Pharmacophore Modeling and 3D-QSAR Study of Indole and Isatin Derivatives As Antiamyloidogenic § Agents Targeting Alzheimer’S Disease

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Article Pharmacophore Modeling and 3D-QSAR Study of Indole and Isatin Derivatives as Antiamyloidogenic § Agents Targeting Alzheimer’s Disease

Rosa Purgatorio 1 , Nicola Gambacorta 1, Marco Catto 1,* , Modesto de Candia 1 , Leonardo Pisani 1 , Alba Espargaró 2, Raimon Sabaté 2 , Saverio Cellamare 1 , Orazio Nicolotti 1 and Cosimo D. Altomare 1

1 Department of Pharmacy-Pharmaceutical Sciences, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy; [email protected] (R.P.); [email protected] (N.G.); [email protected] (M.d.C.); [email protected] (L.P.); [email protected] (S.C.); [email protected] (O.N.); [email protected] (C.D.A.) 2 Institute of Nanoscience and Nanotechnology (IN2UB), Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Joan XXIII 27-31, E-08028 Barcelona, Spain; [email protected] (A.E.); [email protected] (R.S.) * Correspondence: [email protected]; Tel.: +39-080-544-2780 § This paper is dedicated to Prof. Francesco Campagna, who inspired and supervised this project before his retirement. Academic Editor: Derek J. McPhee Received: 15 November 2020; Accepted: 4 December 2020; Published: 7 December 2020

Abstract: Thirty-six novel indole-containing compounds, mainly 3-(2-phenylhydrazono) isatins and structurally related 1H-indole-3-carbaldehyde derivatives, were synthesized and assayed as inhibitors of beta amyloid (Aβ) aggregation, a hallmark of pathophysiology of Alzheimer’s disease. The newly synthesized molecules spanned their IC50 values from sub- to two-digit micromolar range, bearing further information into structure-activity relationships. Some of the new compounds showed interesting multitarget activity, by inhibiting monoamine oxidases A and B. A cell-based assay in tau overexpressing bacterial cells disclosed a promising additional activity of some derivatives against tau aggregation. The accumulated data of either about ninety published and thirty-six newly synthesized molecules were used to generate a pharmacophore hypothesis of antiamyloidogenic activity exerted in a wide range of potencies, satisfactorily discriminating the ‘active’ compounds from the ‘inactive’ (poorly active) ones. An atom-based 3D-QSAR model was also derived for about 80% of ‘active’ compounds, i.e., those achieving ﬁnite IC50 values lower than 100 µM. The 3D-QSAR model (encompassing 4 PLS factors), featuring acceptable predictive statistics either in the training 2 2 set (n = 45, q = 0.596) and in the external test set (n = 14, r ext = 0.695), usefully complemented the pharmacophore model by identifying the physicochemical features mainly correlated with the Aβ anti-aggregating potency of the indole and isatin derivatives studied herein.

Keywords: isatin hydrazones; indole derivatives; antiamyloid agents; multitarget-directed ligands; 3D-QSAR

1. Introduction Alzheimer’s disease (AD) represents a major cause of illness and death in the world, especially in developed countries. Epidemiological data for United States account for almost 6 million Americans (aged 65) currently hit by AD and 122,000 deaths in 2018 [1]. Most recent data for European Union, ≥ collected in the 2019 Yearbook of Alzheimer Europe organization, report about 9 million patients in

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2018, expected to increase to 10 and 16 million in 2025 and 2050, respectively, being Italy the country with the highest prevalence in population (>2%) [2]. Unfortunately, current therapeutic approaches for AD remain still limited to the symptomatic treatment of memory impairment by acetylcholinesterase (AChE) inhibitors, namely donepezil, rivastigmine and galantamine, and of glutamate excitotoxicity by memantine. The lack of disease -resolving therapies for AD still fuels the research in this field, despite poor results so far obtained in clinical trials that have discouraged pharmaceutical industries to pursuing the research for new drugs. There are about 120 trials currently registered in US, almost half of which are from drug repurposing, and most of which addressing disease-modifying targets and/or immunotherapy [3]. On the other hand, alternative hypotheses for the treatment of AD, considering the amyloid aggregation and toxicity, are still pursued mainly in academic settings. Despite many pitfalls registered in preclinical trials [4], there is still high interest in small molecules able to disrupt one or more steps in the amyloid cascade [5]. They may be resumed into a unitary approach known as amyloid hypothesis in AD, encompassing beta amyloid (Aβ) formation from APP precursor [6], Aβ aggregation in the amyloid cascade from toxic oligomers to senile plaques [7], tau protein aggregation [8], and tau hyperphosphorylation [9] as potential therapeutic targets. Considering such a number of pathophysiological events, the so-called multitarget approach, aiming at hitting two or more of AD-related targets with a unique chemical entity, has become the “new paradigm” of researchers in the field [10]. In this context, our research programs have long been focused on multitarget approach having the antiamyloidogenic activity as the central feature [11–14]. Particularly, since 2010 we investigated the chemical space around the isatin/indolinone molecular core [15–17], disclosing potent in vitro inhibitors of beta amyloid aggregation, acting at different stages of the fibrillogenesis [18,19]. Herein, we further proceeded with a ligand-based approach to design novel inhibitors, and evaluated the antiamyloidogenic activity of such new compounds, as well as their potential as multitarget agents for AD. In parallel, we exploited the data of published compounds from our previous screening campaigns to generate a reliable pharmacophore, complemented with a 3D-QSAR model, for Aβ aggregation inhibition by indole/indolone (mainly isatin hydrazones) derivatives. Starting from the hit compound 1 [16,17] (Figure1), the design strategy followed four main synthetic approaches aimed at (a) extending the exploration of the isatin-3-arylhydrazone scaffold; (b) simplifying the molecular structure to smaller isatin derivatives, possibly bearing N-substitution; (c) exploring bioisosteric indole derivatives, namely indole-3-carbaldehyde hydrazones; (d) investigating new indoline- and phthalimide-based analogues. Molecules 2020, 25, 5773 3 of 25

Molecules 2020, 25, 5773 3 of 26 Molecules 2020, 25, 5773 3 of 25

Figure 1. Ligand-based design of new inhibitors of amyloid aggregation. Figure 1.FigureLigand-based 1. Ligand-based design design of of newnew inhibitors inhibitors of amyloid of amyloid aggregation. aggregation.

2. Results 2.and Results Discussion and Discussion

2.1. Chemistry 2.1. Chemistry Hydrazones 2–4 were prepared by condensation of suitable isatin with hydrazine hydrate in Hydrazonesrefluxing 2methanol,–4 were whereas prepared arylhydrazones by condensation 5–9 were ofsynthesized suitable from isatin isatin with or N hydrazine-methylisatin hydrate in refluxingrefluxing methanol,with the corresponding whereas phenylhydrazines arylhydrazones at 5room–9 were temperature synthesized (Scheme 1). from The isatinN-alkylation or NN of-methylisatin-methylisatin 5- methoxyisatin was carried out through different methods giving the derivatives 10–15. The with the the corresponding corresponding phenylhydrazines phenylhydrazines at atroom room temperature temperature (Scheme (Scheme 1). 1The). The N-alkylationN-alkylation of 5- condensation of 10, 14 and 15 with 4-isopropylphenylhydrazine afforded compounds 16–18. of 5-methoxyisatin was carried out through different methods giving the derivatives 10–15. methoxyisatinSubsequently, was carried 16 and 17 outwere throughdemethylated different with BBr 3 methods[16] to 19 and giving 20, respectively the derivatives (Scheme 2). 10–15. The Thecondensation condensation of 10 of, 1014, 14andand 15 15withwith 4-isopropylphenylhyd 4-isopropylphenylhydrazinerazine afforded afforded compounds compounds 1616––1818.. R R R Subsequently, 1616 andand 1717 werewere demethylateddemethylated withwith BBrBBr33 [[16]16] toto 1919 andand1 20202 ,, respectivelyrespectively (Scheme(Scheme2 2).).

2 H H NH2

3 NO2 H NH2 R R1 R2 N R2 O N 4 H H ONH 2 H H N 2 R a R H O O N N 5 3H NOMe2 NH-PhH NH2 R1 R1 6 H H NH-Ph-3-OBnN R O N 2 7 4H H Me NH-Ph-3-OBnH O N R a R H 8 H H NH-Ph-4-OBn O O N N 9 5H HMe NH-Ph-4-OBnMe NH-Ph

R1 R1 6 H H NH-Ph-3-OBn Scheme 1. Synthesis of hydrazones 2–9. Reagents and conditions: a) NH2NH2•H2O, MeOH, reflux or

ArNHNH2, MeOH, room temperature. 7 H Me NH-Ph-3-OBn 8 H H NH-Ph-4-OBn

9 H Me NH-Ph-4-OBn

Scheme 1. Synthesis ofof hydrazoneshydrazones 22––99.. ReagentsReagents andand conditionsconditions:: (a)a) NHNH2NH2•HH2O,O, MeOH, MeOH, reflux reﬂux or 2 2• 2 ArNHNH2,, MeOH, MeOH, room room temperature.

Molecules 2020, 25, 5773 4 of 26 Molecules 2020, 25, 5773 4 of 25 Molecules 2020,, 2525,, 57735773 4 of 25

NH O O NNNH NHN NH O O N N NH O O OO OO HOHO O a a O b O c c HO a b OO OO OO O O O NN N N NN N N N H H R H RR RR R R

RR RR R R Bn 1010 BnBn 1616 BnBn 19 19Bn Bn

11 (CH2)2-Ph 17 cyclo-Pr 20 cyclo-Pr 1111 (CH(CH22))22-Ph-Ph 1717 cyclo-Prcyclo-Pr 20 20cyclo-Prcyclo-Pr

12 (CH(CH2))3-Ph-Ph 18 (CH(CH2))2-N(CH-N(CH3))2 1212 (CH22)33-Ph 1818 (CH2 22)2-N(CH3 23)2 CH -Morpholine 13 CH2-Morpholine 13 CH22-Morpholine 14 cyclo-Pr 1414 cyclo-Pr 15 (CH2)2-N(CH3)2 15 (CH2)2-N(CH3)2 15 (CH2)2-N(CH3)2

2 3 Scheme 2. Synthesis of compounds 10–20.. Reagents and conditions:: (a)(a) DMF,DMF, KK2CO3,, 2-chloro-2-chloro-N,,N- Scheme 2.2. Synthesis of compounds 10–20. Reagents and conditions: (a)(a) DMF,DMF, KK22COCO33,, 2-chloro-N,,N-- dimethylethylamine hydrochloride, rt,rt, oror formaldehyde,formaldehyde, morpholine,morpholine, ethanol,ethanol, reflux;reflux; (b)(b) MeOH,MeOH, 4-4- dimethylethylamine hydrochloride, hydrochloride, rt, rt, or or formaldehyde, formaldehyde, morpholine, morpholine, ethanol, ethanol, reflux; reﬂux; (b) (b) MeOH, MeOH, 4- 3 2 2 isopropylphenylhydrazine,isopropylphenylhydrazine, rt;rt; (c)(c) BBrBBr3,, CHCH2Cl2,, −70 °C. 4-isopropylphenylhydrazine,isopropylphenylhydrazine, rt; rt; (c) (c) BBr BBr3, CH, CH2ClCl2, −70, °C.70 C. 3 2 2 − ◦ The azo-coupling of aryldiazonium salts 22a–c with indolin-2-one 21 [20][20] resultedresulted inin thethe The azo-coupling of aryldiazonium salts 22a–c with indolin-2-one 21 [[20]20] resulted in thethe formation of arylhydrazones 23–25 (Scheme(Scheme 3).3). CompoundCompound 26 waswas synthesizedsynthesized byby thethe condensationcondensation formation of arylhydrazones 23–25 (Scheme3). Compound 26 was synthesized by the condensation of formationof 5-methoxyisatin of arylhydrazones with benzhydrazide 23–25 (Scheme as previously 3). Compound described 26 was[19] (Schemesynthesized 4), while by the the condensation indolin-3- 5-methoxyisatin with benzhydrazide as previously described [19] (Scheme4), while the indolin-3-one of one5-methoxyisatin 27 waswas obtainedobtained with fromfrom benzhydrazide indoxylindoxyl acetateacetate as andandpreviously 4-hydr4-hydroxybenzaldehyde described [19] (Scheme in acidic 4), conditions while the (Scheme indolin-3- 27 was obtained from indoxyl acetate and 4-hydroxybenzaldehyde in acidic conditions (Scheme5). one5). 27 was obtained from indoxyl acetate and 4-hydroxybenzaldehyde in acidic conditions (Scheme 5).

Ar NH Ar Ar NH2 c Ar NH c Ar 2 Ar c Ar 23 Ar NN N NH O Ar NN N Ar 23 a 22a - c NH O a Ar22a NN- c N O 24 O O O 24 N a b N 22ad - c N N b N d H 24 H O H O H O N N b 21 N d N 25 N H H H 25 N 21 25

Scheme 3. Synthesis of 23–25. Reagents and conditions: (a) NH2NH2•H2O, EtOH; (b) NaOH, EtOH, SchemeScheme 3. 3.Synthesis Synthesis of of23 23–25–25. .Reagents Reagents andand conditionsconditions:: (a) (a) NH NH22NHNH2•H2 H2O,2O, EtOH; EtOH; (b) (b) NaOH, NaOH, EtOH, EtOH, H2O, reflux; (c) NaNO2, H2O, HCl 6N, 0 °C; (d) MeOH, CH3COONa, 0• °C. H2HO,2O, reﬂux; reflux; (c) (c) NaNO NaNO2,H2, H22O,O, HCl 6N, 0 0 °C;◦C; (d) (d) MeOH, MeOH, CH CH3COONa,3COONa, 0 °C. 0 ◦ C. Scheme 3. Synthesis of 23–25. Reagents and conditions: (a) NH2NH2•H2O, EtOH; (b) NaOH, EtOH,

H2O, reflux; (c) NaNO2, H2O, HCl 6N, 0 °C; (d) MeOH, CH3COONa, 0 °C. O

NH O N NHO O O a O O O O N ONH N N O N a O N H H O O N 26 N H H SchemeScheme 4. 4.Synthesis Synthesis of of compound compound 2626... ReagentsReagents and and conditions conditions:: :(a)(a) (a) benzhydrazide,benzhydrazide, benzhydrazide, methanol,methanol, methanol, rt.rt. rt. 26 Scheme 4. Synthesis of compound 26. Reagents and conditions: (a) benzhydrazide, methanol, rt.

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O O Molecules 2020, 25, 5773 O O 5 of 26 Molecules 2020, 25, 5773 a 5 of 25 + Molecules 2020, 25, 5773 N O N 5 of 25 H O H O O OH O O O a O OH + a 27 N + N H N NH Scheme 5. SynthesisH of 27. ReagentsOH and conditions: (a) MeOH,H HCl conc., reflux, N2. OH OH The synthesis of indole-3-carbaldehyde hydrazones 28–36 27was accomplished,OH according to 27 previously reported procedures [19], by condensation of the R-substituted indole-3-carbaldehyde Scheme 5. Synthesis of 27. Reagents and conditions: (a) MeOH, HCl conc., reflux, N2. Scheme 5. Synthesis of 27. Reagents and conditions: (a) MeOH, HCl conc., reﬂux,2 N . with arylhydrazineScheme at 5. Synthesisroom oftemper 27. Reagentsature and conditions(Scheme: (a) MeOH,6). TheHCl conc.,reaction reflux, N . of 237 with 4- isopropylphenylhydrazine,TheThe synthesis synthesisThe synthesis of of indole-3-carbaldehyde indole-3-carbaldehydeof indole-3-carbaldehydein the same conditions, hydrazoneshydrazones afforded 282828––36–3636 was wasphthalimidewas accomplished, accomplished, accomplished, monohydrazone according according according to to 38 to previously(Schemepreviouslypreviously 7). reported reported reported procedures procedures procedures [19 [19],], [19], by by condensation by condensation condensation of theof thethe R-substituted R-substitutedR-substituted indole-3-carbaldehydeindole-3-carbaldehyde indole-3-carbaldehyde with arylhydrazinewith witharylhydrazine arylhydrazine at room temperatureat atroom room (Scheme tempertemperature6ature). The (Scheme(Scheme reaction 6). of6). 37TheThewith reactionreaction 4-isopropylphenylhydrazine, of of37 37with with 4- 4- isopropylphenylhydrazine, in the same conditions, afforded phthalimide monohydrazone 38 inisopropylphenylhydrazine, the same conditions, aﬀorded in phthalimidethe same conditions, monohydrazone afforded38 (SchemephthalimideR 7). Rmonohydrazone1 38 (Scheme(Scheme 7). 7). 28 H 4-iPr

R R 29 OCH31 4-CN R R1 28 H 4-iPr 30 H 3-Cl R1 28 H 4-iPr 29 OCH3 4-CN 31 OCH 3-Cl HN 29 OCH3 3 4-CN R 30 H 3-Cl N 1 CHO 3032 HH 3-Cl4-Cl R1 31 OCH3 3-Cl R a R HN N OCH CHO HN 323133H OCH34-Cl3 3-Cl4-Cl R R N a N N H CHO H 3332 OCHH 3 4-Cl4-Cl 34 OCH3 R N a R N N H H 3433 OCHOCH3 3 4-Cl N N N N OCH H H 35 3 N 34 OCH3 35 OCH3 N

N 3536 OCHOCH3 3 36 OCH3 N Cl N Cl

36 OCH Scheme 6. Synthesis of compounds 28–36. Reagents and conditions: (a) arylhydrazine3 HCl, MeOH or Scheme 6. Synthesis of compounds 28–36. Reagents and conditions: (a) (a) arylhydrazine N HCl, HCl,Cl MeOH or EtOH, rt. EtOH, rt. Scheme 6. Synthesis of compounds 28–36. Reagents and conditions: (a) arylhydrazine HCl, MeOH or EtOH, rt.

Scheme 7. Synthesis of compound 38. Reagents and conditions: (a) 4-isopropylphenylhydrazine, MeOH, rt. Scheme 7.7. Synthesis of compound 3838.. Reagents and conditions: (a) (a) 4-isopropylphenylhydrazine, Compounds 40 and 41 were synthesized from 39, prepared under Vilsmeier conditions [21], with MeOH, rt. Schemethe appropriate 7. Synthesis phenylhydrazine of compound in an38 .acid-catalyzed Reagents and reactionconditions (Scheme: (a) 4-isopropylphenylhydrazine, 8). Finally, the synthesis of MeOH,the indolinone rt. 43 was carried out under neat conditions starting from indoxyl acetate and Compounds 4040 andand 4141 werewere synthesized synthesized from from 39, 39prepared, prepared under under Vilsmeier Vilsmeier conditions conditions [21], with [21], with the appropriate phenylhydrazine in an acid-catalyzed reaction (Scheme8). Finally, the synthesis the appropriateCompounds phenylhydrazine 40 and 41 were synthesizedin an acid-catalyzed from 39, prepared reaction under (Scheme Vilsmeier 8). Fi nally,conditions the synthesis [21], with of of the indolinone 43 was carried out under neat conditions starting from indoxyl acetate and thethe indolinone appropriate 43 phenylhydrazine was carried out in anunder acid-catalyzed neat conditions reaction (Schemestarting 8).from Finally, indoxyl the synthesis acetate ofand dihydroxyquinolinedionethe indolinone 43 was 42carried[22] (Scheme out under9). Compounds neat conditions44 [23 starting] and 45 [from24] were indoxyl prepared acetate according and to the quoted references.

Molecules 2020,, 2525,, 57735773 6 of 25

Molecules 2020, 25, 5773 6 of 26 dihydroxyquinolinedione 42 [22][22] (Scheme(Scheme 9).9). CompoundsCompounds 44 [23][23] andand 45 [24][24] werewere preparedprepared according to the quoted references. R

R Cl Br COOH Cl Br COOH a Br O 40 H a O b Br N NH 40 H 41 iPr NH N 41 iPr H N H H HOOC 39

Scheme 8. Synthesis of 40 and 41. Reagents and conditions: (a) DMF, POCl3; (b) R-phenylhydrazine, Scheme 8. Synthesis of 40 and 4141.. Reagents and conditions: (a) (a) DMF, POClPOCl3;; (b) (b) R-phenylhydrazine, acetic acid, EtOH, 0 °C.◦C.

Scheme 9.9. Synthesis of 43.. ReagentsReagents and conditions :: (a)(a) (a) neat,neat, neat, 120120 120 °C.°C.◦C.

2.2. Inhibition of Aβ40 Aggregation 2.2. Inhibition of Aβ40 AggregationAggregation All the newly prepared compounds were tested in a routine in vitro assay of inhibition of Aβ40 All the newly prepared compounds were tested in a routine in vitro assay of inhibition of Aβ40 self-aggregation [25]. For the sake of clarity, compounds and related data are listed in four tables, self-aggregation [25]. For the sake of clarity, compounds and related data are listed in four tables, according to the design strategy above mentioned. Table1 collects all the compounds prepared with according to the design strategy above mentioned. Table 1 collects all the compounds prepared with the aim of extending the exploration of SAR around the arylhydrazone substituent. The most potent the aim of extending the exploration of SAR around thethe arylhydrazonearylhydrazone substituent.substituent. TheThe mostmost potentpotent compounds were found among the N-substituted analogues of 1, namely 18 and 19, while N-cyclopropyl compounds were found among the N-substituted analogues of 1,, namelynamely 18 andand 19,, whilewhile N- derivative 20 resulted poorly active. Naphthylhydrazones 23 and 24 were also found highly active, cyclopropyl derivative 20 resultedresulted poorlypoorly active.active. NaphthylhydrazonesNaphthylhydrazones 23 andand 24 werewere alsoalso foundfound while the quinoline derivative 25, isoster of 23, resulted less potent and the benzhydrazide 26 almost highly active, while the quinoline derivative 25,, isosterisoster ofof 23,, resultedresulted lessless potentpotent andand thethe inactive. The 5-substitution appeared to have contrasting eﬀects on potency; it is worthy of note benzhydrazide 26 almostalmost inactive.inactive. TheThe 5-substitution5-substitution appearedappeared toto havehave contrastingcontrasting effectseffects onon that 5-methoxy analogue of 5-hydroxy derivative 20, previously published [19], returned very higher potency; it is worthy of note that 5-methoxy analogue of 5-hydroxy derivative 20,, previouslypreviously potency than the latter, with IC50 in the submicromolar range. published [19], returned very higher potencypotency thanthan thethe latter,latter, withwith ICIC50 inin thethe submicromolarsubmicromolar range.range.

Table 1. Inhibition data of new isatin derivatives 18–20 andand 23–26..

Aβ40 AggregationAggregation InhibitionInhibition Comp. R R1 RR2 % at 100 μM IC50 ((μM)

18 5-OCH3 1.3 ± 0.1

19 5-OH 1.6 ± 0.3

20 5-OH 26 ± 3

23 H H 4.8 4.8 ±± 0.50.5

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Molecules 20202020,, 2525,, 57735773 66 ofof 2525 MoleculesdihydroxyquinolinedioneMoleculesMolecules 2020 2020, 202025,, 255773, 25, 5773, 5773 42 [22] (Scheme 9). Compounds 44 [23] and 45 [24] were prepared6 of6 25of6 of25 25 accordingdihydroxyquinolinedione to the quoted references. 42 [22][22] (Scheme(Scheme 9).9). CompoundsCompounds 44 [23][23] andand 45 [24][24] werewere preparedprepared dihydroxyquinolinedionedihydroxyquinolinedionedihydroxyquinolinedione 42 42 [22]42 [22] [22](Scheme (Scheme (Scheme 9). 9). Compounds9). Compounds Compounds 44 44 [23]44 [23] [23]and and and45 45 [24]45 [24] [24]were were were prepared prepared prepared according to the quoted references. R accordingaccordingaccording to tothe to the quotedthe quoted quoted references. references. references. R R R R R Cl Cl Br COOH Br a O b Br N NH 40 HR Cl R R R Cl ClCl Cl Br COOH Br Cl Cl Cl 41 iPr Br BrBr COOHNHCOOHCOOH aa Br BrBr N O bb Br Cl 4040 H a a a Br O O O b b Br Br N NN NHNH 40 40H40 H H H b Br H N NNHNH 41 iPr HOOC NH N 41 41i41Pr iPriPr NHNHNH N39N HN NN 41 iPr H HH N NH H H H H HOOCHOOC HOOCSchemeHOOC 8. Synthesis of 40 and 41. 3939Reagents and conditions: (a) DMF, POCl3; (b) R-phenylhydrazine, 39 3939 acetic acid, EtOH, 0 °C. Scheme 8. SynthesisSynthesis ofof 4040 andand 4141.. Reagents and conditions:: (a)(a) DMF,DMF, POClPOCl33;; (b)(b) R-phenylhydrazine,R-phenylhydrazine, SchemeSchemeScheme 8. Synthesis8. 8.Synthesis Synthesis of of40 of 40and 40 and and41 .41 Reagents 41. Reagents. Reagents and and conditionsand conditions conditions: (a): (a) :DMF, (a) DMF, DMF, POCl POCl POCl3; (b)3; 3(b) ;R-phenylhydrazine, (b) R-phenylhydrazine, R-phenylhydrazine, aceticacetic acid,acid, EtOH,EtOH, 00 °C.°C. aceticaceticacetic acid, acid, acid, EtOH, EtOH, EtOH, 0 °C. 0 °C.0 °C.

Scheme 9. Synthesis of 43. Reagents and conditions: (a) neat, 120 °C.

SchemeSchemeScheme 9. Synthesis 9.9. SynthesisSynthesisSynthesis of 43 ofofof. Reagents 434343... ReagentsReagents and conditionsandand conditionsconditions: (a) :neat,:: (a)(a)(a) neat,neat,neat, 120 °C. 120120120 °C.°C.°C. 2.2. Inhibition of Aβ40 SchemeAggregation 9. Synthesis of 43. Reagents and conditions: (a) neat, 120 °C.

2.2.All Inhibition the newly of A βprepared4040 AggregationAggregation compounds were tested in a routine in vitro assay of inhibition of Aβ40 2.2.2.2. Inhibition2.2. Inhibition Inhibition of Aof β ofA40 βA Aggregation40β 40Aggregation Aggregation self-aggregation [25]. For the sake of clarity, compounds and related data are listed in four tables, All the newly prepared compounds were tested in a routine in vitro assay of inhibition of Aβ4040 AllAll theAll the newlythe newly newly prepared prepared prepared compounds compounds compounds were were were tested tested tested in ina in routinea routinea routine in invitro in vitro vitro assay assay assay of ofinhibition of inhibition inhibition of ofA of βA40 βA 40β 40 accordingself-aggregationself-aggregation to the design [25].[25]. ForForstrategy thethe sakesake above ofof mentioned.clarity,clarity, compoundscompounds Table 1 collects andand relatedrelated all the datadata compounds areare listedlisted prepared inin fourfour tables,tables,with self-aggregationself-aggregationself-aggregation [25]. [25]. [25]. For For Forthe the sakethe sake sake of ofclarity, of clarity, clarity, compounds compounds compounds and and andrelated related related data data data are are listedare listed listed in infour in four four tables, tables, tables, theaccording aim of extending to the design the explorationstrategy above of SAR mentioned. around Tablethe arylhydrazone 1 collects all thesubstituent. compounds The prepared most potent with accordingaccordingaccording to tothe to the designthe design design strategy strategy strategy above above above mentioned. mentioned. mentioned. Table Table Table 1 collects 1 collects1 collects all all the all the compoundsthe compounds compounds prepared prepared prepared with with with compoundsthethe aimaim ofof extendingextendingwere found thethe amongexplorationexploration the Nofof-substituted SARSAR aroundaround analogues thethe arylhydrazonearylhydrazone of 1, namely substituent.substituent. 18 and TheThe 19 ,mostmost while potentpotent N- thethe aimthe aim aimof ofextending of extending extending the the explorationthe exploration exploration of ofSAR of SAR SAR around around around the the arylhydrazonethe arylhydrazone arylhydrazone substituent. substituent. substituent. The The Themost most most potent potent potent cyclopropylcompoundscompounds derivative werewere foundfound 20 resultedamongamong thethepoorly N-substituted-substituted active. Naphthylhydrazones analoguesanalogues ofof 1,, namelynamely 23 and 1824 andwereand 19 also,, whilewhile found N -- compoundscompoundscompounds were were were found found found among among among the the theN -substitutedN -substitutedN-substituted analogues analogues analogues of of 1of, 1namely , 1namely, namely 18 18 and18 and and19 ,19 while19, while, while N -N -N- highlycyclopropylcyclopropyl active, derivative derivativewhile the 20 quinoline resultedresulted poorlypoorlyderivative active.active. 25 Naphthylhydrazones,Naphthylhydrazones isoster of 23, resulted 23 andand less 24 potent werewere alsoalsoand found foundthe cyclopropylcyclopropylcyclopropyl derivative derivative derivative 20 20resulted 20 resulted resulted poorly poorly poorly active. active. active. Naphthylhydrazones Naphthylhydrazones Naphthylhydrazones 23 23and 23 and and24 24were 24 were were also also alsofound found found benzhydrazidehighly active, 26while almost the inactive.quinoline The derivative 5-substitution 25,, isosterisoster appeared ofof 23 to,, resultedresultedhave contrasting lessless potentpotent effects andand onthethe highlyhighlyhighly active, active, active, while while while the the thequinoline quinoline quinoline derivative derivative derivative 25 ,25 25isoster, ,isoster isoster of of 23of ,23 23resulted, ,resulted resulted less less lesspotent potent potent and and andthe the the potency;benzhydrazide it is worthy 26 almostalmost of note inactive.inactive. that 5- TheThemethoxy 5-substitution5-substitution analogue appearedappearedof 5-hydroxy toto have havederivative contrastingcontrasting 20, previously effectseffects onon benzhydrazidebenzhydrazidebenzhydrazide 26 26 almost26 almost almost inactive. inactive. inactive. The The The5-substitution 5-substitution 5-substitution appeared appeared appeared to to haveto have have contrasting contrasting contrasting effects effects effects on on on publishedpotency; [19],it is returnedworthy veryof note higher that potency 5-methoxy than analoguethe latter, withof 5-hydroxy IC50 in the derivative submicromolar 20,, previouslypreviously range. potency;potency;potency; it itis it isworthy isworthy worthy of of noteof note note that that that5- methoxy5- 5-methoxymethoxy analogue analogue analogue of of 5-hydroxyof 5-hydroxy 5-hydroxy derivative derivative derivative 20 ,20 previously20, previously, previously Moleculespublished2020, 25 [19],, 5773 returned very higher potencypotency thanthan thethe latter,latter, withwith ICIC5050 inin thethe submicromolarsubmicromolar7 range.range. of 26 publishedpublishedpublished [19], [19], [19], returned returned returnedTable very very 1. very higherInhibition higher higher potency potencydata potency of than ne thanw than theisatin the latter,the derivativeslatter, latter, with with with IC 18 IC50– 20ICin50 50inandthe in the submicromolar23the –submicromolar26 submicromolar. range. range. range.

TableTable 1.1. InhibitionInhibitionInhibition datadatadata ofofof neneneww isatinisatin derivativesderivatives 181818–––202020 andandand 232323–––262626... TableTableTable 1. InhibitionInhibition1. Inhibition data data data of of neof new wne w isatin isatin derivatives derivatives 18 –1820– 20and and 23 –2326–.26 .

Aβ40 Aggregation Inhibition Comp. R R1 R2 Aβ40% Aggregationat 100 μM InhibitionIC50 (μM) Comp. R R1 R2 Aβ4040 AggregationAggregation InhibitionInhibition 40Aβ40 Aggregation Inhibition Comp. R R11 RR22 AβAβ Aggregation40 Aggregation Inhibition Inhibition Comp.Comp. R R R1 R1 R2 R2 % at 100 µM IC50 (µM) Comp.18 5-OCHR 3 R1 R2 % at 100 μM 1.3IC 50±50 0.1((μM) % at% %100at at100 μ 100M μ MμM IC50IC (IC50μ M)(50μ (M) μM) 1818 18 5-OCH5-OCH33 1.3 0.11.31.3 ±± 0.10.1 18 1818 5-OCH5-OCH5-OCH3 3 3 ±1.31.3 ± 1.30.1 ± 0.1 ± 0.1 19 5-OH 1.6 ± 0.3

19 191919 19 5-OH5-OH5-OH5-OH 1.6 1.60.3 ±1.61.6 1.60.3 ±± ± 0.30.30.3 19 5-OH ± 1.6 ± 0.3 20 5-OH 26 ± 3

2020 5-OH5-OH 2626 ±± 33 20 2020 20 5-OH5-OH5-OH 2626 26±3 326 ± 3± 3 ±

23 H H 4.8 ± 0.5

2323 H H 4.8 4.8 ±± 0.50.5 23 2323 23 H H HHH H H H 4.8 4.80.5 4.8 ± 4.80.5 ± 0.5 ± 0.5 Molecules 20202020,, 2525,, 57735773 ± 77 ofof 2525 Molecules 2020, 25, 5773 7 of 25

24 24 H HHH 10 2 10 10 ±± 22 ±

25 25 H HHH 63 635 ± 5 ±

26 26 5-OCH5-OCH33 HHH 12 125 ± 5 3 ±

1 1 0.43 a 0.43 aa QuercetinQuercetin 0.82 a 0.82 aa aa DataData takentaken fromfrom [17].[17]. ValuesValues areare meanmean ±± SEMSEM ((n == 3).3). a Data taken from [17]. Values are mean SEM (n = 3). ± The molecular simplification around the isatin core led to compounds listed in Table 2. In all The molecular simpliﬁcation around the isatin core led to compounds listed in Table2. In all cases, activities were quite far from reference compound 1,, particularlyparticularly forfor unsubstitutedunsubstituted hydrazoneshydrazones cases, activities were quite far from reference compound 1, particularly for unsubstituted hydrazones 2 2 andand 3.. AlsoAlso inin thisthis series,series, N-substituted-substituted derivativesderivatives ofof isatinisatin scorscored the best results, with potencies and 3. Also in this series, N-substituted derivatives of isatin scored the best results, with potencies increasingincreasing fromfrom benzylbenzyl toto phphenethyl and 3-phenylpropyl (10 << 11,, 12).). Disappointingly,Disappointingly, thethe increasing from benzyl to phenethyl and 3-phenylpropyl (10 < 11, 12). Disappointingly, the morpholine morpholine derivative 13,, preparedprepared asas thethe MannichMannich basebase ofof 5-5-methoxyisatin, proved to be a weak derivative 13, prepared as the Mannich base of 5-methoxyisatin, proved to be a weak inhibitor of inhibitorinhibitor ofof aggregation,aggregation, asas wellwell asas thethe cyclopropylcyclopropyl derivativederivative 14.. TheThe substitutionsubstitution atat thethe positionposition 22 ofof aggregation, as well as the cyclopropyl derivative 14. The substitution at the position 2 of isatin, leading isatin,isatin, leadingleading toto condensationcondensation productsproducts 27 andand 44,, aimedaimed atat probingprobing stericsteric effectseffects inin thisthis position.position. to condensation products 27 and 44, aimed at probing steric eﬀects in this position. Both compounds Both compounds retained only a weak activity against Aβ aggregation.aggregation. retained only a weak activity against Aβ aggregation.

Table 2. InhibitionInhibition datadata ofof nenew isatin derivatives 2,, 3,, 10–14,, 27 andand 44..

Aβ4040 AggregationAggregation InhibitionInhibition Code R R11 X Y % at 100 μM IC5050 ((μM) 2 H H N-NH22 O O 17 17 ±± 22 3 5-NO22 HH N-NHN-NH22 O O 11 11 ±± 33

10 5-OCH33 O O 52 ± 1

11 5-OCH33 O O 15 ± 1

12 5-OCH33 O O 19 ± 4

13 5-OCH33 O O 23 ± 5

14 5-OCH33 O O 38 ± 5

27 H H O 69 ± 1

44 H H O 44 ± 1

Molecules 2020, 25, 5773 7 of 25 MoleculesMoleculesMoleculesMoleculesMoleculesMolecules 20202020 2020 20202020 2020,, 25,25 ,,25 2525,, 5773,577325 ,,5773 57735773, 5773 777 of7of7 of of 7of 2525 25of 2525 25

24 H H 10 ± 2 Molecules242424 20202424 24 , 25, 5773 HHHH H H HHHH H H 10 10 10 10 10 7±± 10 ± of ±2±2 2 ±2252 2

24 H H 10 ± 2 25 H H 63 ± 5 2525252525 25 HHHH H H HHHH H H 6363636363 ±± 63± ±5±5 5 ±55 5

25 H H 63 ± 5

26 5-OCH3 H 12 ± 5 2626262626 26 5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH33 33 3 3 HHHH H H 1212121212 ±± 12± ±5±5 5 ±55 5

26 5-OCH3 H 12 ± 5 1 0.43 a 111 11 1 0.430.430.430.430.430.43 aa a a a a Quercetin 0.82 a QuercetinQuercetinQuercetinQuercetinQuercetin 0.820.820.820.820.82 aa a a a a Quercetin1 0.43 a 0.82 a Data taken from [17]. Values are mean ± SEM (n = 3). aa aDataDataa aQuercetinData DataDataa Data takentaken taken takentaken taken fromfrom from fromfrom from [17].[17]. [17]. [17].[17]. [17]. ValuesValues Values ValuesValues Values areare are areare are meanmean mean meanmean mean ±± ± ±SEM±SEM SEM ±SEMSEM SEM (( nn( (n( n=n = (= n=3).=3). 3). =3).3). 3). 0.82 a a Data taken from [17]. Values are mean ± SEM (n = 3). TheTheTheTheTheThe molecular molecular molecular molecularmolecular molecular simplification simplification simplification simplificationsimplification simplification around around around aroundaround around the the the thethe the isatin isatin isatin isatinisatin isatin core core core corecore core led led led ledled led to to to toto compounds compoundsto compounds compoundscompounds compounds listed listed listed listedlisted listed in in in inin Table Tablein Table TableTable Table 2. 2. 2. 2. 2. In In2. In InIn all Inall all allall all cases,cases,cases,cases,cases,cases, activitiesactivities activitiesThe activitiesactivities activities molecular werewere were werewere were simplificationquitequite quite quitequite quite farfar far farfar far fromfrom from fromfrom from around referencereference reference referencereference reference the isatincompoundcompound compound compoundcompound compound core led 11 1 , ,1 1 ,toparticularlyparticularly ,,1particularly particularly particularly,compounds particularly forfor for forlistedfor for unsubstitutedunsubstituted unsubstituted unsubstitutedunsubstituted unsubstituted in Table 2. Inhydrazoneshydrazones hydrazones hydrazones hydrazonesall hydrazones 222 2and2and andcases, 2andand and 33 3 . .3 3 .activitiesAlso Also ..3 Also AlsoAlso. Also inin in inin this this inwere this thisthis this series,series, series,quite series,series, series, far NN N NN-substituted -substitutedfrom -substitutedN-substituted-substituted-substituted reference derivativesderivatives derivatives compoundderivativesderivatives derivatives 1ofof of, ofofparticularly isatinisatin of isatin isatinisatin isatin scorscor scor scorscor scor edforedededed edtheunsubstitutedthe the thethe the bestbest best bestbest best results,results, results, results,results, results, hydrazones withwith with withwith with potenciespotencies potencies potenciespotencies potencies increasingincreasingincreasingincreasingincreasing2increasing and 3. Also from from fromfrom from fromin this benzyl benzyl benzylbenzyl benzyl benzylseries, to to to Nto to to-substitutedph ph phph phenethylenethylphenethylenethylenethylenethyl derivativesand and andand andand 3-phenylpropyl 3-phenylpropyl 3-phenylpropyl3-phenylpropyl 3-phenylpropyl3-phenylpropyl of isatin scor ed ( ( 10(10(10 (the1010( 10 < < < best < < 11 11 <1111 11, , results, 11, ,,12 12 12,12 12).).12). ).). Disappointingly, Disappointingly,). Disappointingly,withDisappointingly, Disappointingly,Disappointingly, potencies the the thethe thethe increasing from benzyl to phenethyl and 3-phenylpropyl (10 < 11, 12). Disappointingly, the morpholinemorpholinemorpholinemorpholinemorpholinemorpholine derivative derivative derivative derivativederivative derivative 13 13 13 1313 ,, 13,prepared ,prepared, prepared prepared,prepared prepared as as as asas the asthe the thethe the Mannich Mannich Mannich MannichMannich Mannich base base base basebase base of of of ofof 5- 5-of 5- 5-5-methoxyisatin,methoxyisatin, methoxyisatin,5-methoxyisatin,methoxyisatin,methoxyisatin, proved proved proved provedproved proved to to to toto be beto be bebe bea a a aaweak weak weak aweakweak weak morpholine derivative 13, prepared as the Mannich base of 5-methoxyisatin, proved to be a weak inhibitorinhibitorinhibitorinhibitorinhibitorinhibitor ofof of ofof aggregation, aggregation, ofaggregation, aggregation,aggregation, aggregation, asas as asas wellwell aswell wellwell well asas as asas thethe asthe thethe the cyclopropylcyclopropyl cyclopropyl cyclopropylcyclopropyl cyclopropyl derivativederivative derivative derivativederivative derivative 1414 14 1414.. 14.TheThe ..The TheThe. The substitutionsubstitution substitution substitutionsubstitution substitution atat at atat the the atthe thethe the positionposition position positionposition position 22 2 2of2of of 2ofof of isatin,inhibitor leading of aggregation, to condensation as well asproducts the cyclopropyl 27 and derivative44, aimed 14 at. The probing substitution steric at effects the position in this 2 ofposition. isatin,isatin,isatin,isatin,isatin,isatin,isatin, leading leading leading leadingleadingleading leading to to to tototo condensation condensationto condensation condensationcondensation condensation productsproducts products products productsproducts products 27 27 27 27 and2727 and27 and and and and 44and ,44 44 44 aimed4444, , 44 ,aimed ,aimed, aimed aimed,aimed aimed at probing at at at atat probing probingat probing probingprobing probing steric steric steric steric effectsstericsteric steric effects effects effects ineffectseffects effects this in inposition. in inin this thisin this thisthis this position. position. position. position.position. position. MoleculesBoth compounds2020, 25, 5773 retained only a weak activity against Aβ aggregation. 8 of 26 BothBothBothBothBothBothBoth compoundscompounds compounds compounds compoundscompounds compounds retainedretained retained retainedretained retained onlyonly only onlyonly only a aa a weak aweakaweak weak aweakweak weak activity activityactivity activity activityactivity activity against againstagainst against againstagainst against Aβ A A aggregation.A AAββ βA β βaggregation.aggregation. aggregation. β aggregation.aggregation. aggregation.

Table 2. Inhibition data of new isatin derivatives 2, 3, 10–14, 27 and 44. TableTableTableTableTableTableTable 2. Inhibition2.2. 2. 2. 2. Inhibition Inhibition2. InhibitionInhibition Inhibition data data datadata data datadata of dataof newofof neof ofof ne ne wofne nene isatinwwisatin neww w isatinisatin wisatin isatinisatin derivativesisatin derivativesderivatives derivatives derivativesderivatives derivatives 2,2 3,,3 2102 ,2 , ,2 102 ,–33 ,,1432 , ,3 –3 ,1010 ,14,103 102710,–– ,–101414 –27–and141414–,, ,1427and27 ,, 27 442727, andand 27.and 44andand and . 4444 44 4444.. .44 .. .

Aβ40 AggregationAβ40 Aggregation Inhibition Inhibition 1 Aβ40 Aggregation Inhibition Code RR R X XYY A4040β40404040 Aggregation Inhibition CodeCode R R11 X Y % atAA A100AβββAβ μAggregationβAggregation Aggregation MAggregation AggregationIC50 (μ InhibitionM)Inhibition Inhibition Inhibition Inhibition CodeCodeCodeCodeCodeCode RRR R R R RRR1R1R 11 R1 1 XXX X X X YYY Y Y Y % at 100 μµM IC IC50 50(μ(M)µM) 2 H H N-NH2 O 17%% %±%% atat2 %at at at 100100 at100 100100 100 μμ μ MμMμM MμM M ICICICICIC5050IC50 50 50(( μ(μ 50 (μ(M)μM)μ M)(M)μM) M) 2 2 HH HH N-NH N-NH2 OO 17 ± 2 222 232 2 5-NOHHHH H H 2 HHHH H H H N-NHN-NHN-NHN-NHN-NHN-NHN-NH2 222 22 2 2 O O O O O O O 11 ± 3 1717 17 17 17 ±± 17± ±2±2 2 ±22 2 3 3 5-NO5-NO2 HH N-NH N-NH2 OO 11 ± 3 333 33 3 5-NO5-NO5-NO5-NO5-NO5-NO222 22 2 2 HHHH H H N-NHN-NHN-NHN-NHN-NHN-NH222 22 2 2 O O O O O O 11 11 11 11 11 ±± 11± ±3±3 3 ±33 3 10 5-OCH3 O O 52 ± 1 10 10 5-OCH5-OCH3 OO O O 52 ± 1 1010101010 10 5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH333 33 3 3 OOOO O O O O O O O O 52 52 52 52 52 ±± 52± ±1±1 1 ±11 1 ±

11 5-OCH3 O O 15 ± 1

11 5-OCH3 O O 15 ± 1 11 1111111111 11 5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH33 33 3 3 OOOOO O O O O O O O O 15 15 15 15 15 ±± 15± ±1±1 1 ±11 1 3 ± 12 5-OCH3 O O 19 ± 4

12 5-OCH3 O O 19 ± 4 12 33333 3 12 12121212 12 5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH3 OOOOO O O O O O O O O 19 19 19 19 19 ±± 19± ±4±4 4 ±44 4 13 5-OCH3 O O 23 ± 5 ±

13 5-OCH3 O O 23 ± 5 13 1313131313 13 5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH33 33 3 3 OOOOO O O O O O O O O 23 23 23 23 23 ±± 23± ±5±5 5 ±55 5 3 14 5-OCH3 O O 38 ± 5 ±

14 5-OCH3 O O 38 ± 5 14 1414141414 14 5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH5-OCH333 33 3 3 OOOOO O O O O O O O O 38 38 38 38 38 ±± 38± ±5±5 5 ±55 5 27 H H O 69 ± 1 ±

27 H H O 69 ± 1 27 2727272727 27 HHOHHHH H H HHHH H H OOOO O O 6969696969 ±± 69± ±1±1 1 ±11 1

44 H H O 44 ± 1 ±

44 H H O 44 ± 1 44 4444444444 44 HHOHHHH H H HHHH H H OOOO O O 4444444444 ±± 44± ±1±1 1 ±11 1 ±

A major objective of this work was to investigate the isosteric variation of hydrazone derivatization from isatin to indole, afforded by condensing indole-3-carbaldehyde with selected arylhydrazines. The Aβ aggregation data in Table3 account for a satisfactory activity profile, partially associated to the favorable effects of 5-methoxy substituent, and likely depending upon the hydrophobic feature of the arylhydrazine moiety. Both isopropyl and chlorine substituents warranted good potencies compared to nitrile, the 4-chloro isomer 32 being the most potent of the molecular subset. As a confirmation of the favorable hydrophobic effects, quinoline derivatives 35 and 36 were much more potent than pyridine congener 34. As already noticed for compound 26, benzhydrazide 45 lost any antiaggregating activity. Molecules 20202020,,, 252525,,, 577357735773 88 ofof 2525 Molecules 2020, 25, 5773 8 of 25 Molecules 2020,, 2525,, 57735773 8 of 25 A major objective of this work was to investigate the isosteric variation of hydrazone MoleculesA major 2020, 25objective, 5773 of this work was to investigatestigate thethe isostericisosteric variationvariation ofof hydrazonehydrazone8 of 25 derivatizationMoleculesA major 2020, 25 from,objective 5773 isatin of to thisindole, work afforded was toby invecondensingstigate the indole-3-carbaldehyde isosteric variation withof hydrazone selected8 of 25 derivatizationA major fromobjective isatin of to this indole, work afforded was to by inve condensingstigate the indole-3-carbaldehyde isosteric variation ofwith hydrazone selected arylhydrazines.derivatizationA major fromTheobjective A isatinβ aggregationaggregation of to this indole, work datadata afforded was inin TableTable to by inve 33 accountaccountcondensingstigate for forthe aa indole-3-carbaldehydesatisfactorysatisfactoryisosteric variation activityactivity profile,ofprofile, hydrazonewith partiallypartially selected derivatizationA major from objective isatin ofto thisindole, work afforded was to by inve condensingstigate the indole-3-carbaldehyde isosteric variation of withhydrazone selected associatedarylhydrazines.derivatization to the fromThe favorable A isatinβ aggregation to effectsindole, data ofafforded 5-methoxy in Table by 3condensing account substituent, for indole-3-carbaldehyde a satisfactoryand likely activity depending withprofile, selectedupon partially the associatedarylhydrazines.derivatization to the Thefrom favorable A isatinβ aggregationaggregation to effectsindole, datadata ofafforded in5-methoxyin TableTable by 33 condensing accountaccount substituent, forfor indole-3-carbaldehyde aa satisfactorysatisfactory and likely activityactivity depending profile,profile,with selectedupon partiallypartially the hydrophobicassociatedarylhydrazines. to featurethe The favorable Aofβ aggregationthe effectsarylhydrazine data of in5-methoxy Table moiety 3 account ..substituent, BothBoth for aisopropylisopropyl satisfactory and likelyandand activity chlorinechlorinedepending profile, substituentssubstituents partially upon the hydrophobicassociatedarylhydrazines. to featurethe The favorable Aofβ aggregationthe effectsarylhydrazine dataof 5-methoxyin Table moiety 3 account substituent,. Both for aisopropyl satisfactory and likely and activity dependingchlorine profile, substituents partiallyupon the warrantedhydrophobicassociated good to feature thepotencies favorable of comparedthe effectsarylhydrazine toof nitrile,5-methoxy moietythe 4-chlorosubstituent,. Both isomer isopropyl and 32 likely being and depending the chlorine most potent uponsubstituents ofthe the warrantedhydrophobicassociated good tofeature thepotencies favorable of the compared effectsarylhydrazine toof nitrile,5-methoxy moiety the 4-chlorosubstituent,.. BothBoth isomerisopropylisopropyl and 32 likely beingbeing andand depending thethechlorinechlorine mostmost potentpotentsubstituentssubstituentsupon oftheof thethe molecularwarrantedhydrophobic subset. good feature potenciesAs a confirmationof thecompared arylhydrazine of to the nitrile, favorable moiety the 4-chloro. hydrophobicBoth isopropylisomer effects, 32 andbeing quinolinechlorine the most derivativessubstituents potent of the35 molecularwarrantedhydrophobic subset.good feature potencies As a confirmationof comparedthe arylhydrazine ofto thenitrile, favorable moiety the 4-chloro. hydrophobicBoth isomerisopropyl effects,32 beingbeingand quinolinechlorine thethe mostmost substituentsderivatives potentpotent ofof the the 35 andmolecularwarranted 36 were subset. goodmuch potencies Asmore a confirmation potent compared than oftopyridine thenitrile, favorable thecongener 4-chloro hydrophobic 34 isomer. As already 32 effects, being noticed thequinoline most for potentcompound derivatives of the 26 35, andmolecularwarranted 36 werewere subset. goodmuchmuch Aspotencies moremore a confirmation potentpotent compared thanthan of topyridinepyridine the nitrile, favorable thecongenercongener 4-chloro hydrophobic 34 isomer.. AsAs alreadyalready 32 effects, being noticednoticed quinolinethe most forfor potent compoundcompoundderivatives of the 26 35,, benzhydrazideandmolecular 36 were subset. much 45 lost As more anya confirmation potentantiaggregating than of pyridine the activity. favorable congener hydrophobic 34. As already effects, quinolinenoticed for derivatives compound 35 26, benzhydrazideandmolecular 36 werewere subset. muchmuch 45 lostlost Asmoremore anyanya confirmation potentpotent antiaggregatingantiaggregating thanthan of pyridinepyridine the activity.activity. favorable congenercongener hydrophobic 34.. AsAs alreadyalready effects, noticednoticedquinoline forfor derivatives compoundcompound 35 26 ,, Moleculesbenzhydrazideand 362020 were, 25 ,much 577345 lost more any potentantiaggregating than pyridine activity. congener 34. As already noticed for compound 269, of 26 benzhydrazideand 36 were much45 lostlost more anyany antiaggregatingantiaggregatingpotent than pyridine activity.activity. congener 34. As already noticed for compound 26, benzhydrazide 45 lost any antiaggregating activity. benzhydrazideTable 45 3.lost InhibitionInhibition any antiaggregating datadata ofof newnew indoindo activity.le-3-carbaldehydele-3-carbaldehyde hydrazoneshydrazones 2828––3636 andand 4545.. Table 3. Inhibition data of new indole-3-carbaldehydeindole-3-carbaldehyde hydrazones 28–36 and 45.. TableTable 3. 3. Inhibition Inhibition data data of of new new indoindole-3-carbaldehydele-3-carbaldehyde hydrazones hydrazoneshydrazones 28 28–36–36 and andand 45 45. .. Table 3. Inhibition data of new indole-3-carbaldehyde hydrazones 28–36 and 45.

Aβ4040 AggregationAggregation InhibitionInhibition Comp. R R1 Comp. R R11 A β Aggregation Inhibition A%β at40 Aggregation10040 μM IC Inhibition5050 ((μM) Comp.Comp. R R R R11 AAββ404040 Aggregation AggregationAggregation Inhibition InhibitionInhibition Comp.Comp. RR R11 Aβ%40 %Aggregationat at100 100 μMµM InhibitionIC50 (μ ICM) (µM) Comp. R R1 %% atat 100 100 μ μMM ICIC50 5050(μ ((M)μM) 50 % at 100 μM IC50 (μM) 2828 H 1818 ±± 22 28 H 18 ± 2 28 2828 H HH 1818 ± ±2 2 18 2 28 H 18 ± 2 ±

2929 5-OCH5-OCH33 5656 ±± 11 2929 5-OCH33 5656 ± ±1 1 29 2929 5-OCH 5-OCH5-OCH333 5656 ± 56± 1 1 1 30 3H ± 13 ± 2 30 H 13 ± 2 3030 H 13 13± 2 ± 2 31303130 5-OCH5-OCHHH 33 139.29.213 ± ± ±±2 0.70.72 30 H 13 2 3131 5-OCH33 9.29.2 ± 0.7 ± 0.7 ± 323131 5-OCH5-OCHH 333 9.28.49.2 ± ±0.7 0.60.7 31 32 5-OCH3H 8.4 ± 0.69.2 0.7 32 H 8.4 ± 0.6 ± 3232 H 8.48.4 ± 0.6 ± 0.6 32 3332 H5-OCHH 33 8.422 ±± 0.62 8.4 0.6 33 5-OCH3 22 ± 2 ± 33 5-OCH3 22 ± 2 33 33335-OCH 5-OCH3 33 22 22± 2 ± 2 22 2 33 5-OCH33 22 ± 2 ±

34 5-OCH33 20 ± 2 34 3434 5-OCH5-OCH5-OCH3 3 2020 ±20± 22 2 34 5-OCH3 20 ± 2 ± 3434 5-OCH33 2020 ± 2± 2 34 5-OCH33 20 ± 2

35 3535 5-OCH5-OCH5-OCH33 1212 ±± 22 12 2 35 5-OCH3 3 12 ± 2 ± 3535 5-OCH33 12 12± 2 ± 2 35 5-OCH33 12 ± 2

36 5-OCH33 13 ± 1 36 363636 5-OCH 5-OCH5-OCH5-OCH3 3 3 131313 ± ±1± 11 13 1 36 5-OCH3 13 ± 1 ± 36 5-OCH3 13 ± 1 36 5-OCH33 13 ± 1

45 454545 5-OCH 5-OCH5-OCH5-OCH3 33 3 000 0 45 5-OCH3 0 45 5-OCH3 0 45 5-OCH33 0

Finally,Finally, a fewa few isomers isomers of of isatin isatin were were exploredexplored in orderorder toto ascertain ascertain the the effe effectscts of of modification modification of of Finally,Finally, a afew few isomers isomers of of isatin isatin werewere exploredexplored inin orderorder to to ascertain ascertain the the effe effectsctscts of of ofmodification modificationmodification of ofof thethe core Finally,core structure, structure, a fewfew while isomerswhile maintaining maintaining of isatin were thethe arylhydrazonearylhydrazone explored in order moietymoiety to ascertain(Table (Table 4). 4). theOnly Only eeffeﬀ compoundects compoundcts of modiﬁcationmodification 40 40 gave gave a ofa thethethe corecoreFinally, core structure,structure, structure, a few whilewhileisomers while maintainingmaintaining maintaining of isatin were thethethe arylhydrazoneexploredarylhydrazone in order moiety moietymoiety to ascertain (Table (Table(Table 4). 4). 4).the Only OnlyOnly effe compound cts compoundcompound of modification 40 40gave gavegave a of aa measurablethemeasurable core structure, IC IC5050 50equalequal equal while toto to 2525 maintaining 25 μ μM,M, while while the 4-isopropyl4-isopropyl arylhydrazone derivativesderivatives moiety 38 38 and (Table andand 41 414 were4).). werewere OnlyOnly found foundfound compoundcompound less lessless potent potentpotent 40 and gave andand a measurablethemeasurable core structure, IC IC50 50equal equal while to to maintaining25 25 μ μM,M, while while the 4-isopropyl4-isopropyl arylhydrazone derivativesderivatives moiety 38 38 (Table and and 41 41 4). were were Only found found compound less less potent potent 40 gaveandgave and aa thethemeasurablethe condensationcondensation condensation IC50 equal equalproductproduct product to to 2543 25 43 μwasµwas wasM,M, whilealmost whilealmostalmost 4-isopropyl 4-isopropyl inactive.inactive.inactive. derivatives 38 and 41 were found less potent and themeasurablethe condensation condensation IC50 equalequal product product toto 2525 43 43 μ was M,was while almost almost 4-isopropyl inactive.inactive. derivatives 38 andand 41 werewere foundfound lessless potentpotent andand the condensation product 43 was almost inactive.inactive. the condensation product 43 waswas almostalmost inactive.inactive.

Molecules 2020, 25, 5773 10 of 26 MoleculesMolecules 20202020,, 2525,, 57735773 99 ofof 2525

TableTableTable 4.4. InhibitionInhibition 4. Inhibition datadata ofof data newnew of derivativesderivatives new derivatives 3838,, 404038,, 4141, 40 andand, 41 4343and.. 43.

AAββ4040 AggregationAggregation Aβ40 Aggregation Inhibition Comp.Comp. StructureStructure InhibitionInhibition % at 100 µM IC50 (µM) %% atat 100100 μμMM ICIC5050 ((μμM)M)

3838 4949 ±± 5549 5 ±

4040 2525 ±± 33 25 3 ±

4141 6363 ±± 6663 6 ±

4343 1717 ±± 3317 3 ±

2.3.2.3. CytoprotectionCytoprotection fromfrom TauTau ToxicityToxicity 2.3. Cytoprotection2.3. Cytoprotection from Tau from Toxicity Tau Toxicity TheThe depositiondeposition ofof aggregatesaggregates ofof hyperphosphoryhyperphosphorylatedlated tautau proteinprotein constitutesconstitutes thethe intracellularintracellular The depositionThe deposition of aggregates of aggregates of hyperphosphory of hyperphosphorylatedlated tau protein tau protein constitutes constitutes the intracellular the intracellular neurofibrillaryneurofibrillary tangles,tangles, aa majormajor histologicalhistological evidenceevidence ofof AD.AD. TheThe rolerole ofof tautau hyperphosphorylationhyperphosphorylation neurofibrillaryneurofibrillary tangles, tangles, a major a major histological histological evidence evidence of AD. of AD.The role The of role tau of hyperphosphorylation tau hyperphosphorylation andand aggregationaggregation inin ADAD hashas establishedestablished bothboth ofof themthem asas aa targettarget forfor therapeutictherapeutic interventionintervention [8],[8], and aggregationand aggregation in AD in has AD established has established both of both them of themas a target as a targetfor therapeutic for therapeutic intervention interventio [8], [8], althoughalthough onlyonly N,N,N',N'N,N,N',N'-tetramethyl-10-tetramethyl-10HH-phenothiazine-3,7-diamini-phenothiazine-3,7-diaminiumum (TRX0237,(TRX0237, leucomethyl-leucomethyl- although only N,N,N0,N0-tetramethyl-10H-phenothiazine-3,7-diaminium (TRX0237, leucomethyl- thioninium),thioninium), thethe reducedreduced formform ofof methylenemethylene blueblue dye,dye, actingacting asas anan inhibitorinhibitor ofof tautau aggregation,aggregation, hashas thioninium),thioninium), the reduced the reduced form of form methylene of methylene blue dye, blue acting dye, as acting an inhibitor as an inhibitor of tau aggregation, of tau aggregation, has reachedreached thethe clinicalclinical phasephase IIIIII [26].[26]. InIn recentrecent years,years, somesome ofof usus developeddeveloped anan efficientefficient cell-basedcell-based assayassay reachedhas the reached clinical the phase clinical III phase [26]. IIIIn [recent26]. In years, recent some years, of some us developed of us developed an efficient an effi cell-basedcient cell-based assay assay ofof tautau aggregation,aggregation, aa simplesimple andand inexpensiveinexpensive methodmethod thatthat providesprovides aa straightforwardstraightforward assayassay toto of tauof tauaggregation, aggregation, a simple a simple and and inexpensive inexpensive method thatthat providesprovides a straightforwarda straightforward assay assay to evaluate to evaluateevaluate thethe putativeputative anti-aggregationanti-aggregation capacitycapacity ofof smsmallall moleculesmolecules [27–29].[27–29]. ThisThis methodmethod reliesrelies onon thethe evaluatethe putativethe putative anti-aggregation anti-aggregation capacity capacity of small of sm moleculesall molecules [27 [27–29].–29]. This This method method relies relies on on the the use of useuse ofof bacteriabacteria overexpressingoverexpressing amyloid-proneamyloid-prone protproteinseins toto monitormonitor thethe amyloidamyloid aggregation,aggregation, thusthus use ofbacteria bacteria overexpressing overexpressing amyloid-prone amyloid-prone proteins prot toeins monitor to monitor the amyloid the amyloid aggregation, aggregation, thus constituting thus constitutingconstituting aa physiologicallyphysiologically relevantrelevant model.model. InIn particular,particular, itit exploitsexploits amyloidamyloid propertiesproperties ofof thethe constitutinga physiologically a physiologically relevant model.relevant In model. particular, In particular, it exploits amyloidit exploits properties amyloid of properties the inclusions of the bodies inclusionsinclusions bodiesbodies (IBs)(IBs) formedformed byby tautau full-lengthfull-length proteinprotein inin bacteriabacteria stainedstained byby thioflavinthioflavin SS (ThS),(ThS), aa inclusions(IBs) formedbodies (IBs) by tau formed full-length by tau protein full-length in bacteria protein stained in bacteria by thioflavin stained by S (ThS),thioflavin a specific S (ThS), amyloid a specificspecific amyloidamyloid dye,dye, toto addressaddress thethe bacterialbacterial amyloidamyloid aggregationaggregation andand thethe effecteffect ofof anti-amyloidanti-amyloid specificdye, amyloid to address dye, the to bacterialaddress amyloidthe bacterial aggregation amyloid and aggregation the effect and of anti-amyloid the effect of compounds anti-amyloid in this compoundscompounds inin thisthis polymerizationpolymerization process.process. compoundspolymerization in this polymerization process. process. FromFrom aa randomizedrandomized screeningscreening ofof ourour in-housein-house library,library, includingincluding fourfour amyloidamyloid aggregationaggregation FromFrom a randomized a randomized screening screening of our of in-house our in-house library, library, including including four fouramyloid amyloid aggregation aggregation inhibitorsinhibitors S22S22,, S23S23,, S34S34 andand S35S35 comingcoming fromfrom aa previousprevious workwork [16],[16], isatinisatin hydrazoneshydrazones S23S23 andand S35S35 inhibitorsinhibitors S22, S23S22,, S34S23 ,andS34 S35and comingS35 coming from a fromprevious a previous work [16], work isatin [16 ],hydrazones isatin hydrazones S23 and S35S23 and emergedemerged asas interestinginteresting inhibitorsinhibitors ofof tautau cytotoxicity,cytotoxicity, withwith ICIC5050 valuesvalues inin thethe micromolarmicromolar rangerange (Table(Table emergedS35 emergedas interesting as interesting inhibitors inhibitorsof tau cytotoxicity, of tau cytotoxicity, with IC50 values with IC in thevalues micromolar in the micromolar range (Table range 5).5). WeWe thenthen decideddecided toto prepareprepare andand test,test, asas analoguesanalogues ofof S23S23,, thethe bis-isatinbis-isatin50 hydrazonehydrazone 44 andand thethe 5-5- 5). We(Table then5 ).decided We then to decided prepare to and prepare test, andas analogues test, as analogues of S23, the of bis-isatinS23, the bis-isatin hydrazone hydrazone 4 and the4 and 5- the unsubstitutedunsubstituted derivativederivative 55,, alongalong withwith thethe 5-unsubstituted5-unsubstituted congenerscongeners ofof S35S35 andand S34S34,, i.e.,i.e., compoundscompounds unsubstituted5-unsubstituted derivative derivative 5, along5, with along the with 5-unsubstituted the 5-unsubstituted congeners congeners of S35 of andS35 S34and, i.e.,S34 compounds, i.e., compounds 66 andand 88.. ToTo confirmconfirm thethe effectseffects ofof NN-substitution-substitution atat thethe isatinisatin nitrognitrogen,en, wewe alsoalso preparedprepared andand testedtested 6 and6 8and. To8 confirm. To confirm the effects the eff ofects N-substitution of N-substitution at the at isatin the isatin nitrog nitrogen,en, we also we prepared also prepared and tested and tested theirtheir NN-methyl-methyl derivativesderivatives 77 andand 99.. FromFrom thethe screening,screening, isatinisatin hydrazonehydrazone 55 emergedemerged asas aa potentpotent their theirN-methylN-methyl derivatives derivatives 7 and7 9and. From9. From the screening, the screening, isatin isatin hydrazone hydrazone 5 emerged5 emerged as a aspotent a potent inhibitorinhibitor ofof tautau aggregationaggregation (IC(IC5050 4.64.6 μμM),M), withwith slightlyslightly higherhigher potencypotency thanthan itsits 5-methoxy5-methoxy congenercongener inhibitorinhibitor of tau of aggregation tau aggregation (IC50 (IC4.6 μM),4.6 µwithM), withslightly slightly higher higher potency potency than thanits 5-methoxy its 5-methoxy congener congener S23S23.. TheThe remainingremaining newnew compoundscompounds50 scoredscored lowlow inhibitioninhibition values,values, despitedespite thethe limitedlimited structuralstructural S23. S23The. remaining The remaining new newcompounds compounds scored scored low inhibition low inhibition values, values, despite despite the limited the limited structural structural variationsvariations performed.performed. OnOn ththee otherother hand,hand, thethe assayassay onon AAββ4040 aggregationaggregation discloseddisclosed thethe strongstrong variations performed. On the other hand, the assay on Aβ40 aggregation disclosed the strong inhibitory inhibitoryinhibitory potencypotency ofof NN-methyl-methyl derivativesderivatives 77 andand 99,, withwith ICIC5050ss inin thethe lowlow toto submicromolarsubmicromolar range.range. inhibitorypotency potency of N-methyl of N-methyl derivatives derivatives7 and 97, and with 9 IC, withs in IC the50s lowin the to low submicromolar to submicromolar range. range. Compared ComparedCompared withwith theirtheir referencereference analoguesanalogues S35S35 andand S34S3450,, wherewhere thethe activitiesactivities ofof metameta werewere slightlyslightly Comparedwith their with reference their reference analogues analoguesS35 and S35S34 and, where S34, thewhere activities the activities of meta ofwere meta slightly were slightly higher than higherhigher thanthan parapara congeners,congeners, 66 andand 88 showedshowed anan oppositeopposite trend,trend, andand bothboth resultedresulted stronglystrongly weakerweaker higherpara thancongeners, para congeners,6 and 8 6showed and 8 showed an opposite an opposite trend, trend, and both and resulted both resulted strongly strongly weaker weaker than their thanthan theirtheir correspondingcorresponding NN-methylated-methylated derivatives.derivatives. Overall,Overall, compoundcompound 99 emergedemerged asas thethe mostmost potentpotent inhibitorinhibitor ofof AAββ4040 aggregationaggregation withinwithin thethe wholewhole seriesseries ofof testedtested herein.herein.

Molecules 2020, 25, 5773 11 of 26 corresponding N-methylated derivatives. Overall, compound 9 emerged as the most potent inhibitor of Aβ40 aggregation within the whole series of tested herein. Molecules 2020, 25, 5773 10 of 25 Molecules 20202020,,, 252525,,, 577357735773 1010 ofof 2525 Table 5. Inhibition of β-amyloid and tau aggregation. Table 5. Inhibition of β-amyloid and tau aggregation. TableTable 5.5. InhibitionInhibitionInhibition ofofof ββ-amyloid-amyloid-amyloid andandand tautautau aggregation.aggregation.aggregation. β a A 40 Inhibition Tau Inhibitiona Code Structure Aβ40 Inhibition Tau Inhibition aa AAββ40 InhibitionInhibitionInhibition TauTau InhibitionInhibition aa CodeCode StructureStructure Aβ4040 InhibitionInhibition Tau Inhibition Code Structure % at 100 µM50 IC50 (µM) % at 10 µM50 IC50 (µM) Code Structure %% atat 100100 μμMM ICIC5050 ((μμM)M) % % atat 1010 μμMM ICIC5050 ((μμM)M) % at 100 μM IC5050 ((μM) % at 10 μM IC5050 ((μM) H HH NHN N N NN b O N N c bbb OO22NN cc c S22S22S22 bb O22N -cc- 15 ±15 3 3 S22 2 O -- 1515 ±± 33 OO ± N O NN HNH H

S23b bb 52 7.7 ± 1.0 S23S23S23 bb 525252 7.7 7.7 7.7 ±±± 1.01.0 7.71.0 1.0 S23 52 7.7 7.7 ±± 1.01.0 ±

S34 bb 20 16 ± 2 S34S34S34b bb 202020 1616 ±± 16 22 2 S34 20 16 ± 2 ±

S35 bb 44 10 ± 2 S35S35S35b bb 444444 10 10 10 ±±± 222 10 2 ±

4 31 ± 3 17 ± 2 444 313131 ±± 33 3 1717 ±± 17 22 2 ± ±

5 22 ± 2 4.6 ± 0.7 555 22 22 22 22±±± 222 2 4.6 4.6 4.6 ±±± 0.70.7 4.60.7 0.7 ± ±

6 45 ± 6 14 ± 2 666 454545 ±± 66 6 1414 ±± 14 22 2 ± ±

7 2.0 ± 0.3 19 ± 2 777 2.0 2.0 2.02.0 ±±± 0.30.30.3 0.3 1919 ±± 19 22 2 ± ±

8 69 ± 3 35 ± 2 888 696969 ±± 33 3 3535 ±± 35 22 2 ± ±

9 0.95 ± 0.04 20 ± 2 999 0.95 0.95 0.950.95 ±±± 0.040.040.040.04 2020 ±± 20 22 2 ± ±

a b aa From cell-based assay. bb Compounds and their data of β-amyloid aggregation are taken from ref. a aa FromFromFrom cell-basedcell-basedcell-based assay.assay.assay.b bb CompoundsCompoundsCompounds andandand theirtheirtheir datadatadata ofofof ββ-amyloid-amyloid aggregationaggregation areare takentaken fromfrom ref.ref. From cell-based assay. Compounds and their data of β-amyloid aggregation are taken from ref. [16] andc presented [16] and presented in Supplementary Materials. Values are mean ± SEM (n = 3) in in vitro assay. ccc No in Supplementary[16][16] andand presentedpresented Materials. inin SupplementarySupplementary Values are mean Materials.SEM (Valuesn = 3) in arein vitromeanassay. ± SEMc No (nn == inhibition= 3)3)3) ininin ininin vitrovitrovitro at 100 assay.assay.assay.µM concentration. cc NoNoNo inhibition at 100 μM concentration. ± inhibitioninhibitioninhibition atatat 100100100 μμMM concentration.concentration. 2.4. Evaluation of Multitarget Activity 2.4.2.4. EvaluationEvaluation ofof MultitargetMultitarget ActivityActivity

Molecules 2020, 25, 5773 12 of 26

2.4. Evaluation of Multitarget Activity The benefits of multitarget approach for the treatment of neurodegenerative diseases are widely recognized, although the pharmacological proof of concept is still controversial [10]. Our research has devoted large attention to dual inhibitors of cholinesterases (ChEs) and monoamine oxidases (MAOs), two major targets in neurodegeneration [30–32]. Acetyl-(AChE) and butyrylcholinesterase (BChE) are responsible for the hydrolysis of neurotransmitter acetylcholine, particularly in brain regions involved in learning and memory processes. AChE inhibitors still remain the first options for the symptomatic treatment of cognitive impairment in AD. Monoamine oxidase isoforms A (MAO A) and B (MAO B) are mitochondrial enzymes responsible for oxidative degradation of amine neurotransmitters and xenobiotics. Selective MAO A inhibitors are second-line drugs in the treatment of depression and mood disorders, while MAO B-selective inhibitors are used in the therapy of Parkinson’s disease and have neuroprotective effects. Isatin is an endogenous biofactor, metabolically derived from indole, largely present in the CNS [33]; isatin derivatives are recognized bioactive molecules for many neurological diseases [34]. 5-Substituted isatins have been described as potent MAO inhibitors [35,36] and recently, N-substituted 3-acylhydrazones of isatin showed interesting dual AChE-MAO B inhibitory activities [37]. The above-cited evidences prompted us to extend the investigation of biological activity to ChEs and MAOs, by testing a limited number of isatin and indole derivatives in in vitro inhibition of human ChEs and MAOs. Compounds were selected according to the following criteria: (a) the most potent inhibitors of Aβ40 aggregation within the structural classes collected in Tables1–3 and5 (i.e., compounds 9, including its isomers 6–8 for the exploration of putative substitution-activity relationships, 18, 19, 23, 11 and 32); (b) the most potent inhibitor of tau toxicity (5); (c) compounds bearing a basic nitrogen, in order to get an efficient interaction with the catalytic site of ChEs, either at the arylhydrazone moiety (35) or Mannich bases at the isatin nitrogen (18 and 13). The data in Table6 assessed a weak inhibitory potency in AChE inhibition ( < 50% at 10 µM), with the valuable exception of benzyloxy isomers 6 and 8 (IC50s in the low micromolar range). Disappointingly, none of compounds with basic substituents was found active. As for BChE inhibition, all compounds were ineffective (data not shown).

Table 6. Inhibition data of acetylcholinesterase and monoamine oxidases.

hAChE a hMAO A a hMAO B a Comp. % Inhib. at 10 µM IC50 (µM) % Inhib. at 10 µM IC50 (µM) % Inhib. at 10 µM IC50 (µM) 5 37 1 0.52 0.04 3.6 0.7 ± ± ± 6 4.2 0.8 23 5 22 4 ± ± ± 7 48 3 29 3 41 1 ± ± ± 8 3.9 0.5 8 3 36 4 ± ± ± 9 48 2 15 3 4.0 0.4 ± ± ± 18 23 1 31 4 47 2 ± ± ± 19 31 1 18 4 46 5 ± ± ± 23 40 3 7.1 0.3 5.3 0.3 ± ± ± 11 47 5 20 4 2.7 0.3 ± ± ± 13 26 1 7 2 4.4 0.1 ± ± ± 32 41 5 0.34 0.01 0.23 0.03 ± ± ± 35 38 2 3.8 0.3 6.5 0.6 ± ± ± Galantamine 0.72 0.05 - - - - ± Saﬁnamide - - 18 3 0.031 0.001 ± ± a Human recombinant enzymes. Values are mean SEM (n = 3). ±

Some positive results came out from MAOs inhibition tests, with indole derivative 32 acting as a strong inhibitor with submicromolar IC50s, although not isoform-selective. Another indole derivative, the quinoline arylhydrazone 35, showed good inhibition of both isoforms, with a slight preference for MAO A. Isatin phenylhydrazone 5 displayed also good inhibitory potency against MAO A, with 7-fold selectivity over MAO B. N-Phenethylisatin 11, in turn, resulted as the best MAO B selective inhibitor, with IC50 in the low micromolar range. Molecules 2020, 25, 5773 13 of 26

Molecules 2020, 25, 5773 12 of 25 2.5. Computational Studies MAO A, with 7-fold selectivity over MAO B. N-Phenethylisatin 11, in turn, resulted as the best MAO WeB selective carried inhibitor, out computational with IC50 in the low ligand-based micromolar range. studies, namely pharmacophore modeling and atom-based 3D-QSAR study, on a pool of 93 already published compounds [15–17,19] (structures and 2.5. Computational Studies related Aβ antiaggregation data in Supplementary Materials) for better addressing the design of the new moleculesWe carried sharing out computational the indole/isatin ligand-based scaffold. studies, The ligand-based namely pharmacophore studies were modeling aimed and at deriving a atom-based 3D-QSAR study, on a pool of 93 already published compounds [15–17,19] (structures and comprehensiverelated Aβ antiaggregation pharmacophore data in hypothesis Supplementary to discern Materials) between for better ‘actives’, addressing i.e., the compounds design of the achieving finitenew IC50 moleculesvalues under sharing the the threshold indole/isatin 100 scaffold.µM concentration, The ligand-based and studies ‘inactives’, were aimed i.e. compoundsat deriving a showing no activitycomprehensive or less than pharmacophore 50% inhibition hypothesis at 100 toµ discerM. Then between pharmacophore ‘actives’, i.e., was compounds then complemented achieving with a 3D-QSARfinite IC study50 values limited underto the the threshold ‘active’ 100 molecules μM concentration, (IC50s < and100 ‘inactivµM),es’, the i.e. purpose compounds being showing to predictively estimateno activity the A orβ inhibitionless than 50% potency inhibition of at new 100 μ indoleM. The/ isatinpharmacophore derivatives was then and complemented ultimately integrating with and a 3D QSAR study limited to the ‘active’ molecules (IC50s < 100 μM), the purpose being to predictively improving the physicochemical information content of the pharmacophore for a prospective design. estimate the Aβ inhibition potency of new indole/isatin derivatives and ultimately integrating and improving the physicochemical information content of the pharmacophore for a prospective design. 2.5.1. Top-Scored Pharmacophore Model Five2.5.1. commonTop-Scored features Pharmacophore were mapped Model as relevant for the pharmacophore generation: two hydrogen bond acceptorsFive common (A), onefeatures hydrophobic were mapped region as relevant (H) and for twothe pharmacophore aromatic rings generation: (R). More two specifically, the featureshydrogen of bond AAHRR acceptors pharmacophore (A), one hydrophobic were mapped region as (H) follows: and two R12 aromatic and H11 rings refer (R). to isatin,More indoline or indandionespecifically, cores, the features A2 and of A1AAHRR refer pharmacophore to the carbonyl were ofisatin mapped or as indandione follows: R12 groups and H11 and refer nitrogen to atom isatin, indoline or indandione cores, A2 and A1 refer to the carbonyl of isatin or indandione groups of hydrazoneand nitrogen moiety, atom respectively, of hydrazone andmoiety, R13 respectively refers to phenyl, and R13 or thiazolerefers to phenyl rings. Moreover,or thiazole rings. some excluded volumesMoreover, were identifiedsome excluded which volumes locate were sterically identified disallowed which locate regions sterically (Figure disallowed2). This regions hypothesis (Figure matched 38 out2). of This 54 hypothesis (70%) ‘actives’ matched and 38 39out out of 54 of (70%) 39 (100%) ‘actives’ ‘inactives’, and 39 out of thus 39 (100%) having ‘inactives’, an overall thus accuracy having equal to 82.80%an (Figureoverall accuracy3). equal to 82.80% (Figure 3).

FigureFigure 2. Pharmacophore 2. Pharmacophore hypothesis hypothesis AAHRR. AAHRR. Orange Orange circles,circles, pinkpink andand greengreen spheres indicate aromatic rings,aromatic hydrogen rings, bonds hydrogen acceptors bonds andacceptors hydrophobic and hydrophobic region features,region features, respectively. respectively. Excluded Excluded volumes are Molecules 2020, 25, 5773 13 of 25 depictedvolumes as translucent are depicted cyanas translucent spheres. cyan spheres.

FigureFigure 3. 3. OverlayOverlay of of actives actives (in (in green) green) and and inactives inactives (in (in gray) gray) shown shown on on the the left-hand left-hand and and right-hand right-hand side,side, respectively. respectively.

This pharmacophore hypothesis was used to predict the 36 newly synthesized compounds (Tables 1–5) by setting a minimum number of at least four matched features. Satisfactorily, about 89% of new designed compounds (i.e., 32 out of 36) complied with the AAHRR pharmacophore hypothesis (Figure 4).

Figure 4. Overlay of the 36 new designed and prepared compounds, based on pharmacophore hypothesis AAHRR. Ligands are rendered as green wireframes.

2.5.2. 3D-QSAR Model We derived an atom-based 3D-QSAR model by employing all the compounds achieving finite IC50 values below the maximum 100 μM concentration tested. The training set was populated by 54 already reported compounds while the external set by 17 newly synthesized compounds. The first 3D-QSAR run which included all the 54 compounds into the training set did not yield any statistically meaningful model within the first five factors (i.e., PLS components). A careful check of the residuals [38,39] revealed a cluster of nine outliers (residuals > 2s): six bearing substituents (both polar and hydrophobic) at the para position of the phenyl ring (i.e., S29, S33, S49, S57 and S59 in Supplementary Materials) or at position 4 of the thiazole ring (S69) of the aryl-hydrazone moiety, and three lipophilic phenyl hydrazones of 3-oxo-3H-indole-2-carbaldehyde (S10, S14, and S15). Excluding relevant discrepancies arising from experimental procedures, as on the other hand confirmed by positive controls and re-determination of some representative ‘old’ compounds, the anomalous deviation from the model of these nine molecules may be likely due to the existence of a nonlinear correlation between the inhibition potency and the ligand hydrophobicity, as noticed by correlation with RP- HPLC parameter in our previous work [19]. In the PLS equation of the 3D-QSAR model there are no square neither cross terms which can account for nonlinear effects. Omitting from the PLS regression the nine outliers, the 3D-QSAR model, encompassing 45 molecules (83%) of the whole training set, was able in predicting quite satisfactorily the IC50 values of 14 (82%) external test set compounds (r2ext = 0.695); three badly predicted compounds were the hydrazone derivatives 30, 31 and 32, sharing the

Molecules 2020, 25, 5773 13 of 25

MoleculesFigure2020 3., 25 Overlay, 5773 of actives (in green) and inactives (in gray) shown on the left-hand and right-hand14 of 26 side, respectively.

This pharmacophore hypothesis was was used to predict the 36 newly synthesized compounds (Tables1 1–5)–5) by by settingsetting aa minimumminimum numbernumber ofof atat leastleast fourfour matchedmatched features.features. Satisfactorily, aboutabout 89%89% of newnew designeddesigned compounds compounds (i.e., (i.e., 32 out32 ofout 36) of complied 36) complied with the with AAHRR the pharmacophoreAAHRR pharmacophore hypothesis hypothesis(Figure4). (Figure 4).

Figure 4. Overlay of the 36 new designed and prepared compounds, based on pharmacophore Figurehypothesis 4. Overlay AAHRR. of Ligands the 36 arenew rendered designed as and green prep wireframes.ared compounds, based on pharmacophore hypothesis AAHRR. Ligands are rendered as green wireframes. 2.5.2. 3D-QSAR Model

2.5.2.We 3D-QSAR derived Model an atom-based 3D-QSAR model by employing all the compounds achieving finite IC50 values below the maximum 100 µM concentration tested. The training set was populated by 54 already We derived an atom-based 3D-QSAR model by employing all the compounds achieving finite reported compounds while the external set by 17 newly synthesized compounds. The first 3D-QSAR IC50 values below the maximum 100 μM concentration tested. The training set was populated by 54 run which included all the 54 compounds into the training set did not yield any statistically meaningful already reported compounds while the external set by 17 newly synthesized compounds. The first model within the first five factors (i.e., PLS components). A careful check of the residuals [38,39] 3D-QSAR run which included all the 54 compounds into the training set did not yield any statistically revealed a cluster of nine outliers (residuals > 2s): six bearing substituents (both polar and hydrophobic) meaningful model within the first five factors (i.e., PLS components). A careful check of the residuals at the para position of the phenyl ring (i.e., S29, S33, S49, S57 and S59 in Supplementary Materials) [38,39] revealed a cluster of nine outliers (residuals > 2s): six bearing substituents (both polar and or at position 4 of the thiazole ring (S69) of the aryl-hydrazone moiety, and three lipophilic phenyl hydrophobic) at the para position of the phenyl ring (i.e., S29, S33, S49, S57 and S59 in Supplementary hydrazones of 3-oxo-3H-indole-2-carbaldehyde (S10, S14, and S15). Excluding relevant discrepancies Materials) or at position 4 of the thiazole ring (S69) of the aryl-hydrazone moiety, and three lipophilic arising from experimental procedures, as on the other hand confirmed by positive controls and phenyl hydrazones of 3-oxo-3H-indole-2-carbaldehyde (S10, S14, and S15). Excluding relevant re-determination of some representative ‘old’ compounds, the anomalous deviation from the model discrepancies arising from experimental procedures, as on the other hand confirmed by positive of these nine molecules may be likely due to the existence of a nonlinear correlation between the controls and re-determination of some representative ‘old’ compounds, the anomalous deviation inhibition potency and the ligand hydrophobicity, as noticed by correlation with RP-HPLC parameter from the model of these nine molecules may be likely due to the existence of a nonlinear correlation in our previous work [19]. In the PLS equation of the 3D-QSAR model there are no square neither between the inhibition potency and the ligand hydrophobicity, as noticed by correlation with RP- cross terms which can account for nonlinear effects. Omitting from the PLS regression the nine outliers, HPLC parameter in our previous work [19]. In the PLS equation of the 3D-QSAR model there are no the 3D-QSAR model, encompassing 45 molecules (83%) of the whole training set, was able in predicting square neither cross terms which can account for nonlinear effects. Omitting from the PLS regression quite satisfactorily the IC values of 14 (82%) external test set compounds (r2 = 0.695); three badly the nine outliers, the 3D-QSAR50 model, encompassing 45 molecules (83%) of extthe whole training set, predicted compounds were the hydrazone derivatives 30, 31 and 32, sharing the 1H-indole core, a much was able in predicting quite satisfactorily the IC50 values of 14 (82%) external test set compounds (r2ext less represented scaffold than 1H-indole-2,3-dione (i.e., isatin) in the training set. = 0.695); three badly predicted compounds were the hydrazone derivatives 30, 31 and 32, sharing the The final 3D-QSAR model has the following features and statistics: four PLS factors; coefficient of determination r2 equal to 0.887 and a standard deviation value to 0.196; leave-one-out coefficient 2 2 of determination q equal to 0.596; external coefficient of determination r ext equal to 0.695 and root mean square error (RMSE) value equal to 0.270. Other statistical parameters could be additionally employed to support the prediction capability of the 3D-QSAR model [40]. We preferred validating the model with a true external set of compounds. The grid-based 3D-QSAR model included the following molecular fields: hydrophobic/non-polar, electron-withdrawing and hydrogen bonds, whose relative contributions were equal to 74.3%, 17.7% and 7.5%, respectively. The remaining 0.5% was due to a miscellaneous field [41]. All statistics related to the first four PLS components are summarized in Table7. Moreover, the correlation between experimental and predicted pIC50 values for training and external test sets is shown in Figure5. Molecules 2020, 25, 5773 14 of 25

1H-indole core, a much less represented scaffold than 1H-indole-2,3-dione (i.e., isatin) in the training set. The final 3D-QSAR model has the following features and statistics: four PLS factors; coefficient of determination r2 equal to 0.887 and a standard deviation value to 0.196; leave-one-out coefficient of determination q2 equal to 0.596; external coefficient of determination r2ext equal to 0.695 and root mean square error (RMSE) value equal to 0.270. Other statistical parameters could be additionally employed to support the prediction capability of the 3D-QSAR model [40]. We preferred validating the model with a true external set of compounds. The grid-based 3D-QSAR model included the following molecular fields: hydrophobic/non- polar, electron-withdrawing and hydrogen bonds, whose relative contributions were equal to 74.3%, 17.7% and 7.5%, respectively. The remaining 0.5% was due to a miscellaneous field [41]. All statistics Moleculesrelated2020 , to25 ,the 5773 first four PLS components are summarized in Table 7. Moreover, the correlation15 of 26 between experimental and predicted pIC50 values for training and external test sets is shown in Figure 5. Table 7. Atom based 3D-QSAR statistics. Table 7. Atom based 3D-QSAR statistics. 2 2 2 # Factors SD r q RMSE r ext # Factors SD r2 q2 RMSE r2ext 1 1 0.3830.383 0.5330.533 0.320 0.320 0.290 0.290 0.651 0.651 2 2 0.2830.283 0.7510.751 0.439 0.439 0.250 0.250 0.754 0.754 3 3 0.2310.231 0.8380.838 0.563 0.563 0.260 0.260 0.720 0.720 4 4 0.1960.196 0.8870.887 0.596 0.596 0.270 0.270 0.695 0.695

FigureFigure 5. Scatter 5. Scatter plot plot showing showing experimental experimental vs.vs. predictedpredicted pIC pIC5050 valuesvalues for for training training (n =( 45,n = red45, solid red solid circles)circles) and and external external (n (=n =14, 14, blueblue solid solid circles) circles) sets, sets, respectively. respectively. Bisector Bisector line is depicted line is depicted in black. The in black. 2 2 The rr2 andand r 2extext coefficientcoeﬃcient values values were were equal equal to 0.887 to 0.887and 0.695, and 0.695,respectively. respectively.

ContourContour maps maps of grid-basedof grid-based descriptors descriptors are reported in in Figure Figure 6 6to to spot spot positive positive or negative or negative molecularmolecular regions regions for for the the activity activity ofof the compounds. compounds. Hydrophobic Hydrophobic substituents substituents at position at position 5 and 6 of 5 and 6 of isatinisatin or indoline indoline cores cores are are detrimental detrimental for activity, for activity, much muchmore than more those than at thosethe ortho at position the ortho of positionthe of thephenyl phenyl ring. ring. Furthermore, Furthermore, bulky bulky substituents, substituents, such as such iso-butyl as iso- orbutyl benzyloxy or benzyloxy groups, at groups, the para at the position of phenyl ring cause a drop of the activity and thus impact the excluded volumes of the para position of phenyl ring cause a drop of the activity and thus impact the excluded volumes of the pharmacophore model. Hydrogen bond donors, such as hydroxyl groups, can enhance the activity pharmacophore model. Hydrogen bond donors, such as hydroxyl groups, can enhance the activity at the position 5 and 6 of the isatin core. The carbonyl groups of 3-indolinone or isatin are beneficial at thefor position activity, 5 while and 6electron-withd of the isatinrawing core. The substituents carbonyl decrease groups th ofe 3-indolinoneactivity when branching or isatin arethe beneﬁcialortho for activity,position while of the electron-withdrawingphenyl ring. substituents decrease the activity when branching the ortho positionMolecules of the 2020 phenyl, 25, 5773 ring. 15 of 25

Figure 6. Cont.

Figure 6. Contour maps rendered as blue and red cubes indicate positive and negative regions for activity. Specifically, panels (a,c,e) show the most active ligands within hydrophobic, HBD and electron withdrawing region, respectively; panels (b,d,f) show the most inactive ligands within hydrophobic, HBD and electron withdrawing regions, respectively. Pharmacophore features and excluded volumes are also reported.

3. Materials and Methods

3.1. General Information Commercial reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy). Melting points (mp) were determined by the capillary method on a Stuart SMP3 electrothermal apparatus (Bibby Scientific, Milan, Italy). IR spectra were recorded using potassium bromide disks on a Spectrum One FT-IR spectrophotometer (Perkin Elmer, Milan, Italy); only the most significant IR absorption bands are reported. 1H-NMR spectra were recorded in DMSO-d6, CDCl3 or acetone-d6 on a Mercury 300 or 500 spectrometer (Varian, Cernusco s. N., Italy). Chemical shifts are expressed in δ (ppm) and the coupling constants J in Hz. The following abbreviations were used: s, singlet; d, doublet; t, triplet; qn, quintuplet; ep, eptuplet; dd, double doublet; td, triplet of doublets; m, multiplet; br s, broad singlet. Chromatographic separations were performed on silica gel 63–200 (Merck, Milan, Italy). ESI-MS was performed with an electrospray interface and an ion trap mass spectrometer (1100 Series LC/MSD Trap System, Agilent, Palo Alto, CA, USA). The sample was infused via a KD Scientific syringe pump at a rate of 10 mL/min. The pressure of the nebulizer gas was 15 psi. The drying gas was heated to 350 °C at a flow of 5 L/min. Full-scan mass spectra were recorded in the mass/charge (m/z) range of 50–800 amu. For some representative compounds, HRMS experiments were performed with a dual electrospray interface (ESI) and a quadrupole time-of-flight mass spectrometer (Q-TOF, Agilent 6530 Series Accurate-Mass Quadrupole Time-of-Flight LC/MS, Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Italy). Compounds 2 [42], 4 [43], 5 [44], 10–12 and 14 [19], 30 [45], 33 [15], 44 [23] and 45 [24] have been prepared according to quoted references and their analytical data agreed with those reported in the literature.

Molecules 2020, 25, 5773 15 of 25

Molecules 2020, 25, 5773 16 of 26

FigureFigure 6.6. ContourContour mapsmaps renderedrendered asas blueblue andand redred cubescubes indicateindicate positivepositive andand negativenegative regionsregions forfor activity.activity. Specifically, Specifically, panels panels (a, c(,ae,)c show,e) show the mostthe most active active ligands ligands within within hydrophobic, hydrophobic, HBD and HBD electron and withdrawingelectron withdrawing region, respectively; region, respectively; panels (b,d ,panelsf) show (b the,d,f most) show inactive the most ligands inactive within ligands hydrophobic, within HBDhydrophobic, and electron HBD withdrawing and electron regions, withdrawing respectively. regions, Pharmacophore respectively. features Pharmacophore and excluded features volumes and are also reported. excluded volumes are also reported. 3. Materials and Methods 3. Materials and Methods 3.1. General Information 3.1. General Information Commercial reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy). MeltingCommercial points (mp) reagents were and determined solvents bywere the purchas capillaryed methodfrom Sigma-Aldrich on a Stuart (Milan, SMP3 electrothermal Italy). Melting apparatuspoints (mp) (Bibby were Scientific, determined Milan, by Italy).the capillary IR spectra meth wereod on recorded a Stuart using SMP3 potassium electrothermal bromide apparatus disks on a(Bibby Spectrum Scientific, One FT-IR Milan, spectrophotometer Italy). IR spectra (Perkin were Elmer,recorded Milan, using Italy); potassium only the bromide most significant disks on IR a Spectrum One FT-IR spectrophotometer1 (Perkin Elmer, Milan, Italy); only the most significant IR absorption bands are reported. H-NMR spectra were recorded in DMSO-d6, CDCl3 or acetone-d6 on a Mercuryabsorption 300 bands or 500 are spectrometer reported. 1H-NMR (Varian, spectra Cernusco were s. recorded N., Italy). in Chemical DMSO-d6 shifts, CDCl are3 or expressed acetone-d in6 onδ (ppm)a Mercury and the300 coupling or 500 spectrometer constants J in (Varian, Hz. The Cernusco following s. abbreviations N., Italy). Chemical were used: shifts s, singlet;are expressed d, doublet; in δ t,(ppm) triplet; and qn, the quintuplet; coupling ep, constants eptuplet; J in dd, Hz. double The doublet;following td, abbreviations triplet of doublets; were used: m, multiplet; s, singlet; br d, s, broaddoublet; singlet. t, triplet; Chromatographic qn, quintuplet; separations ep, eptuplet; were dd, performeddouble doublet; on silica td, geltriplet 63–200 of doublets; (Merck, Milan,m, multiplet; Italy). ESI-MSbr s, broad was singlet. performed Chromatographic with an electrospray separations interface were and performed an ion trap on masssilica spectrometergel 63–200 (Merck, (1100 Milan, Series LCItaly)./MSD ESI-MS Trap was System, performed Agilent, with Palo an Alto, electrospray CA, USA). interface The sample and an wasion trap infused mass via spectrometer a KD Scientific (1100 syringeSeries LC/MSD pump at Trap a rate System, of 10 mL Agilent,/min. The Palo pressure Alto, ofCA, the USA). nebulizer The gassample was 15was psi. infused The drying via a gasKD Scientific syringe pump at a rate of 10 mL/min. The pressure of the nebulizer gas was 15 psi. The was heated to 350 ◦C at a flow of 5 L/min. Full-scan mass spectra were recorded in the mass/charge (dryingm/z) range gas ofwas 50–800 heated amu. to 350 For some°C at representativea flow of 5 L/min. compounds, Full-scan HRMS mass experimentsspectra were were recorded performed in the withmass/charge a dual electrospray (m/z) range interface of 50–800 (ESI) amu. and For a quadrupolesome repres time-of-flightentative compounds, mass spectrometer HRMS experiments (Q-TOF, Agilentwere performed 6530 Series with Accurate-Mass a dual electrospray Quadrupole interf Time-of-Flightace (ESI) and LC a /MS,quadrupole Agilent Technologiestime-of-flight Italiamass S.p.A.,spectrometer Cernusco (Q-TOF, sul Naviglio, Agilent Italy). 6530 Series Compounds Accurate-Mass2 [42], 4 [ Quadrupole43], 5 [44], 10 Time-of-Flight–12 and 14 [19], LC/MS,30 [45], Agilent33 [15], 44Technologies[23] and 45 [Italia24] have S.p.A., been Cernus preparedco sul according Naviglio, to Italy). quoted Compounds references and 2 [42], their 4 analytical[43], 5 [44], data 10– agreed12 and with14 [19], those 30 reported[45], 33 [15], in the 44 literature. [23] and 45 [24] have been prepared according to quoted references and their analytical data agreed with those reported in the literature. 3.2. Chemistry

3.2.1. Synthesis of (Z)-3-Hydrazineylidene-5-nitroindolin-2-one (3) A mixture of 5-nitroisatin (1 mmol, 1 eq) and hydrazine hydrate 55% (2.5 eq) in methanol (1.5 mL) was reﬂuxed for 1 h and then cooled to room temperature, resulting in the precipitation of hydrazone 1 that was ﬁltered, dried and recrystallized from ethanol. Yield: 87%. H-NMR (DMSO-d6) δ 6.99 (s, 1H, H-4), 8.10–8.06 (m, 2 H, H-6 and H-7), 10.18 (d, 1H, Jv = 12.5 Hz, N=NH2), 10.72 (d, 1H, Jv = 12.5 Hz, N=NH2), δ 11.30 (br s, 1H, NH indol.). MS (ESI), m/z = 204.9 (100%) [M H]−. IR (KBr): 3310, 1624, 1 − 1596, 1331 cm− . Mp 217–219 ◦C.

3.2.2. Synthesis of Aryl-hydrazineylidene indolinones 6–9 Compounds 6–9 were synthesized by condensation of N-methylisatin or isatin with suitable arylhydrazine hydrochloride [19]. Molecules 2020, 25, 5773 17 of 26

3-(2-(3-(Benzyloxy)phenyl)hydrazineylidene)indolin-2-one (6) Yield: 60%. 1H-NMR (500 MHz, DMSO-d6) δ 5.12 (s, 2H), 6.67 (dd, 1H, J = 8.1, 2.1 Hz), 6.89 (d, 1H, J = 7.8 Hz), 6.99–6.94 (m, 1H), 7.03 (t, 1H, J = 7.6 Hz), 7.11 (t, 1H, J = 2.1 Hz), 7.27–7.19 (m, 2H), 7.31 (t, 1H, J = 7.3 Hz), 7.38 (t, 2H, J = 7.5 Hz), 7.46 (d, 2H, J = 7.3 Hz), 7.54 (d, 1H, J = 7.5 Hz), 10.99 (s, 1H), 12.66 (s, 1H). HRMS (ESI) m/z + [M + Na] calcd for C21H17N3O2 366.1213; found, 366.1195; [M H]− calcd 342.1248; found, 342.1238. 1 − IR (KBr): 3416, 3162, 1557, 1207 cm− . Mp 182–186 ◦C from EtOH. 3-(2-(3-(Benzyloxy)phenyl)hydrazineylidene)-1-methylindolin-2-one (7) Yield: 55%. 1H-NMR (500 MHz, DMSO-d6) δ 3.23 (s, 3H), 5.12 (s, 2H), 6.68 (dd, 1H, J = 8.1, 2.0 Hz), 6.98 (dd, 1H, J = 8.0, 1.3 Hz), 7.10 (t, 2H, J = 7.3 Hz), 7.13 (t, 1H, J = 2.1 Hz), 7.25 (t, 1H, J = 8.1 Hz), 7.31 (dd, 2H, J = 11.6, 4.1 Hz), 7.38 (t, 2H, J = 7.5 Hz), 7.46 (d, 2H, J = 7.4 Hz), 7.61–7.56 (m, 1H), 12.60 (s, 1H). HRMS (ESI) m/z [M + Na]+ calcd for C22H19N3O2 380.1369; found, 380.1374; [M H]− calcd 356.1405; found, 356.1391. IR (KBr): 3418, 1 − 1676, 1567 cm− . Mp 134–137 ◦C from EtOH. 3-(2-(4-(Benzyloxy)phenyl)hydrazineylidene)indolin-2-one (8) Yield: 54%. 1H-NMR (500 MHz, DMSO-d6) δ 5.06 (d, 2H, J = 5.7 Hz), 6.89 (d, 1H, J = 7.8 Hz), 7.02 (dd, 3H, J = 12.5, 5.8 Hz), 7.19 (td, 1H, J = 7.7, 1.0 Hz), 7.31 (t, 1H, J = 7.3 Hz), 7.39–7.33 (m, 4H), 7.43 (d, 2H, J = 7.3 Hz), 7.50 (d, + 1H, J = 7.3 Hz), 10.93 (s, 1H), 12.73 (s, 1H). HRMS (ESI) m/z [M + Na] calcd for C21H17N3O2 366.1213; found, 366.1206; [M H] calcd 342.1248; found, 342.1239. IR (KBr): 3418, 1680, 1552, 1228, 1174 cm 1. − − − Mp 201–206 ◦C from EtOH. 3-(2-(4-(Benzyloxy)phenyl)hydrazineylidene)-1-methylindolin-2-one (9) Yield: 50%. 1H-NMR (500 MHz, DMSO-d6) δ 3.23 (s, 3H), 5.07 (s, 2H), 7.02 (d, 2H, J = 9.0 Hz), 7.08 (t, 2H, J = 7.2 Hz), 7.33–7.25 (m, 2H), 7.37 (dd, 4H, J = 8.2, 5.3 Hz), 7.43 (d, 2H, J = 7.2 Hz), 7.56–7.50 (m, 1H), 12.67 (s, 1H). HRMS (ESI) m/z + [M + Na] calcd for C22H19N3O2 380.1369; found, 380.1373; [M H]− calcd 356.1405; found, 356.1390. 1 − IR (KBr): 3435, 1667, 1555, 1218 cm− . Mp 126–128 ◦C from EtOH.

3.2.3. Alkylation of 5-Methoxyindoline-2,3-diones 13 and 15

5-Methoxy-1-(morpholinomethyl)indoline-2,3-dione (13)

To a solution of formaldehyde (37% in H2O, 4 eq) and morpholine (2 eq) in ethanol (10 mL) was added 5 mL of ethanoic solution of 5-methoxyisatin (1 mmol, 1 eq). The mixture was reﬂuxed for 2 days and after TLC control, the solvent was evaporated, and the residue crystalized. Yield: 61%. 1 H-NMR (CDCl3) δ 2.61 (t, 4H, J = 4.7 Hz, CH2CH2 morpholine), 3.69 (t, 4H, J = 4.7 Hz, CH2CH2 morpholine), 3.81 (s, 3H, OCH3), 4.41 (s, 2H, N-CH2), 7.02 (d, 1H, Jo = 9.1 Hz, H-7), 7.14–7.18 (m, 2H, + 1 H-4 and H-6). MS (ESI), m/z = 299.0 (100%) [M + Na] . IR (KBr): 2847, 2833, 1738, 1490, 1313 cm− . Mp 107–110 ◦C from EtOH.

1-(2-(Dimethylamino)ethyl)-5-methoxyindoline-2,3-dione (15) A solution in DMF (11 mL) of 5-methoxyisatine (2.8 mmol, 1 eq), 2-chloro N,N-dimethylethylamine hydrochloride (1.3 eq) and potassium carbonate (3 eq) was stirred overnight at room temperature. The mixture was filtered, and the solution was evaporated to afford a red oil that was purified by 1 column chromatography. Yield: 75%. H-NMR (CDCl3) δ 2.29 (s, 6H, N(CH3)2), 2.57 (t, 2H, Jv = 6.9 Hz, CH2), 3.83–3.77 (m, 5H, CH2 and OCH3), 6.87–6.83 (m, 1H), 7.16–7.10 (m, 2H). MS (ESI), m/z = 271.10 + 1 (100%) [M + Na] . IR (KBr): 2943, 1731, 1484, 1163 cm− . Mp 95–97 ◦C from CHCl3/MeOH 90:10 (v/v).

3.2.4. 1-(2-(Dimethylamino)ethyl)-3-(2-(4-isopropylphenyl)hydrazineylidene)-5-methoxyindolin-2-one (18) Compound 18 was synthesized by condensation of 15 with 4-isopropylphenylhydrazine 1 hydrochloride as previously described [19]. Yield: 35%. H-NMR (DMSO-d6) δ 1.14 (d, 6H, Jv = 7.1 Hz, CH(CH3)2), 2.75–2.91 (m, 7H, CH(CH3)2, N(CH3)2), 3.30–3.45 (m, 2H, NCH2CH2N(CH3)2) 3.79 (s, 3H, OCH3), 4.17 (t, 2H, Jv = 6.0 Hz, NCH2CH2N(CH3)2), 6.90 (dd, 1H, Jo = 8.3, Jm = 2.3 Hz, H-6), 7.12–7.26 (m, 4H, H-4, H-7, H-30 and H-50), 7.39 (d, 2H, J = 8.5 Hz, H-20 and H-60), 12.60 (s, 1H, NNH). Molecules 2020, 25, 5773 18 of 26

MS (ESI), m/z = 379.2 (100%) [M H] , 403.2 (100%) [M + Na]+. IR (KBr): 2959, 1670, 1521, 1485 cm 1. − − − Mp 222–224 ◦C from EtOH/H2O.

3.2.5. Synthesis of 5-Hydroxy-isopropylphenyl-hydrazineylidene indolinones 19 and 20 The debenzylation with BBr in dichloromethane at 70 C[16] of compounds 16 and 17 [19] 3 − ◦ gave the 19 and 20, respectively. 1-Benzyl-5-hydroxy-3-(2-(4-isopropylphenyl)hydrazineylidene)indolin-2-one (19). Yield: 20%. 1H-NMR (DMSO-d6) δ 1.19 (d, 6H, Jv = 7.0 Hz, CH(CH3)2), 2.86 (ept, 1H, Jv = 7.0 Hz, CH(CH3)2), 4.94 (s, 2H, CH2-Ph), 6.62 (dd, 1H, Jo = 8.4, Jm = 2.4 Hz, H-6), 6.82 (d, 1H, Jo = 8.4 Hz, H-7), 6.98 (d, 1H, Jm = 2.4 Hz, H-4), 7.24 (d, 2H, J = 8.8 Hz, H-30 and H-50), 7.27–7.36 (m, 7H, H-20, H-6, H-200, H-300, H-400, H-500 and H-600), 9.21 (s, 1H, OH), 12.71 (s, 1H, NNH). MS (ESI), m/z = 383.9 (100%) [M H]−. IR (KBr): 3991, 1 − 3032,1655, 1519, 1164 cm− . Mp 188–190 ◦C from hexane/ethyl acetate 70:30 (v/v). 1-Cyclopropyl-5-hydroxy-3-(2-(4-isopropylphenyl)hydrazono)indolin-2-one (20). Yield: 20%. 1H-NMR (DMSO-d6) δ 0.81–0.86 (m, 2H, CH2cyclo-pr.), 0.96–1.03 (m, 2H, CH2cyclo-pr.), 1.18 (d, 6H, Jv = 7.0 Hz, CH(CH3)2), 2.73 (qn, 1H, Jv = 3.7 Hz, CHcyclopr.), 2.85 (ep, 1H, Jv = 7.0 Hz, CH(CH3)2), 6.72 (dd, 1H, Jo = 8.4, Jm = 2.6 Hz, H-6), 6.94 (d, 1H, Jm = 2.4 Hz, H-4), 6.99 (d, 1H, Jo = 8.4 Hz, H-7), 7.22 (d, 2H, J = 8.6 Hz, H-30 and H-50), 7.31 (d, 2H, J = 8.4 Hz, H-20 and H-60), 9.21 (s, 1H, OH), 12.74 (s, 1H, NNH). MS (ESI), m/z = 333.9 (100%) [M H] . IR (KBr): 3305, 2920, 1656, 1559, 1390, 1190, 1138 cm 1. − − − Mp 222–224 ◦C from hexane/ethyl acetate 75:25 (v/v).

3.2.6. Synthesis of 3-Arylhydrazono-indolin-2-ones 23–25 Compounds 23–25 were prepared from azo coupling of aryldiazonium salts 22a–c with indolin-2-one 21, previously prepared [20]. Aryldiazonium salts were synthesized starting from corresponding amines as described in a previous paper [19]. 1 3-(2-(Naphthalen-1-yl)hydrazono)indolin-2-one (23). Yield: 20%. H-NMR (DMSO-d6) δ 6.97 (d, 1H, Jo = 7.7 Hz, H-7), 7.09 (t, 1H, Jo = 7.7 Hz, Jo = 7.3 Hz, H-5), 7.28 (t, 1H, Jo = 7.7 Hz, H-6), 7.54–7.69 (m, 5H, H-30, H-4´0, H-50, H-60and H-70), 7.82 (d, 1H, Jo = 7.3 Hz, H-4), 7.89 (d, 1H, Jo = 8.4 Hz, H-20), 7.98 (d, 1H, Jo = 8.1 Hz, H-80), 11.23 (s, 1H, NH), 13.79 (s, 1H, NNH). MS (ESI), m/z = 285.8 (100%) [M H]−. 1 − IR (KBr): 3413, 3121, 3049, 1675, 1561, 1196 cm− . Mp 225–230 ◦C from hexane/ethyl acetate 80:20 (v/v). 1 3-(2-(Naphthalen-2-yl)hydrazono)indolin-2-one (24). Yield: 30%. H-NMR (DMSO-d6) δ 6.92 (d, 1H, Jo = 7.7 Hz, H-7), 7.06 (t, 1H, Jo = 7.7 and 7.3 Hz, H-5), 7.25 (td, 1H, Jm = 1.1 Hz, Jo = 7.7 Hz, H-6), 7.36 (t, 1H, Jo = 8.1 and 7.0 Hz, H-60), 7.41 (t, 1H, Jo = 8.1 and 7.0 Hz, H-70), 7.61 (d, 1H, Jo = 7.3 Hz, H-4), 7.71 (dd, 1H, Jm = 1.8 Hz, Jo = 8.8 Hz, H-30), 7.83–7.86 (m, 3H, H-10, H-50 and H-80), 7.93 (d, 1H, Jo = 8.8 Hz, H-40), 11.05 (s, 1H, NH), 12.95 (s, 1H, NNH). MS (ESI), m/z = 285.8 (100%) [M H]−. IR (KBr): 3159, 1 − 1676, 1555, 1223, 1190 cm− . Mp 245–248 ◦C from hexane/ethyl acetate 70:30 (v/v). 1 3-(2-(Quinolin-8-yl)hydrazineylidene)indolin-2-one (25). Yield: 21%. H-NMR (DMSO-d6) δ 6.92 (d, 1H, Jo = 7.5 Hz, H-7), 7.06 (t, 1H, Jo = 7.7 Hz, H-5), 7.26 (t, 1H, Jo = 7.7 Hz, H-6), 7.58–7.67 (m, 4H, H-30, H-40, H-60and H-4), 7.93 (dd, 1H, Jo = 7.0, Jm = 2.2 Hz, H-50), 8.39 (dd, 1H, Jo = 8.4, Jm = 1.6 Hz, H-20), + 8.91–8.93 (m, 1H, H-70), 11.07 (s, 1H, NH), 13.91 (s, 1H, NNH). MS (ESI), m/z = 311.0 (100%) [M + Na] . 1 IR (KBr): 3447, 3152, 1682, 1523, 1198 cm− . Mp > 250 ◦C dec. from CH2Cl2/MeOH 96:4 (v/v).

3.2.7. N0-(5-Methoxy-2-oxoindolin-3-ylidene)benzhydrazide (26) The compound was synthesized by condensation of 5-methoxyisatin and benzhydrazide as 1 previously described [19]. Yield: 37%. H-NMR (DMSO-d6) δ 3.77 (s, 3H, 5-OCH3), 6.87 (d, 1H, J = 8.6 Hz, H-7), 6.96 (dd, 1H, Jo = 8.6, Jm = 2.6 Hz, H-6), 7.13 (d, 1H, Jm = 2.6 Hz, H-4), 7.59 (dd, 2H, Jo = 8.4, Jm = 1.5 Hz, H-50 and H30), 7.63–7.61 (m, 1H, H-40), 7.88 (dd, 2H, Jo = 8.4, Jm = 1.5 Hz, H-20 + and H-60), 11.20 (br s, 1H, NH), 13.90 (br s, 1H, NNHCO). ESI-MS m/z = 318.0 (100%) [M + Na] , 293.8 (100%) [M H] . IR (KBr): 3254, 1704, 1691, 1675, 1486 cm 1. Mp > 250 C from CH Cl /MeOH − − − ◦ 2 2 96:4 (v/v). Molecules 2020, 25, 5773 19 of 26

3.2.8. 2-(4-Hydroxybenzylidene)indolin-3-one (27)

1 Compound 27 was prepared following literature procedures [46]. Yield: 25%. H-NMR (DMSO-d6) δ 6.60 (s, 1H, =CH), 6.85 (d, 2H, Jo = 8.7 Hz, H-30and H-50), 6.87 (t, 1H, Jo = 7.6 Hz, H-5), 7.11 (d, 1H, Jo = 8.3 Hz, H-7), 7.48 (td, 1H, Jo = 8.8, Jm = 1.1 Hz, H-6), 7.54 (d, 1H, Jo = 7.6 Hz, H-4), 7.68 (d, 2H, Jo = 8.7 Hz, H-20and H-60), 9.54 (s, 1H, NH), 9.98 (br s, 1H, OH). MS (ESI), m/z = 235.9 (100%) [M H]−. 1 − IR (KBr): 3311, 1672, 1591, 1509, 1488, 1243 cm− . Mp > 250 ◦C.

3.2.9. Synthesis of Hydrazonomethyl-indoles 28, 29, 31, 32, 34–36 The synthesis of hydrazonomethyl-indoles was performed as previously described [19] by the condensation of corresponding indole carbaldehyde and arylhydrazine. Then the desired compounds were puriﬁed by crystallization or column chromatography. 1 3-((2-(4-Isopropylphenyl)hydrazono)methyl)-1H-indole (28). Yield: 30%. H-NMR (DMSO-d6) δ 1.16 (d, 6H, J = 7.0 Hz, CH(CH3)2), 2.80 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 6.96–7.40 (m, 6H, H-6, H-7, H-20, H-30, H-50 and H-60), 7.59 (s, 1H, H-2), 8.08 (s, 1H, H-8), 8.21–8.24 (m, 1H, H-4), 8.64–8.65 (m, 1H, H-5), 11.31 (s, 1H, NH), 12.79 (s, 1H, NNH). MS (ESI), m/z = 275.9 (100%) [M H]−. IR (KBr): 3436, 2841, 1 − 1644, 1489, 1428, 1223, 1146, 751 cm− . Mp 200–205 ◦C from EtOH. 4-(2-((5-Methoxy-1H-indol-3-yl)methylene)hydrazineyl)benzonitrile (29). Yield: 30%. 1H-NMR (DMSO-d6) δ 3.82 (s, 3H, OCH3), 6.84 (dd, 1H, Jm = 2.6, Jo = 8.8 Hz, H-6), 7.06 (d, 2H, J = 8.6 Hz, H-20 and H-60), 7.31 (d, 1H, Jo = 8.8 Hz, H-7), 7.59 (d, 2H, J = 8.6 Hz, H-30 and H-50), 7.69 (s, 1H, H-2), 7.71 (d, 1H, Jm = 2.6 Hz, H-4), 8.17 (s, 1H, CH=N), 10.55 (s, 1H, NH), 11.33 (s, 1H, NNH). MS (ESI), m/z = 288.8 (100%) [M H] . IR (KBr): 3290, 2220, 1603, 1532, 1290, 1258 cm 1. Mp 227–229 C from EtOH. − − − ◦ 3-((2-(3-Chlorophenyl)hydrazineylidene)methyl)-5-methoxy-1H-indole (31). Yield: 30%. 1H-NMR (DMSO-d6) δ 3.84 (s, 3H, OCH3), 6.66 (dd, 1H, Jm = 2.1, Jo = 8.3 Hz, H-40), 6.82 (dd, 1H, Jm = 2.6, Jo = 8.8 Hz, H-6), 6.86 (dd, 1H, Jm = 2.1, Jo = 8.3 Hz, H-60), 7.11 (t, 1H, Jm = 2.1 Hz, H-20), 7.18 (t, 1H, Jo = 8.3, H-50), 7.30 (d, 1H, Jo = 8.8 Hz, H-7), 7.61 (br s, 1H, H-2), 7.75 (d, 1H, Jm = 2.6 Hz, H-4), 8.09 (s, 1H, CH=N), 10.08 (s, 1H, NH), 11.23 (s, 1H, NNH). MS (ESI), m/z = 297.8 (100%) [M H] , 299.8 (33%) − − [M H] +2. IR (KBr): 3430, 2922, 1614, 1596, 1484, 1210 cm 1. Mp 83–85 C from ethyl acetate/hexane − − − ◦ 50:50 (v/v). 1 3-((2-(4-Chlorophenyl)hydrazono)methyl)-1H-indole (32). Yield: 30%. H-NMR (acetone-d6) δ 7.15 (d, 2H, J = 9.1 Hz, H-20 and H-60), 7.23 (d, 2H, J = 9.1 Hz, H-30 and H-50), 7.13–7.25 (m, 2H, H-6 and H-7), 7.43–7.47 (m, 1H, H-4), 7.59 (s, 1H, H-2), 8.17 (s, 1H, H-8), 8.36–8.42 (m, 1H, H-5), 9.13 (s, 1H, NH), 10.47 (s, 1H, NNH). MS (ESI), m/z = 267.9 (100%) [M H]−, 269.8 (39%) [M H]− + 2. IR (KBr): 3412, 1 − − 3302, 1599, 1497, 1433, 1074, 747 cm− . Anal. calcd for C15H12N3Cl: C, 66.79; H, 4.48; N, 15.58. Found: C, 66.45; H, 5.14; N, 12.11. Mp 117–122 ◦C from ethyl acetate/hexane 50:50 (v/v). 5-Methoxy-3-((2-(pyridin-2-yl)hydrazineylidene)methyl)-1H-indole (34). Yield: 50%. 1H-NMR (DMSO-d6) δ 3.82 (s, 3H, OCH3), 6.64–6.67 (m, 1H, H-40), 6.82 (dd, 1H, Jo = 8.8, Jm = 2.5 Hz, H-6), 7.15 (d, 1H, Jo = 8.2 Hz, H-60), 7.30 (d, 1H, Jo = 8.8 Hz, H-7), 7.60 (br s, 1H, H-2), 7.63–7.66 (m, 1H, H-50), 7.74 (d, 1H, Jm = 2.5 Hz, H-4), 8.04–8.08 (m, 1H, H-30), 8.21 (s, 1H, CH=N), 10.41 (s, 1H, NH), + 11.24 (s, 1H, NNH). MS (ESI), m/z = 264.9 (100%) [M H]−, 289.0 (100%) [M + Na] . IR (KBr): 3439, 1 − 3184, 2998, 1598, 1444 cm− . Mp 185–187 ◦C from EtOH. 1 2-(2-((5-Methoxy-1H-indol-3-yl)methylene)hydrazineyl)quinoline (35). Yield: 50%. H-NMR (DMSO-d6) δ 3.87 (s, 3H, OCH3), 6.84 (dd, 1H, Jo = 8.8, Jm = 2.6 Hz, H-6), 7.20–7.24 (m, 1H), 7.32 (d, 1H, Jo = 8.8 Hz, H-7), 7.50–7.73 (m, 5H), 7.80 (d, 1H, Jm = 2.2 Hz, H-4), 8.17 (d, 1H, Jo = 9.2 Hz), 8.27 (s, 1H, CH=N), + 10.96 (s, 1H, NH), 11.28 (s, 1H, NNH). MS (ESI), m/z = 314.8 (100%) [M H]−, 317.0 (100%) [M + Na] , + 1 − 339.0 (42%) [M+H] . IR (KBr): 3432, 2948, 1607, 1571, 1430 cm− . Mp 233–235 ◦C from EtOH. 7-Chloro-4-(2-((5-methoxy-1H-indol-3-yl)methylene)hydrazineyl)quinoline (36). Yield: 44%. 1H-NMR (DMSO-d6) δ 3.86 (s, 3H, OCH3), 6.87 (d, 1H, Jo = 8.4 Hz, H-6), 7.24–7.26 (m, 1H), 7.36 (d, 1H, Jo = 8.4 Hz, H-7), 7.54 (d, 1H, Jo = 8.4 Hz), 7.65–7.87 (m, 3H), 8.41–8.44 (m, 3H), 8.64 (br s, 1H, CH=N), 11.51 (br s, + + 1H, NNH). MS (ESI), m/z = 348.9 (100%) [M H]−, 373.0 (100%) [M + Na] , 351.0 (16%) [M+H] . 1 − IR (KBr): 3351, 3231, 1615, 1578, 1425 cm− . Mp >255 ◦C from EtOH. Molecules 2020, 25, 5773 20 of 26

3.2.10. 3-(2-(4-Isopropylphenyl)hydrazineylidene)isoindolin-1-one (38) The 3-iminoisoindolinone 37 previously prepared [47] (1 mmol, 1 eq) was dissolved in 5 mL of methanol, then 4-isopropylphenylhydrazine (2.4 eq) was added at room temperature. After stirring overnight, the mixture was ﬁltered and the precipitate was puriﬁed by crystallization. Yield: 60%. 1 H-NMR (DMSO-d6) δ 1.16 (d, 6H, J = 7.0 Hz, CH(CH3)2), 2.80 (ep, 1H, J = 7.0 Hz, CH(CH3)2), 7.03 (d, 2H, J = 8.6 Hz, H-20 and H-60), 7.13 (d, 2H, J = 8.6 Hz, H-30 and H-50), 7.53 (td, 1H, Jo = 7.4, Jm = 1.1 Hz, H-5), 7.68 (td, 1H, Jo = 7.4, Jm = 1.1 Hz, H-6), 7.75 (dd, 1H, Jo = 7.4, Jm = 0.8 Hz, H-4), 7.83 (dd, 1H, Jo = 7.4, Jm = 0.8 Hz, H-7), 9.31 (s, 1H, NH), 10.72 (s, 1H, NNH). ESI-MS m/z = 277.9 (100%) [M–NH]−. 1 IR (KBr): 3351, 2964, 1712, 1612, 1127 cm− . Mp 186–187 ◦C from CHCl3/hexane.

3.2.11. Synthesis of 5-Bromo-3-chloro-2-((2-phenylhydrazineylidene)methyl)-1H-indoles 40 and 41 Compound 39 (0.3 mmol, 1 eq), previously synthesized by conversion of acyclic dicarboxylic to aldehyde under Vilsmeier condition [21], was solubilized in ethanol (1 M). Catalytic amount of acetic acid and suitable arylhydrazine (1.2 eq) were added to the solution and the mixture was stirred for 4 h at 0 ◦C. Then the solvent was evaporated, and the crude product was crystalized or puriﬁed by column chromatography. 1 5-Bromo-3-chloro-2-((2-phenylhydrazineylidene)methyl)-1H-indole (40). Yield: 40%. H-NMR (CDCl3) δ 6.93 (t, 1H, Jo = 7.4 Hz, H-40), 7.12 (d, 2H, Jo = 8.8, H-20 and H-60), 7.22 (d, 1H, Jo = 8.8 Hz, H-7), 7.27–7.35 (m, 3H), 7.72 (br s, 1H, CH=N), 7.82 (s, 1H, H-4), 7.86 (s, 1H, NNH), 8.71 (s, 1H, NH). MS (ESI), m/z = 346.0 (54%) [M H]−, 347.8 (100%) [M H]− +2, 349.7 (25%) [M H]− +4. IR (KBr): 3415, 1 − − − 1630, 1460 cm− . Mp 165–170 ◦C from CHCl3/hexane. 5-Bromo-3-chloro-2-((2-(4-isopropylphenyl)hydrazineylidene)methyl)-1H-indole (41). Yield: 20%. 1 H-NMR (DMSO-d6) δ 1.16 (d, 6H, Jv = 6.9 Hz, CH(CH3)2), 2.80 (ep, 1H, Jv = 6.9 Hz, CH(CH3)2), 7,07–7.12 (m, 4H, H-20, H-30, H-40, H-50 and H-60), 7.28 (dd, 1H, Jo = 8.8, Jm = 1.8 Hz, H-6), 7.36 (d, 1H, Jo = 8.8 Hz, H-7), 7.55 (d, 1H, Jm = 1.8 Hz, H-4), 7.89 (s, 1H, CH=N), 10.64 (s, 1H, NH), 11.66 (s, 1H, NNH). MS (ESI), m/z = 387.8 (73%) [M H]−, 389.7 (100%) [M H]− +2, 391.7 (26%) [M H]− +4. −1 − − IR (KBr): 3382, 2956, 1537, 1515, 1254 cm− . Mp 140–143 ◦C from hexane/ethyl acetate 90:10 (v/v).

3.2.12. 2-(2,4-Dihydroxyquinolin-3-yl)-3H-indol-3-one (43) The dihydroxyquinolinedione 42 [22] (0.5 mmol, 1 eq) was mixed with indoxyl acetate (1 eq) in neat condition and heated to 120 ◦C. After 15 min the mixture was cooled and dissolved with diethyl ether. The organic solvent was decanted and evaporated obtaining the desired compound. Yield: 45%. 1 H-NMR (DMSO-d6) δ 6.90 (t, 1H, Jo = 7.4 Hz), 7.04–7.12 (m, 4H), 7.38 (d, 1H, Jo = 8.3 Hz), 7.58 (t, 1H, Jo = 7.7 Hz), 7.70 (d, 1H, Jo = 7.7 Hz), 10.97 (s, 1H, OH), 11.05 (s, 1H, OH). MS (ESI), m/z = 289.8 (100%) [M H] . IR (KBr): 3385, 1713, 1673, 1614 cm 1. Mp 90–92 C. − − − ◦ 3.3. Biological Assays All assays were performed in 96-well plates (Greiner Bio-One, Kremsmünster, Austria) using an Inﬁnite M1000 Pro plate reader (Tecan, Cernusco s.N., Italy). Inhibition data were obtained as mean SD (for single concentration points) or mean SEM from three independent experiments (for IC s), ± ± 50 using Prism (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA).

3.3.1. Inhibition of Aβ40 Self-Aggregation

Assay of aggregation of Aβ40 (EzBiolab, Carmel, IN, USA) was performed as described [25], using 2% of 1,1,1,3,3,3-hexafluoro-2-propanol as aggregation enhancer and spectrofluorimetric detection of thioflavin T fluorescence as the probe of amyloid aggregation. Molecules 2020, 25, 5773 21 of 26

3.3.2. Inhibition of Cholinesterases Inhibition of human recombinant AChE (2770 U/mg) or BChE from human serum (50 U/mg; both from Sigma Aldrich, St. Louis, MO, USA), was determined as described [48], by applying the Ellman’s spectrophotometric method to a 96-well plate procedure.

3.3.3. Inhibition of Monoamine Oxidases Inhibition of human recombinant monoamine oxidases A (250 U/mg) and B (59 U/mg; microsomes from baculovirus infected insect cells; Sigma Aldrich) was determined as already described [48], measuring the ﬂuorescence of 4-hydroxyquinoline produced by the oxidative deamination of substrate kynuramine.

3.4. In Vitro Cell-Based Assays Compounds for bacterial media were purchased from CondaLab (Madrid, Spain). M9 minimal medium; for 100 mL: 10 mL salts 10 (0.68 g Na HPO , 0.30 g KH PO , 0.05 g NaCl, 0.10 g NH Cl), × 2 4 2 4 4 0.2 mL 1M MgSO4, 0.2 mL 50 mM CaCl2, 2.5 mL 20% glucose and 87.1 mL H2O.

3.4.1. Tau Aggregation Inhibition Assay in Bacterial Cells Escherichia coli competent cells BL21 (DE3) were transformed with pTARA containing the RNA-polymerase gen of T7 phage (T7RP) under the control of the promoter PBAD and pRKT42 vector encoding four repeats of tau protein in two inserts. Because of the addition of the initiation codon ATG in front of gene, the over-expressed protein contains an additional methionine residue at its N terminus. Tau anti-aggregating activities of the target compounds were assessed in E. coli cells, as previously described [27–29]. M9 minimal medium (10 mL) containing 0.5% of glucose, 100 µg/mL of ampicillin and 12.5 µg/mL of chloramphenicol was inoculated with a colony of BL21 (DE3) cells bearing the plasmids. To fresh M9 minimal medium containing 0.5% of glucose, 50 µg/mL of ampicillin, 12.5 µg/mL of chloramphenicol, and 250 µM of ThS, the volume of overnight culture necessary to get a 1:500 dilution was added. The cultures were grown at 37 ◦C and 250 rpm overnight until cell density reached OD600 = 0.6. A volume of 980 µL of the cultures was transferred into 1.5 mL Eppendorf tubes that contained 10 µL of a solution of the target compound in DMSO and 10 µL of arabinose at 25%, leading to a final inhibitor concentration of 10 µM. The resulting cultures were grown at 37 ◦C and 1400 rpm with a Thermomixer (Eppendorf, Hamburg, Germany) overnight. The same amount of DMSO without the target compound was added to the sample as a negative control (maximal amount of tau), whereas non-induced samples (in the absence of arabinose) were prepared as positive controls (absence of tau), and to assess the potential intrinsic toxicity of the target compounds. In addition, the absorbance at 600 nm of these samples was assessed to confirm the correct bacterial growth, discarding potential intrinsic toxicity of compounds. To determine the IC50 values, the same protocol was followed but only modifying the initial concentration of compounds. In order to use different final concentrations of compound, we performed a dilutions banc from a stock solution of 100 mM in MilliQ H2O.

3.4.2. Thioflavin S Steady-State Fluorescence ThS fluorescence and absorbance were tracked using a DTX 800 plate reader Multimode Detector equipped with a Multimode Analysis Software (Beckman-Coulter, Indianapolis, IN, USA). Filters of 430/35 and 485/20 nm were used for the excitation and emission wavelengths, respectively. 535/25 nm filters were also used for the absorbance determination. It should be stressed that ThS fluorescence normalization was carried out considering as 100% the ThS fluorescence of the bacterial cells expressing tau in the absence of compounds and 0% the ThS fluorescence of the bacterial cells non-expressing tau. Molecules 2020, 25, 5773 22 of 26

3.5. Computational Studies For each compound a suitable three-dimensional structure was built by employing MacroModel [49] for energy minimization based on OPLS_2005 force field and by setting a maximum of 100 bioactive conformers as set in ConfGen [50]. Ligprep [51] was employed for determining possible protonation states at physiological pH. The pharmacophore and atom-based 3D-QSAR models were then derived by using Phase [41] after entering the prepared structures along with their respective biological activity values. The ligands were thus designated as actives or inactives based on the IC50 threshold equal to 100 µM. Flexible ligands alignment was automatically performed by Phase. The maximum and minimum number of features was set to 7 and 4, respectively, with a tolerance sphere of 2 Å, provided that at least 50% of actives must match. Excluded volumes were also accounted to better discern inactives for which the clash value was set to 1 and the minimum distance between active surface and excluded volume was set to 1 Å. In doing that, 13 different pharmacophore hypotheses were generated sorted on the values of the Phase Hypo Score, which is a linear combination of different contributes related with site, volume, vector and selectivity scores [52,53]. 3D-QSAR model was based on partial least squares (PLS) regression statistics on grid descriptors (that are HBA, HBD, hydrophobic interactions and electron withdrawing effects) sampled on a lattice with a spacing of 1 Å. The number of optimal PLS factors was equal to 4 given that the increase of q2 was not higher than 5% by adding a further component. For the sake of clarity, only compounds with experimentally measured IC50 values were included in the analysis.

4. Conclusions Building on our previous results, we progressed the decoration of the isatin scaffold to prepare a large number of derivatives displaying antiamyloidogenic activity over a wide range of potency. Five out of the 36 new compounds displayed IC50 values in the low to submicromolar range, being N-substituted isatins 9 (IC50 = 0.95 µM), 18 (1.3 µM) and 19 (1.6 µM) the most potent inhibitors of Aβ aggregation. This feature was in full agreement with our previous observations [19] of the enhancing effect brought by N-substitution at the isatin nitrogen, as herein confirmed by comparing the pairs 6–7 and 8–9. The screening of selected compounds in tau overexpressing cells allowed to disclose an interesting inhibitory activity against tau aggregation, although no apparent correlation was found between this activity and in vitro inhibition of Aβ aggregation. Among the tested compounds, isatin 3-phenylhydrazone 5 scored the best activity (4.6 µM). Taking into account our previous observations assessing the low intrinsic toxicity of isatin scaffold in different cellular assays [17–19], we may conclude that this class of molecules has the potential for further development as antiamyloidogenic agents with safe cytotoxic profile. In this paper, we also expanded the exploration of SAR to indole derivatives, most of them bearing the arylhydrazone moiety (compounds 28–36) and resulting with few exceptions in good inhibitors of Aβ aggregation. Other structural variations based on molecular simplification or shift of hydrazone substituent were not favorable for activity. In the perspective of discovering new multitarget agents for AD, we selected some candidates for their evaluation as inhibitors of AChE and MAOs, two target enzymes involved in neurodegenerative diseases. Satisfactorily, several isatin and indole derivatives displayed interesting MAO inhibition, overall selective (compounds 9, 11, 13) towards the B isoform. Unsurprisingly, isatin hydrazone 5 resulted a potent MAO A inhibitor, since the MAO activity has been reported for different isatin derivatives [35–37]. Conversely, we found indole derivative 32 as the most potent, although nonselective, MAO inhibitor with IC50 values of 0.34 and 0.23 µM for MAO A and MAO B, respectively. Even considering its good potency in inhibition of Aβ aggregation (8.4 µM), the multitarget profile of 32 as dual MAO/Aβ aggregation inhibitor deserves further insights, mainly aiming to improve A/B selectivity. The data obtained from in vitro tests of inhibition of Aβ aggregation prompted us to derive a pharmacophore model for Aβ antiaggregating activity. The molecular shape, lipophilicity of Molecules 2020, 25, 5773 23 of 26 substituents, and the substitution at the isatin nitrogen appeared to be the main determinants for antiamyloidogenic activity. The combined approach based on pharmacophore and 3D-QSAR models allowed us to furnish more confident predictions thus allowing to better address the rational design of new active and more promising compounds.

Supplementary Materials: The following are available online, Chemical structures, SMILES codes, and inhibition values of 93 previously published compounds constituting the training set. Author Contributions: M.C. and C.D.A. conceived the work; M.C., R.S., S.C. and O.N. designed the experiments; R.P., N.G., M.C., M.d.C., L.P. and A.E. performed the experiments; R.P., N.G., M.C. and R.S. wrote the paper; all authors analysed the data and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: M.C. and L.P. acknowledge the financial support from Italian Ministry for Education and Research (Fondo di Finanziamento per le Attività Base di Ricerca, FFABR 2017). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–45 are available from the authors

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