domain: chim sdv

text:
Aurélien Beato

Anthonin Gori

Benjamin Boucherle

Marine Peuchmaur

Romain Haudecoeur

β-Carboline as a Privileged Scaffold for Multitarget Strategies in Alzheimer's Disease Therapy

The natural β-carboline alkaloids display similarities with neurotransmitters that can be favorably exploited to design bioactive and bioavailable drugs for Alzheimer's Disease (AD) therapy. Several AD targets are currently and intensively being investigated, divided in different hypotheses: mainly the cholinergic, the Amyloid β (Aβ) and the Tau hypotheses. To date, only symptomatic treatments are available involving acetylcholinesterase and NMDA inhibitors. Based on plethoric single-target structure-activity relationship studies, the βcarboline scaffold was identified as a powerful tool for fostering activity and molecular interactions with a wide range of AD-related targets. This knowledge can undoubtedly be used to design multitarget-directed ligands (MTDL), a highly relevant strategy preferred in the context of multifactorial pathology with intricated etiology such as AD. In this review, we first individually discussed the AD targets of the β-carbolines and then we focused on the multitarget strategies dedicated to the deliberate design of new efficient scaffolds.

Introduction

The β-carboline alkaloids represent a large group of natural and synthetic heterocyclic amines derived from tryptophan. [START_REF] Cao | β-Carboline alkaloids: biochemical and pharmacological functions[END_REF]2 Probably the most well-studied compounds of this group, harmine 1 and harmaline 2 were originally isolated in the 1840s from the seeds of Peganum harmala L. (Syrian rue), a traditional plant used for various purposes such as an emmenagogue or abortifacient. [START_REF] Nikam | Alkaloids Derived from Tryptophan: Harmine and Related Alkaloids[END_REF] Since their discovery, numerous β-carboline alkaloids were identified, highlighting the wide distribution of this family mainly in plants but also in animal tissues.

These natural products encompass simple β-carbolines and more complex derivatives, such as indoloquinolizidines (e.g., reserpine, mitragynine, yohimbine) and strictosidine-or manzamine-related alkaloids (Figure 1). The β-carboline core is composed of a tricyclic pyrido [3,4-b]indole ring structure, declined in several saturation states: β-carbolines for the unsaturated, fully aromatic scaffold (e.g., harmine 1); dihydro-β-carbolines (e.g., harmaline 2) and tetrahydro-β-carbolines or tryptolines (e.g., tetrahydroharmine 3) for versions with a partially or completely reduced pyridine ring, (the scaffolds are displayed in red, purple and blue, respectively, throughout the review). As such, its structure is reminiscent of human tryptamine-based neurotransmitters, i.e. serotonin and melatonin, and this analogy, together with the enhanced rigidity conferred by an additional ring, have been exploited for the design of bioactive compounds for modulating several central nervous system (CNS) targets. Thus, among their broad spectrum of recorded biological activities (e.g., antimicrobial, antitumor, antiviral, antiparasitic properties), synthetic and natural β-carbolines were most often associated with psychopharmacological effects, interacting with a variety of benzodiazepine, serotonin, imidazoline and dopamine receptors, as well as inhibiting brain enzymes.

Especially, some β-carbolines were reported as best-in-class inhibitors for several biomolecular targets involved in Alzheimer's diseases (AD), or suspected to play a role.

AD is a chronic neurodegenerative disorder first described by the neuropathologist Alois Alzheimer in 1907, leading to a significant number of deaths. Although mainly occurring in elderly populations, the familial AD can affect younger patients. Clinically, the main cognitive symptoms of AD are progressive short-term memory impairment, disturbances of orientation or neuropsychological disorders (e.g., aphasia, agnosia, apraxis). While the pathogenesis of AD is still unclear, due to the complex and multifactorial nature of the disease, several hypotheses have been advanced based on pathological or behavioural changes in AD patients. [START_REF] Abeysinghe | Alzheimer's disease; a review of the pathophysiological basis and therapeutic intervention[END_REF][START_REF] Rao | Alzheimer's disease: emerging trends in small molecule therapies[END_REF][START_REF] Xie | Progress in target drug molecules for Alzheimer's disease[END_REF] Besides the three main hypotheses, namely the cholinergic, amyloid-β (Aβ) and tau protein hypotheses that are regarded as the leading pathological pathways of AD, various other targets have emerged, some of them being interrelated. The cholinergic hypothesis was the first and most studied theory of the disease [START_REF] Abeysinghe | Alzheimer's disease; a review of the pathophysiological basis and therapeutic intervention[END_REF][START_REF] Rao | Alzheimer's disease: emerging trends in small molecule therapies[END_REF][START_REF] Xie | Progress in target drug molecules for Alzheimer's disease[END_REF] and has led to four out of five anti-AD drugs currently on the market (donepezil, rivastigmine, galantamine and tacrine which are acetylcholinesterase (AChE) inhibitors); the fifth being memantine, a N-methyl-Daspartate receptor (NMDA) inhibitor. This hypothesis is based on the utmost role of the neurotransmitter acetylcholine, its decrease being associated with dementia symptoms.

Understandably, research has focused on inhibitors of cholinesterases, serine hydrolase enzymes that can be subdivided into AChE and butyrylcholinesterase (BuChE). The Aβ cascade hypothesis is related to the neurotoxicity and acceleration of the neuronal cell death consecutively to the accumulation of insoluble amyloid plaques. [START_REF] Abeysinghe | Alzheimer's disease; a review of the pathophysiological basis and therapeutic intervention[END_REF][START_REF] Rao | Alzheimer's disease: emerging trends in small molecule therapies[END_REF] These aggregates of Aβ peptides result from the cleavage of the amyloid precursor protein (APP), a transmembrane protein located in neural synapses, by two secretase enzymes, the β-site APP cleaving enzyme 1 (BACE1, or β-secretase) and the γ-secretase. Under normal non-amyloid pathway, APP is sequentially cleaved by α-and γ-secretases leading to soluble and non-toxic APP derivatives.

Conversely, β-and γ-secretases cleavage produces among other products an Aβ42 monomer derivative (the most neurotoxic form of Aβ) known to accumulate in the extracellular environment, ending in amyloid plaques formation and local inflammatory response. The tau hypothesis pinpoints the hyperphosphorylation of tau protein as one of the main pathological pathway of AD. [START_REF] Abeysinghe | Alzheimer's disease; a review of the pathophysiological basis and therapeutic intervention[END_REF][START_REF] Rao | Alzheimer's disease: emerging trends in small molecule therapies[END_REF] This hyperphosphorylation process is a consequence of both the overactivation of kinases [START_REF] Xie | Progress in target drug molecules for Alzheimer's disease[END_REF] (e.g., mitogen-activated protein kinase (MAPK), cyclin-dependent kinase-5 (CDK-5), glycogen synthase kinase-3β (GSK-3β) or dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)) and the inactivation of protein phosphatase or protein tyrosine phosphatase-A. Apart from these three main theories, AD is also related to other pathological hypotheses [START_REF] Rao | Alzheimer's disease: emerging trends in small molecule therapies[END_REF][START_REF] Xie | Progress in target drug molecules for Alzheimer's disease[END_REF] such as the oxidative stress associated with the search of monoamine oxidases (MAO-A and B) inhibitors or metal chelators, and the excitotoxic theory involving for instance phosphodiesterases (PDE), NMDA and gamma-aminobutyric acid (GABA) receptors.

As the pathogenic mechanisms of AD are thus nebulous and multifactorial, with intricated etiology, current drug discovery researches are moving to multitarget strategies with a view to affect several nodes of the complex neurodegenerative network simultaneously. Different approaches have been described to alter the function of several targets at once, among them the multitarget-directed ligands (MTDL) aimed at hybridize pharmacophoric moieties mostly already known as bioactive molecules. [START_REF] Leόn | Recent advances in the multitarget-directed ligands approach for the treatment of Alzheimer's disease[END_REF][START_REF] Guzior | Recent development of multifunctional agents as potential drug candidates for the treatment of Alzheimer's disease[END_REF][START_REF] Maramai | Multitarget therapeutic strategies for Alzheimer's disease: review on emerging target combinations[END_REF] In this line, the tacrine and donepezil scaffolds inspired the design of numerous MTDL. The unique place of natural compounds, such as resveratrol or coumarin, in this strategy has also been repeatedly emphasized.

Similarly, the β-carboline scaffold is at the forefront of the targeting of the myriad of biomolecules involved in AD. Harmine (1) is a reference inhibitor of MAO-A and tauphosphorylation kinase DYRK1A. Tadalafil (8, Figure 1) has been marketed in 2003 as an excellent (and selective) anti-phosphodiesterase-5 (PDE5) agent. Extended β-carbolines were identified as single-digit nanomolar AChE and histone deacetylase (HDAC) inhibitors, and GABAA receptor modulators. Therefore, with the emergence of multitarget strategies against AD, the β-carboline scaffold is naturally well-positioned to become a dedicated platform able to concurrently reach a variety of neurological biomolecules and processes. states (1-3), and derivatives from natural sources (4-7) and drug discovery (8). Thus, we present herein an overview of the β-carboline derivatives (with associated structureactivity relationship (SAR) studies) that have been reported for their ability to inhibit or interfere with the main hypotheses, processes and targets in the frame of AD, namely ChE, BACE1, Aβ fibrillation, tau phosphorylation kinases, MAO, PDE5, glutamate, NMDA, GABAA and 5-hydroxytryptamine (5-HT) receptors, and finally HDAC. After a successive discussion of these targets in individual sections, the last part of the review focuses on multitarget strategies using a β-carboline scaffold deliberately designed for reaching several targets at once, and provides a critical overall analysis of the field together with perspectives for future works.

Acetyl-and butyrylcholinesterase (AChE/BuChE) inhibitors

Acetylcholine, a major neurotransmitter in the CNS, plays a crucial role in the neurological regulation of various functions (e.g., learning, memory, cognition). In AD, its depletion is associated with cognitive deficits, arousing the cholinergic hypothesis in the physiopathology of AD and thus the search for inhibitors of its degrading enzymes, AChE and BuChE. Indeed, some approved drugs (e.g., donepezil and rivastigmin) were put on the market; however, the clinical benefits of these treatments are still low, merely alleviating some of the major symptoms of AD. AChE and BuChE are both present in the brain with different specificities and expression activities. A selective inhibition of AChE is more crucial in the early stage while BuChE inhibition may be critical in the mid to later stages of the pathogenesis.

Nevertheless, AChE, whose natural substrate is acetylcholine, remains a primary target; however, BuChE was recently recognized as a new advanced target to treat AD. BuChE is a non-specific cholinesterase able to hydrolyze various types of natural or toxic esters. Both enzymes are serine hydrolases with a catalytic active site (CAS) located at the bottom of a 20 Å gorge, less restrictive in the case of BuChE, compared to AChE, and a peripheral anionic site (PAS) at the entry of that gorge.

Simple β-carbolines and reduced analogues.

If the β-carboline scaffold by itself clearly appeared as unable to efficiently bind both AChE and BuChE (IC50 >40 µM for 9a) [START_REF] Ghosal | Naturally occurring and synthetic β-carbolines as cholinesterase inhibitors[END_REF][START_REF] Otto | Beta and gamma carboline derivatives as potential anti-Alzheimer agents: a comparison[END_REF] the insertion of simple substituents at various positions only led to moderately active analogues at the micromolar range against both enzymes. 10-22 Indeed, while the introduction of polar groups at position 1 was slightly beneficial in some cases, especially compared with methyl analogues (IC50 = 4.0 µM for 10 vs. >10 µM for 1, Figure 2), the 9-substituted β-carbolines looked more promising. In particular, the 9-propargyl moiety produced the best results, regardless of other positions status: despite 1-methyl, 7-methoxy substitutions, harmine-related compound 11 shared with its naked analogue the same AChE inhibition potency (IC50 = 2.0 and 1.9 µM, respectively). However, the introduction of extended or polar substituents at position 9 of compound 11 gave lower activities in all cases. If isoxazoline derivatives were associated with a ten-fold drop of inhibition potency (IC50 = 11 µM for the most active analogue 12), [START_REF] Filali | Synthesis of new isoxazoline derivatives from harmine and evaluation of their anti-Alzheimer, anti-cancer and anti-inflammatory activities[END_REF] a large series of triazoles and hydrazides was found completely inactive against AChE (IC50 >100 µM). [START_REF] Filali | Synthesis, cytotoxic, anti-lipoxygenase and anti-acetylcholinesterase capacities of novel derivatives from harmine[END_REF] Natural β-carbolines bearing a 9-methoxy group were also marginally active. [START_REF] Zhang | N-methoxy-β-carboline alkaloids with inhibitory activities against Aβ42 aggregation and acetylcholinesterase from the stems of Picrasma quassioides[END_REF] Globally, the functionalization of positions 1 and 7 only provided a limited impact (e.g., IC50 >10 µM for 1, 9a and 9b). Attempts to explore substitutions at positions 3, 4 and 6 failed, as the resulting derivatives were found even less potent than their parent compounds. Partial or total reduction of the pyridine ring afforded comparable results.

The dihydro-β-carboline 2 especially showed similar inhibition values than its fully oxidized counterpart 1, 14,15 while 1-hydroxydihydro-β-carboline was mostly inactive. [START_REF] Fadaeinasab | Cholinesterase enzymes inhibitors from the leaves of Rauvolfia reflexa and their molecular docking study[END_REF] Regarding tetrahydro-β-carbolines, no activity improvement was recorded as well (e.g., IC50 >10 µM for 1 and 3, as well as for 9a and its reduced counterpart), 14,21 except for enantiopure compound 13 (IC50 = 2.5 µM) and for the only 8-substituted analogue, 14, that reached an IC50 of 2.0 µM, [START_REF] Abuhamdah | Elaborate ligand-based modeling coupled with QSAR analysis and in silico screening reveal new potent acetylcholinesterase inhibitors[END_REF] making position 8 of β-carbolines a potentially valuable subject for further studies.

Examining basic interactions between simple β-carbolines and the active site of AChE provided elements of interest. For example, the planar structure of harmane (9b) was found in docking experiments to mainly interact with aromatic residues of the CAS of AChE, especially Trp84 and Phe330. [START_REF] Torres | Structural insights into cholinesterases inhibition by harmane βcarbolinium derivatives: a kineticsmolecular modeling approach[END_REF] A hydrogen bond would also occur between the indole NH and His440, and overall the interaction pattern reported appeared as quite similar to tacrine (a well-known planar inhibitor) interactions. Globally, the piecemeal BuChE inhibition results gave similar trends than for AChE, disregarding a few exceptions. Several 1-substituted compounds, inactive against AChE, were though tested on BuChE, and if compound 15, for example, revealed a promising activity (IC50 = 1.3 µM), submicromolar 1-naphthyl analogue 16 constituted a real breakthrough in the quest of efficient BuChE inhibitors (IC50 = 0.22 µM). [START_REF] Horton | Synthesis and application of β-carbolines as novel multi-functional anti-Alzheimer's disease agents[END_REF] Docking studies showed that 16 seemed to bind in a similar fashion than donepezil, a well-known cholinesterase inhibitor. In particular, the position of 16 allowed the formation of a key hydrogen bond between the indole NH and the OH group of Ser198, as well as π-π stacking interactions with Trp231 and His438. β-Carboliniums and R 2 substituent refinement: towards nanomolar range derivatives. Differences are classically observed in terms of anti-cholinesterase activity between neutral compounds and corresponding ammonium salts. It was often suggested that they could be related to a choline mimic effect, and then several authors focused on developing βcarboliniums for improving β-carboline scaffold affinity. At first glance, the substitution of the pyridine ring at position 2 by a methyl group appeared to yield mixed results for the corresponding pyridiniums. Indeed, if some improvements of activity were recorded, sometimes ten-fold (e.g., IC50 >40 µM for 9a vs. around 5 µM for 17), in other cases the IC50 values remained very similar. [START_REF] Ghosal | Naturally occurring and synthetic β-carbolines as cholinesterase inhibitors[END_REF][START_REF] Tadokoro | N-Methyl-β-carbolinium salts and an N-methylated 8-oxoisoguanine as acetylcholinesterase inhibitors from a solitary ascidian, Cnemidocarpa irene[END_REF] However, when replacing the methyl group by bulkier substituents, more potent compounds were identified, with submicromolar potency. For example, 2-propylated counterpart of 17 reached an IC50 value of 0.85 µM, vs. 4.1-6.7 µM for 17 itself. [START_REF]SAR regarding the effect of β-carbolines on the previously mentioned AD-related targets[END_REF] If the ester derivative 18 eventually showed the best activity among simple substituents at position 2 (IC50 = 0.25 µM), [START_REF] Yang | Peganumine B-I and two enantiomers: new alkaloids from the seeds of Peganum harmala Linn. and their potential cytotoxicity and cholinesterase inhibitory activities[END_REF] the phenyl analogues were also very potent (IC50 = 0.42-0.76 µM, 0.54 µM for 19 and 0.42 µM for 20), [START_REF] Zhou | New 2aryl-9-methyl-β-carbolinium salts as potential acetylcholinesterase inhibitor agents: synthesis, bioactivity and structure-activity relationship[END_REF] especially considering the possibilities of further fine-tuned modifications of the phenyl ring. Indeed, Zhou et al.

generated very recently a systematic catalogue of compounds with various phenyl substitutions in order to determine the most favorable substituent at position 2 of the βcarbolinium scaffold. [START_REF] Zhou | New 2aryl-9-methyl-β-carbolinium salts as potential acetylcholinesterase inhibitor agents: synthesis, bioactivity and structure-activity relationship[END_REF] Among the groups (fluoro, chloro, bromo, iodo, methyl, hydroxyl, methoxy, cyano, etc.) and positions (ortho, meta, para, etc.) evaluated, a few molecules led to inhibition values under 0.30 µM. Compounds 21a and 21b (IC50 = 0.29 µM and 0.24 µM respectively) especially shed light on the potential beneficial role of a para substitution, probably through a steric favorable effect, a feature confirmed by several other comparisons (e.g., IC50 = 0.56 µM for 21c vs. >9 µM for ortho and meta analogues, 0.43 µM for 21d vs. >10 µM for ortho and meta analogues) and by molecular docking of 21a, 21e and their respective meta isomers, that showed inappropriate sterical clash with residue Asn87 in the case of meta substituents. A disubstituted analogue, 22, displayed the best value, almost reaching the two-digits nanomolar range (IC50 = 0.11 µM). It is worth noting that the aforementioned molecules exhibited a remarkable selectivity between AChE and BuChE, unlike the other compounds described so far. Inhibition values against BuChE were systematically around 100 times higher than against AChE (e.g., IC50 = 32-60 µM for 21a-e against BuChE), with a 390-fold difference as the maximum, in the case of a 4'-bromo-2'fluoro derivative (IC50 = 0.30 µM against AChE vs. 116 µM against BuChE). [START_REF] Zhou | New 2aryl-9-methyl-β-carbolinium salts as potential acetylcholinesterase inhibitor agents: synthesis, bioactivity and structure-activity relationship[END_REF] Finally, the partial reduction of the pyridine moiety exerted a global detrimental effect on anti-AChE activity (e.g., IC50 = 1.5-6.8 µM for 23a-c vs. 0.24-0.43 µM for 21a, 21b and 21d). A 100fold drop was even measured when reducing compound 22 into the 3,4-dihydro counterpart 24 (IC50 = 0.11 µM and >10 µM respectively). This phenomenon could be explained by the predicted binding modes of compounds 21a, 21e and 23a in the AChE active sites. In docking experiments, the aromatic core of the β-carbolines was found to be embedded in a group of aromatic residues, such as Trp86, Tyr337, Trp439 and Tyr449. Thus, when partially reducing the pyridine ring, a crucial π-π interaction between this group and the indole ring of Trp86 could be lost, jeopardizing the global affinity of the compounds. 30 Overall, most of these compounds were found active only at concentrations >10 µM, but a couple of structural elements were able to decrease the IC50 values. Once again, the introduction of a positive charge at the nitrogen atom of the quinolizidine part of these molecules gave interesting results: the quinolizidinium salt 25 was 30 times more potent against AChE than the neutral counterpart (IC50 = 4.1 µM and 121 µM respectively, Figure 4). [START_REF] Cao | Monoterpene indole alkaloids from the stem bark of Mitragyna diversifolia and their acetylcholine esterase inhibitory effects[END_REF] Surprisingly, limited modifications of 25 also led to more active derivatives: the reduction of ethylenyl group or the introduction of a hydroxyl group yielded analogues with IC50 values of 5.2 µM and 10 µM, respectively. [START_REF] Cao | Monoterpene indole alkaloids from the stem bark of Mitragyna diversifolia and their acetylcholine esterase inhibitory effects[END_REF] Globally, the glycosylated analogue 26 displayed the best potential in this series but eventually remained in the micromolar range (IC50 = 1.9 µM). [START_REF] Cardoso | Indole glucoalkaloids from Chimarrhis turbinata and their evaluation as antioxidant agents and acetylcholinesterase inhibitors[END_REF] The other disparate compounds did not succeed to improve anti-AChE and anti-BuChE activity, including complex imbricated polycyclic and/or positively charged derivatives. [START_REF] Fadaeinasab | New indole alkaloids from the bark of Rauvolfia reflexa and their cholinesterase inhibitory activity[END_REF][START_REF] Yang | Peganumine B-I and two enantiomers: new alkaloids from the seeds of Peganum harmala Linn. and their potential cytotoxicity and cholinesterase inhibitory activities[END_REF][START_REF] Passos | Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors[END_REF][START_REF] Mroue | Indole alkaloids of Haplophyton crooksii[END_REF][35][START_REF] Geissler | Acetylcholinesterase inhibitors from the toadstool Cortinarius infractus[END_REF] The presence of a fifth fused six-membered ring, common in natural products, appeared as beneficial for anti-AChE and anti-BuChE activities, as witnessed by the good AChE and BuChE inhibition properties of reserpine 4 (IC50 = 1.7 µM and 2.8 µM, respectively, Figure 1). [START_REF] Kashyap | Ajmalicine and reserpine: indole alkaloids as multi-target directed ligands towards factors implicated in Alzheimer's disease[END_REF] Even little substituted analogues reached decent micromolar-range inhibition of BuChE (but not AChE), such as compound 27 (IC50 = 1.0 µM). [START_REF] Liew | Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis[END_REF] Especially, in docking experiments 27 was found to fit deep in the bottom gorge of BuChE, establishing hydrogen bonds with residues Ser198 and His438, and an anchoring π-π stacking with Trp84 at the choline binding site. [START_REF] Liew | Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis[END_REF] Contrariwise, when considering cationic derivatives low micromolar or submicromolar activities against AChE were recorded (e.g., IC50 = 0.78 µM and 1.5 µM for 28 and 29, respectively). [START_REF] Ghosal | Naturally occurring and synthetic β-carbolines as cholinesterase inhibitors[END_REF][START_REF] Perreira | Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission[END_REF] Compound 29 was predicted to bind in the hydrophobic pocket of the PAS, formed by Phe330, Phe331, Tyr334, Tyr121, with a main ππ interaction between the terminal aromatic ring of 30 and Trp279, mostly matching donepezil interaction pattern. [START_REF] Bharate | Total synthesis and anti-cholinesterase activity of marine-derived bis-indole alkaloid fascaplysin[END_REF] The presence of an additional nitrogen atom at position 14, which was associated with the natural evodiamine subclass of β-carbolines, gave rise to an increased potency in terms of BuChE inhibition, while improving the BuChE vs. AChE selectivity of these agents. Indeed, compounds 30 and 31 concomitantly achieved a submicromolar range potency (IC50 = 0.5 µM and 0.88 µM respectively against BuChE) and a 4-7-fold better IC50 against BuChE vs. AChE (IC50 = 3.4 µM and 3.0 µM respectively against AChE). [START_REF] Decker | Novel inhibitors of acetyl-and butyrylcholinesterase derived from the alkaloids dehydroevodiamine and rutaecarpine[END_REF][START_REF] Huang | Identification of a neuroprotective and selective butyrylcholinesterase inhibitor derived from the natural alkaloid evodiamine[END_REF] Extended substitution at position 3 was thus investigated using a carbamate linker and either 5-oxo or 5-deoxo versions of the evodiamine scaffold. Among the twelve analogues reported, all compounds were barely inactive against AChE, especially the 5-oxo derivatives, but some derivatives emerged as good inhibitors of BuChE (IC50 = 1.0-4.0 µM).

More strikingly, compound 32 revealed a very high potential against BuChE, showing an IC50 of 77 nM, the best value found among β-carboline derivatives to date, together with an exquisite BuChE vs. AChE selectivity index of more than 130. [START_REF] Huang | Identification of a neuroprotective and selective butyrylcholinesterase inhibitor derived from the natural alkaloid evodiamine[END_REF] Finally, the impact of position 3 substitution was further scrutinized using a partially or completely oxidized evodiamine scaffold, without the 14-methyl group (i.e., rutaecarpine scaffold). All the compounds tested, bearing an amido bridge linked to various secondary amines, reached submicromolar potency. [START_REF] Wang | Synthesis and evaluation of novel rutaecarpine derivatives and alkaloids derivatives as selective acetylcholinesterase inhibitors[END_REF] Especially, compounds 33 and 34 were associated with IC50 of 29 nM and 10 nM against AChE, while being very selective (IC50 = 0.84 µM and 5.4 µM against BuChE, respectively), a property rationalized by molecular docking.

Aβ peptide production and fibrillation inhibitors

One of the main hallmarks of AD is the accumulation of neurotoxic aggregates of Aβ peptides that could be self-induced or promoted by AChE and finally deposit in the form of amyloid plaques, with various consequences such as the unbalance of intracellular Ca 2+ levels leading to apoptosis or neuronal inflammatory response. The Aβ peptides result from the sequential cleavage of APP by β-site APP cleaving enzyme 1 (BACE1), the initial key enzyme in the amyloidogenic metabolic pathway, and γ-secretase. BACE1 is thus considered as a very interesting target for the development of new drugs for AD treatment. Many forms of Aβ have been previously described such as monomers, soluble oligomers and fibrils with different levels of toxicity. During the aggregation process, Aβ fibrils entrap electrochemically active metal ions, mainly Cu and Zn ions, that can generate reactive oxygen species (ROS). Metal-ion chelators are therefore another research focus within the context of the Aβ cascade hypothesis to reduce the aggregation of Aβ fibrils and thus the generation of ROS.

Accordingly, Aβ aggregation and BACE1 inhibitors, metal chelation and radical scavenging are all attractive approaches that can be independently or simultaneously targeted to disrupt the formation of amyloid peptides and/or plaques.

Structual elements for BACE1 inhibition. Regarding β-carbolines, after virtual screening studies of the Thai medicinal database, the natural product ochrolifuanine E 35 was identified as a BACE1 inhibitor with good predicted pharmacokinetic properties, including blood-brain barrier (BBB) crossing level, but a quite high toxicity. [START_REF] Jiaranaikulwanitch | Triazolyl tryptoline derivatives as β-secretase inhibitors[END_REF] In line with docking studies, (S)-3-(azidomethyl)-2,3,4,9-tetrahydro-1H-pyrido [3,4-b]indole was chemically modulated and, among the twenty-two synthesized compounds, 36 showed the most potent inhibitory activity on BACE1 (IC50 = 1.5 μM, Figure 5). The binding mode of 36 highlighted hydrogen bonding interactions between the two polar hydrogen of the β-carboline core and residues Asp32 and Asp228 of BACE1. The introduction of more polar, bulkier or halogenated substituents generally led to less active compounds. Inhibition of Aβ fibrillation and/or oligomerization. Among others, compounds 16 (Figure 2) and 37, which are BuChE inhibitors, were evaluated at 100 µM for their ability to prevent the formation of Aβ fibrils and soluble Aβ oligomers, described as the more neurotoxic form of Aβ peptides (39% and 58% respectively for 16, 34% and 82% for 37). [START_REF] Horton | Synthesis and application of β-carbolines as novel multi-functional anti-Alzheimer's disease agents[END_REF] Unlike previous studies suggesting that a compound is either a fibril or an oligomer inhibitor, compound 16 is able to inhibit both forms. Some other natural β-carbolines, mainly vinpocetine and reserpine 4 (Figure 1), have been reported in piecemeal studies to have neuroprotective effects linked to Aβ aggregation and/or toxicity. [START_REF] Kashyap | Ajmalicine and reserpine: indole alkaloids as multi-target directed ligands towards factors implicated in Alzheimer's disease[END_REF][START_REF] Pereira | Vinpocetine attenuates the metabolic dysfunction induced by amyloid β-peptides in PC12 cells[END_REF][START_REF] Zhang | Pharmacological basis for the use of evodiamine in Alzheimer's disease: antioxidation and antiapoptosis[END_REF] After identification of the marine fascaplysin 29 (Figure 4) as an Aβ fibrillization inhibitor, the synthetic 9-methyl derivative 38, originally designed as an AChE inhibitor, was recognized as a more potent analogue able to directly interact with Aβ through ionic interactions with the negatively charged residues of Aβ. Finally, 9methylfascaplysin not only inhibited the formation of Aβ fibrillization but also prevented the formation of Aβ oligomers in vitro. [START_REF] Sun | 9-methylfascaplysin is a more potent Aβ aggregation inhibitor than the marine-derived alkaloid, fascaplysin, and produces nanomolar neuroprotective effects in SH-SY5Y cells[END_REF] Natural β-carbolines isolated from Picrasma quassioides were also evaluated for their inhibitory effect on Aβ42 aggregation, and the most active compound 39b, a racemic bis-β-carboline, showed an IC50 value of 10 μM, whereas the N-unsubstituted analogue 39a and other isolated monomeric β-carbolines were inactive or marginally active. [START_REF] Zhang | N-methoxy-β-carboline alkaloids with inhibitory activities against Aβ42 aggregation and acetylcholinesterase from the stems of Picrasma quassioides[END_REF][START_REF] Guo | Effects of Picrasma quassioides and its active constituents on Alzheimer's disease in vitro and in vivo[END_REF]

Tau phosphorylation kinases inhibitors (GSK-3β, DYRK1A, CDK5, CK1/2,

CLK1)

Tau is a hydrophilic microtubule-associated protein that plays an important role in the assembly and stability of microtubules and, therefore, in the normal brain function. In AD, tau is abnormally hyperphosphorylated, resulting in conformational changes and ultimately in the formation of neurofibrillary tangles (NFTs), highly disruptive and toxic intracellular aggregates that deposit in the brain, leading to neuronal dysfunction and death. To prevent the formation of NFTs, considered as the second main marker of Alzheimer's disease, kinase inhibitors were identified or designed. Thus, several studies were dedicated to the inhibition of tau phosphorylation kinases, which mostly belong to the CMGC kinase group, namely GSK-3β, DYRK1A, CDK5, casein kinases 1 and 2 (CK1/2) and dual specificity protein kinase (CLK1), among other minor contributions. [START_REF] Martin | Tau protein kinases: involvement in Alzheimer's disease[END_REF] Especially, harmine 1 was found as one of the most active inhibitors of DYRK1A (IC50 = 22-130 nM, Figure 1), while analogues norharmane 9a and harmane 9b were mostly inactive (IC50 >1.8 µM, Figure 2). [START_REF] Frost | β-Carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's disease-related sites[END_REF][START_REF] Adayev | Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)[END_REF][START_REF] Bain | The selectivity of protein kinase inhibitors: a further update[END_REF][START_REF] Cozza | Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases[END_REF][START_REF] Göckler | Harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation[END_REF][START_REF] Grabher | Library-based discovery of DYRK1A/CLKA inhibitors from natural product extracts[END_REF][START_REF] Mariano | Systematic diversification of benzylidene heterocycles yields novel inhibitor scaffolds selective for Dyrk1A, Clk1 and CK2[END_REF][START_REF] Tahtouh | Selectivity, cocrystal structures, and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B[END_REF][57] However, upon fixing a hydroxy or methoxy group at position 7, some room at position 9 seems to be available for receiving substituents of various sizes, as witnessed by the good potencies of compounds 40 and 41a-c (IC50 = 35-59 nM), 41d being the most promising member of the series (IC50 = 25 nM, Figure 7). [START_REF] Frost | β-Carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's disease-related sites[END_REF][57][START_REF] Drung | Computational & experimental evaluation of the structure/activity relationship of β-carbolines as DYRK1A inhibitors[END_REF][START_REF] Rüben | Selectivity profiling and biological activity of novel β-carbolines as potent and selective DYRK1 kinase inhibitors[END_REF][START_REF] Balint | Structure-based design and synthesis of harmine derivatives with different selectivity profiles in kinase versus monoamine oxidase inhibition[END_REF][START_REF] Kumar | Synthesis and biological validation of a harmine-based, central nervous system (CNS)avoidant, selective, human β-cell regenerative dual-specificity tyrosine phosphorylationregulated kinase A (DYRK1A) inhibitor[END_REF] Only the presence of a 9-carboxylic acid moiety compromised significantly the activity (IC50 = 1.1 µM). Further variations at position 7 revealed a general drop of activity upon methoxy replacement, except for the methyl ester 42a (IC50 = 27 nM, vs. 2.0-9.8 µM for 42b-d). Position 1 was finally modulated by the introduction of substituents of various sizes, with only little impact on the anti-DYRK1A effect (IC50 = 56-110 nM for 43a-c). Contrariwise, the partial reduction of harmine has proved to be unsuccessful (2, IC50 = 4.6-9.0 µM). [START_REF] Frost | β-Carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's disease-related sites[END_REF][START_REF] Adayev | Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)[END_REF] Some of these elements were rationalized by the publication of three crystal structures of β-carboline-DYRK1A complexes with compounds 1, 41d and 44 (PDB code 3ANR, 6UWY and 4YU2, respectively, Figure 8A-C). [START_REF] Kumar | Synthesis and biological validation of a harmine-based, central nervous system (CNS)avoidant, selective, human β-cell regenerative dual-specificity tyrosine phosphorylationregulated kinase A (DYRK1A) inhibitor[END_REF][START_REF] Ogawa | Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A[END_REF][START_REF] Chaikuad | Crystal structure of DYRK1A with harminederivatized AnnH-75 inhibitor[END_REF] The harmine 1 flat structure was found encased in the catalytic site between several hydrophobic residues, such as Ala186, Val173, Phe238, Val306, Leu294 and Leu241, establishing hydrogen bond anchors with the main chain of Leu241 and with the side chain amino group of Lys188. As observed previously, free space occurred around position 9 of harmine 1, and the structures of 41d-DYRK1A and 44-DYRK1A complexes confirmed the possibility of introducing bulky groups without causing major steric clashes. The hydrogen bond set up by the 7-methoxy group could explain the inability of naked counterparts 9a and 9b to exert any significant effect against the enzyme. Interestingly, harmine 1 was described as partially kinase-selective ( Table 1). [START_REF] Frost | β-Carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's disease-related sites[END_REF][START_REF] Adayev | Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)[END_REF][START_REF] Bain | The selectivity of protein kinase inhibitors: a further update[END_REF][START_REF] Cozza | Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases[END_REF][START_REF] Göckler | Harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation[END_REF][START_REF] Grabher | Library-based discovery of DYRK1A/CLKA inhibitors from natural product extracts[END_REF][START_REF] Mariano | Systematic diversification of benzylidene heterocycles yields novel inhibitor scaffolds selective for Dyrk1A, Clk1 and CK2[END_REF][START_REF] Tahtouh | Selectivity, cocrystal structures, and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B[END_REF][57][START_REF] Song | Specific inhibition of cyclin-dependent kinases and cell proliferation by harmine[END_REF][START_REF] Dasappa | Preferential selectivity of inhibitors with human tau protein kinase Gsk3𝛽 elucidates their potential roles for off-target Alzheimer's therapy[END_REF] Especially, when considering Alzheimer's disease-related kinases, it was found active against DYRK1A and CLK1 (IC50 = 22-350 nM and 26-220 nM respectively), but not against CDK5 and CK1/2 (IC50 >1.5 µM). Its activity against GSK-3β, on the other hand, was discordant from study to study (IC50 = 0.35-10 µM). Another β-carboline-based series of pyrrolo [3',4':5,6]indolizino [8,7-b]indole-1,3(2H,8H)-diones, derived from the natural alkaloid granulatimide, was tested against these same five kinases. [START_REF] Deslandes | Synthesis and biological evaluation of analogs of the marine alkaloids granulatimide and isogranulatimide[END_REF] Among them, the 7-hydroxy derivative 45 was of particular interest, as it afforded submicromolar values for both CLK1, DYRK1A and GSK-3β (IC50 = 0.26 µM, 0.20 µM and 0.42 µM respectively).

Other β-carbolines have been developed as dedicated inhibitors of GSK-3β and CDK5, but with poor outputs. However, compound 46 was recently associated with interesting CLK1 inhibition properties (Ki = 0.14 µM) and with exquisite kinase selectivity (IC50 >10 µM against a range of ~50 kinases), making it an excellent candidate in the AD context. The crystal structure of a 46-CLK1 complex (PDB code 6YTG, Figure 8D) revealed a noncanonical binding mode in the active site and the formation of multiple hydrogen bonds between the spiro piperidine moiety, the nitrile group, and residues Lys191, Asp325 and possibly Asn293. In addition, an unusual halogen bond was seen between the 7-chloro group of 46 and residue Glu242. A series of manzamine A (7, Figure 1) derivatives have also been provided as potential inhibitors of GSK-3β. [START_REF] Hamann | Glycogen synthase kinase-3 (GSK-3) inhibitory activity and structure-activity relationship (SAR) studies of the manzamine alkaloids. Potential for Alzheimer's disease[END_REF] Regardless of the modifications made at the indole ring (introduction of hydroxy, ethoxy or arylsulfonyl groups) or at the macrocyclic part (dehydration, insertion of a carbonyl group), the recorded levels of activity remained in all cases marginal (IC50 = 5.4-25 µM), the best inhibitor being 47 (IC50 = 4.8 µM). Finally, a series of β-carbolines was designed and evaluated against CDK5. The inhibition results remained very modest and did not deliver values below 5 µM (IC50 = 5.0 µM for 48). 68 Harmane (9b, Figure 2) and other β-carbolines are well-known as MAO inhibitors, as they constitute natural key ingredients responsible for the lower MAO activity observed in smokers and coffee consumers, resulting in a neuroprotective effect widely documented. [START_REF] Herraiz | Human monoamine oxidase is inhibited by tobacco smoke: βcarboline alkaloids act as potent and reversible inhibitors[END_REF][START_REF] Herraiz | Human monoamine oxidase enzyme inhibition by coffee and βcarbolines norharman and harman isolated from coffee[END_REF] Indeed, 9b was associated in several studies with a submicromolar MAO-A inhibition potential (IC50 = 29-340 nM). [START_REF] Passos | Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors[END_REF][START_REF] Herraiz | Human monoamine oxidase enzyme inhibition by coffee and βcarbolines norharman and harman isolated from coffee[END_REF][71][START_REF] Santillo | Inhibition of monoamine oxidase (MAO) by β-carbolines and their interactions in live neuronal (PC12) and liver (HuH-7 and MH1C1) cells[END_REF][START_REF] Fernandez De Arriba | Inhibition of monoamine oxidase from bovine retina by β-carbolines[END_REF] The analogous harmine (1, Figure 1) was even more potent, showing single-digit nanomolar inhibition values (IC50 = 1.0-5.3 nM), and more efficacy, although still moderate, against MAO-B. [START_REF] Balint | Structure-based design and synthesis of harmine derivatives with different selectivity profiles in kinase versus monoamine oxidase inhibition[END_REF][START_REF] Santillo | Inhibition of monoamine oxidase (MAO) by β-carbolines and their interactions in live neuronal (PC12) and liver (HuH-7 and MH1C1) cells[END_REF][START_REF] Fernandez De Arriba | Inhibition of monoamine oxidase from bovine retina by β-carbolines[END_REF][START_REF] Samoylenko | Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson's disease[END_REF][START_REF] Haider | Isoform selectivity of harmine-conjugated 1,2,3-triazoles against human monoamine oxidase[END_REF] As a high-resolution structure of a 1-MAO-A complex was elucidated by crystallography (PDB code 2Z5X), the structural elements that rule the high inhibition potency are well known (Figure 10A). [START_REF] Son | Structure of human monoamine oxidase A at 2.2-Å resolution: the control of opening the entry for substrates/inhibitors[END_REF] Harmine is located at the active site, interacting with residues Tyr69, Asn181, Phe208, Val210, Gln215, Cys323, Ile325, Ile335, Leu337, Phe352, Tyr407, and Tyr444, and with the cofactor flavin adenine dinucleotide (FAD). A reported π-π interaction between Gln215 and the fivemembered ring of harmine (3.4 Å), together with a water-bridged hydrogen bond network involving the two nitrogen atoms of the β-carboline structure and Tyr69, Asn181 and Gln215 seems to be an important component of the interaction pattern. An overlay of the complex with a MAO-B structure (PDB 1OJ9) [START_REF] Binda | Insights into the mode of inhibition of human mitochondrial monoamine oxidase B from high-resolution crystal structures[END_REF] reveals a major steric clash, as Ile335 is replaced by Tyr326 (black arrow in Figure 10B), and Asn181 becomes Cys172 that no longer allows the formation of a water-bridged hydrogen bond. These elements could explain the significant drop in harmine activity when considering MAO-B inhibition. Unsurprisingly, most of the anti-MAO-A modulation efforts were thus achieved based on the harmine structure.

Monoamine oxidases (MAO-

Introducing various substituents at position 9 always resulted in a loss of anti-MAO-A activity, and the potency even weakened as the substituent size increased (IC50 = 11 nM for 40 vs. 0.74 µM and >10 µM for iso-propyl and benzyl analogues). [START_REF] Balint | Structure-based design and synthesis of harmine derivatives with different selectivity profiles in kinase versus monoamine oxidase inhibition[END_REF] If attempts to replace the 7-methoxy group by carbon-based substituents mostly failed (even if 42c and 42d still reached decent inhibition values, IC50 = 40 nM and 30 nM respectively, Figure 7), the introduction of other 7-alkoxy groups provided derivatives with MAO-A inhibition similar to the parent compound (IC50 <5.0 nM for 49a-49e). [START_REF] Reniers | Synthesis and evaluation of β-carboline derivatives as potential monoamine oxidase inhibitors[END_REF] In addition, compound 49d was notably able to inhibit MAO-B activity one hundred-times more efficiently than harmine (IC50 = 0.22 µM vs. 20 µM). At the other side of harmine, the diversification of position 1 was unsuccessful, IC50 values against MAO-A systematically rising above 1 µM. [START_REF] Balint | Structure-based design and synthesis of harmine derivatives with different selectivity profiles in kinase versus monoamine oxidase inhibition[END_REF] All these observations are consistent with the crystal structure of MAO-A, since position 7 clearly appears as the most tolerant to steric hindrance and especially carbon chains, while positions 1 and 9 are already in close contact with adjacent residues. These elements were confirmed by the limited potential of 9-substituted 7-triazolylmethoxy derivatives reported recently. [START_REF] Haider | Isoform selectivity of harmine-conjugated 1,2,3-triazoles against human monoamine oxidase[END_REF] In all cases, as expected, the functionalization of position 9, with phenylpropyl, propyl, butyl or isobutyl groups, led to derivatives with IC50 values against MAO-A of at least 0.43 µM (IC50 = 0.43 µM for 50b), with sometimes interesting action on MAO-B however (e.g., IC50 = 0.26 µM for 50a). The partial reduction of harmine 1, leading to 3,4-dihydro analogue 2, afforded retained MAO-A inhibition potency (IC50 = 2.5-8.0 nM). [START_REF] Santillo | Inhibition of monoamine oxidase (MAO) by β-carbolines and their interactions in live neuronal (PC12) and liver (HuH-7 and MH1C1) cells[END_REF][START_REF] Fernandez De Arriba | Inhibition of monoamine oxidase from bovine retina by β-carbolines[END_REF][START_REF] Samoylenko | Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson's disease[END_REF][START_REF] Glover | β-Carbolines as selective monoamine oxidase inhibitors: in vivo implications[END_REF] Total reduction of the pyridine ring to give compound 3, as well as reduction of differently substituted β-carbolines

(1-H, 7-OMe, 6-OH, 7-OH) inexorably produced less active molecules, with no specific action on MAO-B as well. [START_REF] Santillo | Inhibition of monoamine oxidase (MAO) by β-carbolines and their interactions in live neuronal (PC12) and liver (HuH-7 and MH1C1) cells[END_REF][START_REF] Fernandez De Arriba | Inhibition of monoamine oxidase from bovine retina by β-carbolines[END_REF][START_REF] Miralles | High-affinity binding of β-carbolines to imidazoline I2B receptors and MAO-A in rat tissues: norharman blocks the effect of morphine withdrawal on DOPA/noradrenaline synthesis in the brain[END_REF][START_REF] Herraiz | Analysis of monoamine oxidase enzymatic activity by reversed-phase high performance liquid chromatography and inhibition by β-carboline alkaloids occurring in foods and plants[END_REF][START_REF] Meller | Tetrahydro-β-carbolines: specific inhibitors of type A monoamine oxidase in rat brain[END_REF] Methyl-pyridinium derivatives of harmine and harmaline were also significantly less active on MAO-A (Ki = 69 nM for 51 vs. IC50 = 1.0-5.3 nM for 1,

and Ki = 140 nM for 52 vs. IC50 = 2.5-8.0 nM for 2), as were all the other 2-methyl cationic analogues. [START_REF] Kim | Inhibition of monoamine oxidase A by β-carboline derivatives[END_REF] Finally, several natural derivatives with more complex structures were tested against MAO-A and MAO-B, but with only limited success. [START_REF] Passos | Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors[END_REF][START_REF] Samoylenko | Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson's disease[END_REF] If compounds 53 and 54 reached IC50 of 0.87 µM and 0.85 µM against MAO-A, the other molecules only yielded values above 1 µM.

Phosphodiesterase type 5 (PDE5) inhibitors

Considering the failing development of effective treatments for AD, focus on other promising strategies highlighted new targets such as PDE. The classification of these enzymes in eleven families, each comprising multiple isoforms and splice variants, mainly relies on their substrate specificity: cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP). These second messengers, respectively arising from ATP or GTP, are important elements in intraneuronal signal transduction cascades. By elevating cyclic nucleotide levels, PDE inhibitors promote gene transcription via the activation of the cAMP response element-binding (CREB), that regulates in turn the expression of genes involved in long-term memory formation. [START_REF] Heckman | Phosphodiesterase inhibitors as a target for cognition enhancement in aging and Alzheimer's disease: a translational overview[END_REF][START_REF] García-Osta | Phosphodiesterase as therapeutic targets for Alzheimer's disease[END_REF] Indeed, the natural β-carboline vinpocetine inhibits PDE1 and shows beneficial effects on memory function in rats. 86 To the best of our knowledge vinpocetine is the only β-carboline described to inhibit PDE1, a dual substrate enzyme able to hydrolyze both cAMP and cGMP. There are few studies of β-carboline on PDE targets other than PDE5. Some β-carbolines inhibiting PDE4 were identified [START_REF] Abdelwaly | Discovery of tetrahydro-β-carboline derivatives as a new class of phosphodiesterase 4 inhibitors[END_REF] but, so far, preclinical studies led on PDE4 inhibitors were hampered by aversive side effects, mainly emesis, because of the lack of selectivity on the four different gene products (PDE4A-D). [START_REF] García-Osta | Phosphodiesterase as therapeutic targets for Alzheimer's disease[END_REF] On the other hand, several FDA-approved drugs, including the β-carboline tadalafil, inhibit PDE5, a cGMP-specific enzyme, for the treatment of male erectile dysfunction. Incidentally, clinical studies have revealed the potential of these inhibitors to enhance memory and cognitive functions strengthening the relevance of this target. [START_REF] García-Barroso | Tadalafil crosses the blood brain barrier and reverses cognitive dysfunction in a mouse model of AD[END_REF] 1-Aryl dihydro-β-carbolines and influence of the R 2 substituent. Several series of simple reduced tricyclic 1-aryl β-carbolines without substitution of the nitrogen at position 2 were evaluated. [START_REF] Abadi | Synthesis of novel tadalafil analogues and their evaluation as phosphodiesterase inhibitors and anticancer agents[END_REF][START_REF] Abadi | molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective[END_REF][START_REF] Abadi | molecular modeling, and biological evaluation of novel tetrahydro-β-carboline hydantoin and tetrahydro-β-carboline thiohydantoin derivatives as phosphodiesterase 5 inhibitors[END_REF][START_REF] Ahmed | synthesis and structure-activity relationship of functionalized tetrahydro-β-carboline derivatives as novel PDE5 inhibitors[END_REF] These compounds were mostly inactive or active at the micromolar range.

Especially, the substitution of the 1-aryl group showed a limited impact on PDE5 inhibition potency: considering derivatives with 1R,3S configuration, the replacement of the 4'bromophenyl group of 55 (IC50 = 3.1 µM, Figure 11) with 4'-bromo or 2',4'-dimethoxy analogues provided activities in a similar order of magnitude (IC50 = 4.2 µM, and 6.4 µM, respectively). However, the absolute configuration of carbon 1 was more discriminating, as only 1R,3S and 1R,3R β-carbolines were found active. The importance of a R configuration at carbon 1 was illustrated by the evaluation of the four diastereoisomers of the 1-(2-bromo)aryl analogues, leading to an activity in the case of 55 and its 1R,3R counterpart (IC50 = 3.1 µM and 5.6 µM respectively), whereas no activity was recorded for the two remaining stereoisomers. The same trend was observed for 1-aryl derivatives bearing different phenyl substituents. The thiophenyl analogue was found inactive however. [START_REF] El-Gamil | Design of novel β-carboline derivatives with pendant 5-bromothienyl and their evaluation as phosphodiesterase-5 inhibitors[END_REF] If the substitution of position 2 by a chloroacetyl moiety did generally not improve the potency of the naked analogues (e.g., IC50 = 2.7 µM for compound 56), the introduction of more complex groups at this position produced two main series of more valuable derivatives. In the first series, phenyl-furoyl β-carbolines were evaluated as racemic mixtures, with a 3,4methylenedioxyphenyl group as the 1-substituent, and all compounds showed anti-PDE5 activity at a submicromolar range. [START_REF] Sui | Synthesis and biological activities of novel βcarbolines as PDE5 inhibitors[END_REF] One compound especially yielded a Ki value below 100 nM (Ki = 78 nM for 57a). A further expansion of substituents of the phenyl-furoyl group provided beneficial effects, when compared with shorter analogues, e.g., 57c vs. 57b (Ki = 64 nM vs. 120 nM), or 58b vs. 58a (Ki = 51 nM vs. 180 nM). The introduction of a piperidyl substituent at the phenyl-furoyl group and the simultaneous replacement of an oxygen atom from the 3,4-methylenedioxy moiety by a carbon provided compound 59, which showed the best activity of the series (IC50 = 8 nM). On the other hand, the reduction of the amido linker was found detrimental. It is worth noting that the evaluation of separated enantiomers of a representative compound confirmed that only the 1R configuration enabled PDE5 inhibition, as the 1R derivative 60 was found at least 150 times more active than the 1S analogue (IC50 = 67 nM and >10 µM respectively). Similar results were obtained from the second series, using a pyrimidine ring as a linker between the β-carbolines and the remote phenyl groups, at position 2 (e.g., Ki = 63 nM for compound 61). [START_REF] Sui | Synthesis and biological activities of novel βcarbolines as PDE5 inhibitors[END_REF] Again, the removal of an oxygen atom from the 3,4-methylenedioxyphenyl group at position 1 led to the most active derivative 62, a dipyridine analogue with a Ki of 4 nM. As the compound was tested as a racemic mixture, it could be supposed that the actual potency of the 1R enantiomer is even higher. Among the other modifications tested, the introduction of a carbonyl group at position 4 of the βcarboline scaffold afforded a beneficial result (Ki = 9 nM for 63 vs. 63 nM for 61), while its reduction in the hydroxy counterpart was unfavorable (Ki >100 nM). A replacement of the 3,4-methylenedioxyphenyl group by a 3,4-dimethoxyphenyl substituent also led to a critical drop of anti-PDE5 activity. Interestingly, the alternative use of the rutaecarpine scaffold for anti-PDE5 activity also produces one compound with IC50 below 100 nM, compound 64 (IC50 = 86 nM), while all other derivatives were found less active. 95 β-Carbolines with a fused hydantoin ring. Starting from the β-carboline scaffold itself, GlaxoSmithKline performed a substructure search in their internal database to find more potent PDE5 inhibitors, and a fused β-carboline-hydantoin analogue was identified as promising, with a good recorded selectivity. [START_REF] Daugan | The discovery of tadalafil: a novel and highly selective PDE5 inhibitor. 1: 5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione analogues[END_REF] Thus, they sought to produce numerous derivatives associated with different modulation points. First, from a stereochemistry point of view, a high number of fused β-carboline-hydantoin racemic mixtures did not allow an easy discrimination between trans and cis forms, showing similar good activities in almost all cases (e.g., IC50 = 10 nM for cis-65a vs. 7 nM for trans-65a, 8 nM for cis-65b vs. 5 nM for trans-65b, 7 nM for cis-65c vs. 3 nM for trans-65c, Figure 12). Contrariwise, as already observed in the 1-aryl β-carboline series, the R configuration (corresponding to the S configuration for sulfur-containing compound 68) of carbon 5, which is the counterpart of carbon 1 in the previous series, appeared as a determinant feature for reaching maximum potency (e.g., IC50 = 330 nM for 66 vs. no activity for the 5-S isomer, 38 nM for 67 vs. 640 nM for the 5-S isomer, 170 nM for 68 vs. no activity for the 5-R isomer). [START_REF] Abadi | molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective[END_REF][START_REF] Ahmed | synthesis and structure-activity relationship of functionalized tetrahydro-β-carboline derivatives as novel PDE5 inhibitors[END_REF][START_REF] El-Gamil | Design of novel β-carboline derivatives with pendant 5-bromothienyl and their evaluation as phosphodiesterase-5 inhibitors[END_REF] In addition, the S configuration at position 11 often promoted anti-PDE5 activity (e.g., IC50 = 0.33 µM for 66 vs. 1.1 µM for the 11-R isomer, 38 nM for 67 vs. 290 nM for the 11-R isomer), but with some discordant data (e.g., IC50 = 0.17 µM for 68 vs. 0.47 µM for the 11-S isomer). As a result, 5R,11S analogues often unveiled the most promising profile for building further fine-tuned iterations. Indeed, modulation efforts at various positions while fixing the absolute configurations afforded the best IC50 values among tested enantiopure derivatives (IC50 = 51 nM for 69a and 40 nM for 69b). [START_REF] Abadi | Synthesis of novel tadalafil analogues and their evaluation as phosphodiesterase inhibitors and anticancer agents[END_REF][START_REF] Abadi | molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective[END_REF][START_REF] Abadi | molecular modeling, and biological evaluation of novel tetrahydro-β-carboline hydantoin and tetrahydro-β-carboline thiohydantoin derivatives as phosphodiesterase 5 inhibitors[END_REF][START_REF] Ahmed | synthesis and structure-activity relationship of functionalized tetrahydro-β-carboline derivatives as novel PDE5 inhibitors[END_REF][START_REF] Ahmed | Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel β-carboline derivatives[END_REF] Regarding the influence of substituents, if the presence of a 4-methoxyphenyl moiety at position 5 was found beneficial overall (IC50 = 5-8 nM for 65b, vs. >20 nM for analogues with chloro or cyano substituents, or with alternative heterocyclic rings), variations of the hydantoin N-substituent did not exert a crucial impact.

Indeed, ethyl, butyl, and even bulkier benzyl, cyclohexyl and ethylpyridine groups provided single-digit nanomolar activities (IC50 <10 nM for 65a-c, among other analogues). Further modulations at these two positions failed to yield derivatives with similar properties.

Especially, dimethoxyphenyl, chlorophenyl, bromophenyl and bromothiophenyl groups introduced at position 5 of various β-carboline-hydantoin stereoisomers (with various Nsubstituents, e.g., ethyl, butyl, chlorophenyl) only generated compounds with IC50 >38 nM.

However, it is worth noting that the best molecules of the series were 5-(bromophenyl) derivatives (67, 69a, 69b). Replacing the hydantoin ring by an analogous 2thioxoimidazolidin-4-one was overall detrimental, even if compound 70 eventually reached the submicromolar range (IC50 = 0.56 µM). Interestingly, only one analogue with 3,4methylenedioxyphenyl substitution at position 5 was tested. [START_REF] Daugan | The discovery of tadalafil: a novel and selective PDE5 inhibitor[END_REF] Despite the partial reduction of their hydantoin scaffold, compounds 71 and 72 revealed a shared promising IC50 of 60 nM.

However, the complete reduction of the hydantoin carbonyl groups yielded a completely inactive compound. β-Carbolines with a fused piperazinedione ring. The expansion of previous β-carbolinehydantoin compounds into piperazinedione analogues gave engaging results. First, the evaluation of pairs of cis and trans racemic mixtures (e.g., IC50 = 5 nM vs. 140 nM for cisand trans-73b, respectively, Figure 13) first revealed a general trend, showcasing the cis configuration as the most favorable. [START_REF] Daugan | The discovery of tadalafil: a novel and selective PDE5 inhibitor[END_REF] Thus, further preliminary studies investigated the substitution of nitrogen 2 of cis β-carboline-piperazinedione derivatives and succeeded to identify racemic, one-digit nanomolar inhibitors of PDE5, such as cis versions of 73a-d, a result highlighting the considerable steric tolerance at this position (IC50 = 5 nM for cyclohexyl 73c and 3 nM for benzyl 73d). All these molecules were associated with the presence of a 3,4-methylenedioxyphenyl group at position 6, which appeared as more advantageous than the 4-methoxyphenyl alternative. Further variations at positions 2 and 6

were performed, including independent analysis of each isolated enantiomer. It seemed that the 6R,12aR configuration issued the best results overall, followed by the isomer 6R,12aS (e.g., IC50 = 3 nM for 74 vs. 1,200 nM for the 12aS isomer, 10 nM for 75 vs. 9 nM for the 12aR isomer). [START_REF] Ahmed | synthesis and structure-activity relationship of functionalized tetrahydro-β-carboline derivatives as novel PDE5 inhibitors[END_REF][START_REF] Ahmed | Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel β-carboline derivatives[END_REF] By contrast, a S configuration at position 6 caused a dramatic loss of activity in each case, with IC50 above 1.0 µM. This work led to the discovery of compound 8

which is known as tadalafil (Figure 1), a drug that was marketed as a PDE5 inhibitor in 2003. [START_REF] Daugan | The discovery of tadalafil: a novel and selective PDE5 inhibitor[END_REF] Tadalafil presented an exquisite PDE5 vs. PDE1-4 selectivity, and a far higher selectivity vs. PDE6 than sildenafil, the molecule mainly used as medicine for PDE5

inhibition. It also had excellent pharmacokinetic properties together with a high metabolic stability, leading to a good in vivo activity. From a structural point of view, the cocrystallization of PDE5 with tadalafil 8 (PDB code 1UDU, Figure 14) allowed to retrieve crucial data about molecular interactions. [START_REF] Sung | Structure of the catalytic domain of human phosphodiesterase 5 with bound drug molecules[END_REF]100 Especially, the pyridine ring is located between Phe820 and Val782, forming a typical P clamp system and an important hydrogen bond with Gln817 side chain. In addition, the flat tetracyclic core of tadalafil is maintained through hydrophobic interactions with Ile768, Ile778, Phe786, Leu804 and Phe820. On the other hand, the 3,4-methylenedioxyphenyl moiety occupies a pocket made of Ala783, Phe786, Phe787, Leu804, Ile813 and Met816. Interestingly, when considering the structure of PDE4 (PDB code 4WCU), 101 the presence of the 3,4-methylenedioxyphenyl group causes a steric clash with M411, potentially explaining the PDE5/PDE4 selectivity observed (IC50 = 5 nM for PDE5 vs. >10 µM for PDE4, a beneficial ratio given the clinical safety issues raised by PDE4 inhibitors). Finally, the amido N-methyl group is located in a very open area, without any close residue. The possibility of introducing more complexity at this position was thus further evaluated. Overall, among the R,R enantiomers tested, only a few showed IC50 values similar to those mentioned above. [START_REF] Abadi | molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective[END_REF][START_REF] Ahmed | synthesis and structure-activity relationship of functionalized tetrahydro-β-carboline derivatives as novel PDE5 inhibitors[END_REF][START_REF] Ahmed | Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel β-carboline derivatives[END_REF][START_REF] Daugan | The discovery of tadalafil: a novel and selective PDE5 inhibitor[END_REF] Other studies focused exclusively on 3,4methylenedioxyphenyl analogues. Interestingly, the tolerance for N-substitution was lesser in this context, and small, aliphatic moieties were found more promising for anti-PDE5 activity than aromatic or extended groups (e.g., IC50 = 16 nM for 76 vs. 5 nM for 8). [START_REF] Daugan | The discovery of tadalafil: a novel and selective PDE5 inhibitor[END_REF]102,103 In addition, the introduction of polar groups, such as OH or NH2, was detrimental, compared to hydrophobic equivalents. 104

Glutamate receptors binders (NMDA, mGluR)

Glutamate, the primary excitatory neurotransmitter present in the CNS, is implicated in several key brain functions such as learning and memory. Glutamate can bind to two families of receptors: the ionotropic glutamate receptors, mainly NMDA receptors, voltage-dependent channels permeable to Ca 2+ that mediate excitatory neurotransmission, and the metabotropic receptors (mGluRs) belonging to the G-protein coupled receptors family and known for their neuroprotection properties. In AD, the over-activation of NMDA receptors through excessive binding of glutamate causes a large influx of Ca 2+ that leads to excitotoxicity, ultimately ending in neuronal degeneration. NMDA antagonists, such as the FDA-approved drug memantine, have demonstrated their neuroprotective role by attenuating incidents of excitotoxicity and thus neuronal death, delivering an efficient symptomatic treatment for AD.

The heterooligomeric NMDA receptors comprise at least two types of subunits: NR1 and NR2, the latter occurring in four distinct subtypes (NR2A-D).

Inhibition of NMDA receptors. β-Carbolines were first identified as potent and selective inhibitors of the NMDA receptor NR2B through a SAR study after rigidification of the primarily identified active skeleton. 106 The activity of this series on NR1/2B subtype appeared to be strongly dependent on the presence of a phenolic hydroxyl group, whether at the A-or B-ring (e.g., IC50 = 50 nM for 79 vs. μM against NR1/2A and NR1/2B, whereas the R enantiomer showed no activity. 107 Both enantiomers of the phenyl analogues, with removed methylene links, were also inactive.

Indeed, the 'V'-like shape of compound 82 seems to foster interactions with the receptor, as corroborated by docking experiments. The two wings of the 'V' are formed by the tetrahydroβ-carboline core and the benzyl group, which interact in a similar manner than memantine or dizocilpine, i.e. with hydrophobic residues Val640, Leu643 and Ala644 (NR2A) or Met641, Val644 and Ala645 (NR1). Another rigidification strategy leading to β-carbolines was initiated after the identification of hit compounds presenting a biological activity on NMDA receptor and displaying both bicyclic lactam and indole moieties. 108 The study focused on nine β-carbolines, varying over their stereochemistry, substitution pattern and lactam-ring size, but the best obtained activity (compound 83, IC50 = 30 µM) remained modest.

Inhibition of mGluR receptors. While the specific involvement of mGluRs in AD pathogenesis is imprecise, their significant role is ascertained by their implication in the modulation of MAPK, directly implicated in the disease process. mGluRs are subdivided into three groups, mGluR1 belonging to group I mGluRs. Since the early 2000s, many reports have shown the neuroprotective effects of the mGluRs modulation, whether by the use of group II mGluR agonists or by group I mGluR antagonists. 109 Notably, β-carbolines designed from previously identified potent and selective pyrroles were evaluated as mGluR1

antagonists. 110 The SAR study highlighted three main parameters responsible for the improvement of their in vitro potency: (1) a H-bond acceptor and/or donor on the unsubstituted lactam ring, (2) a H-bond acceptor group at R 6 and (3) a bulky and lipophilic substituent on the indole ring part and more conveniently at position 6. Structures complying with these criteria exhibited the best activities, and among them 84, 85 (IC50 = 71 nM for both) and 86a (IC50 = 100 nM) were the most promising. For these compounds, in vivo pharmacokinetic properties were evaluated on rats, showing oral bioavailability ranging of 9-12% and a maximum exposure in plasma of 37-145 ng/mL. Nevertheless, the most balanced mGluR1 antagonist regarding both in vitro activity and pharmacokinetic profile is compound 86b (IC50 = 170 nM), which is in addition associated with a suitable level of brain penetration.

γ-Aminobutyric acid receptors (GABAA) ligands

While cholinergic and glutamatergic dysfunctions are well known to be disrupted in AD, the increase or decrease GABA levels in AD patient brains have long been discussed. However, despite some initial controversy, the primary inhibitory neurotransmitter GABA, involved in the maintenance of the excitatory/inhibitory balance in the brain, is undoubtedly implicated in AD pathology. [111][112][113] Indeed, cognitive deficits were related to the dysfunction of presynaptic GABA that acts on GABAA receptors, widely located in the CNS. The increased GABAergic neurotransmission could be circumvented with an agonism at GABAA receptors, thus preventing the neuronal death and the formation of β-amyloid fibrils. 114

Affinity of β-carbolines and influence of the R 3 and R 4 substituents on efficacy. The affinity of β-carbolines for the benzodiazepine binding site of GABAA receptor was initially discovered in the early 1980's. Even elementary β-carbolines with very low substitution levels achieved to fairly interact with GABAA receptor in a ubiquitously used [ 3 H]ligand binding inhibition test (usually classical benzodiazepines were employed, such as diazepam, flunitrazepam or flumanezil). This is especially the case with norharmane 9a and harmane 9b (IC50 = 1.6-8.2 µM and 7.2-25 µM, respectively, Figure 2), while further substitutions and partial or complete saturation of the pyridine ring led to an activity drop (e.g., IC50 >100 µM for 1-3, Figure 1). [115][116][117][118] In contrast, the introduction of an ester group at position 3 produced compounds with far better affinities, in the nanomolar range. β-CCM 87b, β-CCE 87c together with higher homologs 87d and 87e thus shared similar IC50 values of 1-10 nM, and seemingly constitute the optimal steric hindrance window, as both the carboxylic acid 87a and the CH2t-Bu 87f analogues were less active (IC50 = 31-33 µM and 0.75 µM, respectively, Figure 16). 115,117,[119][120][121][122][123][124][125] Interestingly, concomitant substitution of the positions 5 or 6 did not appear to be detrimental in this context, and several derivatives bearing various groups, such as OH, Oi-Pr, NHBn or OBn, managed to retain the same level of affinity (e.g., IC50 = 4 nM for 88a, 10 nM for 88b, vs. 1-19 nM for 87b). 124,126,127 Similarly, when introducing a methoxymethyl substituent at position 3, the affinity values remained in the low nanomolar (e.g., IC50 <10 nM for 89a, 89d, ZK-93423 89e and abecarnil 90) or subnanomolar ranges (e.g., IC50 = 0.9 nM for 89b, 0.5-1 nM for 89c, 0.2-2 nM for 91), [127][128][129][130] even if the best values were yielded by 3-methyl analogue 92 (IC50 = 0.4-0.7 nM). 131 However the presence of a substituent at position 9 dramatically compromised the affinity for GABAA receptor (IC50 >0.29 µM for 9-methyl counterparts of 87b, 89d and 89e; >5.0 µM for other derivatives). 115,117,127,128 A few other chemical groups led to similar affinity upon further pharmacomodulation at position 3. Among them, the propyl ketone 87g, nitrile 87h, isothiocyanate 87i and propoxy 87j yielded the best results (IC50 <11 nM). 117-120 Reduced versions of β-CCM 87b and β-CCE 87c showed far weaker activities (IC50 = 6.0 µM). 115 In addition, the early discovery of β-carboline-based agonists and antagonists prompted research groups to establish enlightening structure-efficacy relationships. One striking feature was the shift of the β-carboline 3-esters efficacy along an inverse agonist-agonist continuum, as the carbon chain of the ester became bulkier (Figure 17). Indeed, 87b and 87c behave as inverse agonists, 87d and 87e as antagonists and higher homologs showed anticonvulsant effects against leptazol-induced seizures, consistent with an agonist action. 132 Several other derivatives with more intrinsic efficacy were identified that fell into one or the other of these categories. Thus, compound 89e appeared as a full agonist of GABAA receptor, whereas 91 was identified as a partial agonist, and 87h, 87j or 92 were acknowledged to be antagonists. In the other hand, very close analogues were seen as inverse agonists, such as 87g or less active 3-methoxy and 3-ethoxy derivatives. 120, While lacking an apparent rationale, this variability in terms of efficacy should derive from changes in molecular interactions, whose complete understanding is scrambled by the absence of accurate structural data and by the diversity of GABAA receptor subtypes. Receptor subtypes selectivity and additional combinations of substituents. The GABAA receptor complex displays high molecular diversity and is made from a combination of various subunits. At least fifteen different subunits have been identified and could explain the diversity of pharmacological effects observed in presence of ligands. 128 In an initial pharmacophoric model, two hydrogen bond donating sites were suggested, termed H1 and H2.

Especially, H2 was thought to correspond to the position 4 of β-carbolines, where the presence of an oxygen atom appeared as beneficial. In addition, the occupation of a lipophilic area at

INVERSE

AGONISTS

ANTAGONISTS AGONISTS

this site seemed to contribute to an agonist activity, as testified by 4-methoxymethyl derivatives 89e and 91. The evaluation of β-carbolines against specific receptor subtypes was a step further towards rationalization at the molecular level. The affinities of some of the best β-carbolines discovered previously for GABAA receptor subtypes α1β3γ2, α2β3γ2, α3β3γ2, α4β3γ2, α5β3γ2 and α6β3γ2 (shortened respectively as α1, α2, α3, α4, α5 and α6 subtypes) were thus assessed. First, all β-carbolines were found far less active against the subtypes α4 and α6 (Ki >1.0 µM in almost all cases), while the remaining four subtypes afforded very low nanomolar Ki values ( Table 2). Interestingly, if globally compounds devoid of 4-substitution showed some selectivity for α1 (e.g., 87b, 87e, 87g, 87j, 88b), 4-methoxymethyl analogues did not discriminate between α1, α2, α3, and α5 subtypes (e.g., 89a, 89d, 89e, 90). 128,[135][136][137][138][139] The residue His105, which is present in subunits α1, α2, α3, and α5, and replaced by an arginine in α4 and α6, was

proposed to play a central role in this selectivity. Indeed, the affinity values of compounds 87b, 87c and 90 for subtype α5 dropped significantly upon mutation of His105 into Arg105 (Ki = 4.4 µM, 1.9 µM and 6.2 µM respectively, vs. 44-68 nM, 27-110 nM and 5-8 nM respectively for the wild type). 137 Further works were undertaken for exploring the selectivity profile of β-carbolines, especially by combining a favorable 3-substituent with diverse groups at positions 6 and 7. Hence, the production of 6,7-dimethoxy analogues afforded surprising pan-subtype inhibitors 93a and 93b, which are quite unique in their ability to bind efficiently to α6 (Ki = 130 nM and 75 nM respectively, Figure 18). 128,135 Introducing other substituents at these positions, such as nitro, isothiocyanate, or ethynyl, provided additional compounds of interest (e.g., 94, 95, 96), but with a more classical selectivity profile, except for 96. The derivatization at position 6 using aryl or heteroaryl moieties, including phenyl, naphthyl, furyl and thiophenyl mostly failed to significantly improve both affinity and selectivity, while a phenyl ester group at position 3 was associated with enhanced α1 selectivity (e.g., for 97, IC50 = 34 nM against α1 vs. >1.0 µM against α2-6). 135,138 Finally, derivative 98 was the only reported example of an active N 2 -substituted β-carboline, revealing especially a good affinity for α1 and α2 subtypes (Ki = 7 nM and 12 nM respectively). 140 Pyridodiindoles and other fused ring systems. Beyond the introduction of simple substituents in the β-carboline scaffold, derivatives including additional fused rings were developed. Especially, pyridodiindoles were conceived as a very rigid and planar platform made of five fused aromatic rings, based on a β-carboline core. Interestingly, the naked pyridodiindole 99a was found as the most active representative of this new class of GABAA ligand (IC50 = 4 nM), while appending various substituents consistently led to weaker activity records (Figure 19). 118,119,141 However, if the introduction of 2'-and 3'-substituents was really prejudicial, similar substitutions at positions 4' and 5' afforded compounds with some retained affinity, as illustrated by the chloro analogues (IC50 = 10 nM and 80 nM for 99b and the 5'-chloro analogue of 99a, respectively, vs. 0.72 µM and 2.2 µM for 2'-and 3'-chloro analogues of 99a, respectively). In particular, at position 4' there seems to be some room for a substituent, regardless of its chemical nature (halogens of various sizes, methoxy, hydroxy, with IC50 <0.019 µM for 99b and 99c) but with limitations in terms of steric hindrance (IC50 >4.0 µM for the benzyl analogue 99d). As previously observed with simple β-carbolines, the substitution of the N 9 with a methyl group led to a substantially decreased activity, while functionalization of the position 6 gave valuable compounds. Especially, by adding nitro or bromo groups, IC50 values were obtained that remained similar to the naked parent 99a (IC50 = 4 nM for 100a and 6 nM for 100b). Finally, when exploring the impact of substituting the indole NH position and position 1 of the β-carboline, detrimental effects were seen in each case. A closer look to the subtype-related potency distribution showed selectivity profiles very similar to those observed with simpler β-carbolines ( Table 2). 135 However, the affinity for subtype α5 was significantly lower in all cases (e.g., Ki = 40 nM against α5 vs. 1 nM against α1-3 for 99a; Ki = 230 nM against α5 vs. <12 nM against α1-

5-Hydroxytryptamine (5-HT) receptors binders

The implications of the serotonergic system, and therefore its neurotransmitter 5-HT, in Alzheimer's disease are diverse and complex, especially due to multiple interactions with other neurotransmitters such as acetylcholine, glutamate or GABA. Serotonin is a monoamine neurotransmitter acting on fourteen pre-and postsynaptic subtypes of receptors divided in seven families (5-HT1 to 5-HT7) and mainly located in brain areas involved in particular in learning and memory. Among them, subtypes 1, 3, 4, 6 and 7 are of particular interest in the AD context. 5-HT4 and 5-HT7 receptor agonists as well as 5-HT1, 5-HT3 and 5-HT6

antagonists have shown pro-cognitive effects in animal studies or clinical trials. 114,142,143 Tetrahydro-β-carbolines and R 1 /R 9 substituents exploration. If tetrahydro-β-carbolines of classical structures, such as 3 (Figure 1), showed only weak activities against 5-HT receptors, improvements were observed upon methoxy or bromo position shifts (e.g., Ki <0.18 µM for 107a and 107b against 5-HT2A and 5-HT2C vs. >10 µM for 3). [144][145][146][147] Especially, 8-bromotetrahydro-β-carboline provided the only inhibition constants below 100 nM, against 5-HT2A and 5-HT2C, while the 6-and 7-methoxy derivatives, mostly inactive against subtypes 1A, 2A and 2C, showed interesting levels of activity against 5-HT2B (Ki >1.0 µM vs. 0.16 µM and 0.39 µM, respectively). The 1-methyl-8-methoxy tetrahydro-β-carboline reached a Ki value of 0.45 µM against 5-HT1A, while all other tested molecules remained at the micromolar range.

Hence, some selectivity was clearly observed upon substituent modifications, allowing the tetrahydro-β-carbolines to preferentially inhibit either subtypes 1A, 2A, 2B or 2C, depending of the position and nature of the chemical groups introduced. In addition, the introduction of benzyl groups at position 1 led to 5-HT2B inhibitors with far better affinities, sometimes in the low subnanomolar range. Especially, 3',4'-dimethoxy derivatives 108a-108d, 109, 110a-b and 111a-b were found to inhibit 5-HT2B with KB values below 0.3 nM, while concomitantly targeting the 2A and 2C subtypes, with a weaker potency however. 148 The best value was recovered from 112, which exhibited a KB of 80 pM, and a good selectivity for 5-HT2B vs. 5-HT2A and 5-HT2C (~100-fold). Interestingly, the substitution pattern of the β-carboline core affected only marginally the 5-HT2B inhibition activities observed, considering either the position (e.g., KB = 80-500 pM for the various methyl derivatives 108a, 109, 110a and 112)

or the nature (e.g., KB = 100-700 pM for 6-substituted compounds 108a-d) of the substituents. Contrariwise, the length of the methylene linker appeared as a crucial feature, as both the lower and the higher homologues led to significantly reduced action. Shifting the 3',4'-dimethoxybenzyl group to position 2 was also unsuccessful. However, the introduction of naphthyl moieties in place of the 3',4'-dimethoxyphenyl part of the molecule afforded results in a similar range of affinity (KB = 400 pM for 113). The natural indoloquinolizidine yohimbine (5, Figure 1), which was found as a potent ligand of 5-HT receptors (Ki = 25-160 nM against 1A, 1B and 1D subtypes, >1.0 µM against 2A and 2C), confirmed the steric tolerance and activity potential of bulky groups at positions 1 and 2 of the scaffold. 149 Dihydro-and fully aromatic derivatives. On the other hand, when considering the 3,4dihydro counterparts of the reduced β-carbolines, various effects were observed. The insertion of the double bond led either to a significant improvement of the potency (e.g., Ki = 0.92 µM vs. >10 µM against 5-HT2C for 115a and its tetrahydro analogue, respectively, Figure 21), to a mere conservation of the efficiency or to an activity drop (e.g., Ki = 0.13 µM vs. 0.47 µM against 5-HT2A for 107a and its oxidized version, respectively). [144][145][146] However, compounds 116a and 116b were the only to reach inhibition values below 100 nM (Ki = 69 nM and 75 nM against 5-HT2C, respectively). A more systematic exploration of 5-HT receptor subtypes provided additional data for representative dihydro-β-carbolines 2 (Figure 1), 115a, and 115b

(Table 3). 146,147 Similar selectivity profiles were seen for both compounds, targeting preferentially subtypes 2B and 7, and to a lesser extent 1A, 2A, 2C and 6. Compound 115b

showed in addition a good potency against 5-HT5A (Ki = 0.85 µM). Further oxidation of the βcarboline pyridine ring afforded completely unsaturated analogues. Once again, the insertion of an additional double bond yielded mixed results: if some derivatives were definitely more active against 5-HT2A (e.g., Ki = 0.15-0.40 µM for 1 and 9b, vs. 0.99-7.8 µM for 2 and 115b), some others were less potent than their dihydro parent (e.g., Ki = 4.2-5.6 µM for 115a vs. >10 µM for the fully aromatic analogue). 144,145,151 There were less variations when considering the 1A and 2C subtypes however. Unlike the tetrahydro-β-carboline series, the activity of fully aromatic analogues against 5-HT2B was hardly the best among 5-HT subtypes, often exceeded by the anti-5-HT2A potency (e.g., 1.4 µM vs. 0.69 µM for 117 and 1.5 µM vs. 0.56 µM for 118). The introduction of heteroaromatic groups at position 1 partially restored a selectivity profile reminiscent of the tetrahydro-β-carbolines, with moderate activities however. 151 Among the evaluated scaffolds, the indole afforded the best results, with for example compounds 119a (Ki = 0.25-0.90 µM against 5-HT2A-C), 119b (Ki = 0.17-0.20 µM against 5-HT2B-C) and 120 (Ki = 0.10-0.81 µM against 5-HT2A-C). However, these molecules were not tested against more AD-relevant 5-HT receptor subtypes.

Histone deacetylase (HDAC) inhibitors

Epigenetic regulation of gene expression via histone modification is a recent field explored in the understanding of AD. HDAC and histone acetyltransferases plays an important role in the acetylation balance of histone for which abnormalities were observed in the pathology of AD.

If the targeting of HDAC was clearly associated with neuroprotective properties in AD models, their action and the precise role of each isozyme is still unknown. Nevertheless, it is now largely accepted that HDAC inhibitors may impact proteins involved in AD, such as Aβ, GSK-3β or tau. To date, several approaches aim at developing pan-HDAC inhibitors, but with expected collateral effects, as some isozymes play important and diverse neurologic functions.

On the other hand, class I HDACs and HDAC6 (class IIb) have notably been proposed as targets of interest for AD treatments. 152,153 As a consequence, literature around β-carboline HDAC inhibitors is still piecemeal. However, the presence of a zinc-chelating hydroxamic acid unit, reminiscently of the structure of SAHA (suberanilohydroxamic acid, or vorinostat, a drug marketed as antineoplastic agent through HDAC inhibition), is a feature shared by all reported β-carboline-based inhibitors. First, introducing the hydroxamic acid function at position 3 of the β-carboline scaffold provided a range of inhibitors. 154 Exploring the chain length of amido-linked derivatives showed that longer arms were favorable, especially when three methylene carbons or more are present (IC50 = 1.4-1.8 µM for 121c-e vs. >3.2 µM for 121a and 121b, Figure 22). Thus, several analogous linear derivatives were tested, with amido or urea links, and led to submicromolar potency (e.g., IC50 = 270 nM for 122). 155 Considering individual HDAC isozymes, these molecules were found more active against HDAC1 and HDAC6 than against HDAC8, but with a small gap (e.g., IC50 = 26 nM against HDAC1 and 34 nM against HDAC6 vs. 160 nM against HDAC8, for 122). Again, compounds bearing shorter carbon chains showed less potential. The introduction of an aromatic link between the hydroxamic acid group and the β-carboline scaffold provided analogues with improved action on HDAC1 (e.g., IC50 = 9.3 nM for 123a vs. 26 nM for 122). The modulation of position 1 produced even better activities, reaching the single-digit nanomolar and subnanomolar ranges against HDAC1, HDAC3 and HDAC6, while exerting low action on HDAC8 (e.g., for 123b, IC50 = 4.7 nM against HDAC1, 0.4 nM against HDAC3 and 0.9 nM against HDAC6 vs. 880 nM against HDAC8, a 200-2000-fold difference). The insertion of a N-substituted amido linker provided various results against nuclear HeLa HDACs, the presence of a 1-(4methoxy)phenyl group being more favorable than the 1-methyl and unsubstituted counterparts (e.g., IC50 = 960 nM for 124a and 340 nM for 124b). 156 The N-substituent was further explored and the presence of terminal amino groups yielded the best inhibition values (IC50 = 92 nM for 124d and 120 nM for 124e). 157 It is worth noting that the evaluation of compound 124c against a series of HDAC isozymes confirmed the potency of these compounds against HDAC1 and HDAC6 (IC50 = 4.1 nM and 2.6 nM respectively) and their inactivity against HDAC8 (IC50 >1.0 µM), but this time also against HDAC2 and HDAC4 (IC50 >1.0 µM).

Alternatively, β-carbolines bearing the hydroxamic acid moiety at position 9 were investigated. A similar trend was observed regarding the potency of aromatic vs. aliphatic links (e.g., IC50 = 1.4 nM for 125a against HDAC6 vs. 550 nM for the analogue bearing a (CH2)5 linker), but the most striking feature was the vastly improved HDAC6 vs. HDAC1 selectivity (e.g., for 125b, IC50 = 2.5 nM against HDAC6 vs. 9700 nM against HDAC1; for 125c, IC50 = 0.9 nM against HDAC6 vs. 4300 nM against HDAC1, an almost 5000-fold gap), which was inexistent with 3-substituted analogues. 158,159 Tetracyclic derivatives shared a similar profile, with sometimes anti-HDAC6 inhibition values at the subnanomolar range (e.g., IC50 = 0.9 nM for 126 and Ki = 0.7 nM for 127), and overall conserved potency no matter the scaffold structure. 160,161 This series was explored further and revealed fairly selective inhibition of HDAC6 among HDAC1, HDAC2, HDAC4, HDAC8 and HDAC10.

Finally, indoloquinolizidine-based compounds showed a broader inhibition spectrum of HDACs, as 128 for example exhibited similar values for HDAC1, HDAC3 and HDAC6 (IC50 = 24 nM, 71 nM and 13 nM respectively). 162 Compound 130 was the only representative of a 2-substituted β-carboline-hydroxamic acid hybrid, with an interesting potency against HeLa HDACs (IC50 = 44 nM). 163

β-Carboline-based multitarget strategies

As previously mentioned, due to the insufficient knowledge of the etiology of AD and its multifactorial nature, a change in the therapeutic paradigm appeared to be essential: the unsuccessful 'one drug-one target' strategy is now increasingly overtaken by the design of MTDL. Indeed, positive results (additive or synergistic, i.e., even better than those obtained for each ligand separately) can be expected by simultaneously interacting on different targets known to be intricated in the complex network of several pathological AD-related events. The MTDL strategy is an alternative to the most classical multiple target approach that relies on the combination of at least two known drugs and can suffer many drawbacks. The attractive MTDL strategy can involve the hybridization of several pharmacophoric moieties, active on different targets, or the design of a molecule able to interact with multiple targets; 7-9 several natural products with these properties have notably been described and could benefit from further investigations in the frame of innovative anti-AD drug development. In this part, we will only focus on the first approach in which a deliberated chemical design is required. The second one, driven by multiple biological tests on isolated or synthesized molecules to specify their bioactive scope, has already been reviewed independently in the previous target-bytarget parts. While the identity of the targets addressed in the MTDL strategies unsurprisingly matches those described above, the target pairs selection for undertaking a de novo design can represent a critical step of the process. In order to interfere at the initial phase of the disease, to slow down its evolution, or to optimally reverse AD progression, acting on the synaptic or neuronal loss would be helpful. In particular, the cholinergic neurons loss in AD patients is massive (almost 80%) [START_REF] Leόn | Recent advances in the multitarget-directed ligands approach for the treatment of Alzheimer's disease[END_REF] and lots of MTDL appropriately embed a scaffold targeting the cholinergic system. Furthermore, the preferred strategies were set up to interfere with both a symptomatic target (mainly receptors) and a disease-modifying target (such as Aβ, tau or neuroprotection), or at least with two different hypotheses of AD, as already mentioned. 164 Based on these elements, four main MTDL strategies involving β-carbolines have been reported so far. Thus, starting from previous results obtained against individual targets, and following a multifunctional approach, researchers developed additional compounds designed to not only target BACE1 but also provide anti-Aβ aggregation, metal complexation and radical scavenging properties. 171 Compound 137 (IC50 >50 μM against BACE1, IC50 = 30 μM against Aβ aggregation, IC50 = 43 μM for antioxidant activity, and 43% of metal chelating capability at 100 μM) is representative of this multifunctional strategy: while retaining the tryptoline core with the two requisite NH groups for BACE1 interaction, it displays an additional phenolic structure playing a role in free radical scavenging but also improving Aβ binding with an evaluated optimal distance of 13-14 Å between the two aromatic moieties. In order to improve the poor inhibitory activity of this series against BACE1, the phenolic part was finally substituted by another more hydrophobic tryptoline moiety; however, the length between the terminal aromatic rings was maintained for the anti-Aβ-aggregation activity (IC50 = 17 μM against BACE1, and 80% of Aβ aggregation inhibition at 100 μM for 138). 172 Targeting AChE and PDE5. While AChE and PDE5 are both involved in memory and cognition processes, the inhibition of these targets implies different and dissociable mechanisms. 173 The design of innovative AChE/PDE5 dual inhibitors potentially producing a synergistic effect on AD thus appears as a relevant MTDL strategy. An initial focus on the repurposing and redeveloping of an existing marketed drug, tadalafil (8, Figure 1), a selective PDE5 inhibitor, was a potential starting point for designing dual inhibitors (IC50 = 26 μM for 8). Reminiscently of previous SAR results on anti-PDE5 tadalafil analogues (which denoted some tolerance at this position, Figure 13), the introduction of a donepezil-inspired As for the tacrines, the optimal distance between the two pharmacophoric moieties is usually fixed at 7-9 carbons with loss of activity for shorter linkages and solubility issues for longer ones. [START_REF]SAR regarding the effect of β-carbolines on the previously mentioned AD-related targets[END_REF]176 By anchoring the linker either at N 2 or N 9 positions of the β-carboline scaffold, two series were developed and reported. The homodimers linked at the N 2 position always kept some anti-ChE activities, often below 200 nM, while the potency against NMDA receptors varied. Especially, compound 143 reached decent ChE inhibition (IC50 = 86 nM and 22 nM against AChE and BuChE, respectively, Figure 26) and promising values against NMDA receptors (IC50 = 2.6 µM and 1.8 µM against NR1/2A and NR1/2B, respectively), despite a longer 12-carbons linker, whereas tetrahydro-β-carboline analogues were globally less active.

The other series produced molecules with much more variability in their AChE/BuChE and NMDA inhibition potencies: if the best AChE inhibitor 144 (IC50 = 0.5 nM) was also the most active against NMDA (IC50 = 1.4 µM and 2.9 µM, respectively), closely related analogues were found to be completely inactive against AChE (IC50 >10 µM) and NMDA (percentage of excitotoxicity at 25 µM around 100%). The activity of some compounds on MAO-A/B was also evaluated but yielded weak values. For reducing the molecular weight of the homobivalent β-carbolines in order to improve bioavailability while retaining both activities against AChE and NMDA, new β-carboline scaffolds were synthesized, but the results were underwhelming. [START_REF] Otto | Beta and gamma carboline derivatives as potential anti-Alzheimer agents: a comparison[END_REF] The best MTDL identified showed only weak activities compared to the homobivalent β-carbolines (IC50 = 0.6 μM against AChE and BuChE, 35 µM and 5 µM against NR1/2A and NR1/2B, respectively, for compound 145). Nevertheless, the SAR studies corroborated the higher potency of quaternary derivatives for NMDA activity and demonstrated that the incorporation of a propargyl moiety, known for its neuroprotective properties in selegiline and rasagiline, has no beneficial effect herein. values below 80 nM for HDAC1 and HDAC6 (Figure 27). While PDE5 inhibition was improved by implementing more flexible and/or shorter linkages between the tadalafil scaffold and the hydroxamic acid head, such modifications dramatically compromised anti-HDAC potency, regardless of the subtype (IC50 = 0.12 µM and 0.14 µM against PDE5 for 147 and 148, vs. 2.5 µM to >20 µM for HDACs subtypes 1, 2 and 6). Similarly, the development

Conclusion and prospects

To date, only symptomatic treatments are available for countering the progression of AD, and the development of new compounds presents higher failure rates, compared to other diseases.

Yet, the initial "one-target-one-compound" paradigm has produced a great number of molecules for addressing individual targets involved in the etiology ofthe disease. But AD is intrinsically multifactorial, and the complex interplay of the pathogenesis has dismissed most of the studies performed with blinkers. The emergence of widely chosen multitargeting compounds, able to concurrently tackle several pathogenetic pathways, is a promising tool to take them off. Apart from classical drug combination and co-formulation, two main polypharmacological approaches have been of increasing importance in the past few years:

the association of two or more ligands through an adequate linker; and the hybridization between active skeletons, often fused or merged to provide compact molecules with optimal size. Both strategies rely on successful stories and FDA-approved drugs in various contexts. In the particular case of AD, some specific limitations appear however. First, the lack of appropriate animal models for AD is critical and makes it difficult to retrieve preclinical data. Second, the necessity of the BBB crossing is a sizeable issue, hampering a full delivery of the biological activity for a wide range of compounds. However, the βcarboline scaffold has several assets. As a natural product family, it is attractive for CNStargeting medicinal development: ~84% of approved drugs for CNS diseases are natural or bioinspired products. Furthermore, since natural derivatives from coffee or tobacco, such as harmane or norharmane, are known to readily exert neurological actions, the BBB crossing could not be an intractable concern for β-carbolines. In addition, the scaffold presents a high structural analogy with native brain molecules, such as neurotransmitters 5-HT or melatonin, indicating a probable capacity to easily transit throughout the brain and to interact with several biomolecules and receptors of interest. Indeed, numerous studies have demonstrated the potential of β-carbolines to target AD-related biomolecules, feeding an impressive and ever-growing SAR meta-analysis. Through this Perspective, we have first tried to draw up the main lines target by target, summarized in Figure 28. The scheme clearly shows that the βcarboline scaffold has been extensively exploited by medicinal chemists for the design of inhibitors of AD-related targets. Both rings have provided positive modifications, in almost every possible substitution positions. Additional fused rings have been added at several points, leading to highly modified analogues, often with very good activity records. While the SAR is overall very complex and hard to condense, several elements deserve to be highlighted. First, convergent favorable components are observed. The position 7 of the scaffold, when receiving an alkyl ether substituent, is able to foster the activity against DYRK1A, MAO, and GABAA to a lesser extent. Similar effects could be seen at position 2, where the substitution the intracyclic nitrogen promotes NMDA, AChE and PDE5 inhibition simultaneously, or at position 1, where the presence of close aryl groups (0-1carbon linker) is a crucial feature for PDE5 inhibition and 5-HT receptor binding. Second, sometimes the structural features required for the activity are not linked to a particular position, providing a flexibility that is precious for MTDL design. This is the case for HDAC targeting, needing the presence of a zinc-chelating head, such as an hydroxamic acid moiety, which can be placed at various positions without compromising the activity. The binding to NMDA receptors is also conditional on the global 'V-like' shape of the compound, achievable through different molecular layouts starting from the initial rather linear skeleton. Finally, in some cases it appears to be possible to reach several targets by arranging independently several parts of the scaffold. As seen for example when examining the aforementioned crystal structures, at least a part of the molecule always points towards an open area, giving some latitude for modifying this position without triggering an activity loss. As such, it would be very engaging to see if the combination of a 5,6-fused heterocycle, for GABAA binding, with a β-carboline core extended at the opposite side with a hydantoin or a piperidine ring, for PDE5 inhibition (and for incidentally consolidating GABAA binding with the amido group thereby introduced at position 3) would deliver a retained activity against both targetsto take just one example. Overall, the meta-analysis performed herein could help researchers in the field to design new β-carbolines able to simultaneously or synergistically tackle multiple AD targets, while keeping a good ability to cross the BBB, for ultimately affording drug candidates of higher potential. By combining the targeting of disease-modifying pathways and symptomatic processes, the next-generation anti-AD agents represent a great promise for the future of AD treatments. The β-carboline platform would be a robust flagship for such an adventure.

leader at Clarins (France). His researches are essentially focused on natural products and bioactive compounds from plants.

Benjamin Boucherle is a Pharm.D. graduated from the University of Grenoble

Figure 1 .

 1 Figure 1. Numbering of the β-carboline scaffold. Structure of representative β-carbolines in various oxidation

Figure 2 .

 2 Figure 2. Selected structures and IC50 of simple substituted β-carbolines as AChE and/or BuChE inhibitors.

Figure 3 .

 3 Figure 3. Selected structures and IC50 of β-carboliniums as AChE and/or BuChE inhibitors.

Figure 4 .

 4 Figure 4. Selected structures and IC50 of natural or bioinspired extended β-carbolines as AChE and/or BuChE inhibitors.

Figure 5 .

 5 Figure 5. Selected structures and IC50 of β-carbolines as BACE1 inhibitors.

Figure 6 .

 6 Figure 6. Selected structures, IC50 and aggregation inhibition percentages of β-carbolines as Aβ aggregation inhibitors.

Figure 7 .

 7 Figure 7. Selected structures of β-carbolines as inhibitors of tau phosphorylation kinases.

Figure 8 .

 8 Figure 8. (A) Crystal structure of 1 (in green) bound to DYRK1A (PDB code 3ANR). (B) Crystal structure of 41d (in mauve) bound to DYRK1A (PDB code 6UWY). (C) Crystal structure of 44 (in blue) bound to DYRK1A (PDB code 4YU2). (D) Crystal structure of 46 (in pink) bound to CLK1 (PDB code 6YTG).

  A/B) inhibitors MAO, which are mitochondrial-associated enzymes, selectively catalyze the degradation of various monoamines including oxidation of amine neurotransmitters such as 5-HT, dopamine, norepinephrine or epinephrine. Studies have recently reported high levels of MAO activity in AD patients and promising results were achieved using MAO inhibitors to improve cognitive ability. Indeed, in AD, activated MAO participate to the neurodegenerative processes both through the increase of hydrogen peroxide and thus oxidative stress and via indirect production of amyloid β peptide in neurons, via the γ-secretase. Selective MAO-B inhibitors are preferred to avoid antidepressant effects and hypersensitive reactions mediated by the inhibition of MAO-A. Selective MAO-A inhibitors are more classically used to treat depression and anxiety by providing emotional stability while MAO-B inhibitors are used in treatment of neurological disease to decrease the production of neurotoxic compounds to enhance the neuronal survival.

Figure 9 .

 9 Figure 9. Selected structures and IC50 (or Ki where indicated) of β-carbolines as MAO-A/B inhibitors.

Figure 10 .

 10 Figure 10. (A) Crystal structure of 1 (in green) bound to MAO-A (PDB code 2Z5X). (B) Overlay of crystal structures of 1 (in green) bound to MAO-A (in gold, PDB code 2Z5X) and of MAO-B (in silver, PDB code 1OJ9). In orange, the FAD cofactor.

Figure 11 .

 11 Figure 11. Selected structures and IC50 (or Ki where indicated) of 1-aryl dihydro-β-carbolines as PDE5 inhibitors.

Figure 12 .

 12 Figure 12. Selected structures and IC50 of β-carbolines with a fused hydantoin ring or analogue as PDE5 inhibitors.

Finally, 6 -

 6 (3,4-methylenedioxyphenyl) compounds bearing (R)pyrrolidine-type substituents at the amido position of the piperazinedione ring were developed as the most potent β-carboline-based PDE5 inhibitors to date. Indeed, these pyrrolidine moieties substituted by various aromatic groups (e.g., pyridine, benzyl, imidazole) were able to bring IC50 to very low nanomolar or subnanomolar values (IC50 = 0.8-2.0 nM for 77a, 77b, 78a and 78b).103,105

Figure 13 .

 13 Figure 13. Selected structures and IC50 of β-carbolines with a fused piperazinedione ring as PDE5 inhibitors.

Figure 14 .

 14 Figure 14. (A) Crystal structure of 8 (in orange) bound to PDE5 (PDB code 1UDU). (B) Overlay of crystal structures of 8 (in orange) bound to PDE5 (in gold, PDB code 1UDU) and of PDE4 (in silver, PDB code 4WCU).

3 . 3

 33 μM for the naked analogue, Figure15), and its position relative to the basic nitrogen atom of the piperidine ring, the three main active compounds (IC50 = 50 nM for 79, 87 nM for 80, and 140 nM for 81) showing similar distances between these two moieties (7.8-8.8 Å). While N 2 -or N 9 -monosubstituted compounds were generally inactive on NMDA, compound 82 yielded IC50 values of 27-28

Figure 15 .

 15 Figure 15. Selected structures and IC50 of β-carbolines as glutamate receptors binders.

Figure 16 .

 16 Figure 16. Selected structures and IC50 of 3-and 4-substituted β-carbolines as GABAA ligands.

Figure 17 .

 17 Figure 17. GABAA efficacy continuum of β-carbolines 3-esters as a function of the ester chain length.

Figure 18 .

 18 Figure 18. Selected structures of second-generation β-carbolines as GABAA ligands.

3

  for 99d). The insertion of an extra intracyclic nitrogen atom at the indole part of 99a induced a loss of activity, irrespectively of its position.135 If compounds 101 and 102 managed to keep a similar activity range against α1, their action against the other subtypes dropped significantly. Other nitrogen positions afforded even less potent compounds against all subtypes. Conversely, the conversion of the β-carboline core into the carbazole counterpart by replacing the nitrogen atom at position 2 by a CH moiety strongly weakened the activity (IC50 = 1.9-2.0 µM vs. 4 nM for 99a), highlighting the crucial importance of the carboline skeleton.118,119 Finally, other classes of heterocyclic-fused β-carbolines were developed, i.e., oxazolo[4,5-g]-β-carbolines and pyrazino[2,3-g]-β-carbolines.129 These molecules, incorporating a classical 4-methoxymethyl group, were found highly potent, hitting the subnanomolar range (IC50 = 0.8-0.9 nM for 103-106) regardless of the structural features embedded (isopropyl ester group or isoxazole ring at position 3, oxazolo or pyrazino groups at positions 5 and 6).

Figure 19 .

 19 Figure 19. Selected structures and IC50 (or Ki where indicated) of fused β-carboline-based aromatic derivatives as GABAA ligands.

  Finally, the substitution of both nitrogen atoms of the tetrahydro-β-carboline scaffold resulted to nanomolar inhibition values, with a modest 1A over 2A selectivity. Methoxyphenyl groups linked by various alkylpiperazyl chains at position 9, together with ketones introduced at position 2, led to Ki values of 20-59 nM against 5-HT1A (a more relevant subtype in the AD context) and 44-340 nM against 5-HT2A, with a very little influence of the structure variations (e.g., Ki = 20 nM and 44 nM for 114 against 1A and 2A subtypes, respectively).150

Figure 20 .

 20 Figure 20. Selected structures and Ki of tetrahydro-β-carbolines as ligands of various subtypes of 5-HT receptors.

Figure 21 .

 21 Figure 21. Selected structures and Ki of dihydro-and fully aromatic β-carbolines as ligands of 5-HT receptors.

Figure 22 .

 22 Figure 22. Selected structures and IC50 (or Ki where indicated) of β-carbolines as inhibitors of HDAC.

Figure 23 .

 23 Figure 23. Selected structures, IC50 and aggregation inhibition percentages of β-carbolines as multitarget agents, inhibitors of AChE/BuChE and Aβ aggregation.

Figure 24 .

 24 Figure 24. Selected structures, IC50 and aggregation inhibition percentages of β-carbolines as multitarget agents, inhibitors of BACE1 and Aβ aggregation.

Figure 25 .

 25 Figure 25. Selected structures and IC50 of β-carbolines as multitarget agents, inhibitors of AChE and PDE5.

Figure 26 .

 26 Figure 26. Selected structures and IC50 of β-carbolines as multitarget agents, inhibitors of AChE/BuChE and NMDA.

Figure 27 .

 27 Figure 27. Selected structures and IC50 of β-carbolines as multitarget agents, inhibitors of PDE5 and HDAC.

(

  2005). He obtained his Ph.D. in Medicinal Chemistry in 2008 under the supervision of Prof. J.-L. Décout, working on CFTR modulators. After a postdoctoral position (2009-2011) focused on protein-protein interaction inhibitors in the Chemistry School of Clermont-Ferrand (France), he moved back to Grenoble for a medicinal chemistry project targeting cystic fibrosis (2012-2015). He is currently lecturer in medicinal chemistry and pharmacognosy at the Faculty of Pharmacy at University Grenoble Alpes. His researches are dedicated to the development of bioactive substances from chemical synthesis or natural products isolation. Marine Peuchmaur is a chemical engineer graduated from Ecole Nationale Supérieure de Chimie de Montpellier (France). She obtained in 2007 her Ph.D. in Chemical Biology from University Grenoble Alpes (France), under the supervision of Dr. Yung-Sing Wong. After two postdoctoral internships at the University of Montpellier (IBMM, France, 2007-2008) and at Trinity College (Dublin, Ireland, 2008-2009), she was hired in 2009 as a lecturer in organic chemistry at the Faculty of Pharmacy (University Grenoble Alpes). Her research work in the Department of Medicinal Chemistry focuses on both phytochemistry and organic synthesis for medicinal chemistry topics (design and synthesis of bioactive molecules or tools for biochemists). She received in 2015 the Accreditation to Supervise Research (HDR). Romain Haudecoeur obtained his Ph.D. in Chemical Biology from University Grenoble Alpes (France) in 2011, under the supervision of Prof. Ahcène Boumendjel. He joined the group of Dr. David Monchaud at the University of Burgundy (Dijon, France) as a postdoctoral associate in the year. He was then appointed as Researcher in 2012 in the Department of Medicinal Chemistry of University Grenoble Alpes and received in 2020 the Accreditation to Supervise Research (HDR). His research work essentially focuses on the rational targeting of biological macromolecules, with the aim of developing new bioinspired, bioactive and smart compounds for modulating the function of proteins such as tyrosinases, viral polymerases and membrane ABC transporters.

Table 1 . IC50 Values Obtained for Harmine (1) Against a Panel of Kinases. In Bold, the Main AD-Related Kinases.

 1

	Kinase	IC50 (µM)	Kinase	IC50 (µM)
	CDK1	17-18	DYRK2	0.12-3.0
	CDK2	33	DYRK3	0.21-0.80
	CDK5	8.0-21	DYRK4	9.5-80
	CDK7	>10	ERK2	>30
	CDK9	0.72	FYN	>250
	CK1	1.5-1.8	GSK-3β	0.35-10
	CK2	>40	LCK	>250
	CLK1	0.026-0.22	LYN	>250
	CLK2	0.12-0.28	MNB	0.15
	CLK3	3.0-10	PIM1	>10
	CLK4	0.024-0.050	PIM3	4.3
	DYRK1A	0.022-0.35	PKA	>250
	DYRK1B	0.028-0.26	PKC	>250

Table 2 . Ki Values Obtained for β-Carbolines Against a Panel of GABAA Subtypes.

 2

	Cd	Ki α1 (nM)	Ki α2 (nM)	Ki α3 (nM)	Ki α4 (nM)	Ki α5 (nM)	Ki α6 (nM)
	87b	2	7	72	1000-1300	44-68	4400
	87c	1	5	6	1000	27-110	2700-3100
	87e	0.7	15	19	1000-3000	110	>5000
	87g	2	16	21	-	1000	>3000
	87j	5	52	69	1000	590	>1000
	88b	5	30	49	-	480	>10000
	89a	0.6	3	5	-	10	1200
	89b	2	5	11	-	19	3000
	89c	0.1	1	2	-	4	480

Table 3 . Ki Values Obtained for β-Carbolines Against a Panel of 5-HT Receptor Subtypes.

 3

	Cd	1A Ki (µM)	1B Ki (µM)	1D Ki (µM)	2A Ki (µM)	2C Ki (µM)	3 Ki (µM)	5A Ki (µM)	6 Ki (µM)	7 Ki (µM)
	2	>10	>10	>10	5.0-7.8	9.4	>10	>10	1.5	5.5
	115a	>10	>10	>10	4.2-5.6	0.92	>10	>10	1.9	3.0
	115b	1.7-3.4	>10	>10	0.99-1.2	1.9	>10	0.85	1.5	0.28

Targeting AChE/BuChE and Aβ aggregation.

  Among MTDL, AChE/BuChE inhibitors represent the largest group, and it is naturally the case for β-carboline-including MTDL. Indeed, besides their catalytic activities, AChE and BuChE possess aggregation-promoting properties (Aβ fibrils and NFTs) involving their PAS. Therefore, in addition to their symptomatic effect, AChE/BuChE inhibitors targeting the PAS or dual-site inhibitors (acting on both CAS and PAS) could simultaneously decrease the rate of formation of Aβ fibrils and thus prevent the progression of AD. To evaluate this effect, Aβ anti-aggregation properties

	in a global higher affinity. Second, rutaecarpine was also derivatized with sulfonamido-linked
	groups. Compound 131 was especially obtained as a selective inhibitor of BuChE (IC50 = 3.6
	μM), able to interact with both the CAS and PAS of BuChE (mainly through hydrogen
	bonding with Thr120 and contacts with Trp82, Gly115, Gly116, Gly121, Pro285, Ser287 or
	have classically been evaluated through two procedures in order to discriminate between self-Tyr332), but unable to efficiently bind AChE (IC50 >100 µM), yielding a molecule with a
	induced Aβ and/or AChE-induced Aβ aggregation inhibition. Moreover, several similar scaffold than 130a-b but with reverse selectivity. 167 Besides its moderate self-induced
	multifunctional AChE/BuChE inhibitors with Aβ anti-aggregation properties feature Aβ aggregation inhibition properties (59% at 100 μM), 131 revealed suitable physicochemical
	additional activities (e.g., antioxidant, neuroprotective, metal chelation). Such a set of properties and antioxidant activity, along with a selective chelation of biometal ion Cu 2+ .
	properties has often been tested for newly synthesized MTDL. 165 Rutaecarpine, a natural Unsurprisingly, hybrids of tacrine (a fragment known to bind to the CAS of AChE) and β-
	product used in Chinese traditional medicine for diverse pharmacological activities, including carboline (an aromatic moiety potentially able to interact with the PAS) behave as mixed-type
	neuroprotection, was used as a template in two independent research developments. First, a ChE inhibitors (e.g., IC50 = 32 nM and 41 nM against AChE and BuChE for 132, IC50 = 22
	SAR analysis performed on a series of 3-aminoalkanamido-substituted 7,8-nM and 40 nM for 133), as suggested by molecular modelling, with good inhibition of Aβ
	dehydrorutaecarpine, inspired from previously reported compounds such as 33 or 34 (Figure aggregation (e.g., 41% and 66% of inhibition of self-induced Aβ1-42 aggregation at 20 μM for
	4), showed some requirements to enhance the multitarget character of the series. Pyridinium 132 and 133). 168 In addition to its ability to cross the BBB, compound 133 also displayed
	substituents especially promoted a dual inhibitory potency against AChE (e.g., IC50 = 0.8 nM good biometal-chelating abilities, a promising inhibition of Cu 2+ -induced Aβ aggregation, and
	and 0.6 nM for 130a and 130b, Figure 23) and Aβ aggregation (e.g., 91-93% of inhibition of moderate antioxidant properties. A similar biological profile was obtained from two distinct
	AChE-induced Aβ1-42 aggregation at 25 μM for 130a and 130b); in return the positive charge series of bivalent β-carbolines incorporating a hydrophilic spacer, designed for solubility
	seemed to compromise antioxidative activity. 166 However, compound 130b was associated purposes. In the first series, containing fully aromatic β-carboline dimers linked by nitrogen 9,
	with additional metal-chelating properties, of great interest in the frame of AD. Interestingly, compound 134 showed modest activity against AChE/BuChE with a slight selectivity for
	the potency of these compounds was found to be variable against BuChE. Hence, if some BuChE (IC50 = 16 μM and 1.8 μM) and good Aβ aggregation inhibition properties (72% at 25
	analogues were able to interact efficiently with both enzymes, some others were very μM). 169 In the second series, which gathers tetrahydro-β-carboline dimers connected through
	selective agents with an AChE vs. BuChE index up to ~3000 (e.g., 130a-b). These nitrogen 2, compound 135 exhibited very similar potency (IC50 = 18 μM and 1.7 μM against
	compounds seem to interact with the PAS of AChE, including hydrophobic contacts with AChE and BuChE, and 83% of inhibition of Aβ aggregation at 25 µM), and exerted extra
	Phe331, Phe330, Tyr334, Tyr121 or Phe290, but with a linker long enough for catching neuroprotective effect in several in vitro studies. But overall, the lack of stereocontrol and the
	additional cation-π interactions with Trp84 and Tyr130 at the CAS of the enzyme, resulting subsequent probable coexistence of several stereoisomers of 135 and derivatives in mixture

  flavin adenine dinucleotide; FDA, Food and Drug Administration; GABA, γaminobutyric acid; GSK-3β, glycogen synthase kinase 3; GTP, guanosine triphosphate; HDAC, histone deacetylase; NFTs, neurofibrillary tangles; mGluR, glutamate metabotropic receptor; NMDA, N-methyl-D-aspartate; NR, NMDA receptor subunits; MAO, monoamine oxidase; MAPK, mitogen-activated protein kinase; MTDL, multitarget-directed ligand; PAS, peripheral anionic site; PDB, Protein Data Bank; PDE, phosphodiesterase; ROS, reactive oxygen species; SAR, structure-activity relationship. hydrophilic side chain potential phosphodiesterase inhibitors. Sci. Pharm. 2016, 84, 428-446. (105) Beghyn, T. B.; Charton, J.; Leroux, F.; Henninot, A.; Reboule, I.; Cos, P.; Maes, L.; Deprez, B. Drug-to-genome-to-drug, step 2: reversing selectivity in a series of antiplasmodial compounds. J. Med. Chem. 2012, 55, 1274-1286. (106) Tamiz, A. P.; Whittemore, E. R.; Woodward, R. M.; Upasani, R. B.; Keana, J. F. W. Structure-activity relationship for a series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4b]indoles : potent subtype-selective inhibitors of N-methyl-D-aspartate (NMDA) receptors. Acta Neurobiol. Exp. 2004, 64, 89-98. (110) Di Fabio, R.; Micheli, F.; Alvaro, G.; Cavanni, P.; Donati, D.; Gagliardi, T.; Fontana, G.; Giovannini, R.; Maffeis, M.; Mingardi, A.; Tranquillini, M. E.; Vitulli, G. From pyrroles to 1-oxo-2,3,4,9-tetrahydro-1H-β-carbolines: a new class of orally bioavailable mGluR1 antagonists. Bioorg. Med. Chem. Lett. 2007, 17, 2254-2259.

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Acknowledgements

The authors are grateful to ANR ("Agence Nationale de la Recherche") for financial support through Labex ARCANE and CBH-EUR-GS (Grant ANR-17-EURE-0003). The authors thank the French MESR ("Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation") for the fellowship grant of A.B. and the company Chanel Parfums Beauté and the ANRT ("Association Nationale Recherche Technologie") for the CIFRE ("Conventions Industrielles de Formation par la Recherche") grant of A.G. (Grant 2017/0042).

prevented any further analysis. In addition, cytotoxicity experiments demonstrated that compounds 134 and 135 are neurotoxic at 10 μM. To address the latter issue, β-carbolinecinnamic acid hybrids were designed based on the previously reported anti-AD properties of cinnamic acid derivatives. Compound 136 inhibited BuChE selectively (IC50 = 21 μM vs. 1.3 μM against AChE and BuChE respectively), consistent with its predicted binding modes at BuChE active site, and with its significant inhibition of Aβ aggregation (72% at 20 μM). 170 Indeed, 136 could interlock in β-sheet, as observed in docking calculations. In addition, its profile includes neuroprotective effects and low neurotoxicity, and in vivo evaluations in AD model mice showed beneficial effects on several behavioral parameters such as restoration of learning and memory functions, without acute toxicity. of ortho-aminoanilide analogues only afforded marginally active molecules (IC50 >2.0 µM against all tested targets). 178 However, a further focus on the activities of all isolated stereoisomers would shed light on the real potential of these structures. Besides, a series of isolated stereoisomers of slightly different structure was reported very recently. 179 Interestingly, some compounds managed to efficiently bind to HDACs while incorporating short linkers, an observation that contradicts previously described results. Even a four-carbon derivative, compound 149a, was able to inhibit both PDE5 (IC50 = 12 nM) and HDAC (IC50 = 131 nM) with decent activity levels; shorter one-, two-and three-carbons analogues failed to efficiently bind HDAC, although their anti-PDE5 activities were exquisite (IC50 = 2.0-20 nM for four molecules). Compound 149b was even more promising (IC50 = 7.0 nM and 61 nM against PDE5 and HDAC, respectively), but anti-PDE5 activity started to drop as the anti-HDAC effect increased with the introduction of a sixth carbon (e.g., IC50 = 126 nM and 9.1 nM against PDE5 and HDAC, respectively, for compound 150). The last molecule also showed some HDAC subtype selectivity, as if HDAC6 and HDAC8 were targeted with IC50 values of 90-200 nM, HDAC1 was associated with far weaker activity (IC50 = 1.65 µM).

These results highlight the need for a balanced linker size and flexibility, as favorable elements for anti-PDE5 and anti-HDAC activities seem to be opposite.

Author information

Notes

The authors declare no competing financial interest.

Biographies

Aurélien Beato is a chemical engineer graduated from Sigma Clermont (France) in cGMP, cyclic guanosine monophosphate; ChE, cholinesterase; CK1/2, casein kinase 1/2; CLK1, dual specificity protein kinase; CNS, central nervous system; CREB, cAMP response element-binding; DYRK1A, dual specificity tyrosine-phosphorylation-regulated kinase 1A;