Contilisant-Belinostat Hybrids: Polyfunctionalized Indole Derivatives as Multineurotarget Drugs for the Potential Treatment of Alzheimer’s Disease

Abstract

In this work, we designed, synthesized, and evaluated two types of multineurotargeting compounds using a pharmacophore merging strategy, aiming to develop potential treatments for Alzheimer’s disease. We combined belinostat, an FDA-approved unselective histone deacetylase (HDAC) inhibitor, with the 5-substituted indole core of contilisant, known for its antioxidant and neuroprotective properties as well as its potent inhibitory activity against monoamine oxidases (MAOs), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Among these, compounds 8c (HDAC1, IC₅₀ = 0.019 μM; HDAC6, IC₅₀ = 0.040 μM; AChE, IC₅₀ = 20.06 μM; BChE, IC₅₀ = 17.10 μM; MAO-B, IC₅₀ = 2.14 μM), and 9c (HDAC1, IC₅₀ = 0.126 μM; HDAC6, IC₅₀ = 0.020 μM; AChE, IC₅₀ = 2.73 μM; BChE, IC₅₀ = 4.03 μM; MAO-B, IC₅₀ = 1.18 μM) emerged as the most promising candidates. These compounds warrant further investigation as potential treatments for Alzheimer’s disease due to their unique inhibition profiles and favorable mode of inhibition.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by the accumulation of amyloid-β (Aβ) peptides and tau, a microtubule-associated protein.¹ This neurodegenerative condition represents 60–80% of the approximately 50 million cases suffering from dementia globally.² Due to demographic changes, it is expected that the number of AD cases will at least double by 2050.² So far, the FDA has approved only four small-molecule drugs for treating AD: the cholinesterase inhibitors donepezil, galantamine, and rivastigmine, and the N-methyl-d-aspartate receptor antagonist memantine. Unfortunately, these drugs are only aimed at improving cognitive and memory impairment rather than acting as disease-modifying agents.³ Additionally, managing AD is difficult due to its complex nature, and there is growing evidence suggesting that targeting multiple disease-relevant pathways may be necessary to enhance therapeutic outcomes.⁴⁻⁶

The most straightforward way to modulate several disease-relevant pathways is the use of different drugs, referred to as combination therapy. Combination therapies exploit the additive or synergistic effects of two or more drugs and are considered essential standard therapies for several complex diseases, including HIV infection, cardiovascular diseases, and cancer.⁷⁻⁹ In contrast, multitarget drugs are single molecules that simultaneously modulate several specific targets.⁸⁻¹⁰ In this way, they combine the therapeutic advantages of polypharmacy (combination therapy) with those of single-molecule drug therapy: multitarget drugs provide a more predictable pharmacokinetic profile compared to drug combinations and reduce the risk of adverse effects caused by chemical or metabolic drug–drug interactions, resulting in simplified therapeutic regimens and better patient compliance.^8,9 In general, there are three pharmacophore combination strategies for the design of multitarget drugs, resulting in “linked”, “fused”, or “merged” pharmacophores”.¹¹ Notably, “merged pharmacophore” multitarget drugs require a certain overlap of the individual pharmacophores in terms of geometry and charge distribution. However, this strategy also allows us to design small, drug-like compounds with low molecular weight and favorable physicochemical properties.¹⁰

Histone deacetylases (HDACs) are enzymes that, among others, remove acetyl groups from histone and nonhistone proteins, including tau.^12,13 The HDAC family encompasses 11 zinc-dependent enzymes (HDACs 1–11) classified into four groups: class I (HDACs 1, 2, 3, and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6 and 10), and class IV (HDAC11).^12,14−16 HDACs are key players in several crucial cellular processes, including chromatin remodeling and gene expression.^12,13,16,17 In this way, HDACs are involved in neurogenesis, neurodevelopment, and the regulation of cognitive and memory-related processes.¹⁸ Accordingly, HDACs have been implicated in a wide range of human diseases, including neurodegenerative diseases such as AD.¹⁹⁻²² In the case of AD, the overexpression of certain HDAC isoforms (e.g., HDAC1 and HDAC6) is closely associated with Aβ deposition and, hence, with disease initiation and progression.^18,23−29 Consequently, HDACs are being investigated as promising therapeutic targets for AD.^5,30,31

A hallmark of AD neuropathology is the loss of cholinergic neurons and a deficit of the neurotransmitter acetylcholine in certain brain regions. Cholinesterase inhibitors increase the synaptic residence time of acetylcholine.³² When adopting the cholinergic hypothesis, acetylcholinesterase (AChE) inhibitors constitute a major treatment for AD. AChE has also been thought to enhance Aβ aggregation and neurotoxicity. Besides the selective retardation of AChE activity, dual inhibition of both AChE and butyrylcholinesterase (BChE) may be beneficial for the symptomatic treatment of AD. BChE inhibitors have been shown to improve cognitive and memory functions in mice, and BChE has been assumed to compensate for AChE in hydrolyzing acetylcholine, particularly in the late stages of AD.^32,33

Rising evidence suggests that the levels of neurotransmitters, other than acetylcholine, are also reduced throughout the stages of AD. In particular, lowered amounts of dopaminergic neurotransmitters are connected with AD pathophysiology.³⁴⁻³⁶ Monoamine oxidases (MAOs), flavin-dependent enzymes localized in the outer mitochondrial membrane, catalyze the oxidative deamination of multiple monoamines, including neurotransmitters such as dopamine and serotonin. The two isoforms of monoamine oxidase, MAO-B and MAO-A, differ in their tissue distribution and substrate and inhibitor preferences.³⁷ While MAO-B predominantly resides in the brain, in particular, in the basal ganglia, MAO-A is ubiquitously present in human tissues. In the course of the conversion of biogenic amines, MAOs produce hydrogen peroxide and further toxic metabolites that are linked to various neurological and psychological disorders.^38,39 In particular, MAO-B has been recognized to play a pivotal role in AD pathogenesis. During aging, the expression of MAO-B increases, which is connected with enhanced neurotransmitter degradation.^38,39 Inhibitors of MAO-B have been receiving increasing attention for their potential use in neurodegenerative disease therapy, owing to their influence on monoamine neurotransmitter metabolism, protection from oxidative stress, and additional neurorescuing effects.³⁵⁻⁴⁰ For example, the neuroprotective activities of MAO-B inhibitors, such as selegiline and safinamide, have frequently been considered beneficial for AD therapy and have inspired the development of compounds simultaneously addressing MAO-B and further AD targets.^{35,36,38,39,41,42}

Previously, we disclosed a series of hybrids of contilisant and the class IIb-selective HDAC inhibitor tubastatin A as new HDAC inhibitor-based multitarget small molecules with in vitro and in vivo activity in neurodegenerative diseases.⁴³ In this study, we conceptualized multineurotargeting compounds capable of interacting with the aforementioned biological targets (HDACs, AChE, BChE, and MAOs). Our molecular design of polyfunctionalized indole derivatives was inspired by the structure of contilisant, an advanced multitarget developmental compound for AD, and that of the HDAC inhibitor belinostat. We introduce novel hybrid compounds with an encouraging inhibitory profile against HDACs 1 and 6, AChE, BChE, and MAO-B.

Results and Discussion

Design and Synthesis

Most HDAC inhibitors follow a “cap”-“linker”-“zinc-binding group” (ZBG) pharmacophore model (see Figure 1, right). Usually, the cap group of HDAC inhibitors protrudes from the hydrophobic channel between the active site of the respective enzyme and its surface and is therefore amenable to diverse chemical modifications. Due to this high structural flexibility of the cap, the HDAC pharmacophore model is particularly suitable for the design of multitarget drugs.^13,44

Figure 1.

Design of multitarget ligands as potential inhibitors of HDACs, monoamine oxidases, and cholinesterases. Left: structure of contilisant. Right: structure of belinostat. Middle: merged pharmacophores yielding polyfunctionalized indole derivatives as potential multitarget drugs.

Target compounds of types I and II were designed based on a pharmacophore merging strategy. We combined belinostat, an FDA-approved unselective HDAC inhibitor, with the 5-substituted indole core of contilisant, known for its antioxidant and neuroprotective properties as well as its potent inhibitory activity against monoamine oxidases and cholinesterases (Figure 1, blue). In detail, the aniline cap of belinostat (Figure 1, black) was replaced by the indole core, yielding the meta-connected derivatives of type I. To increase structural diversity, we also designed para-connected derivatives (type II). Hydroxamate-based HDAC inhibitors without a benzyl or an aminophenyl linker in combination with a T-shaped cap group are usually unselective inhibitors. Thus, to achieve selective inhibition of class I HDAC isoforms such as HDAC1, we also designed compounds with an ortho-aminoanilide ZBG.

The envisaged compounds were synthesized, as summarized in Scheme 1. The commercially or readily available indole derivatives 1a–c were treated with 3-bromobenzenesulfonyl chloride or 4-bromobenzenesulfonyl chloride using sodium hydride as a base to afford the sulfonamides 2a–c and 3a,c. The subsequent Heck reaction of the respective sulfonamides provided the cinnamic acid ethyl esters 4a–c and 5a,c, which were hydrolyzed to key building blocks 6a–c and 7a,c. The desired hydroxamic acid derivatives 8a–c and 9a,c were obtained by an HATU-mediated amide coupling reaction of 6a–c and 7a,c with hydroxylamine. Similarly, HATU-mediated coupling reactions of carboxylic acid intermediates 6a–c and 7a,c with 1,2-phenylenediamine generated the ortho-aminoanilides 10a–c and 11a,c.

Scheme 1. Synthesis of Contilisant-Belinostat Hybrids.

Reagents and conditions: (i) NaH, 3- or 4-bromobenzenesulfonyl chloride (1.1 equiv), DMF, 0 °C to rt, 24 h; (ii) ethyl acrylate (1.2 equiv), triethylamine (1.7 equiv), triphenylphosphine (0.5 equiv), palladium acetate (0.5 equiv), sodium bicarbonate (1.0 equiv), DMF, 80 °C, 24 h; (iii) aq. KOH (4.0 equiv), THF/EtOH (50:50), rt, 4 h; (iv) (a) HATU (1.0 equiv), DIPEA (2.5 or 3.5 equiv), DMF, rt, 15 min, and (b) NH₂OH (1.0 equiv), rt; (v) (a) HATU (1.0 equiv), DIPEA (2.5 or 3.5 equiv), DMF, rt, 15 min, and (b) 1,2-phenylenediamine (1.0 equiv), rt.

Inhibition of HDAC1 and HDAC6

All synthesized compounds were first screened in a fluorogenic assay for their in vitro inhibitory activity against HDAC1 and HDAC6 using an established protocol^45,46 and ZMAL⁴⁷ (Z-Lys(Ac)-AMC) as the substrate. The results are presented in Table 1 and Figures S1 and S2. Compounds bearing a hydroxamic acid as ZBG displayed submicromolar inhibitory activity against HDAC1 and HDAC6. Interestingly, meta-connected derivatives of type I (8a–c) overall showed a slight preference for HDAC1, while the para-connected derivatives of type II (9a,c) were more potent at HDAC6 and showed reduced HDAC1 inhibition (e.g., compounds 8c vs 9c). As expected, compounds bearing an ortho-aminoanilide ZBG (compounds 10a–c and 11a,c) displayed no HDAC6 inhibition. Further, among the ortho-aminoanilide derivatives, only compounds 11a and 11c showed low micromolar activity at HDAC1 (11a: HDAC1, IC₅₀ = 1.24 μM; 11c: HDAC1, IC₅₀ = 2.32 μM). Overall, compound 8c (HDAC1, IC₅₀ = 0.019 μM; HDAC6, IC₅₀ = 0.040 μM) emerged as the most potent HDAC inhibitor of the set, closely followed by compounds 9a (HDAC1, IC₅₀ = 0.086 μM; HDAC6, IC₅₀ = 0.017 μM) and 9c (HDAC1, IC₅₀ = 0.126 μM; HDAC6, IC₅₀ = 0.020 μM).

Table 1. Target Activity of Contilisant-Belinostat Hybrids and Control Compounds against HDAC1, HDAC6, AChE, BChE, MAO-A, and MAO-B.

^aMean value ± standard deviation (SD) of at least two independent experiments.

^bPreincubation of enzyme and inhibitor: 1 h at 20 °C.

^cMean value ± standard error of one duplicate to sextuplicate experiment.

^dMean value ± SD of three independent experiments in duplicate.

^e<33% inhibition at the stated concentration.

^f<50% inhibition at the stated concentration; n.d.; not determined.

Docking Studies

Compounds 8c and 9c were selected for docking studies on HDAC1 and HDAC6. As described before, differences in the connectivity of the cap group may have an impact on the inhibition of the respective HDAC isoforms. The docking poses found for the meta-connected compound 8c suggest positioning of the indole-based cap group near the L1 loop for both HDAC1 (Figure 2a) and HDAC6 (Figure 2b). In contrast, the para-connected derivative 9c exhibits different binding modes depending on the isoform. In HDAC6, 9c interacts with the L1 loop (Figure 2d) similarly to 8c. However, in HDAC1, 9c does not engage with the L1 loop (Figure 2c) and instead approaches the L2 loop. This shift can be explained by the bulky amino acid D⁹⁹ within the L2 loop, hindering the cap group from addressing the L1 loop. In HDAC6, the L2 loop is formed by a less bulky gatekeeper serine residue (S⁵⁶⁸) at this position, enabling both regioisomers to interact with the L1 loop. This may be reflected by the decreased inhibitory activity of compound 9c toward HDAC1 compared to HDAC6.

Figure 2.

Predicted binding modes of compounds 8c (teal-colored) and 9c (coral red-colored) in HDAC1 (a and c, PDB ID: 5ICN) and HDAC6 (b and d, PDB ID: 5EDU). The protein is shown in top view with the L1 (green surface) and L2 loops (light orange-colored surface). The zinc ion appears as a purple-gray sphere.

Extended HDAC Isoform Profile of Contilisant-Belinostat Hybrids 8c and 9c

HDAC1 and HDAC6 have been identified as the most well-validated HDAC targets in AD. Meanwhile, additional studies highlight the neuropathological roles of the class I HDAC isoforms HDAC2 and HDAC3. Overexpression of HDAC2 is linked to cognitive decline in AD, as it impairs synaptic strength and plasticity, while HDAC3 overexpression increases amyloid burden.^48,49 Recognizing the pivotal roles of HDAC2 and HDAC3 in AD progression, we further evaluated the activity of compounds 8c and 9c against these isoforms to gain deeper insights into their HDAC inhibition profiles. The results are summarized in Table 2. Interestingly, compounds 8c and 9c showed more potent in vitro inhibition of HDAC3 compared to that of HDAC1 and HDAC2. This is particularly noteworthy as HDAC3 is the most abundantly expressed class I HDAC isoform in the adult brain and, as such, plays key roles in neurodevelopment, learning, and memory.^{50 −52}

Table 2. Extended HDAC Isoform Profiling of Compounds 8c and 9c.

	IC50 [μM]
Code	HDAC1[a,b]	HDAC2[a,b]	HDAC3[a,b]	HDAC6[a]
8c	0.019 ± 0.0002	0.044 ± 0.006	0.009 ± 0.001	0.040 ± 0.003
9c	0.126 ± 0.008	0.204 ± 0.011	0.053 ± 0.006	0.020 ± 0.0004
vorinostat	0.099 ± 0.011	0.149 ± 0.027	0.070 ± 0.006	0.029 ± 0.004

^aMean ± standard deviation (SD) of at least two independent experiments.

^bPreincubation of enzyme and inhibitor: 1 h at 20 °C.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase

Additionally, we monitored the activity of human AChE and human BChE by means of a photometric assay in the presence of 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) to detect thiocholine, which was released from the substrates acetylthiocholine and butyrylthiocholine, respectively. Noteworthy, only compounds that bear the 3-(piperidin-1-yl)propyl motif (8c, 9c, 10c, and 11c) inhibited cholinesterases (Table 1 and Figure S3). In the case of the hydroxamic acids, meta-substitution was somewhat disfavored over para-substitution (8c vs 9c). The ortho-aminoanilides 10c and 11c exhibited single-digit micromolar IC₅₀ values for both enzymes, irrespective of meta- or para-connection. Overall, our active compounds were slightly less effective than galantamine against AChE (Table 1). In contrast to galantamine, however, they acted as almost equipotent dual AChE/BChE inhibitors.

Similar to the active compounds 8c, 9c, 10c, and 11c, and several known AChE inhibitors,^36,53 the bicyclic ring system of donepezil is connected via five single bonds to the piperidine nitrogen. AChE possesses a deep and narrow gorge, consisting of two binding sites: the peripheral anionic site (PAS) and the catalytic anionic site. The selective AChE inhibitor donepezil interacts with both the PAS and catalytic sites, involving residues that span the length of the active site gorge. The indanone ring is located near W²⁸⁶, a component of the PAS, while the benzyl moiety is directed toward the active site. The piperidine ring of donepezil is engaged in water-mediated hydrogen bonds with midgorge amino acids.⁵⁴ A similar binding mode was expected for our cholinesterase inhibitors, which, however, do not discriminate between the two cholinesterases and also occupy the larger binding pocket of BChE.

Inhibition of Monoamine Oxidases A and B

Activities of human MAO-A and human MAO-B were quantified by a fluorescence-based end-point assay⁵⁵ following enzyme-catalyzed oxidative deamination of the substrate kynuramine to 3-(2-aminophenyl)-3-oxo-propionaldehyde, which then nonenzymatically cyclizes to 4-hydroxyquinoline and is deprotonated by the addition of NaOH to give the fluorescent product. Details regarding the establishment of the assay, including determination of suitable concentrations of enzymes (MAO-A: 1.5 μg mL^–1 and MAO-B: 24 μg mL^–1) and substrate (50 μM)⁵⁶ and characterization of the enzyme–substrate interaction (k_cat/K_M values of 25 000 M^–1 s^–1 (MAO-A) and 1300 M^–1 s^–1 (MAO-B)), are shown in Figures S5–S8 and Table S1. Inhibition of MAO-A and MAO-B was validated by clorgiline, selegiline, and safinamide (Table 1 and Figure S9), whose IC₅₀ values agreed well with reported inhibitory activities.⁵⁷⁻⁵⁹ A screening of the newly synthesized contilisant-belinostat hybrids of type I and type II at a concentration of 10 μM (Figure S10) showed selective inhibition (89%) of MAO-A over MAO-B by compound 11a, whereas compounds 8c, 9c, 10c, and 11c selectively reduced the activity of MAO-B by more than 80%. The inhibitor concentration of 10 μM was chosen to guarantee complete solubility of all investigated contilisant-belinostat hybrids (Figure S4). Detailed investigation of compound 11a, a para-connected derivative of type II bearing an ortho-aminoanilide ZBG and a 5-methoxy-1H-indole, on MAO-A provided an IC₅₀ value of 143 nM (Table 1 and Figure S11a). The respective meta-connected derivative of type I (10a) and derivatives of type II with a hydroxamic acid (9a) or a 5-[3-(piperidin-1-yl)propoxy]-1H-indole (11c) were much less active on MAO-A (IC₅₀ > 10 μM). MAO-B was selectively inhibited by those derivatives of type I (8c and 10c) and type II (9c and 11c) containing a 5-[3-(piperidin-1-yl)propoxy]-1H-indole (Table 1 and Figure S11b–e). Compounds bearing an ortho-aminoanilide ZBG were slightly superior to the respective hydroxamic acids (10c vs 8c and 11c vs 9c), whereas derivatives of type I and type II with the identical substitution pattern were almost equipotent (8c vs 9c and 10c vs 11c). In contrast to the irreversible MAO inhibitor contilisant bearing a propargyl group,⁵⁹ the contilisant-belinostat hybrids 8c and 9c lacking this substituent bind to MAO-B in a reversible manner. The IC₅₀ values of these two derivatives were not affected by increasing incubation periods (0, 30, and 60 min) of enzyme and inhibitor (Figures 3a and S12); reversibility of inhibition was further supported by rapid dilution experiments (Figure S13). Such reversible behavior toward MAO-B has also been observed for cinnamic acid anilides^60,61 and chalcone-derived⁶²⁻⁶⁵ compounds, which share structural similarity with the belinostat-derived moiety of compounds 8c and 9c.

Figure 3.

Inhibition of MAO-B (24 μg mL^–1) by compounds 8c (a, c) and 9c (a, d) and of MAO-A (1.5 μg mL^–1) by compound 11a (b) using the substrate kynuramine (a: 50 μM; b–d: 10–60 μM). Enzyme and inhibitor were incubated for 0–60 min (a) or 30 min (b–d) prior to substrate addition. Data shown are mean values ± SD from three duplicate experiments. (a) IC₅₀ values of compounds 8c and 9c (obtained as shown in Figure S12a,b, respectively) were separately subjected to one-way ANOVA with Tukey’s multiple comparison test, showing no statistically significant difference in the data obtained after different incubation periods (multiplicity adjusted p values of 0.158–0.947 and 0.325–0.789 for compounds 8c and 9c, respectively), thereby proving reversible enzyme–inhibitor interaction. (b–d) Michaelis–Menten plots of enzymatic rates for the determination of the dissociation constant of the compounds (K_i), and their MOI. Data were analyzed by nonlinear regression according to equations of linear noncompetitive inhibition (eq S3, α = 1 and β = 0; b,c) and competitive inhibition (eq S3, α = ∞ and β = 0; d), respectively (see also Supporting Information).

A detailed investigation into the mode of inhibition (MOI) and the dissociation constant of the enzyme–inhibitor complex (K_i) was conducted for MAO-A inhibitor 11a and MAO-B inhibitors 8c and 9c by varying both the substrate and the inhibitor concentrations. Data were initially analyzed by the linear transformations according to Lineweaver–Burk, Hanes–Woolf, Dixon, and Cornish-Bowden (Figures S14–S16), which pointed to both competitive and linear noncompetitive behavior of the compounds. However, an element of uncertainty remained, as less than 75 μM of kynuramine, i.e., [S] < K_M (Table S1), had to be used in these experiments due to a decrease of enzymatic activity at higher substrate concentrations (Figure S7). Although this effect can be described by an equation of substrate inhibition^66,67 (eq S2), it is rather caused by the substrate kynuramine at concentrations of 100 μM and above, absorbing light emitted by the product 4-hydroxyquinoline.^55,66,68

Analysis of the data by nonlinear regression according to eq S3 (Figure 3b–d) provided K_i values in the high nanomolar to low micromolar range and the most likely MOI. While compounds 8c and 11a were characterized as linear noncompetitive inhibitors, compound 9c showed competitive behavior toward the substrate kynuramine, as has been reported for the abovementioned cinnamic acid anilides⁶⁰ and chalcones/chalcone derivatives^63,65 on MAO-A and MAO-B.

Conclusion

In summary, we designed and synthesized a set of ten polyfunctionalized indole derivatives inspired by the structure of contilisant, an advanced multitarget development compound for AD, and the HDAC inhibitor belinostat, and investigated their structure–activity relationships. Overall, the meta-connected hydroxamic acid derivatives of type I (8a–c) showed a slight preference for HDAC1, while the para-connected derivatives of type II (9a,c) were more potent at HDAC6 and showed reduced HDAC1 inhibition (8c: HDAC1, IC₅₀ = 0.019 μM; HDAC6, IC₅₀ = 0.040 μM vs 9c: HDAC1, IC₅₀ = 0.126 μM; HDAC6, IC₅₀ = 0.020 μM). Interestingly, the meta-connected ortho-aminoanilide derivatives (10a–c) showed no HDAC inhibition at all, while the para-connected derivatives of type II (11a,c) emerged as moderate HDAC1 inhibitors. Further, only compounds bearing a 3-(piperidin-1-yl)propyl motif (8c, 9c, 10c, and 11c) were able to inhibit AChE and BChE. Interestingly, in contrast to galantamine, they acted as almost equipotent dual AChE/BChE inhibitors. Similarly, with IC₅₀ values ranging from 0.537 μM (10c) to 2.14 μM (8c), only compounds 8c, 9c, 10c, and 11c were identified as MAO-B inhibitors. Notably, the selected ZBG had a bigger impact on MAO-B inhibition than the compound type: while hydroxamic acid derivatives 8c and 9c showed somewhat less MAO-B inhibition than the ortho-aminoanilide derivatives 10c and 11c, there was no clear trend observable between the meta- and para-connected compound types. Additionally, compound 11a showed a unique inhibition profile within our compound set and emerged – based on our investigation – as a moderately potent dual HDAC1/MAO-A inhibitor (HDAC1, IC₅₀ = 1.24 μM; MAO-A, IC₅₀ = 0.143 μM). Both MAO-A and HDAC1 are overexpressed in prostate and lung cancer and correlate with the progression and prognosis of both cancers.⁶⁹⁻⁷⁵ Therefore, 11a could be a potential lead structure for the future development of multitarget drugs addressing prostate or lung cancer.

Further, selected compounds were investigated concerning their MOI on MAO-B (8c and 9c) and MAO-A (11a), respectively. Experiments revealed that compound 9c was characterized as a competitive inhibitor of MAO-B, while 8c acted as a linear noncompetitive inhibitor of MAO-B. Moreover, in contrast to the irreversible MAO inhibitor contilisant, 8c and 9c bind to MAO-B in a reversible manner, which is preferable in polypharmacology since the isolated, sustained inhibition of only one disease-relevant target can effectively result in a single-target ligand and therefore nullify the synergistic effects of multitarget drugs.

Based on our collective results, 8c and 9c emerged as the most promising multineurotarget drugs. Their unique inhibition profiles and favorable MOI compared to contilisant suggest that both compounds are suitable for further investigation as potential treatments for Alzheimer’s disease.

Experimental Section

For general information and chemistry, data sheets of newly synthesized compounds, experimental procedures concerning the in vitro characterization of final compounds on HDACs, cholinesterases and MAOs, and molecular docking, see the Experimental Section in Supporting Information.

Glossary

Abbreviations

AD

Alzheimer’s disease

AChE

acetylcholinesterase

BChE

butyrylcholinesterase

DIPEA

N,N-diisopropylethylamine

DMF

dimethylformamide

DTNB

5,5′-dithio-bis-2-nitrobenzoic acid

EtOH

ethanol

FDA

Food and Drug Administration

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexa-fluorophosphate

HDAC

histone deacetylase

HIV

human immunodeficiency virus

HPLC

high-performance liquid chromatography

IC₅₀

half maximal inhibitory concentration

IR

infrared

k_cat

turnover number

k_cat/K_M

specificity constant

K_i

dissociation constant of the enzyme–inhibitor complex

K_M

Michaelis constant

MAO-A

monoamine oxidase A

MAO-B

monoamine oxidase B

MOI

mode of inhibition

MS

mass spectrometry

NMR

nuclear magnetic resonance

PAS

peripheral anionic site

SD

standard deviation

THF

tetrahydrofuran

ZBG

zinc binding group

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00709.

• Figures and Tables; general information and chemistry; data sheets of newly synthesized compounds; experimental procedures concerning the in vitro characterization of final compounds on HDACs, cholinesterases, and MAOs, and molecular docking; NMR spectra (¹H and ¹³C), IR spectra, and MS spectra of newly synthesized compounds; HPLC traces of final compounds (PDF)

Author Contributions

^∇ L.S.H. and M.T.P. contributed equally to this work and share the first authorship. *M.P., M.G., J.M.C., and F.K.H. contributed equally to this work and share the senior and corresponding authorship. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

J.M.C. thanks AEI (Government of Spain; Grant PID2019–105813RB-C21) for support. S.E., V.K., and M.P. are grateful to the Faculty of Applied Natural Sciences, TH Köln – University of Applied Sciences, for financial support. The work of L.S.H., M.G., and F.K.H. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – GRK2873 (494832089).

The authors declare no competing financial interest.