Insights into Structure-Activity Relationships of 3

molecules Article

Insights into Structure-Activity Relationships of 3-Arylhydrazonoindolin-2-One Derivatives for Their Multitarget Activity on β-Amyloid Aggregation and Neurotoxicity Rosa Purgatorio 1 , Modesto de Candia 1 , Annalisa De Palma 2 , Francesco De Santis 2 , Leonardo Pisani 1 ID , Francesco Campagna 1 , Saverio Cellamare 1 , Cosimo Damiano Altomare 1 and Marco Catto 1, * ID 1

2

*

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via E. Orabona 4, I-70125 Bari, Italy; [email protected] (R.P.); [email protected] (M.d.C.); [email protected] (L.P.); [email protected] (F.C.); [email protected] (S.C.); [email protected] (C.D.A.) Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari “Aldo Moro”, via E. Orabona 4, I-70125 Bari, Italy; [email protected] (A.D.P.); [email protected] (F.D.S.) Correspondence: [email protected]; Tel.: +39-080-544-2780

Received: 11 May 2018; Accepted: 24 June 2018; Published: 26 June 2018

Abstract: Despite the controversial outcomes of clinical trials executed so far, the prevention of β-amyloid (Aβ) deposition and neurotoxicity by small molecule inhibitors of Aβ aggregation remains a target intensively pursued in the search of effective drugs for treating Alzheimer’s disease (AD) and related neurodegeneration syndromes. As a continuation of previous studies, a series of new 3-(2-arylhydrazono)indolin-2-one derivatives was synthesized and assayed, investigating the effects of substitutions on both the indole core and arylhydrazone moiety. Compared with the reference compound 1, we disclosed equipotent derivatives bearing alkyl substituents at the indole nitrogen, and fairly tolerated bioisosteric replacements at the arylhydrazone moiety. For most of the investigated compounds, the inhibition of Aβ40 aggregation (expressed as pIC50 ) was found to be correlated with lipophilicity, as assessed by a reversed-phase HPLC method, through a bilinear relationship. The N1 -cyclopropyl derivative 28 was tested in cell-based assays of Aβ42 oligomer toxicity and oxidative stress induced by hydrogen peroxide, showing significant cytoprotective effects. This study confirmed the versatility of isatin in preparing multitarget small molecules affecting different biochemical pathways involved in AD. Keywords: beta-amyloid aggregation inhibitors; indolin-2-ones; Alzheimer’s disease; quantitative structure-activity relationships; multitarget activity

1. Introduction Alzheimer’s disease (AD) is the most common cause of age-related neurodegenerative pathologies. AD represents a serious challenge for health systems, physicians and caregivers, because of its disabling course and the limited efficacy of pharmacological therapies [1]. The treatments approved for AD, namely the restoration of cholinergic transmission by means of acetylcholinesterase inhibitors [2], and the neuroprotection from glutamate excitotoxicity exerted by memantine [3], are only symptomatic and do not meet the clinical need for effective disease-modifying drugs. A typical feature of AD consists in the deposition of extracellular β-amyloid (Aβ) peptide aggregates (amyloid plaques), starting from cholinergic neurons of hippocampus and then Molecules 2018, 23, 1544; doi:10.3390/molecules23071544

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A typical feature of AD consists in the deposition of extracellular β-amyloid (Aβ) peptide aggregates (amyloid plaques), starting from cholinergic neurons of hippocampus and then progressively extending to the whole brain cortex [4]. Aβ peptide derives from proteolysis of amyloid progressively extending to the whole brain cortex [4]. Aβ peptide derives from proteolysis of precursor protein (APP) catalyzed by β- and γ-secretases, and is formed as 40- (Aβ40 ) or 42-mer amyloid precursor protein (APP) catalyzed by β- and γ-secretases, and is formed as 40- (Aβ40) or (Aβ42 ). Aβ monomers in AD brains aggregate to soluble oligomers, that in turn lead to intermediate 42-mer (Aβ42). Aβ monomers in AD brains aggregate to soluble oligomers, that in turn lead to protofibrils and finally to the deposition of amyloid plaques [5]. The formation of such oligomeric intermediate protofibrils and finally to the deposition of amyloid plaques [5]. The formation of such and prefibrillar species is correlated with the neurotoxicity in the AD brain, which represents a major oligomeric and prefibrillar species is correlated with the neurotoxicity in the AD brain, which causal factor of the cognitive impairment and the synaptic loss in AD patients [6–8]. represents a major causal factor of the cognitive impairment and the synaptic loss in AD patients [6–8]. The amyloidogenesis of Aβ occurs since the early stages of the disease insurgence, so The amyloidogenesis of Aβ occurs since the early stages of the disease insurgence, so that that preventing the oligomerization and/or fibrillization process could represent a promising preventing the oligomerization and/or fibrillization process could represent a promising disease-modifying treatment for AD. Despite the number of research findings in this field, only disease-modifying treatment for AD. Despite the number of research findings in this field, only a a very small number of molecules have reached the preclinical stage, and no one entered therapy very small number of molecules have reached the preclinical stage, and no one entered therapy so so far [9]. However, many small molecules acting as disruptors of protein-protein interactions have far [9]. However, many small molecules acting as disruptors of protein-protein interactions have demonstrated potential in inhibiting Aβ aggregation [10]. Among them, indole derivatives such demonstrated potential in inhibiting Aβ aggregation [10]. Among them, indole derivatives such as as melatonin [11], fluorinated indoles [12], hydroxyindoles [13] (Figure 1) displayed the structural melatonin [11], fluorinated indoles [12], hydroxyindoles [13] (Figure 1) displayed the structural features for an efficient antiaggregating activity. Particularly, they act as intercalators in hydrophobic features for an efficient antiaggregating activity. Particularly, they act as intercalators in interactions between Aβ side chains, including aromatic π-stacking interactions [14–17]. As recently hydrophobic interactions between Aβ side chains, including aromatic π-stacking interactions [14– shown by our structure-activity relationship (SAR) studies [16,18], these hydrophobic interactions 17]. As recently shown by our structure-activity relationship (SAR) studies [16,18], these taking place between Aβ and many classes of small molecules could be reinforced by polar interactions hydrophobic interactions taking place between Aβ and many classes of small molecules could be and/or hydrogen bond (HB) formation. reinforced by polar interactions and/or hydrogen bond (HB) formation.

Figure 1. Structures of indole derivatives endowed with amyloid anti-aggregating properties. Figure 1. Structures of indole derivatives endowed with amyloid anti-aggregating properties.

From previous studies [19–22], 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone (1, Figure 1) previous [19–22], 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone (1,(AChE), Figure 1)with was was From identified as a studies promising inhibitor of Aβ aggregation and acetylcholinesterase identified as a promising inhibitor of Aβ aggregation and acetylcholinesterase (AChE), with IC in 50 s of IC50s in the submicromolar and low μM range, respectively. Herein, we synthesized a number the submicromolar and low µMevaluated range, respectively. synthesized number of congeners congeners of compound 1, and the in vitroHerein, activitywe as inhibitors of a Aβ 40 aggregation. With of compound 1, and evaluated the in vitro activity as inhibitors of Aβ aggregation. With the 40 the aim of extending the SAR exploration, the new derivatives were designed in order to undertake aim of extending the SAR exploration, the new derivatives were designed in order to undertake a more systematic exploration (Figure 2) of: (i) the 3-arylhydrazone moiety, by introducing a number a more systematic exploration 2) of:the(i)aryl the 3-arylhydrazone moiety, by introducing a of diverse substituents and/or (Figure modifying substituent (compounds 4–11, Scheme 1; number of diverse substituents and/or modifying the aryl substituent (compounds 4–11, Scheme 1; compounds 13–18, Scheme 2); (ii) the length and the chemical nature of the linker (compounds 19 compounds 13–18, Scheme the length either and the nature of the linker24,(compounds 19 and 21–23, Scheme 3); (iii) 2); the(ii) substitution onchemical the nitrogen (compounds 26, 28, 32–34; and 21–23, Scheme 3); (iii) the substitution either on the nitrogen (compounds 24, 26, 28, 32–34; Scheme 4) and the benzene moiety of the indole ring (compounds 36–39, Scheme 5). SARs, including Scheme 4) and the benzene moiety of the indole ring (compounds 36–39, Scheme correlation of antiaggregating activity with lipophilicity, were investigated, and 5). for SARs, one ofincluding the most correlation of antiaggregating activity with lipophilicity, were investigated, and for one of the potent Aβ40 aggregation inhibitors (compound 28) cytoprotection from toxic Aβ42 oligomersmost in a potent Aβassay inhibitors (compound from toxic Aβ42stress oligomers a 40 aggregation cell-based was evaluated. Moreover, taking28) intocytoprotection account the role of oxidative in AD in and cell-based assay was evaluated. Moreover, taking into account the role of oxidative stress in AD and other neurodegenerative diseases [23], the antioxidant property of 28 against hydrogen peroxide other neurodegenerative diseases [23], the antioxidant property of 28 against hydrogen peroxide insult insult in SH-SY5Y cells was also tested. in SH-SY5Y cells was also tested.

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variation of substitution in para position Molecules 2018, 23, 1544

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(hetero)aromatic rings


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variation of substitution

substituent(s) in in para position 5, 6 and 7 position

N

NH

O

substituent(s) in 5, 6 and 7 position

N H

NH

N

(hetero)aromatic rings

length and chemical nature of linker

substitution on indole nitrogen

length and chemical nature of linker

Figure 2. Summary of the investigated structural modifications of isatin-3-arylhydrazones.

O 2. Results and Discussion

N H

2.1. Chemistry

substitution on New 5-methoxy-3-arylhydrazonoindolin-2-ones 4–11 were prepared by condensation of indole nitrogen 5-methoxyisatin with the corresponding (hetero)arylhydrazines (compounds 4–7, 9, 11) or alternatively via azo coupling of aryldiazonium salts 3a,b with 5-methoxyindolin-2-one 2 [24] to Figure 2. Summary of the the investigated modifications isatin-3-arylhydrazones. 2. Summary investigatedstructural structural1). modifications of of isatin-3-arylhydrazones. yield theFigure compounds 8 andof10, respectively (Scheme O 2. Results and Discussion

Ar

i

H3CO

O N H

2.1. Chemistry

4

NH2

8

CN

W

New 5-methoxy-3-arylhydrazonoindolin-2-ones 4–11 were prepared by condensation of 5-methoxyisatin with the corresponding (hetero)arylhydrazines (compounds 4–7, 9, 11) or Ar 9 CF3 5 with 5-methoxyindolin-2-one alternatively via azo coupling of aryldiazonium salts 3a,b 2 [24] to N N NH iv iii ii yield the compounds 8 and 10, respectively H3CO (Scheme 1). O

O

H3CO

N2

iW

O N H W

H3CO

N H

CF3

3b (W = N)

10 CF3

4

2 CH) (W = 3a NH

O

Ar

6

N

8

CN 11

7

N

N

N H

v

Ar 9 CF3 5 Cl N N NH iv and conditions:CO(i) ArNHNH2, MeOH, room temperature; (ii) NH2NH2∙H2O, iii ii Scheme 1. Reagents 3 ArNHNH , MeOH, room temperature; (ii) NH NH ·H O, EtOH; Scheme 1. Reagents and conditions:H(i) 2 2 2 2 EtOH; (iii) NaOH, EtOH, H2O, reflux; (iv) NaNO2, HCl, 0 °C; (v) MeOH, CH3COONa, O (v) ◦ C. 0 °C. (iii) NaOH, EtOH, H2 O, reflux; (iv) NaNO2 , HCl, 0 ◦ C; MeOH, CH3 COONa, 0 CF 3 N H Thiazolylhydrazones N13–18 were synthesized through the Hantzsch reaction [25] of 2 6 10 bromomethyl ketonesWwith 2-(5-methoxy-2-oxoindolin-3-ylidene)hydrazinecarbothioamide 12 [26] 2

CF3

(Scheme 2).

3a (W = CH) 3b (W = N)

H3CO O N H

N

11

7

N

N

v

2

Cl

Scheme 1. Reagents and conditions: (i) ArNHNH2, MeOH, room temperature; (ii) NH2NH2∙H2O, EtOH; (iii) NaOH, EtOH, H2O, reflux; (iv) NaNO2, HCl, 0 °C; (v) MeOH, CH3COONa, 0 °C.

Thiazolylhydrazones 13–18 were synthesized through the Hantzsch reaction [25] of


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Scheme 2. Reagents and conditions: (i) thiosemicarbazide, isopropanol, room temperature; (ii) (bromomethyl) alkyl or aryl ketone, EtOH, room temperature.

Hydrazones 19, 21, and 22 were prepared through condensation of the corresponding carbaldehydes and hydrazines, while compound 23 was synthesized by condensation of 5-methoxyisatin with 3-chlorobenzohydrazide. The 3-arylidenehydrazono-5-methoxyindolin-2-one Scheme 2. Reagents thiosemicarbazide, isopropanol, roomroom temperature; (ii) Scheme 2. Reagentsand andconditions: conditions:(i) (i) thiosemicarbazide, isopropanol, temperature; derivatives 21 and 22 required the preparation of 5-methoxyisatin-3-hydrazone 20 [27], which was (bromomethyl) alkyl or aryl ketone, EtOH, room temperature. (ii) (bromomethyl) alkyl or aryl ketone, EtOH, room temperature. then reacted with 4-isopropyl or 3-chlorobenzaldehyde, respectively (Scheme 3).

Hydrazones 19, 21, and 22 were prepared through condensation of the corresponding carbaldehydes and hydrazines, while compound 23 was synthesized by condensation of HN 5-methoxyisatin with 3-chlorobenzohydrazide. The 3-arylidenehydrazono-5-methoxyindolin-2-one N derivatives 21 and 22 requiredCHO the preparation of 5-methoxyisatin-3-hydrazone 20 [27], which was H CO H34-isopropyl CO 3 then reacted with or 3-chlorobenzaldehyde, respectively (Scheme 3). i N H

N H

CHO

19 N

HN H3CO

i

H3CO R

N H

N H

ii

H3CO

N NH2

19 iii

O

O

O

H3CO

ii

iv

O O N H

N H

20

N H

N N

O N H

H3CO

H3CO

H3CO

N NH2

Cliii

21 R = 4-CH(CH3)2 N N 22 R = 3-Cl H3CO

O

H3CO

O

N O H N NH

N H

20

O N H

R

Cl

21 R = 4-CH(CH3)2 22 R = 3-Cl

O

23

N NH Scheme 3. and conditions: (i) 4-isopropylphenylhydrazine hydrochloride, hydrochloride, MeOH, room ivand H3conditions: CO Scheme 3. Reagents Reagents (i) 4-isopropylphenylhydrazine 2 NH 2 , AcOH, reflux; (iii) R-C 6 H 4 CHO, MeOH, reflux; reflux; (iv) temperature; (ii) NH O MeOH, room temperature; (ii) NH2 NH2 , AcOH, reflux; (iii) R-C6 H4 CHO, MeOH, N 3-chlorobenzohydrazide, MeOH, room temperature. (iv) 3-chlorobenzohydrazide, MeOH, room temperature. H

23 Scheme 3. Reagents and conditions: (i) 4-isopropylphenylhydrazine hydrochloride, MeOH, room temperature; (ii) NH2NH2, AcOH, reflux; (iii) R-C6H4CHO, MeOH, reflux; (iv) 3-chlorobenzohydrazide, MeOH, room temperature.


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The alkylation of the indole and/or hydrazone nitrogens was accomplished through different Moleculesmethods, 2018, 23, 1544 starting from commercial 5-methoxyisatin or the previously reported compound 1 [20]

5 of 23

(Scheme 4), which provided a series of N -alkyl-substituted derivatives 24, 26, 28, 32–34. 1

N NH

H3CO

i

N N

H3CO

O

O

N H

N

24

1

O

H3CO

ii

O

N NH

H3CO

O

N

i

N

26

25

O

H3CO

iii

O

H3CO

ii

N NH

H3CO

O

O N H

O

N

N

27

28

iv

O

H3CO

ii

N NH

H3CO

O

O

N (CH2)n

N (CH2)n

29 n = 1 30 n = 2 31 n = 3

32 n = 1 33 n = 2 34 n = 3

Scheme 4. Reagents and conditions: (i) Methyl iodide, K2CO3, DMSO, room temperature; (ii)

Scheme 4. Reagents and conditions: (i) Methyl iodide, K2 CO3 , DMSO, room temperature; 4-isopropylphenylhydrazine hydrochloride, MeOH, room temperature; (iii) Cyclopropylboronic (ii) 4-isopropylphenylhydrazine hydrochloride,bipyridine, MeOH, room 50 °C; temperature; (iv) Br(CH2)nC6(iii) H5, KCyclopropylboronic 2CO3, DMSO, acid, Na2CO3, Cu(OAc)2, 1,2-dichloroethane, ◦ acid, Naroom 2 COtemperature. 3 , Cu(OAc)2 , 1,2-dichloroethane, bipyridine, 50 C; (iv) Br(CH2 )n C6 H5 , K2 CO3 , DMSO, room temperature. Molecules 2018, 23, x

6 of 22 Finally, compounds 36–39 were synthesized by condensation of 4-isopropylphenylhydrazine hydrochloride with isatins 35a [28] and 35d [29] (Scheme 5), previously prepared through the Sandmeyer reaction [30], and commercial isatins 35b and 35c. Synthesis and characterization of compounds 15 [31], 25 [32] and 29 [33] have been already reported.

N NH

O

i, ii R

NH2

O R

35a 35b 35c 35d

N H

O R

R = 5-n-butyl R = 7-Br R = 5-NaSO3 R = 5,6-

iii

O

O

36 37 38 39

N H R = 5-n-butyl R = 7-Br R = 5-NaSO3 R = 5,6-

O

O

Scheme 5. Reagents and conditions: (i) CCl3CH(OH)2, Na2SO4∙10H2O, H2O, HCl conc., NH2OH∙HCl,

Schemereflux; 5. (ii) Reagents and 80 conditions: (i) CCl3 CH(OH)2 , Nahydrochloride, H2 O, room HCl conc., 2 SO4 ·10H2 O,MeOH, °C; (iii) 4-isopropylphenylhydrazine H2SO4 conc., ◦ NH2 OHtemperature. ·HCl, reflux; (ii) H2 SO4 conc., 80 C; (iii) 4-isopropylphenylhydrazine hydrochloride, MeOH, room temperature. 2.2. Inhibition of Amyloid Aggregation In vitro inhibition of Aβ aggregation was assessed through a ThT fluorescence-based method [18], with the use of 2% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as aggregation enhancer. In this medium-throughput assay, we preferred using Aβ40 peptide, being more manageable than Aβ42 and less prone to the formation of preaggregates [18]. Samples of Aβ were co-incubated with test molecules in PBS at 100 μM concentration and the antiaggregating activities were measured after 2 h

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2. Results and Discussion 2.1. Chemistry New 5-methoxy-3-arylhydrazonoindolin-2-ones 4–11 were prepared by condensation of 5-methoxyisatin with the corresponding (hetero)arylhydrazines (compounds 4–7, 9, 11) or alternatively via azo coupling of aryldiazonium salts 3a,b with 5-methoxyindolin-2-one 2 [24] to yield the compounds 8 and 10, respectively (Scheme 1). Thiazolylhydrazones 13–18 were synthesized through the Hantzsch reaction [25] of bromomethyl ketones with 2-(5-methoxy-2-oxoindolin-3-ylidene)hydrazinecarbothioamide 12 [26] (Scheme 2). Hydrazones 19, 21, and 22 were prepared through condensation of the corresponding carbaldehydes and hydrazines, while compound 23 was synthesized by condensation of 5-methoxyisatin with 3-chlorobenzohydrazide. The 3-arylidenehydrazono-5-methoxyindolin-2-one derivatives 21 and 22 required the preparation of 5-methoxyisatin-3-hydrazone 20 [27], which was then reacted with 4-isopropyl or 3-chlorobenzaldehyde, respectively (Scheme 3). The alkylation of the indole and/or hydrazone nitrogens was accomplished through different methods, starting from commercial 5-methoxyisatin or the previously reported compound 1 [20] (Scheme 4), which provided a series of N1 -alkyl-substituted derivatives 24, 26, 28, 32–34. Finally, compounds 36–39 were synthesized by condensation of 4-isopropylphenylhydrazine hydrochloride with isatins 35a [28] and 35d [29] (Scheme 5), previously prepared through the Sandmeyer reaction [30], and commercial isatins 35b and 35c. Synthesis and characterization of compounds 15 [31], 25 [32] and 29 [33] have been already reported. 2.2. Inhibition of Amyloid Aggregation In vitro inhibition of Aβ aggregation was assessed through a ThT fluorescence-based method [18], with the use of 2% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as aggregation enhancer. In this medium-throughput assay, we preferred using Aβ40 peptide, being more manageable than Aβ42 and less prone to the formation of preaggregates [18]. Samples of Aβ were co-incubated with test molecules in PBS at 100 µM concentration and the antiaggregating activities were measured after 2 h of incubation at 25 ◦ C. For compounds showing >80% Aβ40 aggregation inhibition, IC50 s were determined. The already reported antifibrillogenic activity of the indolylhydrazones [19,20] allowed us to identify (3Z)-5-methoxy-1H-indole-2,3-dione 3-[(4-isopropylphenyl)hydrazone] (1, Figure 1) as a hit compound, displaying strong inhibition of Aβ40 aggregation (IC50 0.43 µM) [20]. Compound 1 proved to be quite stable at pH 7.4 and 37 ◦ C (half-life 24 h) and to inhibit AChE (IC50 6.25 µM) [22]. An early objective of the present study was to replace the 4-iPr substituent with phenyl groups bearing polar and apolar electron-withdrawing substituents (compounds 4–6, Table 1). Such modifications determined a drop of the activity, and only the 30 ,50 -bis(trifluoromethyl) derivative 6 showed IC50 in the low micromolar range (9.9 µM), which resulted however 25-fold less potent than compound 1. The replacement of the hydrazone phenyl ring with a number of other aromatic or heteroaromatic rings was carried out for investigating the effects of bulkiness and/or additional π-π interactions on the anti-aggregating potency. The bioisosteric replacement of the phenyl group with pyrid-2-yl (7) resulted in a much lower activity (IC50 90 µM), whereas the 1-naphthyl moiety in compound 8 did recover a fair potency (IC50 28 µM), indicating that additional aromatic interactions may improve the antiaggregating potency. In contrast, quinolyl analogues 9 and 10, (similarly to the pyrid-2-yl congener 7), showed lower potency, and the 7-chloroquinolin-4-yl derivative 11 resulted a very weak inhibitor.

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Table1.1.Aβ AβAntiaggregating AntiaggregatingActivity ActivityofofCompounds Compounds1 1and and4–11. 4-11. Table Ar NH N H3CO O N H

Comp. 1 4 5 6 7 8 9 10 11

IC50 (µM) or (% Inhibition @ 100 µM) 1

Ar

Comp.

1

4

4-(CH(CH3 )2 )C6 H4 Ar 4-CNC6 H4 4-CF3 C6 H4 3,5-(CF3 )2 C6 H3 3)2)C6H4 4-(CH(CH Pyrid-2-yl 1-Naphthyl Quinolin-2-yl Quinolin-8-yl 4-CNC6H4 7-Cl-quinolin-4-yl 1

5

0.43 ± 0.04 IC50 (µM) or (% Inhibition @ 100 µM) 1 (16 ± 3) (57 ± 5) 9.9 ± 0.8 0.43 ± 0.04 90 ± 5 28 ± 2 (67 ± 4) (16 ± (58 3) ± 4) (14 ± 2)

Data are mean ± SEM of three independent experiments.

4-CF3C6H4

(57 ± 5)

Further information on the bioisosteric replacement of phenylhydrazone ring was achieved with thiazolylhydrazones 13–18 (Table 2). An exploration of the substituents on the thiazole moiety clearly 6 4-phenylthiazole 9.9indeed ± 0.8 proved to be a potent inhibitor 3,5-(CF3)2C6H3derivative 15, which indicated a preference for of Aβ aggregation (IC50 1.2 µM), apparently due to additional hydrophobic/aromatic interactions Moleculesby 2018, x 2 of 18 attained the23,phenyl group at the C4 position of the thiazole ring. 7

Pyrid-2-yl

90 ± 5

Table Aβ Antiaggregating Activity Compounds 13-18. Table 2. 2. Aβ Antiaggregating Activity ofof Compounds 13–18. 8

1-Naphthyl

R

28 ± 2 R1

S

9

Quinolin-2-yl

N

(67 ± 4)

N NH H3CO

10

Quinolin-8-yl

O N H

(58 ± 4)

R R1 IC50 (µM) or (% Inhibition @ 100 µM) 1 11 7-Cl-quinolin-4-yl (14 ± 2) 13 Comp. H R CH 13 ± 3 @ 100 µM) 1 R1 2 Cl IC50 (µM) or (% Inhibition 14 CH3 CH3 (43 ± 1) 1 Data are mean ± SEM of three independent experiments. 15 H C6 H5 1.2 ± 0.2 16 H 30 -OHC6 H4 16 ± 2 13 13 ± 3 H CH2Cl 17 30 -OCH3 C6 H 34number ±6 The replacement ofH the hydrazone phenyl ring with a of other aromatic or 4 0 -BrC H 18 H 3 40 ± 2 6 4 heteroaromatic rings was carried out for investigating the effects of bulkiness and/or additional π-π Comp.

Data are mean ± SEM ofThe threebioisosteric independent experiments. interactions on the anti-aggregating potency. replacement of the phenyl group with 14 CH3 (43 ± 1) CH3 pyrid-2-yl 0 (7) resulted in a much lower activity (IC50 90 μM), whereas the 1-naphthyl moiety in The 3 -substituted congeners of 4-phenylthiazol-2-yl derivatives 16–18, regardless the compound 8 did recover a fair potency (IC50 28 µM), indicating that additional aromatic interactions physicochemical feature of the meta substituent, retained antiaggregating activity in the micromolar 15 1.2analogues ± 0.2 H C6H5 In contrast, quinolyl may improve the antiaggregating potency. 9 and 10, (similarly to the range, but resulted 8-to-30-fold less potent than 15, thereby suggesting critical steric requirements pyrid-2-yl congener 7), showed lower potency, and the 7-chloroquinolin-4-yl derivative 11 resulted a for these derivatives. In contrast, smaller alkyl substituents, namely methyl or chloromethyl in very weak inhibitor. compounds 13 and 14,16respectively, displayed 16 ± 2the 4-chloromethyl derivative 13 H 3′-OHC6H4contrasting effects, with Further information on the bioisosteric replacement of phenylhydrazone ring was achieved retaining a fair anti-aggregating potency (IC50 13 µM), and 4,5-dimethyl analogue resulting a very with thiazolylhydrazones 13–18 (Table 2). An exploration of the substituents on the thiazole moiety weak inhibitor. clearly indicated a preference 4-phenylthiazole derivative 15, proved to be a potent H for 3′-OCH 3C6H 4 34which ± 6stepindeed According to our17 investigation strategy (Figure 2), the following was aimed at exploring the inhibitor of Aβ aggregation (IC50 1.2 µM), apparently due to additional hydrophobic/aromatic effects on the inhibition of Aβ aggregation of a few variations of the linker (length and the chemical interactions attained by the phenyl group at the C4 position of the thiazole ring. nature) between the two structural moieties (Table 3). 1

18

H

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3′-BrC6H4

40 ± 2

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The 3′-substituted congeners of 4-phenylthiazol-2-yl derivatives 16–18, regardless the physicochemical feature of the meta substituent, retained antiaggregating activity in the micromolar


Table 3. Aβ Antiaggregating Activity of Compounds 19 and 21–23.





Molecules 2018, 23, 1544 Table 3. Aβ Antiaggregating Activity of Compounds 19 and 21–23. Molecules 2018, 23, x

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Table3.3.Aβ AβAntiaggregating AntiaggregatingActivity ActivityofofCompounds Compounds19 19and and21–23. 21–23. Table Comp.

R

L

IC50 (µM) or (% inhibition @ 100 µM) 1

Comp.

R

L


Comp.

R

L


Comp. Comp. 19

R R

19 Comp.

L L

R

L

19 19

21

23

IC50 (µM) or 41 (%±inhibition @ 100 µM) 1 3

41 ± 3 IC50 (µM) or (% inhibition @ 100 µM) 1 41 ± 3

19

41 ± 341 ± 3

21 19 21

4-CH(CH3)2

N N

4-CH(CH3)2

N N

6.7 ± 0.2 41 ± 3 6.7 ± 0.2

21

4-CH(CH 4-CH(CH 3 )2 3)2

N N

6.7 ±6.7 0.2± 0.2

21 22 22


22 21 22 22 23 23 22 23

4-CH(CH3)2 3-Cl 3-Cl3-Cl 4-CH(CH 3-Cl 3)2

3-Cl 3-Cl3-Cl

N N N N N N N N N N

O N N O N NH

6.7 ± 0.2 11 ± 2 11 ±11 2 ±2 6.7 ± 0.2 11 ± 2 11 ± 2 (31 ±(31 3) ± 3)

3-Cl (31 ± 3) N ONH 3-Cl 11 ± 2 N N O 3-Cl are mean ± SEM (31 ± 3) Data N NHof three independent experiments. 1 Data are mean ± SEM of three independent experiments. 23 3-Cl (31 ± 3) NH NSEM A comparison of the1data that of the hit compound 1 revealed a 100-fold decrease Datain areTable mean3±with of three independent experiments. 1

Owas changed The introduction of alkyl and phenylalkyl groups indole N and methylation of the 1 Data of potency (from 0.43 to 41 µM), when linker inatthethe 1H-indole derivative 19, likely due to are mean ± SEM of three independent experiments. hydrazone NH in the hit structure 1 were then investigated (Table 4). Inhibition data highlighted The introduction of alkyl and phenylalkyl groups at the indole N and methylation of the the lack of intramolecular hydrogen bond (IMHB) between the hydrazone NH and the carbonyl O 23 3-Cl (31 ± 3) NH 1 Data N of three independent experiments. are mean ± SEM small bulky on the 1H-indole nitrogen of 1the were tolerated, thatmethylation the N-methyl hydrazone NH in thealkyls hit structure 1phenylalkyl were then investigated (Table 4). Inhibition data highlighted atthat position 2and of isatin, which favors co-planarity between two aromatic moieties. The inhibitory The introduction of alkyl and groups at the indole Nso and of(26), the N-cyclopropyl (28) and N-phenylalkyl (32–34) derivatives resulted almost equipotent with the(26), hit thatThe small and bulky alkyls onlow the 1H-indole nitrogen 1atwere tolerated, so that the N-methyl activity was maintained in the range byofthe aza-derivatives 21 (IC 6.7 µM), that is hydrazone NH in the hit structure 1phenylalkyl were then investigated (Table 4). N Inhibition data highlighted 50 introduction of alkyl andmicromolar groups the indole and methylation of the 1 compound 1 in the submicromolar range (IC 50 s ranging from 0.53 to 0.83 µM). A nonlinear relation Data are mean ± (32–34) SEM of three independent experiments. N-cyclopropyl (28) and N-phenylalkyl derivatives resulted almost equipotent with the hit a strict analog of 1, and 22 (IC 11 µM), that is the 3-chloro congener of 21, whereas a sharp drop that small and bulky alkyls on the 1H-indole nitrogen of 1 were tolerated, so that the N-methyl (26), 50 hydrazone NH in the hit structure 1 were then investigated (Table 4). Inhibition data highlighted compound 1 in the submicromolar range (ICnitrogen 50 s derivatives ranging 0.53 to 0.83 µM). Athe nonlinear relation between the antifibrillogenic potency and lipophilicity of the N-alkyl groups exists for these of activity was observed foronthe carboxyhydrazide derivative 23. The antiaggregating potency of N-cyclopropyl (28) and N-phenylalkyl (32–34) resulted almost equipotent with the hit that small and bulky alkyls the 1H-indole of 1from were tolerated, so that N-methyl (26), The introduction of alkyl and phenylalkyl groups at the indole N and methylation of the compounds, the N-benzyl derivative 32 being the most potent one within the subset (IC 50 0.53 µM). between the antifibrillogenic potency and lipophilicity of the N-alkyl groups exists for these compounds to be related to (IC their lipophilicity, as assessed logP calculated compound 121–23 in(28) theappears submicromolar range 50s ranging from 0.53 toalmost 0.83by µM). A values nonlinear relation N-cyclopropyl and N-phenylalkyl (32–34) derivatives resulted equipotent with the hit hydrazone NH inN-benzyl the hitofstructure 1and were then investigated (Table 4). Inhibition data In contrast, the potency hydrazone nitrogen (24) resulted in a0.83 more than decrease in compounds, the derivative 32 being the most potent one within the subset (IChighlighted 50 0.53 µM). with the ACDLab software (4.97, 4.43 3.53 for 21, 22 and 23, respectively). between the antifibrillogenic and lipophilicity of the groups exists for these compound 1 methylation in the submicromolar range (IC 50s ranging from 0.53 toN-alkyl µM). A 10-fold nonlinear relation that small and bulky alkyls on the 1H-indole nitrogen of 1 were tolerated, so that the N-methyl (26), the inhibition potency compared with the respective non-methylated compound (26), which proves In contrast, methylation of the hydrazone nitrogen (24) resulted in a more than 10-fold decrease The the introduction of alkyl and phenylalkyl at indole N groups andsubset methylation of thein compounds, the N-benzyl derivative 32and being thegroups most potent one within the (IC50for 0.53 µM). between antifibrillogenic potency lipophilicity of the the N-alkyl exists these N-cyclopropyl (28) and N-phenylalkyl (32–34) derivatives resulted almost equipotent with the hit the inhibition potency compared with the respective non-methylated compound (26), which proves that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position [22] hydrazone inN-benzyl the hit structure 1 were then investigated (Table Inhibition data highlighted that In contrast,NH methylation of the hydrazone nitrogen (24)potent resulted inwithin a more 10-fold in compounds, the derivative 32 being the most one4). thethan subset (IC50decrease 0.53 µM). compound 1 in the submicromolar range (IC 50 s ranging from 0.53 to 0.83 µM). A nonlinear relation and/or may introduce a steric effect that hinders hydrazone NH from acting as HB-donor (HBD) in that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position [22] small and bulky alkyls on the 1H-indole nitrogen of 1 were tolerated, so that the N-methyl (26), the inhibition potency compared with the respective non-methylated compound (26), which proves In contrast, methylation of the hydrazone nitrogen (24) resulted in a more than 10-fold decrease in between theintroduce antifibrillogenic potency and lipophilicity ofNH thefrom N-alkyl groups exists for these the interaction withand the peptide. and/or may aAβ steric effect that hinders hydrazone acting as HB-donor (HBD) in N-cyclopropyl (28) N-phenylalkyl derivatives resulted almost equipotent with the that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position [22] the inhibition potency compared with the(32–34) respective non-methylated compound (26), which proves compounds, the N-benzyl derivative 32 being the most potent one within the subset (IC 50 0.53 µM). thecompound interaction the Aβ peptide. hit 1with inofthe range (IC from 0.53 to 0.83 µM). A nonlinear and/or may introduce asubmicromolar steric effect thatprevent hinders hydrazone NH from acting as O HB-donor (HBD) 50 s ranging that N-methylation hydrazone N may IMHB formation with carbonyl at 2-position [22]in In contrast, methylation of the hydrazone nitrogen (24) resulted in a more than 10-fold decrease in Table 4. Aβ Antiaggregating Activity of Compounds 24, 26, 28 and 32-34. relation between the antifibrillogenic potency and lipophilicity of the N-alkyl groups exists for these the interaction with the Aβ peptide. and/or may introduce a steric effect that hinders hydrazone NH from acting as HB-donor (HBD) in the inhibition potency with the respective (26), which proves Table compared 4. Aβ Antiaggregating Activity of non-methylated Compounds 24, 26,compound 28 and 32-34. compounds, the N-benzyl the interaction with the Aβderivative peptide. 32 being the most potent one within the subset (IC50 0.53 µM). that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position Table 4.of Aβthe Antiaggregating Activity of Compounds 24,a26, 28 and 32-34. In contrast, methylation hydrazone nitrogen (24) resulted in more than 10-fold decrease[22] in and/or may introduce a steric effect that hinders hydrazone NH from acting as HB-donor (HBD) in the inhibition potency compared with the respective (26), which proves Table 4. Aβ Antiaggregating Activity of non-methylated Compounds 24, 26,compound 28 and 32-34. the interaction with the Aβ peptide. that N-methylation of hydrazone N may prevent IMHB formation with carbonyl O at 2-position [22]

and/or may introduce a steric effect that hinders hydrazone NH from acting as HB-donor (HBD) in Table 4. Aβ Antiaggregating Activity of Compounds 24, 26, 28 and 32-34. the interaction with the Aβ peptide.

O N R

Comp.

Molecules 2018, 23, 1544


R

R1

IC50 (µM) 1

24 CH3 1124, ± 226, 28 and 32–34. CH3 Table 4. Aβ Antiaggregating Activity of Compounds

26

CH3

H

28

Cyclopropyl

HN

H3CO

9 of 23

4 of 18

0.81 ± 0.11

N

0.80 ± 0.13

R1 O

N

32 Comp.

Benzyl R

HR R1

0.53 ± 0.10 IC50 (µM) 1

24 Comp. CH CH 11 1± 2 33 2-Phenylethyl H 0.61 0.03 3 R R31 IC50 ±(µM) 26 CH3 H 0.81 ± 0.11 28 Cyclopropyl H 0.80 ± 0.13 32 Benzyl H 0.53 ± 0.10 34 3-Phenylpropyl H 3 0.8311± ±0.20 24 CH 2 CH3 33 2-Phenylethyl H 0.61 ± 0.03 34 3-Phenylpropyl H 0.83 ± 0.20 1 Data are mean ± SEM of three independent experiments. 1 Data are mean ± SEM of three independent experiments. 26 H 0.81 ± 0.11 CH3

Finally, compounds compounds36–39, 36–39, bearing diverse lipophilic and hydrophilic substituents the Finally, bearing diverse lipophilic and hydrophilic substituents on the on indole indole ring5), (Table werefor tested for theoneffects on Aβ aggregation. The introduction n-butyl ring (Table were5), tested the effects Aβ aggregation. The introduction of n-butylof (36) or Br (36) (37) 28 respectively, Cyclopropyl Hdetrimental, 0.80 ± 0.13leading in the latter case to or Br (37) at 5and 7-position, resulted at 5- and 7-position, respectively, resulted detrimental, leading in the latter case to complete loss of completeThe lossevidence of activity. evidence of an of enhancing rolesubstituents of hydrophilic substituents (particularly activity. of The an enhancing role hydrophilic (particularly 5,6-dihydroxy 5,6-dihydroxy derivatives) for an efficient inhibition of Aβ 40 aggregation, emerging from our derivatives) for an efficient inhibition from our previous works [20,21], 32 of AβBenzyl H emerging 0.53 ± 0.10 40 aggregation, previous works [20,21], was endeavored by introducing a net the negative charge with the sulfonate salt was endeavored by introducing a net negative charge with sulfonate salt 38 and a protecting 38 andfor a protecting group for catechol derivatives, i.e., the substituent, 5,6-methylenedioxy substituent, in 39. group catechol derivatives, i.e., the 5,6-methylenedioxy in 39. Both modifications Both modifications resulted poorly effective, and the two compounds showed inhibitory potencies 33 2-Phenylethyl H 0.61 ± 0.03 resulted poorly effective, and the two compounds showed inhibitory potencies much lower than that much lower than of compound 1. that of compound 1. 34 Antiaggregating 3-Phenylpropyl 0.83 ± 0.20 36-39. Table5.5.Aβ Aβ ActivityHof of Compounds Compounds Table Antiaggregating Activity 36–39. 1


Finally, compounds 36–39, bearing diverse lipophilic and hydrophilic substituents on the indole ring (Table 5), were tested for the effects on Aβ aggregation. The introduction of n-butyl (36) N NH or Br (37) at 5- and 7-position, respectively, resulted detrimental, leading in the latter case to O of hydrophilic substituents (particularly R complete loss of activity. The evidence of an enhancing role N H 5,6-dihydroxy derivatives) for an efficient inhibition of Aβ40 aggregation, emerging from our previous works [20,21], was endeavored by introducing a net negative Comp. R % Inhibition @ 100charge µM 1 with the sulfonate salt 38 and a protecting group for catechol derivatives, i.e., the 5,6-methylenedioxy substituent, in 39. Comp. R % Inhibition 36 5-n-Butyl 55 @ ± 100 1 µM 1 Both modifications resulted showed inhibitory potencies 37 poorly effective, 7-Br and the two compounds