Synthesis and in silico analysis of the quantitative structure–activity relationship of heteroaryl–acrylonitriles as AChE inhibitors

Journal of the Taiwan Institute of Chemical Engineers 59 (2016) 45–60

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Synthesis and in silico analysis of the quantitative structure–activity relationship of heteroaryl–acrylonitriles as AChE inhibitors ✩ Pedro De-la-Torre a,b,∗, Adriana V. Treuer a, Margarita Gutierrez a, Horacio Poblete b,c, Jans H. Alzate-Morales b, Jorge Trilleras d, Luis Astudillo-Saavedra a, Julio Caballero b,∗∗ a

Organic Synthesis Laboratory and Biological Activity (LSO-Act-Bio), Institute of Chemistry of Natural Resources, Universidad de Talca, Casilla 747, Talca, Chile Center for Bioinformatics and Molecular Simulation, Universidad de Talca, 2 Norte 685, Casilla 721, Talca, Chile Institute of Computational Comparative Medicine, Nanotechnology Innovation Center of Kansas State, and Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506-5802, USA d Heterocyclic Compounds Research Group, Chemistry Program, Universidad del Atlántico, Km 7 vía Puerto Colombia, Barranquilla, Colombia b c

a r t i c l e

i n f o

Article history: Received 5 April 2015 Revised 16 July 2015 Accepted 20 July 2015 Available online 17 August 2015 Keywords: Heteroaryl–acrylonitriles Quantitative structure–activity relationships CoMSIA Knoevenagel condensation Cholinesterases

a b s t r a c t Alzheimer disease (AD) is a neurodegenerative disorder that causes damages in brain due to factors such as oxidative stress, low-levels of the neurotransmitter acetylcholine, β -amyloid protein aggregation, etc. It is necessary the design of novel efficient drugs for AD treatment to counteract the increase of people suffering from AD. Recently, heteroaryl–acrylonitrile derivatives have emerged as a new family of acetylcholinesterase inhibitors (AChEIs). The analysis of the structure–activity relationship of these compounds could help to elucidate the main molecular features that contribute to the activity of these compounds. In this paper, we performed 3D-QSAR analyses through a Comparative Similarity Indices Analysis (CoMSIA) to determine the keyfactors for the activity of E/Z-heteroaryl–acrylonitriles reported in literature and novel derivatives that are reported in this work for the first time. The novel derivatives were synthetized in order to enlarge the library of compounds available in literature. They were synthetized via microwave-assisted Knoevenagel reaction and their biological activities as AChE/BuChE inhibitors were explored by the Ellman’s spectrophotometric method. The best CoMSIA model included both electrostatic and hydrogen bond donor fields (CoMSIA-ED model) and provided the best statistical results with a highest Q2 value of 0.901. The model also had satisfactory predictions of external compounds. Our in silico study provided a new tool for predicting the affinity of heteroaryl–acrylonitriles as AChEIs to the scientific community. It can be used for guiding the design and synthesis of novel, selective, and more potent AChEIs. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction Alzheimer disease (AD) is one of the most common forms of dementia in elderly people. It is a neurodegenerative disease that causes progressive damage to the central nervous system, which is manifested by cognitive deterioration and behavioral disorders. These effects are related to the neuronal cell death, due to the oxidative stress caused by the extracellular formation of senile amyloid-β peptide (Aβ ) plaques [1,2] and hyperphosphorylated-tau oligomers

✩ This article is dedicated to the memory of Professor Luis Astudillo Saavedra, for his great passion for friendship and fraternity. ∗ Correspondence author at: Organic Synthesis Laboratory and Biological Activity (LSO-Act-Bio), Institute of Chemistry of Natural Resources, Universidad de Talca, Casilla 747, Talca, Chile. Tel.: +56 71 2200389; fax: +56 71 2200448. ∗∗ Corresponding author. Tel.: +56 71 2201662; fax: +56 71 2201561. E-mail addresses: [email protected], [email protected] (P. De-la-Torre), [email protected], [email protected] (J. Caballero).

[3], which entail the decreased levels of acetylcholine (ACh) and proteins such as acetylcholinetransferase (ChAT: involved in the synthesis of ACh) and acetylcholinesterase (AChE: responsible for the hydrolysis of the same neurotransmitter at nerve–nerve synapses and neuromuscular junctions) [4]. The worldwide cost of dementia care is currently over US$600 billion, or around 1% of global GDP: with the increase of people suffering from AD; these statistics could increase twice in the next 20 years [5–7]. Currently, there are two drug classes approved by FDA [8]. The first one, cholinesterase inhibitors, restore the levels acetylcholine, (for instance, Aricept:donepezil [9], Exelon:rivastigmine tartrate [10], Razadyne:galantamine [11], Cognex:tacrine [12,13], which was removed from the market in 2012). The second class is composed by the agonists of the N-methyl-d-aspartate (NMDA) receptors (for instance: Namenda:memantine) [14]. Both classes are used to treat cognitive symptoms such as memory loss, confusion, and problems with thinking and reasoning of early Alzheimer’s disease [8].

http://dx.doi.org/10.1016/j.jtice.2015.07.022 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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P. De-la-Torre et al. / Journal of the Taiwan Institute of Chemical Engineers 59 (2016) 45–60

Fig. 1. E/Z-acrylonitrile derivatives with inhibitory activity for AChE reported.

Scheme 1. Synthesis of novel 3-cyanoacetyl-indole-acrylonitriles 3a–j.

Many kinds of heterocyclic derivatives have been reported with potent AChE inhibitory activity [9–13,15]; however, many of them have been shown adverse effects and problems of bioavailability [16,17]. Therefore, it is necessary the development of new scaffolds with potential applications for the treatment of AD. Currently, E/Z-acrylonitrile derivatives have attracted the attention as new AChE inhibitors [18,19]. We previously reported that E-heteroaryl– acrylonitrile derivatives with different substituents at position 3 and with the 2-(benzo[d]thiazol-2-yl)acetonitrile substituent at position 2 (Fig. 1, compound A), have demonstrated competitive and selective inhibition for AChE over BuChE [18]. Molecular docking studies showed that these compounds are accommodated along the AChE gorge, adopting some interactions that were previously described for the AChE–donepezil complex [18,20] and other compounds described in wet or dry laboratories [21]. Recently, Parveen et al. reported that Z-heteroaryl–acrylonitrile derivatives with the 4-NO2 phenyl substituent at position 2 (Fig. 1, compound B), have displayed the strongest inhibition for AChE [19], ratifying the potential of acrylonitrile scaffold for the design of novel AChEIs (Fig. 1).

Despite the above, the structure–activity relationship of E/Zheteroaryl–acrylonitrile derivatives as AChEIs is not available yet. This approach could lead to the understanding of the structural factors related with the bioactivity of these compounds in detail. Computational methods that describe the structure–activity relationship of compounds (mainly QSAR) have been increasingly used for understand the causes of the high or poor activity of inhibitors and predict novel compounds taking into account physico-chemical and structural factors. More in detail, QSAR modeling has been considered a powerful tool to design new and potent inhibitors due to its capability to explain the trend of the biological activity for a series of compounds using a low computational cost. In this sense, a QSAR study was developed for modeling activities of heteroaryl–acrylonitriles as AChEIs with the purpose to detect the physico-chemical requirements in the molecular groups that are related to a high AChE inhibitory activity. Particularly, we constructed comparative molecular similarity indices analysis (CoMSIA) models to predict and interpret the AChE inhibitory activities of a set of heteroaryl–acrylonitriles that have been reported previously and we include some novel compounds. The novel compounds were synthesized and were evaluated as AChE/BuChE inhibitors (Schemes 1 and 2, Tables 1 and 2, respectively). Our goal is the understanding of the structural factors involved in differential AChE inhibitory activities of heteroaryl– acrylonitriles. 2. Materials and methods 2.1. Compounds and biological data sets Molecular structures used in this study were collected from the paper of Parveen et al. (12 compounds) [19] and from the paper reported by our group (21 compounds) [18]. Furthermore, the library of compounds was enlarged by the synthesis and inhibitory activities of two new derivative sets (Schemes 1 and 2). Finally, we determined cholinesterase inhibitory activities of the previously reported compounds 4a–j [22] (their biological activities were not published before). Electrophorus electricus AChE inhibitory potencies of the novel compounds were evaluated by using galantamine as the reference compound. The activity profiles were also evaluated against BuChE,

Scheme 2. The synthesis steps for the compounds 3k–p. The compound 3k was prepared using the following conditions a: (4a) HCl Conc/methanol (5:5), SnCl2 x2H2 O, 80 °C, reflux. (3l) compound 3k (1 mmol), Bn bromide (1 mmol), toluene (1 mL), TEA (0.2 mL), MWI, 150 W, 75 °C. (3m) compound 3k (1 mmol), MWI, Bn bromide (2.1 mmol), toluene (1 mL), TEA (0.2 mL), MWI, 150 W, 75 °C. (3n) compound 3k (1 mmol), CH3 COOH (1 mL), acetic anhydride (2 mmol), 80 °C, MWI, 150 W. (3o) compound 3k (1 mmol), THF (1 mL), maleic anhydride (1 mmol), MWI, 323 K, 50 °C. For the preparation of the derivative 3p was used the protocol reported in the literature [22].

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47

Table 1 Structures, reaction parameters and AChE inhibitory activities of the compounds 3a–j.

R

O

2

N N H Compound

R2

Yield (%)

Time (min)

IC50 (μM)a AChE

3a

3b

N 3c

SI BuChE

73

60

170 ± 0.1

1091 ± 0.1

6.4

86

40

299 ± 0.1

>1399 ± 0.5

>4.7

77

16

274 ± 0.1

>1307 ± 0.1

>4.8

47

25

62 ± 0.1

>1411 ± 0.3

>22.76

78

15

451 ± 0.1

>1357 ± 0.4

>3

63

25

78 ± 0.0

87 ± 0.0

1.11

51

1

437 ± 0.2

>1830 ± 0.4

>4.19

80

2

292 ± 0.0

929 ± 0.2

3.18

90

60

410 ± 0.1

1759 ± 0.3

4.29

87

60

888 ± 0.0

747 ± 0.6

0.84

0.54 ± 0.7

8.80 ± 0.5

O

O

O 3d

O CH3 O

3e

O

O 3f

O

O

H2N 3g

N 3h

N 3i

O 3j

O

O

O Galantaminec

16

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Table 2 Chemical structures, reaction parameters and AChE and BuChE inhibitory activities for compounds 3k–p and 4a–j.

N N S Compound

R

3k

3l

NH

O

Ph

N

O

NH

O

Yield (%)

AChE

90

70

144 ± 1.0

10

53

390 ± 1.4

ndb

ndb

17

51

366 ± 1.3

ndb

ndb

15

55

134,9 ± 0.9

ndb

ndb

20

82

330.72 ± 1.3

ndb

ndb

20

52

96.05 ± 0.7

>1437,53

>15

4

81

130 ± 0.8

527,6 ± 2.0

4.05

9

56

291.65 ± 1.1

>1437,53 ± 2.2

>4.93

3

92

434.36 ± 1.4

>1437,53 ± 1.8

>3.31

6

80

138.23 ± 0.6

759.10 ± 1.5

5.49

2

78

434.14 ± 0.9

395.17 ± 1.8

0.91

BuChE 835,7 ± 1.3

5.8

O

3o

NH

O

O

O 3p

N

N

OH

O O

4a

NO2

O 4b

O

O

4c

N

4d

4e

Time (min)

SIc

Ph

3n

O Cl

IC50 (μM)

Ph

3m

Cl

Reactions parameters

NH2

O

O

R.

N

N

O

N (continued on next page)

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49

Table 2 (continued) Compound

4f

R

O

Reactions parameters

IC50 (μM)

Time (min)

Yield (%)

AChE

4

50

413.05 ± 1.5

425.49 ± 2.0

4

49

371.47 ± 1.2

371.70 ± 1.8

3

62

361.57 ± 1.0

153.48 ± 1.1

0.42

5

60

543.42 ± 1.7

>1437,53 ± 1.8

2.64

8

94

693,92 ± 1.3

>1437,53

2.07

SIc BuChE 1.03

O 4g

O

1

O 4h

O CH3 O

4i

O

O 4j

N Galantamine

0.54 ± 0.7

8.80 ± 0.5

16.29

a

Compound Set 1 (3a–p). The compounds 4a–j (compound set 2) were extracted from Ref. [22] and its inhibitory activities are informed for the first time in this article. b Not determined (nd). c Selectivity index for AChE.

in order to gain insight about inhibitor’s selectivities. Both inhibitory activities were evaluated by the method of Ellman et al. [23]. The chemical structures and experimental activities are shown in Tables 1 and 2. 2.2. Synthetic methodologies and structural identification In this section, we inform the synthesis of novel heteroaryl– acrylonitriles via Knoevenagel condensation between substituted aldehydes and different methylene-active compounds in order to obtain structural diversity at position 2 and 3 on acrylonitrile scaffold. Compounds in Schemes 1 and 2 were prepared by using cyanoacetyl– indole (1a) and 2-(benzo[d]thiazol (1b) as methylene-active systems, respectively. The reactions were monitored by thin-layer chromatography (TLC) with visualization using UV light; TLC was done on pre-coated silica gel 60 F254 plates (Merck). All the compounds were obtained in good purities and yields in short reaction times, and were characterized using IR spectroscopy, NMR spectroscopic techniques, and high-resolution mass spectrometry. All solvents were of analytical grade. Nuclear Magnetic Resonance (RMN) spectra were recorded in diluted CDCl3 and DMSO-d6 solutions. 1 H NMR spectra were recorded on a Bruker AM 400 instrument. The chemical shifts are reported as parts per million (ppm) and multiplicities are designated as singlet (s), doublet (d), triplet (t), quadruplet (q), multiplet (m). IR spectra, KBr pellets, 500–4000 cm−1 were recorded on a Thermo

Nicolet NEXUS 670 FT-IR spectrophotometer. Melting points were recorded on a Buchi apparatus and are uncorrected. High-resolution mass spectrometry ESI-MS and ESI-MS/MS analyses were conducted in a high-resolution hybrid quadrupole (Q) and orthogonal timeof-flight (TOF) mass spectrometer (Waters/Micromass Q-TOF micro, Manchester, UK) with a constant nebulizer temperature of 100 °C. The experiments were carried out in positive ion mode, and the cone and extractor potentials were set at 10 and 3.0 V, respectively, with a scan range of m/z 105–600. MS/MS experiments were carried out by mass selection of a specific ion in Q1, which was then submitted to collision-induced dissociation (CID) with helium in the collision chamber. The product ion MS analysis was accomplished with the high-resolution orthogonal TOF analyzer. The samples were dissolved in acetonitrile and were directly infused into the ESI source, via a syringe pump, at flow rates of 5 μL/min, via the instrument’s injection valve. The synthetic procedures and the spectroscopic data of the new compounds are described as follows. 2.2.1. Procedure for the preparation of the (E)-cyanoacetyl)-3-arylacrylonitriles 3a–j A mixture of cyanoacetyl indole 1 (1.0 mmol) and aldehydes 2a– j (1.0 mmol) in ethanol were subjected to microwave irradiation (maximum power 300 W during 1–60 min at controlled temperature of 100 °C) using a focused microwave reactor (CEM DiscoverTM ). The consumption of the precursors over time was monitored us-

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ing TLC. Later, the mixture was concentrated under vacuum and the residue was purified by flash column chromatography using ethyl acetate–petroleum ether (3:7) or dichloromethane (CHCl2 ) as eluent to obtain the pure compounds. (The chemical structures and experimental activities are shown in Table 1). (E)-3-(1,1 -biphenyl-4-yl)-2-(1H-indol-3-ylcarbonyl)acrylonitrile (3a). Yellow solid. Yield 73%. mp 260–265 °C. IR (cm−1 ): 3250 (NH), 2956, 2910, 2841, 2215 (CN), 1742, 1699 (C=O), 1600, 1561, 1410, 1212, 1166, 1107, 1008, 839. 1 H NMR (CDCl3 ): δ ppm 12.30 (s, 1H, NHIndole ), 8.48 (s, 1H, H2 Indole ), 8.28 (s, 1H, H–C=olefinic ), 8.19 (d, 1H, H4 Indole , J = 8.1 Hz), 8.16 (d, 2H, Ho , J = 8.1 Hz), 7.92 (d, 2H, Ho  , J = 8.3 Hz), 7.80 (d, 2H, Hm , J = 7.3 Hz), 7.56–7.50 (m, 3H, H7 Indole , Hm  ), 7.43 (t, 1H, Hp  , J = 7.3 Hz), 7.31–7.25 (m, 2H, H5 -H6 Indole ); EI-MSMS (m/z): 348.4395 (M+ , 25), 347.5207 (35), 333.5748 (35), 299.6628 (55), 227.9975 (55), 218.0821 (100). (E)-2-(1H-indol-3-ylcarbonyl)-3-(4-morpholin-4-ylphenyl) acrylonitrile (3b). Orange solid. Yield 86%. mp 228–230 °C. IR (cm−1 ): 3214 (NH), 2952, 2913, 2844, 2249 (CN), 1640 (C=O), 1521, 1439, 1372, 1237, 1125, 901, 752. 1 H NMR (CDCl3 ): δ ppm 12.15 (s, 1H, NHIndole ), 8.41 (s, 1H, H2 Indole ), 8.17 (d, 1H, H4 Indole , J = 7.1 Hz), 8.12 (s, 1H, H–C=olefinic ), 8.00 (d, 2H, Ho , J = 8.8 Hz), 7.53 (d, 1H, H7 Indole , J = 7.3 Hz), 7.28–7.21 (m, 2H, H5 -H6 Indole , J1 =14.6 Hz, J2 =6.5 Hz), 7.09 (d, 2H, Hm , J = 9.1 Hz), 3.74–3.72 (m, 4H, =N-CH2-morpholine ), 3.39-3.36 (m, 4H, -O-CH2-morpholine ). EI-MSMS (m/z): 380.2800 (M + Na+ , 5), 379.2542 (30), 378.2674 (100), 347.4937 (10), 313.5975 (35), 221.9283 (28), 170.1648 (20). (E)-2-(1H-indol-3-ylcarbonyl)-3-(6-isopropyl-4-oxo-4Hchromen-3-yl)acrylonitrile (3c). yellow solid. Yield 77%. mp 245–248 °C. IR (cm−1 ): 3384 (NH), 2963, 2918, 2360 (CN), 1741 (C=O), 1650, 1515, 1475, 1315, 1240, 902, 825, 762. 1 H NMR (CDCl3 ): δ ppm 12.34 (s, 1H, NHIndole ), 9.19 (s, 1H, CHChromone ), 8.43 (d, 1H, H2 Indole , J = 2.9 Hz), 8.19 (d, 1H, H4 Indole , J = 6.6 Hz), 8.14 (s, 1H, H–C=olefinic ), 7.96 (d, 1H, CHChromone , J = 1.5 Hz), 7.83–7.81 (m, 1H, CHChromone ), 7.72 (d, 1H, CHChromone , J = 8.6 Hz), 7.57–7.52 (dd, 1H, H7 Indole , J1 = 13.3 Hz, J2 = 8.2 Hz), 7.32–7.25 (m, 2H, H5 -H6 Indole ), 3.12–3.05 (dt, 1H, CH–CH3, J1 = 13.8 Hz, J2 = 6.9 Hz), 1.26 (d, 6H, CH3 , J = 6.9 Hz). EI-MSMS (m/z): 405.2950 (M + Na+ , 12), 386.4733 (25), 363.4074 (40), 348.5117 (50), 347.5027 (98) 333.6012 (100), 259.8290 (90), 234.0452 (75). (E)-2-(1H-indol-3-ylcarbonyl)-3-(6-methyl-4-oxo-4H-chromen3-yl)acrylonitrile (3d). Yellow solid. Yield 47%. mp 270–275 °C. IR (cm−1 ): 3385 (NH), 2962, 2923, 2287 (CN), 1743 (C=O), 1622, 1480, 1310, 1168, 820, 758. 1 H NMR (CDCl3 ): δ ppm 12.34 (s, 1H, NHIndole ), 9.18 (s, 1H, CHChromone ), 8.43 (s, 1H, H2 Indole , J = 2.93 Hz), 8.18 (d, 1H, H4 Indole , J = 7.09 Hz), 8.14 (s, 1H, H–C=olefinic ), 7.93 (d, 1H, CHChromone ), 7.69 (q, 2H, CHChromone , J = 8.72 Hz), 7.56 (d, 1H, H7 Indole , J = 7.58 Hz), 7.28 (ddd, 2H, H5 -H6 Indole , J1 = 13.33 Hz, J2 = 6.85 Hz, J3 = 6.72 Hz), 2.45 (s, 3H, CH3 ). EI-MSMS (m/z): 377.2350 (M + Na+ , 5), 375.2210 (100), 354 (M+ , 5), 347.5117 (22), 330.6881 (50), 302.7889 (48), 299.6460 (12), (32), 228.0121 (15), 211.0656 (45). (E)-2-(1H-indol-3-ylcarbonyl)-3-(6-ethyl-4-oxo-4H-chromen3-yl)acrylonitrile (3e). Yellow solid. Yield 78%. mp 220–222 °C. IR (cm−1 ): 3247 (NH), 2958, 2919, 2848, 2215 (CN), 1731 (C=O), 1624, 1480, 1429, 1314, 1237, 1114, 825, 748. 1 H NMR (CDCl3 ): δ ppm 12.33 (s, 1H, NHIndole ), 9.18 (s, 1H, CHChromone ), 8.43 (d, 1H, H2 Indole , J = 2.93 Hz), 8.19 (d, 1H, H4 Indole , = 7.34 Hz), 8.14 (s, 1H, H–C= olefinic ), 7.94 (s, 1H, CHChromone ), 7.77–7.66 (m, 2H, CHChromone ), 7.56 (d, 1H, H7 Indole , J = 7.34 Hz), 7.29 (ddd, 2H, H5 -H6 Indole , J1 = 13.82 Hz, J2 = 7.09 Hz, J3 = 6.97 Hz), 2.76 (q, 2H, -CH2 -CH3, J1 = J2 = J3 = 7.58 Hz), 1,22 (t, 3H, -CH3, J1 = J2 = 7.58 Hz). EI-MSMS (m/z): 407.1346 (M + K+ , 12), 387.4518 (32), 386.4638 (100), 368 (M+ , 12), 358.6179 (35), 330.6706 (50), 302.7973 (40), 272.9109 (52), 229.0925 (52), 218.0892 (30), 202.8962 (20). (E)-2-(1H-indol-3-ylcarbonyl)-3-(2-amino-4-oxo-4H-chromen3-yl)acrylonitrile (3f). Ivory solid. Yield 63%. mp 208–210 °C. IR (cm−1 ): 3243 (NH), 2958, 2924, 2347 (CN), 1621 (C=O), 1577, 1474,

1334, 1230, 1212, 1157, 874, 755. 1 H NMR (CDCl3 ): δ ppm 12.22 (s, 1H, NHIndole ), 10.07 (d, 1H, H4 Indole , J = 6.0 Hz), 9.59 (s, 2H, NH2-Chromone ), 8.37 (d, 1H, H2 Indole , J = 3.2 Hz), 8.12 (s, 1H, H–C=olefinic ), 8.01 (dd, 1H, CHChromone , J1 = 7.7 Hz, J2 = 1.4 Hz), 7.73–7.69 (m, 1H, CHChromone ), 7.50 (d, 1H, H7 Indole , J = 6.0 Hz), 7.44-7.38 (m, 2H, CHChromone ), 7.26–7.19 (m, 2H, CHChromone ). EI-MSMS (m/z): 383.3941 (M+ , 30), 363.3983 (28), 347.5027 (100), 326.5736 (26), 229.0999 (45), 211.0726 (48), 202.9306 (22). (E)-2-(1H-indol-3-ylcarbonyl)-3-pyridin-3-ylacrylonitrile (3g). Ochre solid. Yield 51%. mp 206–208 °C. (cm−1 ): 3380 (NH), 2955, 2918, 2847, 2380 (CN), 1728 (C=O), 1629, 1424, 1157, 1107, 1024, 874, 775. 1 H NMR (CDCl3 ): δ ppm 12.38 (s, 1H, NHIndole ), 8.81 (d, 2H, CHm-pyridine , J = 5.9 Hz), 8.50 (s, 1H, H2 Indole , J = 3.2 Hz), 8.22 (d, 1H, H4 Indole , J = 7.1 Hz), 8.13 (s, 1H, H–C= olefinic ), 7.88 (d, 2H, CHo-pyridine , J = 5.6 Hz), 7.56 (d, 1H, H7 Indole , J = 6.4 Hz), 7.32-7.26 (m, 2H, H5 - H6 Indole ), 2.45 (s, 3H, CH3 ). EI-MSMS (m/z): 273.7332 (M+ , 12), 272.7275 (50), 212.0408 (15), 211.0585 (100), 194.1137 (15). (E)-2-(1H-indol-3-ylcarbonyl)-3-pyridin-4-ylacrylonitrile (3h). Yellow solid. Yield 80%. mp 200–203 °C. IR (cm−1 ): 3423 (NH), 2959, 2914, 2854, 2388 (CN), 1634 (C=O), 1515, 1418, 1377, 1246, 1156, 1007, 821, 761. 1 H NMR (CDCl3 ): δ ppm 12.38 (s, 1H, NHIndole ), 8.81 (d, 2H, CHm-pyridine , J = 5.9 Hz), 8.50 (s, 1H, H2 Indole , J = 3.2 Hz), 8.22 (d, 1H, H4 Indole , J = 7.1 Hz), 8.13 (s, 1H, H–C=olefinic ), 7.88 (d, 2H, CHo-pyridine , J = 5.6 Hz), 7.56 (d, 1H, H7 Indole , J = 6.4 Hz), 7.32–7.26 (m, 2H, H5 -H6 Indole ). EI-MSMS (m/z): 302.7805 (100), 273.7173 (M+ , 12), 272.7434 (28), 265.8257 (14), 228.0194 (20), 218.892 (22). (E)-3-(2-furyl)-2-(1H-indol-3-ylcarbonyl)acrylonitrile (3i). Light brown solid. Yield 90%. mp 207–209 °C. IR (cm−1 ): 3337 (NH), 2926, 2845, 2020 (CN), 1598 (C=O), 1418, 1242, 1120, 1019, 932, 741. 1 H NMR (CDCl3 ): δ ppm 12.23 (s, 1H, NHIndole ), 8.47 (s, 1H, H2 Indole ), 8.18 (s, 2H, H4 Indole , H5furane ), 8.08 (s, 1H, H–C= olefinic ), 7.54 (d, 1H, H7 Indole , J = 7.1 Hz), 7.45 (d, 1H, H3furane ), 7.28 (m, 2H, H5 -H6 Indole , J1 = 13.5 Hz, J2 = 7.2 Hz, J3 = 6.0 Hz), 6.85 (m, 1H, H4furane ). EI-MSMS (m/z): 261.7786 (M+ , 18), 255.0331 (60), 228.0121 (45), 218.0821 (68), 212.0699 (88), 211.0656 (98), 194.1272 (100), 176.2525 (52), 139.2957 (25). (E)-3-[5-(acetoximethyl)-2-furyl]-2-(1H-indol-3-ylcarbonyl) acrylonitrile (3j). Ivory solid. Yield 87%. mp 160–165 °C. IR (cm−1 ): 3339 (NH), 2956, 2915, 2849, 2217 (CN), 1750 (C=O), 1628, 1514, 1408, 1427, 1237, 1159, 1024, 897, 770. 1 H NMR (CDCl3 ): δ ppm 10.93 (s, 1H, NHIndole ), 8.25 (s, 1H, H2 Indole ), 7.99 (d, 1H, H4 Indole , J = 6.4 Hz), 7.71 (s, 1H, H–C=olefinic ), 7.23 (d, 1H, H7 Indole , J = 6.9 Hz), 7.11 (d, 1H, H3furane , J1 = 3.4 Hz), 6.96–6.90 (m, 2H, H5 -H6 Indole ), 6.47 (d, 1H, H4furane , J = 3.4 Hz), 2.18 (s, 2H, CH2 ), 1.70 (m, 3H, CH3 ). EI-MSMS (m/z): 334.3383 (M+ , 10), 308.6147 (65), 299.6460 (25), 273.7652 (20), 251.9041 (25), 244.9930 (30), 211.0585 (100). 2.2.2. Procedure for the preparation of the (E)-2-(benzo[d]thiazol-2-yl)3-arylacrylonitriles 3k–p The compounds were prepared using the derivative 4a from Ref. [22] as starting material. The synthetic procedures and the spectroscopic data for 3k–p are described as follows. Procedure for the preparation of the (E)-3-(5-(4-aminophenyl)furan2-yl)-2-(benzo[d]thiazol-2-yl)acrylonitrile (3k): a mixture of 4a (1 mmol) in a solution of HCl concentrate: Methanol (5:5), was stirred at room temperature for 5 min. Later, Tin (II) chloride dihydrate (SnCl2 . 2H2 O, 0.25 mmol) was added and heated under reflux for 1.5 h at 80 °C. The consumption over time of 4a was monitored using TLC and the excess of HCl in the reaction mixture was neutralized with saturated solution of K2 CO3 (dropwise) and subsequently the organic phase was extracted with ethyl acetate (2 × 20 mL). The organic extract was dried over Na2 SO4 , and was filtered and concentrated under vacuum. Later, the residue was purified by flash column chromatography using ethyl acetate–petroleum ether (3:7) or dichloromethane (CH2 Cl2 ) as eluent to afford pure 3k. Solid violet, yield 70%, mp 224– 226 °C. IR (KBr) cm−1 : 3377, 2963, 2927, 2213 (CN), 1724, 1600, 1270.

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NMR (400 MHz, CDCl3 ): δ 8.02 (d, 1H, H4 BT ), 7.99 (s, 1H, H–C=), 7.89 (d, 1H, J = 7.83 Hz, H7 BT ), 7.71 (d, 2H, J = 8.56 Hz, Ho ), 7.51 (t, 1H, H6 BT ), 7.40 (t, 1H, H5 BT ), 7.21 (d, 1H, H3furanyl ), 6.75 (d, 2H, J = 8.56 Hz, Hm ), 6.71 (d, 1H, J = 3.91 Hz, H4furanyl ), 3.96 (s, 2H, ArNH2 ). EI-MS (m/z): 344.0778 (M+ , 9.07), 316.1995 (30.03), 229.1430 (17.62), 202.2823 (100.00). Procedure for the preparation of the acrylonitrile 3l–o: (E)2-(benzo[d]thiazol-2-yl)-3-(5-(4-(benzylamino)phenyl)furan-2-yl) acrylonitrile (3l): a mixture of 3k (1 mmol), benzyl bromide (1 mmol) in toluene (1 mL) and catalytic amounts of trietylamine (0.2 mL), was exposed to microwave radiation for 10 min at 75 °C and a maximum power of 150 W using a focused microwave reactor (CEM DiscoverTM , Mathews, NC, USA). Subsequently, water was added to this solution (30 mL) and the product was extracted with ethyl acetate (2 × 30 mL). The organic phase was dried over Na2 SO4 and, filtered and concentrated under vacuum. Later, ethanol (10 mL) and water (aprox 5 mL) were added to the residue; the solid was filtered to give pure compound 3l. Solid red, yield 53%, mp 200–202 °C. IR (KBr) cm−1 : 3583, 3388, 2955, 2211 (CN), 1586, 1444. 1 H NMR (400 MHz, CDCl3 ): δ 8.13 (d, 1H, J = 8.07 Hz, H4 BT ), 8.09 (s, 1H, H–C=), 8.00 (d, 1H, J = 8.07 Hz, H7 BT ), 7.66 (d, 2H, J = 8.32 Hz, Ho ), 7.54 (t, 1H, H6 BT ), 7.47 (d, 1H, J = 3.92 Hz, H3furanyl ), 7.44 (m, 1H, Hp -Bn ), 7.38 7.31 (m, 4H, Hm -Bn and Ho -Bn ), 7.24 (t, 1H, H5 BT ), 7.05 (d, 1H, J = 3.67 Hz, H4furanyl ), 7.01 (t, 1H, NH-Bn), 6.72 (d, 2H, J = 8.31 Hz, Hm ), 4,36 (d, 2H, J = 5.87 Hz, CH2 ). EI-MS (m/z): 433.5101 (M+ , 9), 413.5598 (23), 332.5612 (100), 304.5133 (84), 229.3041 (50), 163.0074 (17.23). (E)-2-(benzo[d]thiazol-2-yl)-3-(5-(4-(dibenzylamino)phenyl)furan2-yl)acrylonitrile (3m): Was obtained by the procedure of 3l, using 2.1 mmol of benzyl bromide. In this case, was obtained a mixture of 3l and 3m. The compound 3m was obtained as a solid red wine, yield 51%, mp 198–200 °C. IR (KBr) cm−1 : 2958, 2918, 2866, 2204 (CN), 1444, 1380, 1195. 1 H NMR (400 MHz, CDCl3 ): δ 8.00 (d, 1H, J = 8.32 Hz, H4 BT ), 7.94 (s, 1H, H–C=), 7.87 (d, 1H, J = 7.83 Hz, H7 BT ), 7.70 (d, 2H, J = 9.05 Hz, Ho -phenyl ), 7.48 (t, 1H, H6 BT ), 7.38– 7.33 (m, 6H, Hm -Bn , Hp -Bn , H5 BT ), 7.29 (d, 2H, J = 7.10 Hz, Ho -Bn ), 7.25–7.23 (m, 3H, Ho -Bn , Hp -Bn ), 7.18 (d, 1H, H3furanyl ), 6.71 (d, 1H, J = 3.91 Hz, H4furanyl ), 6.81 (d, 2H, J = 9.05 Hz, Hm -phenyl ), 6.66 (d, 1H, J = 3.66 Hz, H3furanyl ), 4.72 (s, 4H, CH2 ). EI-MS (m/z): 523.6691 (M+ , 9), 507.6829 (13), 463.6231 (12), 360.6132 (19), 333.5671 (24), 332.5612 (98), 304.5133 (100), 273.3572 (5), 229.2967 (22), 163.0198 (8.57). (E)-3-(5-(4-(acetamido)phenyl)furan-2-yl)-2-(benzo[d]thiazol-2-yl) acrylonitrile (3n): a mixture of 3k (1 mmol), acetic anhydride (2 mmol), acetic acid (2 mL) was heated under reflux for 2 h (or under MWI at 80 °C and 150 W for 15 min). Later, water was added (10 mL) and the aqueous phase was extracted with ethyl acetate (2 × 30 mL). The organic phase was concentered under vacuum until 10 mL. The compound was filtered to give pure compound 3n. Solid red wine, yield 55%, mp 248–250 °C. IR (KBr) cm−1 : 3345, 3118, 2953, 2209 (CN), 1738, 1668, 1601. 1 H NMR (400 MHz, DMSO-d6 ): δ 10.17 (s, 1H, NH), 8.19 (s, 1H, H–C=), 8.15 (d, 1H, J = 7.83 Hz, H4 BT ), 8.04 (d, 1H, J = 8.07 Hz, H7 BT ), 7.88 (d, 2H, J = 8.81 Hz, Ho ), 7.74 (d, 2H, J = 8.56 Hz, Hm ), 7.56 (t, 1H, H6 BT ), 7.50–7.45 (m, 2H, H5 BT and H3furanyl ), 7.26 (d, 1H, H4furanyl ), 2.07 (s, 3H, CH3 ). EI-MS (m/z): 363 (M+ , 8), 321 (13), 261 (8), 210 (100), 193 (42), 152 (27), 115 (20). (E)-3-(4-(5-((E)-2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)furan-2-yl) phenylamino)acrylic acid (3o): a mixture of 3k (1 mmol), maleic anhydride (1 mmol), THF (2 mL), catalyst-free, was exposed to microwave radiation for 20 min at 50 °C and a maximum power of 150 W using a focused microwave reactor (CEM DiscoverTM , Mathews, NC, USA). Later, ethanol was added (10 mL), filtered and washed with hexane/ethanol (7:3). The solid was purified by column chromatography using CH2 Cl2 as eluent to afford pure compound 3o. Solid red, yield 82%, mp (nd). IR (KBr) cm−1 : 3221, 3073, 2904, 2216 (CN), 1584. 1 H NMR (400 MHz, DMSO-d6 ): δ 15.03 (s, 1H, COOH), 8.16 (s, 1H, H–C=), 1H

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8.12 (d, 1H, J = 7.83 Hz, H4 BT ), 8.01 (d, 1H, J = 8.07 Hz, H7 BT ), 7.89 (d, 2H, J = 8.31 Hz, Ho ), 7.78 (d, 2H, J = 8.55 Hz, Hm ), 7.55 (m, 3H, H6 BT , H5 BT and H3furanyl ), 7.26 (d, 1H, J = 3.18 Hz, H4furanyl ), 6.26 (d, 1H, J = 13.21 Hz, CO-C=H), 5.86 (d, 1H, J = 13.20 Hz, H=C-CO2 H). (E)-2-(benzo[d]thiazol-2-yl)-3-(4-(4-(pivaloyl)piperazin-1-yl) phenyl)acrylonitrile (3p). The compound was obtained using the same protocol reported in literature [22]. Orange solid, yield 52%, mp 191–192 °C. 1 H NMR (400 MHz, DMSO-d6 ): δ 8.18 (s, 1H, H–C=), 8.13 (d, 1H, J = 7.83 Hz, H4 BT ), 8.03–7.99 (m, 3H, H7 BT and Ho ), 7.54 (t, 1H, H6 BT ), 7.45 (t, 1H, H5 BT ), 7.09 (d, 2H, J = 9.05 Hz, Hm ), 3.45 (s, 8H, N-(CH2 )2 -N), 1.42 (s, 9H, CO-O-(CH3 )3 ). EI-MS (m/z): 446.5621 (M+ , 15.02), 346.3857 (100.00), 261.9745 (32.69). 3. CoMSIA models CoMSIA models were prepared as described in previous literature [24] using the Sybyl 7.3 software of Tripos [25]. All the molecules were aligned and minimized using the software Maestro [26] taking the acrylonitrile scaffold as template. For 3D-QSAR calculations, the compound sets were divided into training set (39 compounds) and test set (10 compounds). This split was performed in a way that both sets represent equally well the chemical and biological properties of the whole data set. The molecules of the training set were placed in a rectangular grid extended beyond 4 A˚ in each direction from the coordinates of each molecule. The interaction energies between a probe atom (a sp3 hybridized carbon atom with +1 charge) and all compounds were computed at the surrounding points, using a volume-dependent lattice with 2.0 A˚ grid spacing. Then, standard Sybyl parameters were used for a partial least squares (PLS) analysis. The numbers of components in PLS models were optimized by using the Q2 value, obtained from leaveone-out (LOO) cross-validation procedure using SAMPLS [27] method. The number of components was increased in order to verify the best model by using the Q2 per added component. For CoMSIA, similarity is expressed in terms of steric occupancy, electrostatic interactions, local hydrophobicity, Hbond donor and acceptor properties, using a 0.3 attenuation factor. 4. Results and discussion In this section, we discuss our results in 3 sections (chemistry, biological activities, and CoMSIA results). 4.1. Chemistry We report the synthesis of compounds 3a–j, corresponding to a novel library of 3-cyanoacetyl-indole-acrylonitriles obtained by microwave-assisted Knoevenagel-condensation (Scheme 1, Table 1). The reactions were performed in a focused microwave reactor (CEM Discoverۛ) at 300 watts and 100 °C of temperature. The compounds were obtained with high purity and moderate to high yields (51–90%) at different reaction times (1–60 min). Regarding the reactivity of the aldehydes that were used as reactants, it is worth noting that the electronic effects of the electron-releasing and electron-withdrawing substituents at position R2 of the derivatives had a minor effect on product yields, as can be seen in Table 1. We observed in a few cases that compounds obtained from 3formylchromone and 3-formylpyridine had lower yields, while products containing furanyl substituents had the higher yields. However, the same behavior was not observed for the reduction of the reaction time, since the longest time reactions were obtained with furfuraldehydes. Long reaction times obtained in this synthesis could be due to the low solubility of 3-cyanoacetylindole in ethanol and the low reactivity of some aldehydes. High temperatures (100 °C) instead of more efficient methodology reported recently [22] were used for them. In order to enlarge the library of compounds reported by our group in Refs. [18,22] and additionally obtain a wide range of biological activities, we explored the microwave-assisted synthesis of

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compounds 3k–p (Scheme 2). First, we synthesized the compound 4a by using the protocol reported in literature [22], and then we modified the structure of this compound by successive reactions performed on R1 substituent at position 4 of the 5-(4-R1 -phenyl)furan2-yl group. The 4-NO2 group (4a) was transformed by catalytic reduction to the corresponding NH2 group (3k) [28,29]. Later on, the compound 3k was used as precursor to obtain the libraries of compounds 3l–o by modification of the NH2 substituent using a focused microwave reactor [30,31]. The use of MWI allowed obtaining efficiently the derivatives 3l–o with good yields and short reaction times. In all cases, 1 H NMR analysis of all the novel derivatives revealed a single olefinic proton consistent with the formation of unique isomer E or Z. However, the derivatives were assigned to have thermodynamically more stable E configuration by considering E-acrylonitriles previously reported with similar structural characteristics [18,22,32–34]. 4.2. Biological activities The inhibitory potencies of the compounds 3a–j, 3k–p, and 4a–j as AChEIs were evaluated against Electrophorus electricus AChE; galantamine was used as reference compound. The selectivity index (SI) was calculated considering the inhibition of the derivatives over butyrylcholinesterase (BuChE). Both inhibitory activities were evaluated by the method of Ellman et al. [23]. The results were summarized in Tables 1 and 2. Compounds 3a–c, 3e, and 3g–j showed poor inhibition against AChE due to the presence of chemical substituents such as 3-(4 biphenyl) (3a; IC50 = 170 μM), 3-(4 -morpholino-phenyl) (3b; IC50 = 299 μM), 3-[4 -isopropyl-4H-chromen-3-yl] (3c; IC50 = 274 μM), 3-[4 -ethyl-4H-chromen-3-yl] (3e; IC50 = 451 μM), pyridinyl (3g and 3h; IC50 = 437 and 292 μM, respectively), and furanyl (3i and 3j, IC50 = 410 and 888 μM, respectively). However, the change of the isopropyl (3c, IC50 = 274 μM) and ethyl (3e, IC50 = 451 μM) substituents by methyl (3d, IC50 = 62 μM) group at position 6 in the chromone ring increased the inhibitory activity and the selectivity index for AChE. Thereby, the most selective and active AChE inhibitory in vitro activity was observed for the compound 3d. Interestingly, the compound 3d (IC50 = 62 μM for AChE) has the highest selectivity index (SI > 23) for AChE over BuChE when is compared with the selectivity of galantamine as a reference compound (SI = 16). When isopropyl and ethyl substituents at position 6 are removed and amino group (NH2 ) is added at position 2 of the chromone ring (compound 3f, IC50 = 78 μM), the activity did not vary significantly respect to compound 3d (62 μM); although the SI significantly decreases to 1.11. We suggest that 3d could be accommodated along the gorge of AChE and its binding mode within the AChE binding site would share some common characteristics with the AChE–donepezil complex [35] (see supplementary data, Fig. S1). The chemical structures and the inhibitory activities for 3k–p and the compounds 4a–j from Ref. [22] are reported in Table 2. Compounds 3k, 3n, and 4a showed a slight inhibition with IC50 values of 144, 134.9 and 130 μM, respectively. With the reduction of the NO2 group (4a, IC50 = 130 μM) to NH2 (3k, IC50 = 144 μM) or acetylation (3n, IC50 = 135 μM) the activity did not vary significantly; but the modifications of 3k at position 4 by bulky and hydrophobic substituents decreased the activity (3l: IC50 = 390 μM, 3m: IC50 = 366 μM, and 3o: IC50 = 330.72 μM). The more active compound in Table 2 is 3p (IC50 = 96.05 μM), which contains 4-(4Boc-piperazin-1-yl)phenyl substituent. 3p is a long shaped molecule and also we expected that its binding mode within the AChE binding site would share some common characteristics along the gorge of AChE, as was previously reported (see supplementary data, Fig. S2 and Table S1). The compounds 4b–c and 4e–j also showed poor inhibition against AChE due to the presence of chemical substituents such

Fig. 2. Atom-by-atom superposition used for CoMSIA analysis. The acrylonitrile scaffold was used as the building block for the alignment.

as furanyl-5-methyl acetate (4b, IC50 = 291.65 μM), quinolinyl (4c, IC50 = 434.36 μM), imidazolyl (4e, IC50 = 434.14 μM), 3-chromenyl (4f–i, IC50 = 413.05, 371.47, 361.57, and 543.42 μM), and pyridinyl (4j, IC50 = 693.92 μM). However, 4d showed a slight inhibition with IC50 value of 138 μM due to the presence of the methylendioxy substituent coupled with the quinoline scaffold. The results of in vitro BuChE inhibition for novel compounds show that the majority of them displayed poor inhibition against BuChE. Compound 3p displayed a poor activity against BuChE; thereby, this compound showed a moderate selectivity for AChE over BuChE (selectivity index > 15) when is compared with the selectivity of galantamine (selectivity index = 16.29). 4.3. CoMSIA results Fig. 2 shows the alignment carried out for the molecules used to generate the CoMSIA columns using Maestro software [26]. The stepwise development of CoMSIA models using SAMPLS and different field combinations [36,37] is presented in Table 3. The predictability of the models was the most important criteria for the election of the best model. CoMSIA methodology has the advantage of exploring all the fields related with the activities of the compounds, namely steric (S), electrostatic (E), hydrophobic (H), H-bond donor (D) and H-bond acceptor (A), and the possible combinations of fields included in the model, as shown in Table 3. With this methodology, predictive models were obtained using individual fields and different field combinations. In general, all the CoMSIA models (with only one field and combined fields) had high predictability because Q2 values were higher than 0.80 together with standard deviations lower than 0.59, except the model CoMSIA-D that showed the less reliable statistics (Q2 = 0.076, S = 1.384). The best predictability as indicated by the highest Q2 value of 0.901 was obtained for model CoMSIA-ED including electrostatic and H-bond donor coupled-fields by using three components. CoMSIA-ED showed contributions of the electrostatic and H-bond donor fields of 80.4 and 19.7%, respectively, with a very low standard deviation (S = 0.393) together with a high Fischer ratio (F = 144.81). Addition of more fields did not produce an improvement of Q2 value in the internal validation, since Q2 values were maintained between 0.842 and 0.896. Models were generated using training set and the CoMSIA-ED model was also used to predict the inhibitory activities for the AChEIs of the test set. Predictions of log(1/IC50 ) values for the test set are shown in Table 4. The correlations between the calculated and experimental values of log(1/IC50 ) for training set (model predictions and LOO cross-validation predictions) and test set are shown in Fig. 3. Considering classical QSAR statistics, our results indicate that this model was able to describe the test set variance with a high predictability (Rtest 2 = 0.924). However, it is clear that our model, instead of correlating, properly distributes the studied compounds into two groups. In this sense, the model CoMSIA-ED really identifies

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53

Table 3 Stepwise development of CoMSIA models by using SAMPLS and different field combinations.a NC is the number of components from PLS analysis, Q2 and Scv are the correlation coefficient and standard deviation of the leave-one-out (LOO) crossvalidation. CoMSIA

Q2

NC

Scv

Fields included in the model Steric

S E H D A SE SH SD SA ED EA HD HA DA SED SEA SHD SHA SDA HDA CoMSIA-All

4 2 3 3 4 2 3 6 3 3 3 5 4 5 3 4 4 4 5 5 3

0.857 0.900 0.880 0.076 0.838 0.898 0.888 0.851 0.853 0.901 0.870 0.872 0.869 0.842 0.896 0.861 0.880 0.868 0.847 0.872 0.872

0.553 0.448 0.498 1.384 0.587 0.453 0.482 0.582 0.551 0.452 0.520 0.530 0.528 0.585 0.464 0.543 0.505 0.528 0.579 0.531 0.516

Electrostatic

Hydrophobic

H-bond donor

x x x x x x x x x

x x x x x x

x x x x

x x x x x

x x

x

x

x x x

x x

x x x

x x x x x

x x x

x x

Table 4 Experimental and predicted inhibitory activities (log(1/IC50) of heteroaryl– acrylonitrile inhibitors of AChE by using CoMSIA-ED models. No.

Compound

3aa,b

log(1/IC50 ) Experimental

CoMSIA-ED Predicted

–2.230

–2.463

–2.475

–2.463

–2.437

–2.455

–1.792

–2.455

–2.654

–2.455

–1.892

–1.876

N

H N

O 3ba

O

N

H N

N

O 3ca

N

H N

O 3da

N

H N

3e

O 3f

a

O

O

O

N H2N

H N

O

O N

H N

O

O

O a

H-bond acceptor

O

O (continued on next page)

54

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Table 4 (continued) No.

3ga,b

Compound

N

H N

log(1/IC50 ) Experimental

CoMSIA-ED Predicted

–2.640

–2.755

–2.465

–2.688

–2.612

–2.447

–2.948

–2.447

–2.158

–2.173

–2.591

–2.421

–2.563

–2.582

–2.130

–2.328

–2.519

–2.332

–1.982

–2.554

N O 3ha

H N

N N O

3i

a

H N

N O O

3ja

H N

N

O

O O O

3kc

N N

NH2

O S

3lb,c

N N

NH

O S

3mc

N N

N

O S

3nc

N N

NH

O

O

S 3oc

N

O

O

N

OH

NH

O S

3pc

N N S

N

N

O O (continued on next page)

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Table 4 (continued) No.

Compound

4a

N N

log(1/IC50 ) Experimental

CoMSIA-ED Predicted

–2.113

–2.588

–2.464

–2.574

–2.637

–2.883

–2.140

–2.882

–2.637

–2.797

–2.616

–2.581

–2.569

–2.581

–2.558

–2.581

–2.735

–2.581

–2.841

–2.815

NO 2

O S

4b

N O N

O

O S

4c

N

Cl

N

Cl

N

N S 4d

N

O

N

O S

4e

N N N

N S

4f

N

O

N S 4g

O

N

O

N S 4h

O N

O

N S 4i

O N

O

N S 4j

O

N N N S

(continued on next page)

55

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Table 4 (continued) No.

Compound

4k

N

log(1/IC50 ) Experimental

CoMSIA-ED Predicted

–3.105

–2.891

–3.071

–2.561

–2.844

–2.561

–2.702

–2.590

–2.481

–2.520

–2.012

–2.882

–1.806

–2.574

–3.380

–2.939

–3.547

–2.574

–3.431

–2.589

N O

N S 4l

N

N O

N S 4mb

N

N O

N

F S

4n

O

N

N

N S 4ob

N N N

N H

S 4pb

N N

N S 4q

N N

O

Cl

S 4r

N N

Cl

S 4s

N N

N N

O S

4t

N N S

(continued on next page)

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Table 4 (continued) No.

Compound

4ub

N N

log(1/IC50 ) Experimental

CoMSIA-ED Predicted

–3.392

–2.592

0.698

0.415

0.537

0.421

0.568

0.418

0.638

0.418

0.366

0.597

0.602

0.364

0.455

0.716

0.522

1.161

0.420

0.859

N S

5a

N

O O O

O 2N 5b

N O O

O 2N 5c

N

O

O O 2N 5d

N

O O

O 2N 5e

N NO 2 O 2N

5f

N

N

O 2N 5g

N

OH O

O 2N 5hb

N

OH OH OH

O 2N 5i

N

OH

OH O 2N (continued on next page)

57

58

P. De-la-Torre et al. / Journal of the Taiwan Institute of Chemical Engineers 59 (2016) 45–60 Table 4 (continued) No.

Compound

5jb

N

CoMSIA-ED Predicted

0.244

0.430

0.508

0.429

0.657

0.921

O

O 2N 5k

O

log(1/IC50 ) Experimental

N

O Br O

O 2N 5lb

N H2N

O 2N

O

O

a

Compound sets 1. Test-set compounds. c Compound sets 2. Derivatives 4a–j were extracted from Ref. [22] and its inhibitory activities are informed for the first time in this article. Derivatives 4k–u and 5a–l were extracted from Refs. [18,19], respectively. b

Table 5 Rm 2 test on external validation for model CoMSIA-ED. Statistical parameter

Value

Rtest 2 R0 2 (Rtest 2 – R0 2 )/Rtest 2 k R 0 2 (R test 2 – R 0 2 )/R test 2 k Rm 2

0.924 0.918 0.006 0.985 0.903 0.023 0.973 0.811

Fig. 3. Scatter plot of the experimental activities versus predicted activities for model CoMSIA-ED: (•) training set predictions, () LOO cross-validated predictions, (×) test set predictions.

the chemical features that distinguish compounds in the most active group from compounds in the less active group. Rm 2 test was additionally applied to the test set [38]. Under this test we had to ensure the following: (i) correlation coefficient Rtest 2 between the predicted and observed activities of compounds from an external test set was close to 1 (Rtest 2 > 0.6), (ii) at least one of the correlation coefficients for regressions through the origin (predicted versus observed activities, or observed versus predicted activities), i.e. R0 2 or R 0 2 was close to Rtest 2 [(Rtest 2 − R0 2 )/Rtest 2 ] or [(Rtest 2 − R0 2 )/Rtest 2 ] < 0.1), (iii) at least one slope (k or k ) of regression lines through the origin was close to 1 (it corresponds to R0 2 or R 0 2 that is closer to Rtest 2 ); k or k should satisfy: 0.85 ≤ k ≤ 1.15, or 0.85 ≤ k ≤ 1.15, (iv) a high value of Rm 2 (Rm 2 > 0.5) was obtained, where Rm 2 accounts for the difference between values of R0 2 and Rtest 2 : Rm 2 = Rtest 2 × [1 – (Rtest 2 − R0 2 )1/2 ]. Table 5 shows that all the abovementioned points were satisfied. Fig. 4 depicts the contour maps for CoMSIA electrostatic (E) and H-bond donor (D) derived from model CoMSIA-ED. The most active

Fig. 4. CoMSIA contour maps for AChEIs (CoMSIA-ED model). Compound 5a is shown inside the field. Cyan isopleths indicate regions where H-bond donor groups enhance the activity, while purple isopleths indicate regions where H-bond donor groups disfavor the activity. Blue isopleths indicate regions where an increase of positive charges enhances the activity, and red isopleths indicate regions where more negative charges are favorable for activity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

studied compound 5a is included as a Ref. [19]. The cyan and purple isopleths represent areas where H-bond donor groups favored and disfavored the AChE inhibitory activity, respectively. A big cyan isopleth is located in front of the trimethoxyphenyl group of 5a. Some of the most active compounds contain H-bond donor groups in this zone

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such as compounds 5g, 5h, and 5i; in fact, the predictions of these compounds were overestimated using model CoMSIA-ED (Table 4). As mentioned before, the electrostatic field has a higher contribution in the model CoMSIA-ED. The big red and blue isopleths enclose the region of the structure of 5a due to the NO2 group strongly deactivating (electron withdrawing). The big red and blue isopleths located at the region of the nitrophenyl group are related with the negative density of the oxygens of the NO2 group and the mesomeric effects caused by the NO2 group, which create regions of positive charge around the phenyl ring (Fig. 4). These features are included in the most active compounds 5a–l, but are not preset in the remaining compounds. Other red and blue isopleths enclose positions 2 and 5 of the trimethoxyphenyl group of 5a. The red isopleth indicates that carbonyl group is important at position 4 in the derivatives that contain the chromone ring (5j–l) and the blue isopleth indicates that amino group at position 2 of the chromenyl ring in 5l has an important contribution to the inhibitory activity. The same group is found in compound 3f (the 2-amino-chromenyl substituent); however, this compound shows a low activity when it is compared with 5l. The big difference in activities is due to the above-mentioned red and blue isopleths located at nitrophenyl region of 5a (see supplementary data, Fig. S3). According to this analysis, the most important features identified in model CoMSIA-ED are the electrostatic isopleths located in the zone occupied by the substituent at position 2 of acrylonitrile. The inclusion of a nitro group or other electron withdrawing substituent in this zone should be considered in the future to obtain novel active compounds. The CoMSIA model reported here captured the molecular features contained in the compounds from the group of Parveen et al. [19], and they should be considered in the future design of acrylonitrile derivatives as AChE inhibitors. The main identified characteristic is the inclusion of substituents with an electron-withdrawal effect to aromatic groups at position 2 of the acrylonitrile. 5. Conclusions In summary, heteroaryl–acrylonitrile derivatives previously reported in literature and novel compounds of the same family have been studied by using 3D-QSAR analysis. The novel compounds were synthetized by Knoevenagel reaction using microwave-assisted organic synthesis (MAOS) and they were evaluated as AChE and BuChE inhibitors. Inhibition assays and 3D-QSAR analyses showed that the inhibitory activities of heteroaryl–acrylonitrile derivatives were influenced mainly by electrostatic properties of the substituents at position 2 and 3 on acrylonitrile scaffold and the presence of Hbond donor groups. Excellent statistical results were obtained in the QSAR analysis; the model CoMSIA-ED provided the most significant correlation of electrostatic and H-bond donor fields versus the AChE inhibitory activities with a Q2 value of 0.901. The contour plots may help to identify relevant regions where electron withdrawing/ donating groups play an important role in the activity against the studied target. The information obtained in this study provides the tools for the prediction of the affinity of heteroaryl–acrylonitriles and the rational basis for the structural design, development, and synthesis of novel, potent, and selective heteroaryl–acrylonitriles as AChEIs. For future design of new derivatives it is suggested to take into account the coupling of substituents with an electron-withdrawal effect to the (4nitrophenyl)acetonitrile, 2-(benzo[d]thiazolyl, and the cyanoacetylindole substituents in order to increase the bioactivity of this family of compounds as AChEIs. Conflict of interest The authors have no conflict of interest to declare.

59

Acknowledgments PD thanks the Doctoral Program of Applied Sciences at Universidad de Talca, as well as the Chilean International Cooperation Agency (AGCI) and CONICYT-Chile for a doctoral fellowship (Folio Beca 63130202). MG and LAS thank FONDECYT project 1100481, HP thanks FONDECYT project 3140288, JC thanks FONDECYT project 1130141.

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