency so-called burst mode. Data from animal models support clinical observations showing that diltiazem has positive effects on AD-induced pathology. Ca²⁺ channel blockers, such as diltiazem, protect neurons from Aβ-induced influx of Ca²⁺ ions and downstream toxicity as well as decrease the amyloid content by facilitating the clearance rate.[258,259] Excessive neuronal Ca²⁺ influx contributes to neuronal dysfunction and degeneration that underlies cognitive disturbances in AD. Neuronal cultures incubated with Aβ peptides led to an enhanced Ca²⁺ entry.[260] Furthermore, endogenous accumulation of oligomeric Aβ led to an upregulated expression of L-type VOCCs (Cav1.2).[259] Diltiazem improved survival rate and decreased Ca²⁺ intracellular concentration by blocking L-type Ca²⁺ channels in vitro. Diltiazem protected MC65 neuroblastoma cells from the toxicity mediated by the APP C-terminal fragment by improving cell survival.[261]

In addition, the Aβ_25-35 fragment decreased both secreted APP and Aβ while promoted cell-associated APP in chick sympathetic neurons. Enhanced APP secretion with no variations in the amount of cell-associated APP occurred in response to diltiazem.[262] The accumulation of exogenous Aβ significantly attenuated cell viability and proliferation (determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) in primary hippocampal neuronal cultures, which was reversed by exposure to diltiazem.[13] This study also provided evidence supporting that Aβ toxicity shows an early Ca²⁺-independent phase and a late Ca²⁺-dependent phase. Spontaneous alternation behavior, a reliable measure of short-term of spatial working memory, was significantly increased in mice treated with diltiazem.[263] Chronic exposure to aluminum or any other of its forms in the drinking water has been associated with higher risk of developing AD.[264,265,266] Aluminum accumulation has been detected in the brain of AD cases, where causes neurofibrillary degeneration in neurons.[267] Bouras et al. found a specific increased content of aluminum within NFTs in both the inferior temporal cortex and hippocampus of individuals with AD.[268] Aluminum induces Aβ protein conformational modifications and aggregation (including amyloid fibrillation) either in vitro or in vivo.[269,270,271,272] Aluminum chloride has been reported to cause dementia (AD accounts for the ~70% of cases) but administration of diltiazem reversed learning and memory deficits, AChE upregulation, and oxidative damage in mice.[273] Ginkgo biloba augmented the bioavailability of diltiazem by modulating cytochrome P450 3A activity in both the rat liver and small intestine.[274]

It has been reported that Cav1.2 and Cav1.3 L-type VOCCs are susceptible of degeneration during the progression of PD.[275,276,277] During spontaneous action potentials, L-type VOCCs contribute to Ca²⁺ oscillations in the soma and dendrites of constantly active midbrain DA neurons. The extended opening of L-type VOCCs throughout autonomous pacemaking induces prolonged cytoplasmic Ca²⁺ trafficking and its overload in SN DA neurons, leading to mitochondrial oxidative stress.[275,277] L-type VOCCs-mediated alteration of the steady-state of DA levels is responsible for causing downstream oxidative stress and α-syn-induced toxicity.[278] In contrast, some studies suggest that administration of Ca²⁺ channel blockers (including diltiazem) could be associated with the development of parkinsonian symptoms, particularly, in subjects that already had subclinical idiopathic PD pathology.[279,280] Despite there is a lack of research investigating the potential antioxidant effect of diltiazem in AD or PD, it has been found that its intraperitoneal injection decreased the levels of nitrite (an indirect assessment of nitric oxide) and MDA (a lipid peroxidation marker) and upregulated the activity of several antioxidant enzymes such as reduced glutathione, catalase, and superoxide dismutase in a streptozotocin-induced rat model of diabetes.[281] High-performance liquid chromatography analysis showed a significant decline of MDA content in perfused rabbit hearts treated with diltiazem.[282] Oral supplementation with diltiazem for two weeks reduced thiobarbituric acid-reactive species (TBARS, a by-product of LPO) and nitrite content and upregulated superoxide dismutase (SOD) and reduced glutathione enzymatic activities in an aluminum chloride mouse model of AD.[273] Because of their autonomous pacemaking activity coupled to action potentials, SN DA neurons have particularly high energetic (ATP) demands and may be subjected to elevated levels of basal oxidative stress.[283]

Likewise, the anti-inflammatory effect of diltiazem in patients with AD or PD has not yet been examined. Diltiazem produced a dose-dependent inhibitory effect on T-cell proliferation.[284] Individuals undergoing cardiopulmonary bypass surgery displayed at increase in IL-6 content, which was significantly attenuated following diltiazem administration.[285] Enhanced levels of the anti-inflammatory cytokine IL10 were observed in subjects with unstable angina receiving diltiazem.[286] Moreover, intraperitoneally injected diltiazem reduced IL-10 and TNF-α (but no nitrite/nitrate and IL-6) plasma concentration in BALB/c mice injected with bacterial lipopolysaccharide (LPS).[287] Synthetic Aβ_25-35 fragments added to human fetal brain cell cultures activates microglia and increases the levels of intracellular Ca²⁺ in a time-dependent fashion.[288] Treatment with the L-type VSCCs antagonist diltiazem resulted in a transmembrane diminished Ca²⁺ influx that abolishes microglial stimulation. Cav1.2 VOCCs (particularly α1 subunit) are highly expressed in reactive astrocytes of mice overexpressing Aβ protein precursor (AβPP), which is specifically linked to an Aβ plaque deposition.[289] Cav1.2 channels are also expressed in microglia. Variations in the balance between M1 (pro-inflammatory) and M2 (anti-inflammatory) microglial phenotypes are related to PD. It has been demonstrated that diltiazem-induced blockade of L-type VOCCs promotes pro-inflammatory M1 transition and decreases anti-inflammatory M2 macrophage polarization in mouse microglia-derived MG6 cells, resulting in an upregulated iNOS mRNA expression and downregulated arginase levels.[290] In addition, knockdown of microglial Cav1.2 led to behavioral impairment, DA neuron degeneration in the SN of MPTP-injected mice compared to control group, and reduce polarization toward the M1 phenotype.

In summary, the L-type VOCC diltiazem is a Ca²⁺ antagonist that can be used to treat dementia and dementia-related diseases due to its capability of improving cognitive deficits, such as learning and memory capacity. The drug also improves neuron survival and exhibits both antioxidant and anti-inflammatory properties. Diltiazem can cause nausea and vomiting, pulmonary oedema, and renal failure. Diltiazem, together with verapamil, are the most toxic Ca²⁺ channel blockers in overdose and their toxicity is associated with significant cardiovascular collapse, metabolic problems (e.g., hyperglycemia) and vascular smooth muscle tone deficits.

The first racemic synthesis of diltiazem (2-(2-dimethylaminoethyl)-5-(4-methoxyphenyl)-3-oxo-6-thia-2-azabicyclo[5.4.0]undeca-7,9,11-trien-4-yl]ethanoate) 3 was described in 1990.[291] The first asymmetric total synthesis of (+)-diltiazem was reported by Naito and co-workers, using a diastereoface differentiating nucleophilic addition as the key step to create the two contiguous stereogenic centers.[292] Schwartz used the separation of diastereomeric glycidic esters (15 and 16) by direct crystallization as the key step, based on their marked difference in solubility.[293] Enantiomerically pure glycidic ester 15, the required isomer for the synthesis of (+)-diltiazem 3, was the major product obtained (54% yield) (Scheme 4).

In 1994, Jacobsen et al. used a manganese-catalyzed asymmetric epoxidation of cinnamate esters for the preparation of an enantiomerically pure key intermediate 18 in the synthesis of 3 (Scheme 5).[294]

To date, more synthetic routes to prepare (+)-diltiazem 3 from a chiral epoxide intermediate have been reported. The enantiomerically pure epoxide has been obtained by the introduction of a chiral auxiliary in the reaction[295,296], utilization of Yang’s catalyst[297], addition of metal complexes[298] or by direct acquisition.[299]

A useful synthetic methodology employed to induce chirality in the product is the utilization of lipase[300,301] or baker’s yeast catalysed reactions.[302] The most recent approach employed for total synthesis of (+)-diltiazem 3 was reported by Chen and colleagues in 2022.[303] Racemic keto ester 19, obtained in two steps from commercially available reagents, is converted into enantiomerically pure hydroxy ester 20 by ketoreductase-catalyzed dynamic reductive kinetic resolution. Two conventional steps (ring closure and ring opening) are further taken to produce an intermediate 21, which is readily subjected to intramolecular acid-catalyzed amidation. Two additional steps from intermediate benzothiazepinone 22 are required to obtain (+)-diltiazem 3. This chemoenzymatic synthesis involves eight steps to obtain (+)-diltiazem 3 in ~45% overall yield (Scheme 6).

4. Latrepirdine

Latrepirdine (Dimebon) is a carboline that blocks H1 histamine receptor (H1R) activity. A broad spectrum of effects on neurologically relevant targets was proposed based on various cell- and animal-based studies. The compound is a potent reversible and competitive inhibitor of both AChE and butyrylcholinesterase, which leads to elevated acetylcholine content and concomitant boosting of cognitive performance.[304,305] Besides its anti-histaminic properties, latrepirdine can modulate a wide range of other neurotransmitter receptors (such as DA, serotonin, glutamate, α-adrenergic, and imidazole).[306,307] Studies carried out in rat cerebral neurons confirmed the inhibitory effect of latrepirdine on NMDA receptors and involve the polyamine site of the NMDA NR2B subunit (the target for histamine) as a potential binging site.[304,308] Latrepirdine has also been shown to interact with Ca²⁺ channels; for example, its administration effectively blocked the activity of L-type VOCCs (IC₅₀ = 57 μM) in cerebellar granule neurons.[309] A pilot clinical trial carried out in patients with mild-to-moderate AD given latrepirdine for 8 weeks showed an important reduction of neuropsychiatric symptoms (depression) and a significant cognitive function enhancement together with a lack of hematological and biochemical disturbances.[304] Drug safety and efficacy were also examined in moderate-to-severe AD subjects (NCT00912288). Data from a phase II randomized, double-blind, placebo-controlled clinical study showed a significant improvement in cognition, function, and behavioral outcome following 60 mg/day latrepirdine for 26 and 52 weeks (6 months extension phase) in individuals with mild-to-moderate AD with no adverse effects recorded.[310] CONNECTION and CONTACT phase III trials (NCT00838110) failed to show significant improvement in any primary or secondary outcome measures of cognition in patients treated with latrepirdine.[311] A phase II randomized, placebo-controlled trial of latrepirdine showed a protective effect against cognitive impairment in subjects with mild-to-moderate Huntington’s disease.[312] Results from the double-blind, placebo-controlled Phase III HORIZON trial (NCT00920946) revealed that latrepirdine failed to achieve statistical significance for co-primary endpoints in individuals with mild-to-moderate Huntington's disease. A meta-analysis study revealed that latrepirdine did not ameliorate overall cognition, but the molecule enhanced the neuropsychiatric inventory scale, used to assess psychopathology in dementia patients.[306]

Cerebellar granule neurons incubated with the Aβ_25-35 peptide displayed morphological alterations, cell loss, and dysregulated intracellular Ca²⁺ homeostasis.[309] Nevertheless, exposure to latrepirdine preserved neurons against Aβ-mediated toxicity through inhibition of the L-type VOCCs current. Intraperitoneal administration of latrepirdine reversed learning and memory loss following chronic partial deprivation of cerebral cholinergic functions, which causes dementia in rats.[305] Latrepirdine had the capability of modulating cognitive ability in 5xFAD mice, though it was unable to mitigate Aβ-associated pathology.[313] P301S tau mice consistently performed better on both the inverted grid and accelerating rotarod tests after latrepirdine treatment, suggesting that the molecule is associated with enhanced motor performance.[314] Treatment with latrepirdine 15 min prior scopolamine-induced memory impairment significantly ameliorated delayed matching-to-sample task accuracy (to assess memory) in young adult and aged Rhesus macaques.[315] Treatment with latrepirdine in the drinking water for 4 months resulted in improved cognitive function in TgCRND8 mice although did not modify neither Aβ₄₀ or Aβ₄₂ content, suggesting APP processing as a direct target of the drug.[316] FDG-PET studies revealed an increased cerebral glucose utilization in aged mice in response to latrepirdine exposure.[317] Augmented succinate dehydrogenase activity, ΔΨm, and ATP synthesis were normalized following latrepirdine administration in differentiated SH-SY5Y cells and primary neuron cultures from mouse cerebral cortex with no differences in the mtDNA copy number, thereby suggesting a restoration of mitochondrial function.[318] Preservation of mitochondrial shape, mass, and respiratory chain complex activities were seen following latrepirdine treatment in cells incubated with Aβ.[319] The compound can also preserve mitochondrial function by targeting the mPTP opening induced by the Aβ_25-35 peptide and MPP⁺ (1-methyl-4-phenylpyridinium).[320] Mitochondrial accumulation of Ca²⁺ induces depolarization of the mitochondrial membrane and, if large and sustained enough, a subsequent irreversible opening of the mPTP. Moreover, no changes were observed in pivotal metabolic enzymes, suggesting that latrepirdine’s neuroprotective effect is independent of promoting mitochondrial energy metabolic pathways.[309]

It has been shown that short-term exposure to latrepirdine causes: (i) Aβ accumulation and increased secretion of APP metabolites in mouse N2a neuroblastoma cells overexpressing APPsw; (ii) higher levels of Aβ₄₂ in isolated cortical synaptoneurosome preparations from TgCRND8 mice; and (iii) enhanced Aβ₄₀ levels in the hippocampal interstitial fluid of Tg2576 AD mice.[321] Either Ca²⁺ or Aβ_25-35 toxic fragment promoted mitochondrial LPO but administration of latrepirdine inhibited oxidative damage to lipids.[320,322] In addition, the molecule has been shown to prevent LPO induced by tert-butyl hydroperoxide in rat brain mitochondrial homogenates.[323] In addition, administration of the drug diminished the content of hyperphosphorylated tau-positive dystrophic neurons in the mouse spinal cord while no changes in the levels of inflammatory markers were detected. Latrepirdine can also have a pro-autophagic activity, with a selective autophagic elimination of protein aggregates. Latrepirdine induces MTOR- and ATG5-dependent autophagy, which results in decreased levels of intracellular APP metabolites, including Aβ in the neocortex and hippocampus of TgCRND8 AD mice.[324] Latrepirdine can also stimulate autophagy-mediated degradation of α-syn in differentiated SH-SY5Y neurons and in the mouse brain.[325] Immunohistochemical analyses found a significant decrease in the number of TDP-43-like inclusions in cells transfected with TDP-43.[326] α-Syn pathogenic and toxic effect was markedly reduced in S. cerevisiae, SH-SY5Y cells, and the mouse brain through a selective autophagic protein degradation.[325] Administration of latrepirdine attenuated methamphetamine- (but not MPTP) induced cytotoxicity in mice in a body temperature-independent manner, suggesting a neurotoxin-specific protective effect.[13] However, the authors only measured the concentration of DA in the mouse striatum, with a lack of assessment of other additional key factors associated with neurodegenerative processes. Increased lifespan and motor performance parallel to reduced γ-syn aggregation, number of proteinaceous inclusions, and the inflammatory response were observed in transgenic mice overexpressing γ-syn treated with latrepirdine in the drinking water.[314] In contrast, exposure to the drug failed to protect DAergic neurons in C. elegans and mouse models of PD.[327] Fourteen-old-month α-syn transgenic mice receiving latrepirdine did not exhibit neurochemical, behavioral, or histopathological variations relative to the control group. Noteworthy, these mice recapitulate some pathological features typically manifested at the early onset of the disease.[328]

In summary, psychiatric symptoms (such as depression) and learning and cognitive measures were restored in AD patients undergoing latrepirdine treatment, in part due to its ability to increase acetylcholine concentration by inhibition of either AChE or histamine receptors. Latrepirdine may elicit neuroprotective activity by promoting mitochondrial function and clearance of a range of intracellular inclusions through the stimulation of autophagy. The molecule may also regulate several targets involved in AD pathology, such as neurotransmitter receptor activity, stabilization of Ca²⁺ signaling, and LPO. Toxicological studies have shown that latrepirdine is safe and a well-tolerated drug. A dosage exceeding the therapeutic range by 100 times for a period of 2 months did not cause any physiological changes or pathology in guinea pigs, rats, or dogs.[304,329]

Latrepirdine (2,3,4,5-tetrahydro-2,8-dimethyl-5-[2-(6-methyl-3-pyridinyl)ethyl]-1H-pyrido[4,3-b] indole) 4 was originally synthesized using the Fischer-indole reaction.[330] A more recent synthesis reported by Zheng et al. employed p-toluidine 23 and 2-methyl-5-vinylpyridine 24 as commercial starting materials and achieved the desired product 4 in 16% yield over four reactions steps.[331] Moreover, cyclization of compound 26 and 1-methylpiperidin-4-one 27 was carried out with 80% HAc in a one-pot two steps reaction instead of using benzene and HCl/EtOH (Scheme 7).

The synthesis of latrepirdine 4 has also been performed using the chemistry of ruthenium (III) catalysts in a concise and efficient manner.[332] The reaction comprised six steps, which required three purification processes only with an ~47% overall yield. The key step involved the stereoselective formation of γ-carboline 32 from ortho-substituted aryl azide 31 catalysed by RuCl₃·nH₂O (Scheme 8).

5. Nifedipine

Nifedipine (Procardia) is a first-generation dihydropyridine Ca²⁺ channel blocker used to treat hypertension and to control angina pectoris. Nifedipine is a selective antagonist of the L-type VOCCs that plays an essential role in neuronal processes triggered by membrane depolarization, thereby contributing to Ca²⁺-mediated events activated by signaling pathways and diverse stimuli, including neurotransmitter release, rhythmic firing, gene expression, etc.[333] There is growing evidence that nifedipine may be an effective therapeutic agent for the treatment of neurodegenerative diseases, including AD and PD. APOE ε4 peptide-induced increase in the concentration of intracellular Ca²⁺ and transcriptional activity of cAMP-response element-binding protein (CREB) in rat hippocampal neuronal cultures was reduced after administration of nifedipine, suggesting that L-type VOCCs are involved in neuron responses to APOE ε4.[334] In culture of primary mouse hippocampal neurons, Aβ enhanced the content of cytosolic Ca²⁺ and concomitant phosphorylation of serine-880 in the AMPA-selective glutamate receptor 2 (GluR2), which leads to attenuated synaptic activity.[335] In contrast, exposure to nifedipine diminished Ca²⁺ levels and GluR2 phosphorylation and increased cell surface GluR2. It has been reported that Aβ-mediated toxicity promotes the activation of the Ca²⁺-calmodulin kinase II (CaMKII)/AMP-activated protein kinase (AMPK) pathway.[336,337] Exposure to nifedipine mitigated Aβ toxicity by preserving cell viability in SH-SY5Y cells stably transfected with an empty vector or expressing the cellular prion protein, indicating that Ca²⁺ influx via L-type VOCCs is involved in Aβ-induced neurotoxicity.[338] Administration of nifedipine significantly diminished the content of secreted Aβ_1-42 and key components of the gamma secretase complex (e.g., PS-1) in H4 neuroglioma cells overexpressing APP, without triggering cell death.[339] Primary CNS neurons cultured from PS1-deficient mice showed higher susceptibility to oxidative stress but exposure to nifedipine increased the survival rate, suggesting that L-type VOCCs-mediated Ca²⁺ influx via was responsible for the neuronal loss.[340]

It has been suggested that deficits in APP^−/− mice are associated with alterations in Ca²⁺ homeostasis. Treatment with nifedipine (but not the NMDAR blocker 2-amino-5-phosphonovaleric acid) restored post-hypoxic related damage, leading to an improvement of neuron functional deficiencies such as population spike amplitude and depolarized resting ΔΨm in hippocampal slices from mice lacking APP.[341] To determine the specific pathways involved in altered Ca²⁺ homeostasis, primary cortical neuron cultures from 3xTg-AD or APPsw mice were incubated with intracellular Aβ.[342] Quantitative measurements displayed a marked boost in resting [Ca²⁺]_cyt concentration (further enhanced by extracellular Ca²⁺ efflux) that was blocked by nifedipine, suggesting that elevated Ca²⁺ influx occurs through L-type VOCCs. An age-dependent increase of L-type VOCC amplitude was described in CA1 pyramidal neurons (but not CA3 or dentate granule neurons) in 3xTgAD mice relative to wild-type mice, consistent with the notion that CA1 neurons are prone to p-tau/NFT pathology due to an excessive Ca²⁺ trafficking. Nevertheless, Ca²⁺ current was limited by nifedipine, which attributes to the L-type VOCCs the enhanced intracellular Ca²⁺ pool.[343] The stromal interaction molecule 1 (STIM1) is a type I transmembrane protein that plays a pivotal role in Ca²⁺ influx. Stim1 positively modulates Orai1 channels for store-operated Ca²⁺ entry and negatively modulates L-type VOCCs. STIM1 protein levels were decreased in the medium frontal gyrus of patients diagnosed with AD.[344] STIM1-KO differentiated cells displayed an elevated Ca²⁺ entry in response to membrane depolarization, which was nifedipine-sensitive. Primary hippocampal neuronal cultures from 5xFAD mice exposed to nifedipine inhibit abnormal VOCC and store operated Ca²⁺ (SOC) entry, a process regulated mainly by STIM1. In contrast, STIM2 was responsible to modulate depolarization-mediated Ca²⁺ entry through VOCCs into cells with full Ca²⁺ stores.[345] Intracellular Ca²⁺ responses to membrane depolarization were potentiated by the V337M mutant tau in diverse cell cultures, which were inhibited following administration of nifedipine. These findings suggest that upregulation of L-type VOCCs-mediated Ca²⁺ influx results from destabilization of microtubules triggered by tau mutations.[346] Late insoluble (but not soluble or early insoluble) tau aggregates activated sensitive Ca²⁺ channels, thereby increasing Ca²⁺ signaling, cell loss, and oxidative damage (O₂^•− levels) in primary cultures of rat cortical neurons and astrocytes.[347] The rate of ROS production was significantly diminished in the presence of the NADPH oxidase inhibitor AEBSF (2-aminoethyl) benzenesulfonyl fluoride hydrochloride), indicating that ROS formation was NADPH oxidase-dependent. In addition, administration of nifedipine limited tau-mediated Ca²⁺ influx, generation of ROS, and overall toxicity. The data demonstrated that: (i) tau effect depends on its aggregation state, and (ii) late insoluble aggregates incorporate into the membranes, resulting in an ionic current alteration and VOCC stimulation.

The subthalamic nucleus (STN) has been proposed to play a central role in the disrupted function of the basal ganglia circuitry associated with PD. Distinct activity patterns, such as increased burst firings, have been observed in STN neurons and represent a pathognomonic electrophysiological feature linked to PD. High-frequency stimulation of the STN or pharmacological blockade of the subthalamopallidal network in monkeys and rats improved motor symptoms.[348,349,350] Patch-clamp studies performed in rat brain slices showed that around 50% of STN neurons display the intrinsic property of switching from single-spike activity to burst-firing mode.[351] Treatment with nifedipine caused an irreversible reduction in burst frequency and abolished burst firing. Furthermore, the role of VOCCs was investigated in STN neurons. Based on nifedipine effects on the frequency and current curve, it was established that both short- and long-duration rebound bursting neurons contain nifedipine-sensitive Cav1.2-1.3 channels, which only contribute to rebound activity in STN neurons with long-lasting rebounds.[352] High-frequency stimulation-induced oscillations in the STN resulted at least in part from Ca²⁺ entry through the high-threshold potential nifedipine-sensitive L-type VOCCs.[353] Pasternak et al. reported that nifedipine was not linked to significantly lower risk of developing PD, but the small sample size undermined the findings.[354] Nifedipine exposure did not have a significant effect on DA neuron survival but exhibited a stimulatory impact on neurite length. The actions of cholinesterases such as AChE and butyrylcholinesterase on Ca²⁺ conductance may be responsible for the trophic effect on neurite outgrowth in embryonic ventral midbrain cultures.[355] Nifedipine limited both glutamate- and NMDA-related CREB phosphorylation. Stimulation of D1 receptors or cAMP pathway in primary striatal neuron cultures produced cytosolic accumulation of Ca²⁺ that interacted with nifedipine, resulting in Ca²⁺-mediated CREB phosphorylation and c-fos gene expression.[356]

Injection of nifedipine into the dorsal striatum did not affect apomorphine-induced rotational behavior, indicating that it had no effect on DAergic transmission.[357] In a different study, microinjection of 6-OHDA resulted in a significant upregulation of mRNA levels of the Cav1.2 Ca²⁺ channel α1 subunit in the ipsilateral SN.[358] Subcutaneous injection of 3.5 mg/kg nifedipine significantly decreased apomorphine-induced rotation and partially restored striatal DA concentration in 6-OHDA-lesioned rats. These findings imply that L-type VOCCs are directly connected with DA neurodegeneration. Nifedipine prevented nobiletin- (a natural polymethoxy flavonoid extracted from the fruit peel of citrus) induced DA release in the CA1 region of the hippocampus of MPTP-injected mice.[359] The high-threshold Ca²⁺ spike (HTS) and the slow oscillatory potential (SOP) are diverse Ca²⁺ conductances that play an important role in the generation of action potentials in SN DA neurons. While nifedipine showed a slight inhibitory effect on HTS, the molecule was able to block SOP. Moreover, nifedipine steadily abolished the spontaneous firing pattern.[360] Quinpirole is a D2 receptor agonist that inhibits Ca²⁺ current in both 6-OHDA-lesioned and reserpine-injected rats.[361] However, quinpirole inhibitory effect was reversed by nifedipine. These data suggest that DA-depletion leads to a rearrangement of the high voltage-activated (HVA) Ca²⁺ current profile, an outcome also observed in monogenic forms of PD (DJ1 mice). Rotenone-treated SH-SY5Y cells incubated with nifedipine showed a concentration-dependent decrease in Ca²⁺ trafficking, suggesting that the neurotoxin activates the L-type VOCCs opening.[362] The results also demonstrated that Ca²⁺ is involved in rotenone-mediated apoptosis and ROS production. Pretreatment with nifedipine increased cell survival, synaptic vesicle exocytosis, and neurite outgrowth as well as mitigated DA release and generation of ROS (using the dichlorodihydrofluorescein diacetate (DCFH-DA) probe) in PC12 treated with rotenone.[363] These results indicated that intracellular Ca²⁺ plays an important role in rotenone-induced DA toxicity. Quantitative assessment in organotypic sagittal vibrosections from p10 rat brain, showed that L-type VOCC inhibitor nifedipine do not exert a neurotoxic activity on DAergic or cholinergic neurons.[364] Even though nifedipine did not protect cholinergic neurons, the drug counteracted axotomy-mediated DA neuron degeneration in the SN but not in the ventral tegmental area, possibly by regulating proinflammatory cytokine release.

In summary, nifedipine is an antagonist of L-type VOCCs antagonist involved in Aβ and tau pathology, neurotoxin-induced DA degeneration, Ca²⁺ homeostasis and signaling, synaptic function, oxidative insult, and apoptotic cell death. The primary manifestation of nifedipine-related toxicity is hypotension secondary to loss of systemic vascular resistance. Subacute and subchronic toxicity studies indicated that oral administration of nifedipine has a safety profile at doses of up to 50 mg/kg in rats over a period of thirteen weeks. In dogs, no damage was detected up to 100 mg/kg dosage for a period of four weeks. Rats tolerated daily intravenous administration of 2.5 mg/kg nifedipine over a period of three weeks while dogs tolerated up to 0.1 mg/kg nifedipine for six days. An overdose of nifedipine can induce severe hypotension, systemic vasodilation, and reflex tachycardia.

The first total synthesis of nifedipine 5 was reported in 1989 by Singh, who used an acid-catalyzed reaction of an enamine with two perhydro-heterocycles.[365] Other synthetic procedures were reported in the following years using different methodologies.[366,367,368,369,370] Solid phase synthesis was also applied [371,372] as well as one-pot solvent-free synthesis.[373,374,375] The most common synthetic procedure to obtain pyridines is using the Hantzsch reaction.[376,377,378,379,380,381,382,383] The preparation of 1,4-dihydropyridines requires two equivalents of β-keto ester, an aldehyde and nitrogen donor. Sudalai and co-workers utilized dimethylmalonate 35 as the β-keto ester, 2-nitro-benzaldehyde 36 for the aldehyde and ammonium acetate as the nitrogen donor (Scheme 9).[384]

This reaction has a great number of modifications, such as the utilization of different homogeneous[385,386,387,388] or heterogeneous[389,390,391] catalysts, and the variation of the solvent.[392] In 2021, nifedipine (3,5-dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) 5 was synthesised through a photoinduced iron-catalysed ipso-nitration via single-electron transfer.[393] Aryl iodine 37 changed the iodine to nitro substituent to obtain 5 with good yield induced by photocatalysis (Scheme 10).

The first flow multicomponent synthesis of nifedipine 5 was reported elsewhere.[394] Methanol solutions of compounds 36, 38 and 39 were loaded to a heated 10 mL stainless steel coil reactor (150 °C) at 0.167 mL min⁻¹ to obtain nifedipine 5 in 71% yield (Scheme 11).

More recently, Xiang et al. described a green methodology for the synthesis of nifedipine 1.[395] A metal-free multicomponent cycloaddition of ketones with an ammonium cation under a CO₂ atmosphere is used to obtain the desired dihydropyridine derivatives. Thus, using methyl acetoacetate 38 and o-nitrobenzaldehyde 36, in the presence of ammonium chloride (NH₄Cl) and CO₂ in aqueous solution, 5 was obtained in 32% yield (Scheme 12).

6. Nimodipine

Nimodipine (Nimotop) is a Ca²⁺ channel blocker utilized to prevent brain damage as a consequence of reduced blood flow. The molecule antagonizes the Cav1.2-1.3 L-type VOCCs, showing a greater affinity for the Cav1.2 channel.[396] As a highly lipophilic compound, nimodipine can easily cross the BBB and reaches elevated concentrations in the CSF. The drug has specific binding affinity for dihydropyridine receptors, in contrast to nifedipine that shows predominantly peripheral effects. Nimodipine has been proposed to be of potential therapeutic utility in clinical trials of subjects with AD or PD. The beneficial effects attributable to nimodipine are related to clinical symptoms, cognitive function, and overall physical activity. The drug was shown to be more effective in primary degenerative dementia rather than in multi-infarct dementia.[397] A multicenter, double-blind, placebo controlled, randomized clinical trial of nimodipine (30 mg/kg tris in die) for 3 months demonstrated its efficacy to prevent behavioral, cognitive or affective impairments in patients with primary degenerative dementia of the Alzheimer's type.[398,399] An identical dose of nimodipine resulted in a marked improvement of the global functional state in individuals with mental deterioration of degenerative or vascular origin.[400] A review article of several double-blind, placebo-controlled clinical trials using nimodipine demonstrated its beneficial properties in elderly subjects suffering from a cognitive impairment syndrome.[401] The first randomized, double-blind, controlled trial focusing on subcortical vascular dementia was carried out in 2005 and concluded that nimodipine might exert a protective effect against cardiovascular comorbidities.[402]

Clinical observations have been confirmed by experimental research. Nimodipine possess the potential to reverse learning and memory defects in both young and aged hypertensive rats.[403,404] Bilateral injection of Aβ_1-42 in the entorhinal cortex of rats led to delayed acquisition in a spatial reference memory task and decreased excitatory transmission. Long-term treatment with nimodipine prevented aged-related attenuation of learning and memory through modulation of synaptosome Ca²⁺-binding proteins.[405] Administration of nimodipine improved reversal spatial learning impairment and synaptic current defects associated with Aβ pathology.[406,407] Age-related increase of the L-type Ca²⁺ channel protein α1D in the CA1 region of the hippocampus correlated with higher working memory defect, but nimodipine-treated rats for several months exhibited lower α1D immunoreactivity and improved spatial working memory.[408] Nimodipine ameliorated scopolamine-induced cognitive decline via its regulatory effect on the brain-derived neurotrophic factor (BDNF) and acetylcholine, which were elevated in both the cortex and hippocampus.[409] Nimodipine also ameliorated memory failure in aged rhesus monkeys.[410] Sustained depolarization-mediated increase of cytosolic levels of Ca²⁺ (but not released from the ER) produce substantial amounts of intraneuronal Aβ_1-42 and cell death in primary rat cortical neuron cultures. Exposure to nimodipine reduced Ca²⁺ influx and associated Aβ_1-42 accumulation.[411] Nimodipine restored the neurophysiological responses of ghrelin on the ΔΨm in young but not older neurons from Tg2576 female mice incubated with the oligomeric Aβ_1-42 fragment, indicating that Aβ-mediated intracellular Ca²⁺ dysregulation is only reversible during the early stages of Aβ pathology.[412] MTT assay and morphometric cell counting showed that treatment with nimodipine protected both rat cortical and hippocampal neurons from Aβ_25-35-induced cell death.[413] In primary rat hippocampal neuron cultures, Aβ_25-35 fragment triggered reversible enhancement in intracellular Ca²⁺ content and bursts of action potentials, which were exacerbated after extracellular Mg²⁺ removal. Treatment with the L-type blocker nimodipine inhibited Aβ_25-35-mediated toxic effects in both cortical and hippocampal neuronal cultures.[414,415] A different study using the same cultures confirmed that addition of nimodipine limited Aβ_25-35-related Ca²⁺ uptake and apoptotic effect.[416,417] Patch-clamp recording studies demonstrated that nimodipine inhibits Aβ_25-35 peptide excessive Ca²⁺ current density. Nevertheless, exposure to nimodipine did not mitigate inhibit oxidative damage associated to Aβ_25-35. These results suggest that Aβ significantly enhances Ca²⁺ trafficking via nimodipine-sensitive L-type VSCCs, which leads to free radical-induced neuronal loss.

Okadaic acid promoted phosphorylation of tau by increasing Ca²⁺ influx through L-type VOCCs, since nimodipine attenuates phospho-tau levels in SH-SY5Y neuroblastoma cells.[418] H4/APPsw cells or primary neuronal cultures derived from the cerebellum of Tg2576 AD mice incubated with nimodipine displayed an augmented Aβ₄₂ secretion. In addition, a comparable effect was described in Tg2576 mice injected with nimodipine, which showed a marked increase in Aβ₄₂ plasma concentration. Thus, the drug may modulate the release of Aβ₄₂ through an elusive mechanism rather than its capacity to inhibit Ca²⁺-influx pathways.[419] Driving tau expression in M4/6 neurons led to increased sensitivity of Ca²⁺ transients to nimodipine. This effect was ablated in M4/6 neurons co-expressing tau and Ca-α1D-RNAi.[420] Moreover, the authors demonstrated that knockdown of the Drosophila L-type Ca²⁺ channel Ca-α1D reverses tau-induced olfactory memory abnormalities by restoring Ca²⁺ handling. Elevated Ca²⁺ concentration aggravated Aβ-mediated behavioral impairment and defective chemotaxis, resulting in a shortened lifespan in C. elegans overexpressing Aβ_1-42.[421] Exposure to nimodipine extended lifespan and rescued motor lesions, synaptic deficiencies, and DAergic degeneration in worms. Furthermore, nimodipine limited Aβ aggregation via through upregulation of glutathione S-transferase activity, resulting in an attenuation of oxidative damage. LPS-treated rats exhibited important memory deficits, enhanced synaptosomal Ca²⁺ uptake, and microglial (but not astrocytic) stimulation with subsequent cytokine storm but administration of nimodipine reversed LPS-induced toxicity.[422] A dose-dependent depletion of Aβ-stimulated IL-1β content and release was detected following nimodipine exposure in both the N13 microglia cell line and cultures of primary mouse microglia together with reduced cell loss.[423] In addition, the molecule mitigated Aβ-induced intracellular accumulation of IL-1β in mice receiving a stereotaxic hippocampal injection of oligomeric Aβ_1-42 peptide.

A more recent study reported that nimodipine combined with piracetam results in a significant improvement of cognitive abilities and quality of life scores in patients with vascular dementia after an ischemic stroke.[424] Sadleir et al. showed that oral administration of nimodipine to 5XFAD mice did not have a beneficial effect on amyloid pathology, since the molecule did not prevent neuritic dystrophy or reduce cortical or hippocampal Aβ content.[425] However, the treatment did not exacerbate the AD phenotype, suggesting a safety and tolerability profile. It has been described that iron uptake, which competes with Ca²⁺ for entry into neurons through the L-type VOCCs, can be inhibited with nimodipine in a dose-dependent fashion in potassium chloride stimulated neuronal cells.[426] The data suggest that, under cellular iron overload conditions, iron uptake occurs through L-type VOCCs. Subcutaneous delivery of nimodipine reversed behavioral abnormalities, preserved the number of DAergic neurons in the locus coeruleus, and attenuated microglial activation in LPS-infused rats.[427] Exposure to nimodipine blocked DA neuron pacemaker activity, which can be restored by virtual Cav1.3 channels.[428] In contrast, virtual NMDAR were not capable of restoring regular pacing in nimodipine-silenced DA neurons. In vitro and in vivo studies demonstrates that incubation with nimodipine abolishes autonomous pacemaking in DA neurons and the underlying ΔΨm oscillations.[178]

Primary culture of cerebellar granule neurons incubated with MPP⁺ led to prominent cell death but nimodipine limited the effect of the neurotoxin.[429] MPP⁺ elevated cytosolic Ca²⁺ content and induced cell death parallel to mitochondrial depolarization and fragmentation in vitro.[430] However, exposure to nimodipine diminished Ca²⁺ levels, improved cell survival rate, and restored mitochondrial morphology. Nimodipine also ameliorated MPTP-induced behavioral phenotype and limited striatal DA depletion, SN neuronal loss, and oxidative insult without regulating MAO-B activity. The data indicated that nimodipine has the ability to improve mitochondrial function and integrity and involves L-type VOCCs in MPTP-mediated nigrostriatal DA neurodegeneration. In a different study, the same authors reported defective function in proteins involved in the modulation of intracellular Ca²⁺ homeostasis, including calbindin and calpain. This abnormal function was rescued by nimodipine treatment.[431] A different report showed that nimodipine had no effect on behavioral impairment and striatal DA depletion but preserved DA neurons from death in marmosets injected with MPTP.[256] Similar findings were observed in MPTP-treated mice, in which treatment with nimodipine protected DA neurons in the SN but no changes were found in the striatum.[255]

Nimodipine simultaneously upregulated DA release whilst suppress AChE release in both rat cerebral cortex and striatum.[432] Nimodipine prevented dendritic spine loss and behavioral abnormalities but not associated rotational asymmetry in DA-grafted rats.[433] Moreover, exposure to nimodipine did not impact DA graft survival but promoted graft reinnervation of striatum. Continuous-release pellets of nimodipine prevented locomotor disturbances, in unilateral 6-OHDA mesencephalic lesions.[434] Intrastriatal injection of 6-OHDA in rats caused a significant loss of retrogradely fluorogold and DAergic labelled neurons in the SN at 1-month post-injection. Nimodipine treatment failed to improve behavioral phenotype or nigrostriatal DA degeneration.[435] Exposure to nimodipine increased survival of SN (but not VTA) DA neurons in axotomy-induced rat model.[364] Although the precise mechanism remains elusive, the ability of the drug to counteract the inflammatory processes may be crucial for mitigating axotomy-induced neurodegeneration. Knockdown of Homer1, a postsynaptic density scaffold protein that regulates synaptic plasticity and Ca²⁺ signaling, preserved DA neurons from MPP⁺ toxicity.[436] This protective effect was linked to attenuated Ca²⁺-mediated ROS generation, which in turn, was dependent on the modulatory activities on ER Ca²⁺ trafficking and release through plasma membrane Ca²⁺ channels. Nimodipine significantly decreased the amount of NO• and LPS-activated microglia, which releases pro-inflammatory mediators such as interleukin-1β (IL-1β), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α).[437] In addition, in the absence of microglia, pretreatment with nimodipine did not exert a neuroprotective effect against MPP⁺-mediated DA toxicity. Nimodipine also downregulated DA uptake in neuron-glia cultures from mice lacking functional NADPH oxidase (an enzyme involved in the production of O2^•−) incubated with LPS. Taken together, these findings indicate that nimodipine protects DAergic neurons by mitigating the inflammatory response and by inhibiting NADPH oxidase-ROS signaling pathway.

In summary, the L-type Ca²⁺ channel blocker antagonist nimodipine protects from Aβ and presenilin pathology and from LRRK2- and α-syn-induced toxicity. The molecule ameliorates behavioral outcome, increases synaptic transmission and neuron survival, improves mitochondrial function, and attenuates oxidative stress and inflammation. Nimodipine is, in general, well tolerated although sensations of warmth and skin reddening can occur. High concentrations of the drug can result in reduced blood pressure, headache, nausea, muscle weakness, and gastrointestinal complaints.[397,398,399] Isolated CNS symptoms, such as insomnia, tachycardia, and increased motor activity have been reported. Nimodipine exhibits a low incidence of severe side-effects.

A solid-phase synthesis was reported by Gordeev in 1996 for the preparation of nimodipine (3-(2-Methoxyethyl) 5-propan-2-yl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) 6 and other bioactive dihydropyridines.[438] Condensation of the immobilized N-tethered enamino component 42 with the corresponding 2-benzylidene α-keto ester 43 provided the conjugated enamine 44. This intermediate was treated with trifluoroacetic acid to obtain the free enamino ester which spontaneously cyclizes in solution to afford the desired product 6 (Scheme 13).

More recently, due to a considerable demand of nimodipine in the Russian market, Pharm. Sintez Co. has developed a new technology to produce the drug in pilot batches.[439] The approach includes the production of 1-methylethyl-3-amino-crotonate 48 and 2-methoxyethyl-2-(3-nitrobenzyl-idene)acetoacetate 49. Cyclocondensation of both compounds, 48 and 49, employing iPrOH as the solvent and in the presence of hydrochloric acid, afforded the final product 6 in high yield and purity (Scheme 14).