Tetrahydroquinoline-isoxazole/isoxazoline hybrid compounds as potential cholinesterases inhibitors: Synthesis, enzyme inhibition assays, and molecular modeling studies

International Journal of

Molecular Sciences

Article

Tetrahydroquinoline-Isoxazole/Isoxazoline Hybrid

Compounds as Potential Cholinesterases Inhibitors:

Synthesis, Enzyme Inhibition Assays, and Molecular

Modeling Studies

Yeray A. Rodríguez Núñez ¹ , Margarita Gutíerrez *, Jans Alzate-Morales

2,

3

,

Francisco Adasme-Carreño ³ , Fausto M. Güiza , Cristian C. Bernal and

4

,

Arnold R. Romero Bohórquez *

1

Laboratorio Síntesis Orgánica, Doctorado en Ciencias Mención Investigación y Desarrollo de Productos

Bioactivos, Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, Talca 3460000,

Chile; yenuro30@gmail.com

Laboratorio Síntesis Orgánica, Instituto de Química de Recursos Naturales, Universidad de Talca,

Casilla 747, Talca 3460000, Chile

2

3

Center for Bioinformatics, Simulations and Modeling, Faculty of Engineering, University of Talca,

2

Norte 685, Casilla 721, Talca 3460000, Chile; jalzate@utalca.cl (J.A.-M.);

francisco.adasme@gmail.com (F.A.-C.)

4

Grupo de Investigación de Compuestos Orgánicos de Interés Medicinal (CODEIM), Parque Tecnológico

Guatiguará, Universidad Industrial de Santander, A.A. 678, Piedecuesta 681011, Colombia;

fausto.marin1@correo.uis.edu.co (F.M.G.); cristian.bernal@correo.uis.edu.co (C.C.B.)

Correspondence: mgutierrez@utalca.cl (M.G.); arafrom@uis.edu.co (A.R.R.B.)

*

ꢀ

ꢁꢂꢀꢃꢄꢅꢆꢇ

Received: 27 November 2019; Accepted: 4 December 2019; Published: 18 December 2019

ꢀ

ꢁꢂꢃꢄꢅꢆ

Abstract: A series of 44 hybrid compounds that included in their structure tetrahydroquinoline (THQ)

and isoxazole/isoxazoline moieties were synthesized through the 1,3-dipolar cycloaddition reaction

(

1,3-DC) from the corresponding N-allyl/propargyl THQs, previously obtained via cationic Povarov

reaction. In vitro cholinergic enzymes inhibition potential of all compounds was tested. Enzyme

inhibition assays showed that some hybrids exhibited signiﬁcant potency to inhibit acetylcholinesterase

(

AChE) and butyrylcholinesterase (BChE). Especially, the hybrid compound 5n presented the more

eﬀective inhibition against AChE (4.24 M) with an acceptable selectivity index versus BChE (SI: 5.19),

while compound 6aa exhibited the greatest inhibition activity on BChE (3.97 M) and a signiﬁcant

µ

selectivity index against AChE (SI: 0.04). Kinetic studies were carried out for compounds with greater

inhibitory activity of cholinesterases. Structure–activity relationships of the molecular hybrids were

analyzed, through computational models using a molecular cross-docking algorithm and Molecular

Mechanics/Generalized Born Surface Area (MM/GBSA) binding free energy approach, which indicated

a good correlation between the experimental inhibition values and the predicted free binding energy.

Keywords: Alzheimer’s disease; cholinesterase inhibitors; cross-docking and MM/GBSA free binding

energy; hybrid compounds

1

. Introduction

Alzheimer’s disease (AD) is the most common form of dementia that aﬀects over 30 million

people, most of whom are elderly [

which is characterized by loss of memory and the gradual decline in cognitive functions resulting in

behavior changes [ ]. Knowledge about its causes and mechanisms has grown enormously in the last

1]. AD is a major public health problem in developed countries,

2

decades. For instance, extracellular deposits of

β-amyloid plaques (A

β), the formation of intracellular

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www.mdpi.com /journal /ijms

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neuroﬁbrillary tangles (NFT), decreased activity of choline acetyltransferase enzyme, and oxidative

stress are some of the pathological features of AD [3,4].

There is currently no cure for AD, although there are some drugs available that can delay the

disease progression at certain stages of illness [5]. The most signiﬁcant strategy of palliative treatment

for AD has been the use of cholinergic drugs, which counteract cognitive dysfunction by inhibition of

the enzyme AChE, thus increasing the levels of the neurotransmitter acetylcholine, regulating synaptic

functions [6].

The active site of AChE (CAS) is composed of a catalytic triad (Ser-His-Glu), found at the bottom

of ~20 Å deep gorge lined by a high presence of aromatic residues [

the peripheral anionic site (PAS) that is located at 18 Å away from the active site [

with a supportive role in the cholinergic process. It has received important attention because once it

is inhibited, cognitive functions are improved [ ]. Although AChE and BChE share some structural

7

], and another binding site named

8]. BChE is an enzyme

9

similarities, including the presence of a catalytic center within the gorge, ligand binding modes of

BChE diﬀer signiﬁcantly from those of AChE due to the absence of some aromatic residues located

mainly in the acyl binding pocket at midgorge. Thereby, diﬀerent compounds can speciﬁcally inhibit

one of those enzymes, or they are also able to exert a dual binding action, depending on amino acid

recognition and interactions generated within the active site [10,11].

Commercial drugs like donepezil, rivastigmine, galantamine, and tacrine have demonstrated

eﬃcacy in the treatment of patients with a mild-to-moderate AD, although the therapeutic use of

tacrine has been discouraged due to its hepatotoxicity eﬀects [12]. Generally, these drugs bind at a

speciﬁc point of the active site, but recent research is looking for compounds having an action at several

points of the active site in order to increase their bioactivity. Seeking to obtain new compounds that

can recognize more than one binding spot in the same therapeutic target, tacrine, galantamine, and

donepezil have been used as scaﬀolds to produce a new series of hybrid compounds for improving the

inhibitory activity against cholinergic enzymes, and at the same time, improve their pharmacological

properties [13–16]. Curcumin–tacrine hybrids have been synthesized as multifunctional compounds

that interact with the catalytic active site and the peripheral anionic site of AChE, showing positive

inhibitory eﬀects even more potent than individual tacrine [17]. Similarly, the synthesis of molecular

hybrids from donepezil and ebselen, a drug capable of inhibiting iron-induced tau phosphorylation,

has been described. The new hybrid compounds showed good dual inhibitory activity against AChE

and BChE [16]. Thus, the molecular hybridization synthetic approach can be applied to obtain new

molecules from the combination of two or more pharmacophoric structures that are present in bioactive

compounds [18] (Figure 1).

Figure 1. Hybrid compounds from commercial drugs tested on cholinergic enzymes.

With all the above in mind, our previously reported work has shown the synthesis and

anticholinesterase activity of N-allyl/propargyl THQs obtained by an eﬃcient and mild methodology

based on the cationic Povarov reaction (a domino Mannich/Friedel–Crafts reaction) [19] and cytotoxicity

on HeLa and HepG2 cancer cell for some THQ–isoxazole hybrids [20,21]. As a further step in the

development of new molecules with improved cholinergic activity, in the present study, we describe

the successful synthesis of THQ–isoxazoline/isoxazole hybrids through the 1,3-DC reaction, as well as

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their in vitro inhibitory activity evaluation against AChE and BChE. Additionally, their ADME-Tox

properties (absorption, distribution, metabolism, excretion, and toxicity), potential binding modes,

and binding aﬃnity against AChE and BChE were predicted using computational tools like ensemble

docking and MM/GBSA calculations.

2

. Results and Discussion

2

.1. Chemistry

Taking advantage of the reactivity of the propargyl and allyl fragments present in the THQ

compounds that were previously synthesized via the cationic Povarov reaction [19], the synthesis of

new THQ–isoxazoline/isoxazole hybrids was carried out using a gentle and eﬃcient process based on

1

,3-dipolar cycloaddition. In this reaction, nitrile oxides (formed in situ from corresponding aldoximes)

were used as dipole and the propargyl and allyl THQs as dipolarophiles. In both cases, reactions

occur easily and with good yields in anhydrous DCM in the presence of Et N and NaOCl at room

3

temperature (Scheme 1). New hybrid compounds THQ–isoxazoline (series 5) and THQ–isoxazole

synthesized (series 6) contain a methylene bridge that keeps the two N-heterocyclic rings together.

Scheme 1. Synthesis of new THQ–isoxazoline (5a-p) and THQ–isoxazole (6a-ab) hybrid compounds.

The hybrid compounds of series 5a-p were obtained as a mixture of a pair of non-separable

diastereoisomers through column chromatography. In this case, all compounds are viscous oils, and

the reaction takes place with good yield after puriﬁcation (63–77%; Supplementary Table S1). Whereas,

hybrid compounds of series 6a-ab were obtained mostly as viscous oils, and some as stable solids, with

good and excellent reaction yields after column chromatography (40–95%; Table S2).

In this report, 40 hybrid compounds with four (4) diﬀerent substituent groups at the C-6 position of

the THQ scaﬀold were synthesized. In addition, when the aryloxime 2 was 3,4-dimethoxybenzaldehyde

oxime, four other (4) new hybrid compounds with substituent groups at C-6 and C-8 positions of

THQ were obtained. All THQ–isoxazole/isoxazoline molecular hybrids were obtained as regioisomers

3

,5-disubstituted and in all of them were possible systematically characterized by spectroscopic

1

13

techniques, including IR, ESI-MS, H-NMR, and C-NMR.

In the case of IR spectra, typical bands for carbonyl group C=O were observed (1656–1681 cm ¹).

−

The band associated with the vibration of the C–O bond of methoxy groups from the starting aldoximes

−

1

(

2

1122–1178 cm ) was also observed. Besides, the vibration band of the aromatic C–H bonds at

−

832–2976 cm was identiﬁed easily. H NMR and C NMR spectroscopy in CDCl solution

3

1

13

were carried out, and all signals for each individual H–atom and C–atom were properly assigned.

1

All H-NMR spectra of the THQ–isoxazole/isoxazoline hybrids synthesized showed a similar patron,

which was characterized by the presence of several well-deﬁned signal groups. At high ﬁeld (chemical

shifts between 1.80–3.60 ppm) the signals corresponding to the methylenic protons of the 2-pyrrolidone

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fragment and the piperidine ring were characterized. The signal corresponding to the chiral proton

of the THQ ring was observed around 5.40 ppm. Signals for aromatic protons were observed and

clearly diﬀerentiated between 6.31–7.70 ppm. In the case of molecular hybrids belonging to the

THQ–isoxazole 6 series, a single signal was observed between 6.29–6.63 ppm, corresponding to the

proton of the isoxazole ring, which gives strong evidence of the successful formation of the new

heterocyclic. Similarly, THQ-isoxazoline 5 molecular hybrids showed a signal around 4.89–5.21 ppm

indicating the formation of the isoxazoline ring. Furthermore, the lack of signal for protons from

the propargyl and the allyl fragments was additional evidence that the 1,3-DC reaction was carried

out satisfactorily.

2

.2. Cholinesterase Biological Activity

All synthesized hybrid compounds were evaluated in vitro as dual AChE/BChE inhibitors. The

IC₅₀values were calculated by means of regression analysis. Results are expressed as averaged

µM ± SEM values, and compared using ANOVA analysis. A p-value of less than 0.05 was considered

signiﬁcant. Details for pharmacological experiments are described in the experimental section as well

as in our previous report [19].

Results of enzyme inhibition assays of THQ–isoxazoline hybrids 5a-p on AChE and BChE showed

that the hybrid 5k (R = OCH ) and hybrids 5n, 5o, and 5p (R = Cl) presented good inhibitory action

1

3

1

against AChE (IC₅₀value < 15.26

µM). Compound 5n is highlighted with an IC₅₀value of 4.24 µM.

These THQ-isoxazoline hybrids also showed high selectivity for AChE enzyme over BChE (SI values

of 16.8, 5.19, 6.35, and 8.61, respectively) (Table 1).

Table 1. Inhibitory activity of THQ–isoxazoline hybrids 5 on AChE and BChE.

Comp. 5

R₁

R₂

R₃

R₄

IC₅₀AChE (µM)^aIC₅₀BChE (µM)^a

SI^b

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

H

OCH₃

H

77.37 ± 0.01

95.14 ± 0.08

100.65 ± 0.05

49.82 ± 0.03

189.10 ± 0.01

16.31 ± 0.01

99.71 ± 0.01

37.41 ± 0.03

82.04 ± 0.11

93.66 ± 0.006

15.26 ± 0.02

109.26 ± 0.01

30.10 ± 0.01

4.24 ± 0.02

81.90 ± 0.04

65.59 ± 0.006

N.D

1.06

0.69

-

OCH₃OCH₃

OCH₃OCH₃OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

H

13.66 ± 0.01

31.40 ± 0.01

23.36 ± 0.005

31.49 ± 0.005

63.72 ± 0.001

146.37 ± 0.03

144.71 ± 0.004

255.82 ± 0.01

43.67 ± 0.02

19.09 ± 0.01

22.00 ± 0.02

83.65 ± 0.01

105.78 ± 002

8.80 ± 0.5

0.27

0.17

1.43

0.32

1.70

1.79

1.55

16.8

0.40

0.63

5.19

6.35

8.61

16.29

CH₃

OCH₃

H

OCH₃

H

OCH₃

H

OCH₃OCH₃OCH₃

OCH₃OCH₃OCH₃OCH₃

Cl

H

OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

13.17 ±0.02

12.28 ± 0.01

0.57 ± 0.10

Galantamine

a

Values are the average from three independent experiments. ^bSelectivity index (SI) for AChE is deﬁned as IC50

(

BChE)/IC50 (AChE).

Conversely, results indicated that THQ–isozaxole hybrids 6a (R = H), 6h (R = OCH ),

1

3

6

r

(R = CH CH ), and hybrids 6z and 6aa (R = Br) showed a very high inhibitory activity against BChE,

1

2

3

1

with IC₅₀values (<7.18

µM) even better than that found for the reference compound galantamine

(

IC₅₀= 8.80 µM), a commercial drug widely used in the palliative treatment of AD. Hybrid 6aa,

a molecular hybrid with a bromine atom at C-6 in the THQ ring and 3,4-dimethoxyphenyl group at

the C-3 of isoxazole moiety, was the most active compound (IC₅₀= 3.97 M). Notably, this hybrid

µ

compound showed high selectivity for BChE, being about 24 times more potent against BChE than for

the AChE enzyme (Table 2).

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Table 2. Inhibitory activity of THQ–isoxazole hybrids 6 on AChE and BChE.

Comp. 6

R₁

R₂

R₃

R₄

R₅

AChE (µM)^a

BChE (µM)^a

SI^b

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

ab

H

OCH₃

H

Cl

H

Cl

H

Cl

H

Cl

H

104.74 ± 0.01

42.44 ± 0.01

22.07 ± 0.01

37.84 ± 0.01

30.43 ± 0.02

54.71 ± 0.02

72.94 ± 0.02

52.76 ± 0.01

84.51 ± 0.01

54.69 ± 0.01

20.19 ± 0.02

92.01 ± 0.05

13.27 ± 0.01

8.98 ± 0.01

4.36 ± 0.03

27.25 ± 0.003

47.96 ± 0.01

18.51 ± 0.01

79.81 ± 0.06

370.12 ± 0.03

78.16 ± 0.01

4.33 ± 0.004

213.22 ± 0.01

149.00 ± 0.02

73.26 ± 0.01

14.11 ± 0.02

18.42 ± 0.01

24.67 ± 0.01

313.82 ± 0.01

46.84 ± 0.01

108.28 ± 0.01

7.18 ± 0.004

22.28 ± 0.004

N.D

17.74 ± 0.01

83.75 ± 0.01

46.92 ± 0.01

163.12 ± 0.01

84.91 ± 0.01

5.81 ± 0.003

3.97 ± 0.005

54.09 ± 0.01

8.80 ± 0.5

0.04

0.64

2.17

0.49

2.62

6.76

1.07

0.08

2.52

2.72

3.63

0.15

1.39

2.75

3.88

1.11

1.43

0.38

0.83

-

0.15

1.97

2.79

6.57

0.87

0.48

0.04

0.36

16.29

OCH₃OCH₃OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

H

CH₃

OCH₃

Cl

H

OCH₃

H

OCH₃

H

OCH₃

H

80.89 ± 0.04

41.95 ± 0.11

75.95 ± 0.04

18.83 ± 0.003

26.82 ±0.002

66.77 ± 0.01

116.63 ± 0.01

42.48 ± 0.004

16.83 ± 0.002

24.83 ± 0.003

97.84 ± 0.01

12.19 ± 0.005

96.1 ± 0.01

CH CH₃

H

OCH₃

H

2

CH CH₃

2

CH CH₃OCH₃OCH₃

2

CH CH₃OCH₃OCH₃OCH₃

2

F

H

OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

H

OCH₃OCH₃

OCH₃OCH₃OCH₃

F

Br

H

OCH₃

H

148.31 ± 0.004

0.57 ± 0.10

Galantamine

a

Values are the average from three independent experiments. ^bSelectivity index (SI) for AChE is deﬁned as IC50

(

BChE)/IC50 (AChE).

As can be seen (Tables 1 and 2), a signiﬁcant decrease in IC₅₀values and consequently a better

inhibitory activity of these hybrid compounds are observed when diﬀerent substituents groups are

introduced into the THQ scaﬀold, especially halogens (Cl and Br), and methoxy groups into aryl

fragment on C-3 position of isoxazoline or isoxazole ring. However, beyond the diﬀerent substituents

groups on the hybrid molecules and their impact on IC₅₀values, it is important to rescue the synergistic

eﬀect generated by the two fused heterocyclic rings, in order to inhibit the biological activity of

cholinergic enzymes selectively. Most of the new hybrid compounds turned out to be eﬀective

inhibitors of cholinergic enzymes with IC₅₀values spanning a µM range. The new designed hybrid

compounds are better inhibitors of cholinergic enzymes when compared to our previous series [19

]

and, in some cases, more active than the reference drug, galantamine.

Based on the previously obtained results, and with the aim to assess the kinetic model of

AChE/BChE inhibition of the target compounds, the most active compounds 5n and 6aa were subjected

to kinetics studies. For this purpose, the rate of enzyme activity was measured at eight diﬀerent

concentrations of substrate ATC/BTC. The obtained Michaelis–Menten parameters along with the

Lineweaver–Burk plots allow us to conclude that the compound 5n (Figure 2a) displayed a mixed-type

inhibition against AChE with variation in Km and Vmax values. However, the compound 6aa

(Figure 2b) showed a competitive type inhibition against BChE with variation in Km values.

The analysis of inhibition modality is a piece of relevant information in the early stages of design

and drug discovery, showing an approximation to the type of bind with the active site. As an example,

competitive inhibitors bind exclusively to the free enzyme form, while non-competitive or mixed

type inhibitors bind with some aﬃnity to the free enzyme, as the enzyme–substrate complex. Thus,

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the non-competitive modality can be an advantage in vivo when the physiological context exposes

the enzyme to high substrate concentrations. Although the clinical advantage of non-competitive

inhibition has been recognized, there are a very large number of drugs in clinical use today that are

competitive enzyme inhibitors, and which can be related to historical approaches for drug discovery

that have been focused on active site-directed inhibitors [22

from tetrahydroquinolines that act via competitive (6aa) and mixed (5n) mechanism. The mechanistic

diﬀerences between compounds 5n, 6aa, and galantamine could be assigned to the interactions with

Trp84, Tyr334, and Phe330, which were observed only for these two tetrahydroquinolines derivatives

,23]. Here, we presented two hybrids

π–π

with higher inhibitory potency. These results highlight the importance of the

ligands and thus can be used for designing new AChEIs.

π–π interaction for the

(

a)

(b)

Figure 2. The Lineweaver–Burk plot of AChE/BChE (0.02 U) with acetylthiocholine/butyrylthiocholine

substrate in the absence and presence of inhibitor 5n (a) and 6aa (b), respectively.

2

.3. Binding Modes and Aﬃnities Against AChE

The binding modes of the synthesized compounds within the AChE active site were predicted by

means of ensemble docking, where each ligand is docked into multiple protein structures, as a low-cost

alternative to model protein ﬂexibility during docking. As the chirality of the scaﬀold was unknown,

all possible enantiomers (120 in total) were considered in the docking calculations. Thousands of

docking poses were obtained and they were clustered and then rescored by the MM/GBSA method.

The selected cluster of representative poses with chiralities R and RS are presented in Figure 3a. The

predicted docking pose for galantamine within AChE active site (using PDB code: 1ODC), as reference

compound, was also obtained. It is observed that docked compounds adopted approximately the same

position within the active site. The binding modes encompass four X-ray structures with PDB codes

1

ODC, 1ZGC, 2CKM, and 2CEK, which exhibit distinct conformations of the side chains of the amino

acids Trp279, Phe330, and His440. The THQ moiety was located at the anionic site near the bottom

of the active site gorge, which is composed by the amino acids Trp84, Tyr130, Phe330, and Phe331

(magenta in Figure 3), occupying the same space as known compounds such as huprine (PDB code

1

E66) [24]. The terminal phenyl group extends to the peripheral anionic site (PAS, orange) at the top of

the gorge formed by the amino acids Tyr70, Asp72, Tyr121, Trp279, and Tyr334, resembling the position

of alkylene-linked heterodimers of tacrine (PDB code 1ZGC) [25]. This binding orientation is similar to

the docking poses obtained for another THQ derivative series reported in a previous work [19]. With

respect to galantamine, it established a cation–π interaction with Phe330 and π–π stacking interactions

with residue Trp84, and as mentioned above, our compounds occupied the same space within active

site but they also extended their isoxazole and phenyl rings towards the PAS going through acyl

pocket (residues Phe288 and Phe290) (Figure 3c). Potentially improvement in the interactions with

residues composing the PAS site, where there exist some structural diﬀerences between AChE and

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BChE, would allow us to achieve better speciﬁcity in a next series of hybrid compounds. It is worth

mentioning that it was not possible to obtain analogous docking poses for the four compounds having

a chlorine atom at position 8 of the THQs (substituent at R in Scheme 1), probably due to steric

5

clashes with side chain of residue Tyr334 that may have prevented them to enter the active site in the

proper orientation. In general, the predicted docking poses seem to agree with the binding modes

reported for known AChE inhibitors, which are placed in well-known pockets described as relevant

for inhibitory potency. On the other hand, we also performed molecular docking of compounds 5n

and 6aa, as the most actives against AChE and BChE, respectively, within the BChE active site with

the aim to understand their potential selectivity on these molecular targets. In Figures S1 and S2 are

shown the most favorable poses for compounds 5n and 6aa within BChE and AChE binding sites.

As can be seen, the poses adopted by those compounds resemble the ones obtained for all set of

compounds against AChE active site. Speciﬁcally, 6aa (IC₅₀BChE (

kcal/mol) = 8.203), presented stacking interactions with residues Trp82 and His438; meanwhile,

compound 5n (IC₅₀BChE ( M) = 22.00, and a docking score (kcal/mol) = 7.681), only establishes one

stacking interaction with Trp82 (see Figure S1). With respect to the high selectivity of compound

aa (SI = 0.04) against BChE, it is diﬃcult to drawn conclusions using only binding energies obtained

µM) = 3.97), with a docking score

(

−

π–π

µ

−

π–π

6

with molecular docking. However, it could be argued that structural diﬀerences at the PAS site of

BChE, which is conformed by less bulky residues like Asn68, Gln119 and Ala277, when compared

with PAS site in AChE (residues Tyr70, Tyr121, and Trp279) would be involved in an easier access of

compound to gorge in conjunction to better physicochemical properties due to bromide substituent at

R (see Figures S1 and S2). In the case of 5n compound

´

s selectivity against AChE, there is a good

correspondence between computational docking calculations and biological experiments. For instance,

the IC₅₀AChE ( M) = 4.24 and IC₅₀BChE ( M) = 22.00 values compared well with the corresponding

aﬃnities in terms of docking scoring energies that are 10.461 and 7.861 kcal/mol, respectively. The

trend in aﬃnity can also be reproduced by docking calculations, which means that compound 5n

has more favorable binding energy within AChE binding site ( 10.461 kcal/mol) than compound 6aa

8.127 kcal/mol). This binding energy diﬀerence could be due to diﬀerences at the PAS site, as was

mentioned above, but also to a structural diﬀerence at hydrophobic patch, where AChE has a Phe330

and BChE an Ala328. Compound 5n establishes a stacking interaction with Phe330 that cannot

1

µ

−

(−

π–π

be established at BChE active site (see Figures S1 and S2). In any case, the diﬀerences in residues

conforming to the PAS site, the acyl pocket, and to a lesser extent the hydrophobic patch, will be taken

into account when designing new hybrid compounds against these cholinesterases.

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Figure 3. Predicted binding modes for studied compounds within the AChE active site. The most

populated pose cluster is shown in ( ), and the identiﬁed intermolecular interactions are depicted in

b). Compounds are shown in ball-and-stick representation with cyan carbons. Relevant amino acids

a

(

are shown in tubes in the following coloring code: catalytic triad in yellow, anionic site in magenta,

acyl pocket in green, and peripheral anionic site in orange. Secondary protein structure is depicted

as white ribbons. (c). Predicted binding mode for galantamine (shown with carbons in plum) within

AChE active site.

Similarly, the predicted free binding energies (

∆

Gpred) through the MM/GBSA method agreed well

with the experimental energies (

∆Gexpt, converted from the IC₅₀values), showing a good correlation

2

(R = 0.83), as evidenced in Figure 4. More importantly, the most and least active compounds were

correctly ranked among the 40 compounds.

expt

Figure 4. Scatter plot of the experimental activities (IC ) converted to free binding energies (∆G )

5

0

2

vs. predicted free binding energies (∆G_p_r_e_d). Solid line is shown for the linear regression ﬁt (R = 0.83).

The speciﬁc protein–ligand interactions observed in the representative docking poses are

summarized in Table 3. A single ligand is shown within the AChE active site in Figure 3b as

an illustrative example. Several

the THQ and terminal phenyl moieties and the aromatic cavity formed by the side chains of Trp84,

Trp279, Phe330, and Tyr334. Particularly, the -stacking interaction in sandwich conformation with

residue Phe330 seems to be an anchor point, as all compounds exhibited such interaction with a very

short distance (3.56 0.38 Å), which suggest a strong attraction. Similarly, most ligands also established

-stacking interactions with either Trp84 (in parallel-displaced conformation) or Tyr334 (in T-shaped

conformation), or both, with average distances of 4.10 0.18 Å and 4.55 0.62 Å, respectively. These

same non-covalent interactions are observed in the X-ray structure of huprine [24]. Some compounds

7) also presented a -stacking interaction between the terminal phenyl group and the side chain of

Trp279, with a distance of 4.92 0.45 Å. Additionally, hydrogen bonds (H-bonds) were identiﬁed

between the methoxy substituents at the terminal phenyl moiety and the amine of the Trp279 side

chain in 11 out of the 40 ligands with an average distance of 3.17 0.28 Å. Furthermore, minor

π-stacking interactions were identiﬁed between the aromatic rings of

π

±

π

±

(

π

±

H-bonds were observed with one aromatic hydrogen of Phe330 side chain, either via either the carbonyl

oxygen or oxygen of the pyrrolidone moiety, with average distances of 2.68 ± 0.13 Å and 1.99 ± 0.30 Å,

respectively. These results suggest that the synthesized ligands interact with AChE primarily by

π

-stacking and other hydrophobic interactions, whereas H-bonds only help to further stabilize the

binding of these inhibitors.

Int. J. Mol. Sci. 2020, 21, 5

9 of 25

Table 3. Speciﬁc protein–ligand interactions, and their occurrence (%) and averaged distances, obtained

from molecular docking experiments.

Interaction Type Residues

N

Distance (Å)

Trp279

11 (28%)

8 (20%)

13 (33%)

3.17 ± 0.28

2.68 ± 0.13

1.99 ± 0.30

4.10 ± 0.18

4.92 ± 0.45

3.56 ± 0.38

4.55 ± 0.62

†

H-bond

Phe330

‡

Trp84

24 (60%)

7 (18%)

40 (100%)

24 (60%)

Trp279

Phe330

Tyr334

π-stacking

H-bond distances were measured between the acceptor and hydrogen atoms, whereas

π

-stacking interaction

distances were measured between the centers of mass of the aromatic rings.

carbonyl oxygen of the ligands and one of the aromatic hydrogens of Phe330.

oxygen of the isoxazole moiety of the ligands and one of the aromatic hydrogens of Phe330.

†

Aromatic H-bond between the

‡

Furthermore, an in silico analysis of the ADME pharmacokinetic properties (Supplementary

Table S3) indicates that there are no signiﬁcant violations of Lipinski’s rule since the calculated

descriptors are within the normal ranges (Log p = 3.181–4.881, HB acceptors = 5.500–9.700, and

HB = 0.0 donors), except 6o, 6aa, and 6ab compounds that have a molecular weight over 500 atomic

mass units (502.396, 512.402, and 542.428 g/mol, respectively). With respect to the predicted qualitative

oral absorption, a value of 1, 2, or 3 indicates low, medium, or high absorption, respectively. This

prediction was made through the analysis of the adequate values of diﬀerent descriptors, such as the

number of probable metabolic reactions, the number of rotatable bonds, log p, solubility and cellular

permeability. Most of the compounds had a value of 3, so they could present a good absorption at the

oral level. In addition, membrane penetration and distribution of these compounds in organisms are

given by parameters such as PSA and solubility, which were analyzed and are within the acceptable

range deﬁned for human use. In general, after calculating the in silico values of the diﬀerent properties,

it can be inferred that these new THQ–isoxazole/isoxazoline hybrids have characteristics that classify

them as potential drugs. Table 4 shows the values of descriptors of the most active compounds.

Table 4. Main descriptors calculated for the most active THQ–isoxazole/isoxazoline hybrid compounds.

M.W.

g/mol)

log p

(o/w)

Mol.

Vol (Å )

Acceptor

HB

Donor

HB

log S^f

Oral Abs.^g

5 Rule^h

Comp.

PSA^e

a

3 b

c

d

(

5

6

n

h

z

439.941

403.480

482.376

512.402

3.661

3.798

4.399

4.528

2.020

1.312.201

1.272.946

1.328.457

1.411.174

903.565

7.450

6.250

7.000

5.200

0.000

1.000

61.604

61.931

61.908

66.358

44.892

−4.601

−4.608

−5.547

−5.805

−2.163

3

0

1

0

6

aa

Galantamine 287.358

a

6.5); ^bTotal solvent accessible volume in cubic angstroms using a probe with a

log p for octanol/water (

−

2.0–

−

radius of 1.4 Å. ^cEstimated number of H-bonds that would be donated by the solute to water molecules in an

d

aqueous solution; Estimated number of H-bonds that would be accepted by solute from water molecules in an

aqueous solution; ^eVan der Waals surface areas of polar nitrogen and oxygen atoms; Predicted aqueous solubility,

f

−3

g

log S, S in mol dm

Violations number of Lipinski’s rule of 5.

( 6.5–0.5); Qualitative human oral absorption predicted: 1, 2 or 3 for low, medium or high;

−

h

3

. Materials and Methods

3

.1. Chemistry

All reagents were purchased from Merck (Darmstadt, Germany), J.T. Baker, and Sigma and

1

13

Aldrich Chemical Co. and they were used without puriﬁcation. H NMR and C NMR were recorded

at 400 and 100 MHz, respectively, on a Bruker Ultrashield-400 spectrometer. The chemical shifts (

δ

)

are reported in parts per million (ppm), using CDCl as solvent with TMS as an internal standard.

3

The coupling constant (J) are reported in Hertz (Hz). A FT-IR Bruker Tensor 27 spectrophotometer

coupled to Bruker platinum ATR cell was used to obtain IR spectra. Mass spectra were recorded

Int. J. Mol. Sci. 2020, 21, 5

10 of 25

using a Bruker Daltonics ESI-IT Amazon X spectrometer with direct injection, operating in full scan

◦

at 300 C and 4500 V in the capillary. High-resolution mass spectrometry ESI-MS analyses were

conducted in a high-resolution hybrid quadrupole (Q) and orthogonal time-of-ﬂight (TOF) mass

spectrometer (Waters/Micromass Q-TOF micro, Manchester, UK) with a constant nebulizer temperature

◦

of 100 C. The elemental analysis of the diﬀerent compounds was performed in ThermoScientiﬁc

CHNS-O analyzer equipment (Model Flash 2000, Thermo Scientiﬁc, Waltham, MA, USA). Melting

points (uncorrected) were determined on an Electrothermal IA9100 melting point apparatus. The

progress of reaction was monitored by TLC, puriﬁcation of compounds was performed by column

chromatography using silica gel as support. Solvents employed were of analytical grade.

3

.1.1. General Procedure for the Synthesis of Aldoximes

A mixture of appropriate aromatic aldehyde 1 (1 mmol) hydroxylamine hydrochloride (1.1 mmol),

and sodium carbonate (1.1 mmol) was dissolved in MeOH and stirred at room temperature for 2 h.

The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 15 mL). The

organic layer was separated and dried (Na SO ). The respective aldoxime 2 was obtained by solvent

×

2

4

removal under vacuum.

3

.1.2. General Procedure for Isoxazole/Isoxazoline Synthesis

A mixture of N-Allyl (3) or N-propargyl (4) THQ (1 mmol) and the appropriate aldoxime 2 was

dissolved in 8 mL of DCM and stirred for 10 min. Triethylamine (1 mmol) was added and the reaction

◦

was cooled to 0 C and stirred. 8 mL of NaClO was added dropwise. The reaction mixture was

stirred 3–4 h at room temperature. Water was added and ethyl acetate was used to extract. The

organic layer was separated and dried with NaSO . The solvent was removed and the ﬁnal product

4

puriﬁed by column chromatography using appropriate ethyl acetate and petroleum ether mixture to

provide isoxazolines 5 and isoxazoles 6 compounds. NMR spectra of compounds can be found in the

Supplementary Materials (Figures S3–S22).

0 0

-phenyl-5-[(4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-4,5

dihydroisoxazole (5a): 74% yield; Yellow oil; IR (ATR): 1664, 1605, 1502, 1357 cm . H NMR (400 MHz,

3

−

1 1

0

CDCl₃)

δ

(ppm): 77.63–7.60 (8H, m), 7.40–7.37 (8H, m), 7.18–7.15 (2H, m), 5.41 (2H, q, J = 7.2 Hz, 4 -H),

4

.80–4.73 (2H, m), 3.54–3.44 (6H, m), 3.32–3.28 (4H, m), 3.24–3.16 (4H, m), 3.05–2.99 (2H, m), 2.45–2.39

13

(

1

7

1

3

2

4H, m), 2.01–1.91 (4H, m), 1.49–1.47 (4H, m). C NMR (100 MHz, CDCl₃)

74.75, 156.73, 146.21, 138.31, 131.56, 130.18, 129.60, 129.34, 128.78 (2C), 126.88 (2C), 126.50, 126.30,

9.96, 57.88, 57.51, 48.32, 42.43, 38.53, 31.41, 17.96, 16.48. Diastereomer : 174.75, 156.68, 146.21, 138.07,

31.56, 130.16, 129,61, 129.30, 128.78 (2C), 126.88 (2C), 126.48, 126.27, 79,96, 57.86, 57.50, 48.28, 42.41,

δ (ppm). Diastereomer α:

β

+

8.53, 31.41, 17.96, 16.42. ESI-MS (m/z): 376 [M + H] , 398 [M + Na] , 414 [M + K] , 763 [2M + Na] ,

+

91 [(M + H)-C H NO] , 160 [(M + H)-C H N O] . Anal. Calcd. for C H N O : C, 73.57; H, 6.71;

4

6

13 15

2

23 25

3

2

N, 11.19%. Found: C, 72.45; H, 6.59; N, 10.89%.

0

3-(4-methoxyphenyl)-5-[4-((2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-4,5-

−

1 1

dihydroisoxazole (5b): 64% yield; Yellow oil; IR (ATR): 1668, 1608, 1512, 1355 cm . H NMR (400 MHz,

CDCl₃) (ppm): 7.63–7.58 (4H, m), 7.24–7.20 (2H, m), 7.04 (2H, m), 6.93–6.87 (4H, m), 6.76 (2H, m),

.58 (2H, m), 5.34 (2H, q, J = 7.5 Hz), 5.21–5.11 (2H, m), 3.82 (3H, s ), 3.81 (3H, s ), 3.60–3.23 (14H,

m), 3.10–3.01 (2H, m), 2.51–2.42 (4H, m), 2.08–1.97 (4H, m), 1.58–1.52 (4H, m). C NMR (100 MHz,

CDCl₃) (ppm). Diastereomer : 174.74, 156.94, 145.45, 130.82, 129.86, 128.04, 128.32 (2C), 127.49,

26.50, 122.12, 114.27 (2C), 113.19, 81.87, 57.29, 55.45, 48.22, 47.0, 43.37, 38.78, 31.38, 18.17, 16.88.

Diastereomer : 174.67, 156.58, 144.93, 130.60, 129.77, 128.01, 128.32 (2C), 127.45, 126.16, 121.98, 114.27

δ

6

α

β

13

δ

α

1

β

+

(

4

2C), 113.02, 81.70, 56.79, 55.45, 47.33, 46.3, 42.83, 38.11, 31.17, 17.99, 16.75. ESI-MS (m/z): 406 [M + H] ,

28 [M + Na] , 444 [M + K] , 833 [2M + Na] , 321 [(M + H)-C H NO] , 190 [(M + H)-C H N O] .

4 6 13 15 2

+

Anal. Calcd. for C H N O : C, 71.09; H, 6.71; N, 10.36%. Found: C, 72.08; H, 6.62; N, 10.08%.

2

4

27

3

0 0

3

-(3,4-dimethoxyphenyl)-5-[4-((2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-4,5-

dihydroisoxazole (5c): 65% yield; Yellow oil; IR (ATR): 1666, 1602, 1514, 1366 cm . H NMR (400 MHz,

−1 1

Int. J. Mol. Sci. 2020, 21, 5

11 of 25

CDCl₃)

δ

(ppm): 7.37 (1H, s

α

), 7.35 (1H, sβ), 7.23–7.14 (2H, m), 7.02–6.97 (2H, m), 6.85–6.91 (4H, m),

6

.75–6.69 (4H, m), 5.40 (2H, q, J = 7.4 Hz), 5.08–4.99 (2H, m), 3.90 (6H, s

m), 3.25–3.01 (6H, m), 2.42–2.38 (4H, m), 2.03–1.90 (4H, m), 1.47–1.44 (4H, m). C NMR (100 MHz,

CDCl₃) (ppm). Diastereomer : 174.71, 156.86, 151.00, 149.19, 147.20, 129.68, 128.29, 128.22, 122.38,

20.50, 118.60, 113.17, 110.53, 108.66, 79.76, 56.02 (2C), 48.54, 48.27, 47.18, 42.27, 38.25, 31.68, 17.94, 16.49.

Diastereomer : 174.42, 156.47, 150.83, 149.12, 147.20, 129.35, 128.29, 128.35, 122.10, 120.50, 118.60,

13.17, 110.45, 108.60, 79.42, 56.02 (2C), 48.54, 48.17, 47.18, 42.21, 38.25, 31.40, 17.90, 16.38. ESI-MS

α), 3.89 (6H, sβ), 3.56–3.37 (10H,

13

δ

α

1

β

1

+

(m/z): 436 [M + H] , 458 [M + Na] , 474 [M + K] , 893 [2M + Na] , 351 [(M + H)-C H NO] , 220

4 6

[

(M + H)-C H N O]+. Anal. Calcd. for C H N O : C, 68.95; H, 6.71; N, 9.65%. Found: C, 68.39;

13

15

2

25 29

3

4

H, 6.59; N, 9.89%.

-(3,4,5-trimethoxyphenyl)-5-[4-((2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-

0

3

−1

1

4

,5-dihydroisoxazole (5d): 65% yield; Red oil; IR (ATR): 1666, 1606, 1514, 1357 cm . H NMR

(

400 MHz, CDCl₃)

2H, s

δ

(ppm): 7.36–7.30 (2H, m), 7.14 (2H, m), 7.01–6.99 (2H, m), 6.88 (2H, s

α

), 6.86

0

β

), 6.63 (2H, m), 5.37 (2H, q, J = 6.5 Hz, 4 -H), 4.94–4.80 (2H, m, 5-H), 3.89 (6H, s), 3.85 (6H, s),

3

.84 (6H, s), 3.60–3.40 (8H, m), 3.39–3.23 (6H, m), 3.05–2.97 (2H, m), 2.46–2.35 (4H, m), 2.16–1.99 (4H,

13

m), 1.53–1.49 (4H, m). C NMR (100 MHz, CDCl₃)

δ

(ppm). Diastereomer

α: 175.48, 155.32, 153.50

(

2C), 144.87, 140.12, 128.05, 127.12, 124.94, 121.65, 121.45, 112.58, 104.19 (2C), 79.88, 61.32, 56.55 (2C),

5

4.74, 48.90, 47.88, 43.12, 38.42, 31.19, 18.34, 16.94. Diastereomer β: 175.39, 155.32, 153.42 (2C), 144.75,

1

39.95, 127.94, 127.08, 124.87, 121.58, 121.38, 112.45, 104.12 (2C), 79.64, 61.29, 56.50 (2C), 54.70, 48.75,

+

4

7.80, 42.95, 38.40, 31.12, 18.29, 16.78. ESI-MS (m/z): 466 [M + H] , 488 [M + Na] , 504 [M + K] , 953

2M + Na]⁺, 381 [(M + H)-C H NO] , 250 [(M + H)-C H N O] . Anal. Calcd. for C H N O :

+

[

4 6 13 15 2 26 31 3 5

C, 68.95; H, 6.71; N, 9.65%. Found: C, 68.45; H, 6.65; N, 9.82%.

0 0

-[(6-methyl-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-phenyl-4,5-

dihydroisoxazole (5e): 65% yield; Orange oil; IR (ATR): 1664, 1600, 1496, 1357 cm . H NMR

5

−

1

(

400 MHz, CDCl₃)

1H, dd , J = 8.4, 2.0 Hz), 6.72 (1H, d

.37(1H, dd , J = 9.4, 5.8 Hz), 5.31(1H, dd

.25–3.01 (6H, m), 2.51–2.46 (4H, m), 2.20 (3H, s

δ

(ppm): 7.68–764 (6H, m), 7.42–7.39 (4H, m), 6.93 (1H, dd

, J = 2.0 Hz), 6.67 (1H, d , J = 2.0 Hz), 6.54 (2H, d, J = 8.4 Hz),

, J = 9.4, 5.8 Hz), 5.09–5.01 (2H, m), 3.56–3.37 (10H, m),

), 2.19 (3H, s ), 2.14–2.05 (4H, m), 1.98–1.90 (4H, m).

(ppm). Diastereomer : 175.62, 156.71, 144.02, 130.35, 129.46, 129.38,

28.87 (2C), 128.84, 126.77 (2C), 126.05, 119.73, 111.47, 79.86, 55.35, 48.96, 48.07, 44.39, 38.23, 31.63, 26.97,

0.40, 18.43. Diastereomer : 175.51, 156.70, 143.50, 130.28, 129.33, 128.99, 128.84 (2C), 128.51, 126.77

2C), 125.90, 119.51, 111.27, 79.43, 54.96, 48.79, 47.95, 43.67, 37.92, 31.58, 26.71, 20.38, 18.34. ESI-MS

α, J = 8.4, 2.0 Hz), 6.91

β

α

β

5

3

α

β

α

β

1

3

C NMR (100 MHz, CDCl₃)

δ

α

1

2

β

(

+

m/z): 390 [M + H] , 412 [M + Na] , 428 [M + K]+, 801 [2M + Na] , 305 [(M + H)-C H NO] , 243

4

6

+

[

(M + H)-C H NO] , 160 [(M + H)-C H N O] . Anal. Calcd. for C H N O : C, 74.01; H, 6.99; N,

9

8

14 17

2

24 27

3

2

1

0.79%. Found: C, 73.76; H, 6.87; N, 10.54%.

0

3-(4-methoxyphenyl)-5-[(6-methyl-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-

−

1

methyl]-4,5-dihydroisoxazole (5f): 70% yield; Orange oil; IR (ATR): 1676, 1606, 1514, 1359 cm .

1

H NMR (400 MHz, CDCl₃)

, J = 8.9 Hz), 6.72 (2H, dd, J = 8.6, 2.7 Hz), 6.63(1H, d

, J = 2.7 Hz), 6.50 (1H, d J = 8.6 Hz), 6.49 (1H, d , J = 8.6 Hz), 5.39 (1H, dd

1H, dd , J = 9.8, 6.5 Hz), 5.08–4.99 (2H, m), 3.89 (3H, s ), 3.83 (3H, s ), 3.58–3.35 (10H, m), 3.24–3.01

6H, m), 2.51–2.45 (4H, m), 2.22 (3H, s ), 2.21 (3H, s

), 2.11–2.03 (4H, m), 2.01–1.92 (4H, m). ¹³C NMR

100 MHz, CDCl₃) (ppm). Diastereomer : 174.78, 155.10, 150.90, 144.195, 129.44, 128.94 (2C), 128.44,

27.37, 122.12, 121.16, 114.41 (2C), 113.88, 82.01, 57.12, 55.52, 47.20, 46.77, 43.55, 38.72, 31.34, 27.80,

9.30, 18.37. Diastereomer : 174.56, 154.88, 150.90, 143.75, 129.23, 128.94 (2C), 128.34, 126.88, 122.07,

21.16, 114.18 (2C), 113.88, 81.79, 56.46, 55.47, 47.20, 46.77, 42.61, 37.68, 31.18, 27.80, 19.30, 17.75. ESI-MS

δ

(ppm): 7.62 (2H, d

α

, J = 8.9 Hz), 7.61 (2H, d

β

, J = 8.9 Hz), 6.93 (2H, d

, J = 2.7 Hz), 6.61 (1H,

, J = 9.2, 5.6 Hz), 5.34

α,

J = 8.8 Hz), 6.91 (2H, d

β

α

d

β

α

,

β

α

(

β

α

β

α

β

δ

α

1

(

[

β

+

m/z): 420 [M + H] , 442 [M + Na] , 458 [M + K] , 861 [2M + Na] , 335 [(M + H)-C H NO] , 243

4 6

(M + H)-C H NO ] , 190 [(M + H)-C H N O] . Anal. Calcd. for C H N O : C, 71.57; H, 6.97;

+

1

0

10

2

14 17

2

25 29

3

N, 10.02%. Found: C, 72.06; H, 6.83; N, 10.29%.

Int. J. Mol. Sci. 2020, 21, 5

12 of 25

0 0

-(3,4-dimethoxyphenyl)-5-[(6-methyl-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-

methyl]-4,5-dihydroisoxazole (5g): 63% yield; Orange oil; IR (ATR): 1666, 1598, 1508, 1365 cm .

3

−1

1

H NMR (400 MHz, CDCl₃)

δ

(ppm): 7.40 (2H, s), 7.07–7.00 (4H, m), 6.92 (1H, dd

, J = 8.6, 2.0 Hz), 6.70 (1H, d , J = 2.0Hz), 6.66 (1H, d , J = 2.0 Hz), 6.53 (2H, d, J = 8.6 Hz), 5.34

, J = 9.5, 5.8 Hz), 5.30 (1H, dd , J = 7.4, 5.4 Hz), 5.04–4.99 (2H, m, 5-H), 3.90 (6H, s ), 3.89 (6H,

), 2.18 (3H, s ), 2.11–2.04 (4H,

(ppm). Diastereomer : 175.68, 156.47, 150.98,

49.20, 143.98, 129.32, 128.93, 127.48, 126.00, 120.46, 119.68, 111.51, 110.51, 108.61, 79.65, 56.09 (2C),

α, J = 8.6, 2.0 Hz), 6.90

(

s

1H, dd

β

α

β

1H, dd

α

β

α

β

), 3.50–3.38 (10H, m), 3.24–3.03 (6H, m), 2.50–2.47 (4H, m), 2.19 (3H, s

α

β

13

m), 1.99–1.94 (4H, m). C NMR (100 MHz, CDCl₃)

δ

α

1

4

8.90, 48.10, 47.19, 43.70, 38.09, 31.61, 26.94, 20.38, 18.34. Diastereomer

β: 175.56, 156.47, 150.93, 149.18,

1

4

9

43.47, 129.19, 128.44, 127.35, 125.84, 120.43, 119.45, 111.27, 110.47, 108.61, 79.26, 56.02 (2C), 48.77, 47.99,

+

7.14, 43.54, 37.84, 31.56, 26.68, 20.36, 18.30. ESI-MS (m/z): 450 [M + H] , 472 [M + Na] , 488 [M + K] ,

+

21 [2M + Na] , 365 [(M + H)-C H NO] , 243 [(M + H)-C H NO ] , 220 [(M + H)-C H N O] .

4

6

11 12

3

14 17

2

Anal. Calcd. for C H N O : C, 69.47; H, 6.95; N, 9.35%. Found: C, 70.12; H, 6.79; N, 9.61%.

2

6

31

3

4

0

5

-[(6-methyl-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-(3,4,5-

trimethoxyphenyl)-4,5-dihydroisoxazole (5h): 68% yield; Orange oil; IR (ATR): 1664, 1598,

−

1

508, 1369 cm . H NMR (400 MHz, CDCl₃)

J = 2.6 Hz), 6.71 (1H, d , J = 2.6 Hz), 6.64 (1H, dd

.55 (2H, dd, J = 8.8 Hz), 5.39 (1H, dd , J = 9.6, 6.0 Hz), 5.33 (1H, dd

m, 5-H), 3.90 (6H, s), 3.89 (6H, s), 3.87 (6H, s), 3.58–3.35 (10H, m), 3.23–3.00 (6H, m), 2.53–2.45 (4H,

m), 2.23 (3H, s ), 2.21 (3H, s

), 2.14–2.06 (4H, m), 2.01–1.94 (4H, m). ¹³C NMR (100 MHz, CDCl₃)

ppm). Diastereomer : 175.88, 155.40, 153.56 (2C), 144.10, 139.40, 129.70, 128.74, 125.65, 124.22, 121.41,

11.54, 104.12 (2C), 79.94, 61.08, 56.43 (2C), 55.77, 47.63, 47.20, 43.60, 38.02, 31.58, 26.46, 20.65, 18.41.

: 175.62, 155.40, 153.49 (2C), 143.69, 139.40, 129.70, 128.74, 125.65, 124.22, 121.41, 111.54,

04.06 (2C), 78.22, 61.04, 56.38 (2C), 54.31, 47.58, 46.75, 42.88, 37.52, 31.23, 26.46, 20.65, 17.85. ESI-MS

δ

(ppm): 6.92 (2H, s

, J = 8.8, 2.6 Hz), 6.63 (1H, dd

, J = 7.4, 5.6 Hz), 5.05–4.98 (2H,

α

), 6.90 (2H, s

β

), 6.73 (1H, d

α,

β

α

β, J = 8.8, 2.6 Hz),

6

α

β

α

β

δ

(

1

α

Diastereomer

1

(

[

β

+

m/z): 480 [M + H] , 502 [M + Na] , 518 [M + K] , 981 [2M + Na] , 395 [(M + H)-C H NO] , 250

4 6

+

(M + H)-C H N O] . Anal. Calcd. for C H N O : C, 67.62; H, 6.94; N, 8.76%. Found: C, 68.23; H,

14 17 2 27 33 3 5

6

.83; N, 8.54%.

-[(6-methoxy-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-phenyl-4,5-

0

5

−1 1

dihydroisoxazole (5i): 77% yield; Red oil; IR (ATR): 1668, 1606, 1502, 1357 cm . H NMR (400 MHz,

CDCl₃) (ppm): 7.67–7.64 (4H, m), 7.41–7.37 (6H, m), 6.73 (1H, dd , J = 8.7, 2.6 Hz), 6.71 (1H, dd

J = 8.7, 2.6 Hz), 6.59 (2H, d, J = 8.7 Hz), 6.51 (1H, d , J = 2.6 Hz), 6.46 (1H, d , J = 2.6 Hz), 5.37 (1H,

dd J = 9.6, 6.0 Hz), 5.33 (1H, dd , J = 8.4, 5.6 Hz), 5.06–4.99 (2H, m), 3.70 (3H, s ), 3.69 (3H, s ),

.53–3.35 (10H, m), 3.25–3.05 (6H, m), 2.49–2.44 (4H, m), 2.17–2.05 (4H, m), 1.98- 1.90 (4H, m). C NMR

100 MHz, CDCl₃) (ppm). Diastereomer : 175.48, 156.65, 151.37, 140.30, 130.22, 129.33, 128.78

2C), 126.69 (2C), 120.95, 114.16, 113.57, 112.55, 79.47, 55.86, 55.52, 48.84, 48.09, 43.41, 37.93, 31.44,

6.60, 18.28. Diastereomer : 175.48, 156.65, 151.37, 140.30, 130.22, 129.33, 128.78 (2C), 126.69 (2C),

20.95, 114.16, 113.57, 112.55, 79.47, 55.86, 55.52, 48.84, 48.09, 43.41, 37.93, 31.44, 26.60, 18.28. ESI-MS

δ

α

β,

α

β

α

,

β

α

β

13

3

(

2

1

δ

α

β

+

(m/z): 406 [M + H] , 428 [M + Na] , 444 [M + K] , 833 [2M + Na] , 321 [(M + H)-C H NO] , 259

4 6

+

[(M + H)-C H NO] , 160 [(M + H)-C H N O ] . Anal. Calcd. for C H N O : C, 71.09; H, 6.71; N,

9 8 14 17 2 2 24 27 3 3

1

0.36%. Found: C, 70.78; H, 6.67; N, 10.12%.

-(4-methoxyphenyl)-5-[(6-methoxy-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-

0

3

−1 1

methyl]-4,5-dihydroisoxazole (5j): 70% yield; Red oil; IR (ATR): 1664, 1602, 1508, 1369 cm . H NMR

400 MHz, CDCl₃) (ppm): 7.62 (4H, d, J = 8.8 Hz), 6.91 (2H, d , J = 8.8 Hz), 6.90 (2H, d J = 8.8 Hz),

.72 (1H, dd , J = 8.9, 2.6 Hz), 6.70 (1H, dd , J = 8.9, 2.6 Hz), 6.59 (1H, d , J = 2.6 Hz), 6.57 (1H, d

J = 2.6 Hz), 6.50 (1H, d , J = 8.9 Hz), 6.49 (1H, d , J = 8.9 Hz), 5.39 (1H, dd , J = 9.6, 6.0 Hz), 5.33 (1H,

dd , J = 7.4, 5.6 Hz), 5.05–4.97 (2H, m, 5-H), 3.83 (3H, s ), 3.82 (3H, s ), 3.70 (3H, s ), 3.69 (3H, s ),

.50–3.38 (10H, m), 3.22–3.09 (6H, m), 2.49–2.46 (4H, m), 2.16–2.07 (4H, m), 1.98–1.94 (4H, m). C NMR

100 MHz, CDCl ) δ (ppm). Diastereomer α: 175.64, 156.63, 155.08, 151.49, 140.84, 128.32 (2C), 122.11,

(

6

δ

α

β,

α

β

α

β,

α

β

α

β

α

β

α

β

13

3

(

3

1

21.25, 115.99, 114.21 (2C), 112.81, 111.97, 79.52, 55.94, 55.61, 55.22, 48.94, 47.39, 43.40, 38.50, 31.51, 26.89,

1

8.42. Diastereomer β: 175.54, 156.28, 154.93, 151.37, 140.32, 128.30 (2C), 122.02, 120.96, 115.76, 113.63

Int. J. Mol. Sci. 2020, 21, 5

13 of 25

(

[

2C), 112.59, 111.64, 79.19, 55.79, 55.46, 54.13, 48.27, 46.94, 42.79, 38.26, 31.29, 26.66, 18.34. ESI-MS

+

m/z): 436 [M + H] , 458 [M + Na] , 474 [M + K] , 893 [2M + Na] , 351 [(M + H)-C4H6NO] , 259

+

(M + H)-C H NO ] , 190 [(M + H)-C H N O ]+. Anal. Calcd. for C H N O : C, 68.95; H, 6.71;

10 10 2 14 17 2 2 25 29 3 4

N, 9.65%. Found: C, 68.78; H, 6.54; N, 9.41%.

-(3,4-dimethoxyphenyl)-5-[(6-methoxy-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-

0

3

−

1 1

methyl]-4,5-dihydroisoxazole (5k): 76% yield; Red oil; IR (ATR): 1666, 1602, 1502, 1357 cm . H NMR

400 MHz, CDCl₃) (ppm): 7.39 (2H, d, J = 2.0 Hz), 7.02 (1H, dd , J =8.3, 2.0 Hz), 7.00 (1H, dd J = 8.3,

, J = 8.3 Hz), 6.83 (2H, d , J = 8.3 Hz), 6.73 (1H, dd , J = 9.0, 2.8 Hz), 6.71 (1H,

, J = 9.0, 2.8 Hz), 6.58 (2H, d, J = 9.0 Hz), 6.50 (1H, d, J = 2.8 Hz), 6.46 (1H, d , J = 2.8 Hz), 5.39 (1H,

), 3.69 (3H,

), 3.54–3.36 (10H, m), 3.27–3.04 (6H, m), 2.50–2.45 (4H, m), 2.17–2.05 (4H, m), 1.99–1.92 (4H, m).

(

2

dd

δ

α

β,

.0 Hz), 6.85 (2H, d

α

β

α

β

dd, J = 9.6, 6.0 Hz), 5.33 (1H, dd, J = 8.4, 5.6 Hz),5.05–4.97 (2H, m), 3.90 (12H, s), 3.70 (3H, s

α

s

β

3

1

C NMR (100 MHz, CDCl₃)

22.26, 121.31, 120.47, 114.46, 113.97, 112.84, 110.53, 108.63, 79.67, 56.07, 55.95, 55.86, 49.06, 48.29, 44.18,

8.40, 31.56, 26.89, 21.18, 18.43. Diastereomer : 175.58, 156.48, 151.42, 150.96, 149.22, 140.35, 122.19,

21.00, 120.44, 114.23, 113.60, 112.60, 110.49, 108.62, 79.37, 56.03, 55.94, 55.64, 48.92, 48.18, 43.51, 38.18,

δ (ppm). Diastereomer α: 175.68, 156.49, 151.53, 151.00, 149.22, 140.81,

1

3

1

3

β

+

1.52, 26.66, 20.91, 18.37. ESI-MS (m/z): 466 [M + H] , 488 [M + Na] , 504 [M + K] , 953 [2M + Na] ,

81 [(M + H)-C H NO] , 259 [(M + H) C H NO ] , 220 [(M + H)-C H N O ] . Anal. Calcd. for

+

4

6

11 12

3

14 17

2

C H N O : C, 67.08; H, 6.71; N, 9.03%. Found: C, 67.41; H, 6.59; N, 9.29%.

2

6

31

3

5

0 0

5-[(6-methoxy-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-(3,4,5-

trimethoxyphenyl)-4,5-dihydroisoxazole (5l): 68% yield; Red oil; IR (ATR): 1670, 1597, 1504, 1369 cm ¹.

−

1

H NMR (400 MHz, CDCl₃)

, J = 8.7, 2.8 Hz), 6.59 (1H, d

.47 (1H, d , J = 2.8 Hz), 5.40 (1H, dd

2H, m), 3.87 (12H, s), 3.86 (3H, s ), 3.85 (3H, s

.25–3.05 (6H, m), 2.50–2.45 (4H, m), 2.17–2.07 (4H, m), 2.01–1.97 (4H, m). C NMR (100 MHz, CDCl₃)

(ppm). Diastereomer : 175.69, 156.59, 153.43 (2C), 151.57, 140.77, 139.96, 124.91, 121.40, 114.43,

δ

(ppm): 6.88 (2H, s

, J = 8.7 Hz), 6.57 (1H, d

, J = 9.6, 5.9 Hz), 5.33 (1H, dd

), 3.70 (3H, s ), 3.69 (3H, s

α

), 6.86 (2H, s

β

),6.72 (1H, dd

, J = 8.7 Hz), 6.50 (1H, d

, J = 8.6, 5.6 Hz), 5.07–5.00

), 3.54–3.37(10H, m),

α

, J = 8.7, 2.8 Hz), 6.71

(

6

(

3

1H, dd

β

α

β

α,

J = 2.8 Hz),

β

α

β

α

β

α

β

1

3

δ

α

1

13.95, 112.87, 104.02 (2C), 79.94, 61.08, 56.38 (2C), 55.95, 55.84, 49.06, 48.27, 44.15, 38.35, 31.58, 26.87,

8.44. Diastereomer : 175.57, 156.59, 153.39 (2C), 151.47, 140.33, 139.90, 124.85, 121.10, 114.20, 113.56,

12.58, 104.02 (2C), 79.68, 61.08, 56.38 (2C), 55.92, 55.62, 48.93, 48.18, 43.46, 38.14, 31.48, 26.65, 18.35.

β

+

ESI-MS (m/z): 496 [M + H] , 518 [M + Na] , 534 [M + K] , 1013 [2M + Na] , 411 [(M + H)-C H NO] ,

2

6

4

6

+

59 [(M + H)-C H NO ] , 250 [(M + H)-C H N O ] . Anal. Calcd. for C H N O : C, 65.44; H,

12 14 4 14 17 2 2 27 33 3 6

.71; N, 8.48%. Found: C, 65.21; H, 6.60; N, 8.29%.

0 0

-[(6-chloro-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-phenyl-4,5-

5

dihydroisoxazole (5m): 74% yield; Yellow oil; IR (ATR): 1676, 1597, 1497, 1356 cm . ¹H NMR

−1

(

6

3

400 MHz, CDCl₃)

1H, dd , J = 8.9, 2.6 Hz), 6.83 (1H, d

.53 (1H, d , J = 8.9 Hz), 5.34 (1H, dd

.58–3.37 (10H, m), 3.27–3.01 (6H, m), 2.52–2.44 (4H, m), 2.16–2.04 (4H, m), 1.98–1.91 (4H, m). C NMR

δ

(ppm): 7.67–7.62 (4H, m), 7.42–7.38 (6H, m), 7.04 (1H, dd

, J = 2.6 Hz), 6.79 (1H, d , J = 2.6 Hz), 6.55 (1H, d

, J = 9.8, 5.7 Hz), 5.29 (1H, dd , J = 8.8, 5.4 Hz), 5.05–4.98 (2H, m),

α

, J = 8.9, 2.6 Hz), 7.03

β

α

β

α

, J = 8.9 Hz),

β

α

β

13

(

100 MHz, CDCl ) δ (ppm). Diastereomer α: 175.64, 156.67, 144.64, 130.39, 129.22, 128.84 (2C), 128.47,

3

127.57, 126.69 (2C), 121.50, 121.32, 112.51, 79.45, 55.17, 48.80, 47.85, 43.90, 38.13, 31.36, 26.28, 18.30.

Diastereomer : 175.57, 156.67, 144.15, 130.33, 129.16, 128.82 (2C), 128.38, 127.22, 126.69 (2C), 121.36,

β

+

1

4

21.12, 112.23, 79.07, 54.87, 48.78, 47.77, 43.32, 37.85, 31.31, 26.14, 18.23. ESI-MS (m/z): 410 [M + H] ,

+

32 [M + Na] , 448 [M + K] , 841 [2M + Na] , 325 [(M + H)-C H NO] , 160 [(M + H)-C H ClN O] .

4

6

13 14

2

Anal. Calcd. for C H ClN O : C, 67.39; H, 5.90; N, 10.25%. Found: C, 66.98; H, 5.84; N, 9.99%.

2

3

24

3

2

0

5

-[(6-chloro-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-(4-

methoxyphenyl)-4,5-dihydroisoxazole (5n): 64% yield; Yellow oil; IR (ATR): 1673, 1604, 1497,

−

1 1

1

7

356 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.59 (2H, d

, J = 8.8, 2.6 Hz), 7.03 (1H, dd , J = 8.8, 2.6 Hz), 6.92 (2H, d

J = 9.0 Hz), 6.83 (1H, d , J = 2.6 Hz), 6.79(1H, d , J = 2.6 Hz), 6.55 (1H, d

J = 8.8 Hz), 5.35 (1H, dd , J = 9.7, 5.6 Hz), 5.30 (1H, dd

α

, J = 9.0 Hz), 7.58 (2H, d

, J = 9.0 Hz), 6.90 (2H, d

, J = 8.8 Hz), 6.53 (1H, d

β, J = 9.0 Hz),

3

.04 (1H, dd

α

β

α

β

,

α

β

α

β

α

β

, J = 8.7, 5.2 Hz), 5.02–4.95 (2H, m), 3.83 (3H,

Int. J. Mol. Sci. 2020, 21, 5

14 of 25

s

(

3

1

α

), 3.82 (3H, s

4H, m). ¹³C NMR (100 MHz, CDCl₃)

2C), 128.45, 127.57, 121.76, 121.45, 121.25, 114.22 (2C), 112.51, 79.11, 55.49, 55.23, 48.85, 47.95, 43.99,

8.46, 31.46, 26.37, 18.39. Diastereomer : 175.56, 156.22, 144.17, 128.53, 128.50 (2C), 128.34, 127.22,

21.69, 121.34, 121.09, 114.17 (2C), 112.19, 78.73, 55.49, 54.95, 48.85, 47.85, 43.42, 38.21, 31.40, 26.20, 18.32.

β

), 3.58–3.33 (10H, m), 3.27–3.00 (6H, m), 2.53–2.45 (4H, m), 2.16–2.04 (4H, m), 2.00–1.94

δ

(ppm). Diastereomer : 175.63, 156.22, 144.80, 128.53, 128.50

α

β

+

ESI-MS (m/z): 440 [M + H] , 462 [M + Na] , 478 [M + K] , 901 [2M + Na] , 355 [(M + H)-C H NO] ,

1

4

6

+

90 [(M + H)-C H ClN O] . Anal. Calcd. for C H ClN O : C, 65.52; H, 5.96; N, 9.55%. Found: C,

13 14 2 24 26 3 3

6

5.98; H, 5.88; N, 9.41%.

-(4,5-dimethoxyphenyl)-5-[(6-chloro-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-

0

3

−

1 1

methyl]-4,5-dihydroisoxazole (5o): 75% yield; Yellow oil; IR (ATR): 1674, 1599, 1499, 1369 cm . H

NMR (400 MHz, CDCl₃) (ppm): 7.38 (2H, s), 7.07–6.98 (4H, m), 6.85 (1H, dd , J = 8.6, 2.6 Hz), 6.83

1H, dd , J = 8.6, 2.6 Hz), 6.83 (1H, d , J = 2.6 Hz), 6.79 (1H, d , J = 2.6 Hz), 6.55 (1H, d , J = 8.6 Hz),

.53 (1H, d , J = 8.6 Hz), 5.35 (1H, dd , J = 9.7, 5.6 Hz), 5.29 (1H, dd , J = 8.8, 5.3 Hz), 5.03–4.96 (2H,

m), 3.90 (6H, s ), 3.89 (6H, s ), 3.57–3.39 (10H, m), 3.24–3.02 (6H, m), 2.53–2.45 (4H, m), 2.19–2.05

4H, m), 1.99–1.94 (4H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm). Diastereomer : 175.74, 156.49,

51.10, 149.26, 144.70, 128.54, 127.65, 122.09, 121.56, 121.38, 120.50, 112.60, 110.56, 108.62, 79.33, 56.07

: 175.66, 156.49, 151.06, 149.26,

δ

α

(

6

β

α

β

α

β

α

β

α

β

(

1

δ

α

(

2C), 55.24, 48.86, 47.96, 43.99, 38.34, 31.44, 26.36, 18.38. Diastereomer β

1

4

44.21, 128.45, 127.28, 122.02, 121.43, 121.19, 120.47, 112.28, 110.53, 108.62, 78.98, 56.04 (2C), 54.98, 48.86,

+

7.87, 43.43, 38.10, 31.39, 26.21, 18.33. ESI-MS (m/z): 470 [M + H] , 492 [M + Na] , 508 [M + K] , 961

+

[2M + Na] , 385 [(M + H)-C H NO] , 220 [(M + H)-C H ClN O] . Anal. Calcd. for C H ClN O :

4 6 13 14 2 25 28 3 4

C, 63.89; H, 6.01; N, 8.94%. Found: C, 64.18; H, 6.13; N, 9.11%.

0

5

-[(6-chloro-4-(2 -oxopyrrolidin-1 -yl)-3,4-dihydroquinolin-1(2H)-yl)-methyl]-3-(3,4,5-

trimethoxyphenyl)-4,5-dihydroisoxazole (5p): 75% yield; Yellow oil; IR (ATR): 1674, 1597,

−

1 1

1

501, 1370 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.05 (1H, dd

), 6.83 (1H, d , J = 2.6 Hz), 6.79 (1H, d

, J = 9.8, 5.4 Hz), 5.30 (1H, dd , J = 9.2, 5.2 Hz), 5.07–4.99 (2H, m), 3.88 (6H,

s), 3.87 (6H, s), 3.86 (6H, s), 3.57–3.40 (10H, m), 3.22–3.02 (6H, m), 2.52–2.46 (4H, m), 2.19–2.04 (4H, m),

.01–1.97 (4H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm). Diastereomer : 175.76, 156.60, 153.49 (2C),

44.66, 140.11, 128.55, 127.64, 124.75, 121.64, 121.47, 112.61, 104.07 (2C), 79.64, 61.13, 56.39 (2C), 55.26,

8.93, 47.97, 43.96, 38.31, 31.46, 26.36, 18.40. Diastereomer : 175.65, 156.60, 153.49 (2C), 144.16, 139.99,

28.44, 127.25, 124.67, 121.55, 121.29, 112.27, 104.07 (2C), 79.33, 60.54, 54.39 (2C), 54.95, 48.87, 47.85,

α

, J = 8.9, 2.6 Hz), 7.03 (1H, dd

β,

3

J = 8.9, 2.6 Hz), 6.88 (2H, s

d, J = 8.9 Hz), 5.37 (1H, dd

α

), 6.86 (2H, s

β

α

β, J = 2.6 Hz), 6.55 (2H,

α

β

2

1

4

1

4

δ

α

β

+

3.37, 38.09, 31.37, 26.20, 18.32. ESI-MS (m/z): 500 [M + H] , 522 [M + Na] , 538 [M + K] , 1021 [2M +

Na] , 415 [(M + H)-C H NO] , 250 [(M + H)-C H ClN O] . Anal. Calcd. for C H ClN O : C,

+

4

6

13 14

2

26 30

3

5

6

2.46; H, 6.05; N, 8.40%. Found: C, 62.01; H, 6.18; N, 8.56%.

0 0 0 0 0 0 0

-phenyl-5-((4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)isoxazole (6a):

3

−

1 1

5

5% yield; Orange oil; IR (ATR): 3465, 2931, 1670, 1497, 1438, 1167 cm . H NMR (400 MHz, CDCl )

δ

3

(

1

4

ppm): 7.76–7.72 (2H, m), 7.43–7.40 (3H, m), 7.10 (1H, tdd, J = 7.8, 1.6, 0.7 Hz), 6.89 (1H, td, J = 7.4,

.1 Hz), 6.70 (1H, dd, J = 7.4, 1.0 Hz), 6.66 (1H, d, J = 8.6 Hz), 6.37 (1H, s), 5.41 (1H, dd, J = 9.2, 5.4 Hz),

.62 (1H, d, J = 17.5 Hz), 4.56 (1H, d, J = 17.5 Hz), 3.60–3.39 (2H, m), 3.27–3.10 (2H, m), 2.51–2.47 (2H,

13

m), 2.23–2.05 (2H, m), 2.05–1.95 (2H, m). C NMR (100 MHz, CDCl ) δ (ppm): 175.72, 170.00, 162.51,

3

1

4

7

44.97, 130.15, 128.97 (2C), 128.83, 128.80, 127.91, 126.87 (2C), 120.11, 117.11, 111.96, 100.38, 48.20, 47.97,

+

7.71, 43.83, 31.5, 26.70, 18.35. ESI-MS (m/z): 289.1 [M-C H NO] , 374.1 [M + H] , 396.1 [M + Na] ,

4

8

+

69.1 [2M + Na] . Anal. Calcd. for C H N O : C, 73.97; H, 6.21; N, 11.25%. Found: C, 73.49; H, 6.34;

23

3

2

N, 11.25%. The NMR and ESI-MS data match the previously reported data [21].

0 0 0 0 0 0 0

-(4-methoxyphenyl)-5-((4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-

yl)methyl)isoxazole (6b): 65% yield; Red oil; IR (ATR): 2961, 1672, 1422, 1250, 1017 cm .

3

−1

1

H NMR (400 MHz, CDCl₃)

δ (ppm): 7.67 (2H, d, J = 8.9 Hz), 7.09 (2H, td, J = 7.4, 1.2 Hz), 6.92 (2H, d,

J = 8.9 Hz), 6.88 (1H, td, J = 7.7, 1.4 Hz), 6.68 (1H, dd, J = 7.4, 0.9 Hz), 6.64 (1H, d, J = 8.5 Hz), 6.32 (1H,

s), 5.40 (1H, dd, J = 9.4, 5.6 Hz), 4.59 (1H, d, J = 17.7 Hz), 4.53 (1H, d, J = 17.7 Hz), 3.81 (3H, s), 3.59–3.37

2H, m), 3.27–3.09 (2H, m), 2.48 (2H, td, J = 7.7, 1.0 Hz), 2.21–2.02 (2H, m), 2.08–1.91 (2H, m). ¹C NMR

3

(

Int. J. Mol. Sci. 2020, 21, 5

15 of 25

(100 MHz, CDCl₃)

δ

(ppm): 175.70, 169.68, 162.02, 161.01, 144.97, 128.67, 128.24 (2C), 127.82, 121.27,

1

20.04, 117.60, 114.18 (2C), 111.93, 100.10, 55.38, 48.14, 47.94, 47.64, 43.79, 31.47, 26.65, 18.31. ESI-MS

+

(m/z): 426.1 [M + Na] , 829.1 [2M + Na] . Anal. Calcd. for C H N O : C, 71.44; H, 6.25; N, 10.41%.

24

25

3

Found: C, 72.01; H, 6.12; N, 10.18%.

0

3-(3,4,5-trimethoxyphenyl)-5-((4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-

−1 1

yl)methyl)isoxazole (6c): 75% yield; Brown oil; IR (ATR): 3437, 2939, 1668, 1421, 1236, 1124 cm . H

NMR (400 MHz, CDCl₃) (ppm): 7.31–7.27 (1H, m), 7.06–6.92 (2H, m), 6.85–6.81 (1H, m), 6.64 (1H,

d, J = 10.1 Hz), 6.56 (1H, d, J = 7.1 Hz), 6.32 (1H, s), 5.47–5.34 (1H, m), 4.64 (1H, d, J = 16.7 Hz), 4.51

1H, d, J = 16.7 Hz), 3.91 (6H, s), 3.87 (3H, s), 3.70–3.36 (2H, m), 3.35–3.12 (2H, m), 2.56–2.44 (2H, m),

.25–1.89 (4H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 174.98, 169.49, 162.41, 153.65 (2C), 143.46,

39.65, 128.61, 127.12, 124.11, 122.55, 121.96, 113.17, 104.05 (2C), 100.49, 61.02, 56.44 (2C), 49.98, 48.41,

δ

(

2

δ

1

4

+

7.82, 43.53, 31.24, 26.38, 18.33. ESI-EM (m/z): 486.0 [M + Na] , 520.1 [M + Cl + Na] . Anal. Calcd for

C H N O : C, 67.37; H, 6.31; N, 9.07%. Found: C, 67.78; H, 6.17; N, 8.81%. The NMR and ESI-MS

26

29

3

5

data match the previously reported data [21].

0 0 0 0 0 0 0 0

-((6 -methyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-

5

phenylisoxazole (6d): 72% yield; Red oil; IR (ATR): 2952, 1667, 1421, 1285, 1093 cm . ¹H NMR

−

1

(

400 MHz, CDCl₃)

δ (ppm): 7.76–7.72 (2H, m), 7.43–7.40 (3H, m), 6.91 (1H, dd, J = 8.4, 2.1 Hz), 6.71

1H, d, J = 1.8 Hz), 6.58 (1H, d, J = 8.4 Hz), 6.36 (1H, s), 5.38 (1H, dd, J = 9.0, 5.4 Hz), 4.59 (1H, d,

J = 17.4 Hz), 4.54 (1H, d, J = 17.4 Hz), 3.56–3.36 (2H, m), 3.27–3.10 (2H, m), 2.52–2.48 (2H, m), 2.19 (3H,

13

s), 2.24–2.15 (2H, m), 2.10–1.95 (2H, m). C NMR (100 MHz, CDCl₃) δ (ppm): 175.66, 170.18, 162.52,

1

4

42.82, 130.15, 129.37, 128.98 (2C), 128.90, 128.51, 127.04, 126.90 (2C), 120.18, 112.23, 100.41, 48.23, 47.83,

7.87, 43.93, 31.56, 26.98, 20.46, 18.41. ESI-MS (m/z): 410.1 [M + Na] , 797.2 [2M + Na] . Anal. Calcd

+

for C H N O : C, 74.39; H, 6.50; N, 10.84%. Found: C, 74.78; H, 6.37; N, 10.61%. The NMR and

24

25

3

2

ESI-MS data match the previously reported data [21].

0 0 0 0 0 0 0 0

-(4-methoxyphenyl)-5-((6 -methyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-

3

−

1 1

yl)methyl)isoxazole (6e): 64% yield; Red oil; IR (ATR): 2957, 1664, 1425, 1251, 1176 cm . H NMR

400 MHz, CDCl₃) (ppm): 7.72 (2H, dd, J = 6.1, 2.1 Hz), 7.10 (1H, d, J = 1.9 Hz), 6.94 (2H, dd, J = 6.1,

.9 Hz), 6.89–6.85 (1H, m), 6.71 (1H, d, J = 1.8 Hz), 6.59 (1H, s), 6.32 (1H, s), 5.36 (1H, dd, J = 9.2, 7.2 Hz),

(

δ

1

4

2

1

5

.38 (1H, d, J = 16.5 Hz), 4.31 (1H, d, J = 16.5 Hz), 3.84 (3H, s), 3.39–3.22 (2H, m), 3.20–2.96 (2H, m),

.54–2.43 (2H, m), 2.21 (2H, s), 2.12–1.81 (4H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 175.84, 171.14,

62.22, 161.99, 142.23, 133.44, 130.68, 129.08, 128.23 (2C), 127.71, 126.88, 121.46, 114.31 (2C), 100.75,

5.39, 48.69, 47.70, 46.96, 43.03, 31.21, 26.33, 20.60, 18.16. ESI-MS (m/z): 474.1 [M + Cl + Na]+, 925.1

2M + 2Cl + Na]+. Anal. Calcd for C H N O : C, 71.92; H, 6.52; N, 10.06%. Found: C, 73.72; H, 6.68;

δ

[

25

27

3

N, 9.86%. The NMR and ESI-MS data match the previously reported data [20].

0

5

-((8 -chloro-6 -methyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-

(

1

3,4-dimethoxyphenyl)isoxazole (6f): 58% yield; Beige solid; IR (ATR): 2966, 1656, 1429, 1255, 1143,

−

016 cm . H NMR (400 MHz, CDCl ) δ (ppm): 7.40 (1H, d, J = 8.3 Hz), 7.30 (1H, dd, J = 8.3, 2.0

3

1

Hz), 7.11 (1H, d, J = 2.1 Hz), 6.91 (1H, d, J = 8.3 Hz), 6.73 (1H, t, J = 1.0 Hz), 6.63 (1H, s), 5.38 (1H, dd,

J = 8.8, 7.1 Hz), 4.38 (1H, d, J = 16.3 Hz), 4.33 (1H, d, J = 16.3 Hz), 3.94 (3H, s), 3.91 (3H, s), 3.31–3.21

(

2H, m), 3.24–3.00 (2H, m), 2.51–2.45 (2H, m), 2.23 (3H, s), 2.06–1.95 (2H, m), 1.94–1.84 (2H, m).

1

3

C NMR (100 MHz, CDCl₃)

δ (ppm): 175.75, 171.34, 162.42, 150.62, 149.30, 142.30, 133.53, 130.73,

1

2

29.18, 127.78, 126.96, 121.75, 120.03, 111.05, 109.24, 100.78, 56.09, 56.02, 49.82, 47.70, 46.98, 43.05, 31.28,

1.41, 20.67, 18.24. ESI-MS (m/z): 482.0 [M + H] , 504.1 [M + Na] , 985.1 [2M + Na] . Anal. Calcd for

+

C H ClN O : C, 64.79; H, 5.86; N, 8.72%. Found: C, 64.58; H, 5.75; N, 8.61%

2

6

28

3

4

0 0 0 0 0 0

3-(3,4,5-trimethoxyphenyl)-5-((6 -methyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-

0

−1

1

(2 H)-yl)methyl)isoxazole (6g): 63% yield; Red oil; IR (ATR): 2935, 1668, 1421, 1124, 1001 cm . H

NMR (400 MHz, CDCl₃) (ppm): 6.96 (2H, s), 6.91 (1H, dd, J = 8.4, 2.0 Hz), 6.7 (1H, d, J = 2.0 Hz), 6.56

1H, d, J = 8.4 Hz), 6.32 (1H, s), 5.40 (1H, dd, J = 9.2, 5.6 Hz), 4.62 (1H, d, J = 17.6 Hz), 4.51 (1H, d,

J = 17.6 Hz), 3.90 (6H, s), 3.86 (3H, s), 3.58–3.37 (2H, m), 3.28–3.12 (2H, m), 2.55–2.47 (2H, m), 2.19

3H, s), 2.23–2.06 (2H,m), 2.05–1.95 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 175.75, 170.33,

δ

(

δ

Int. J. Mol. Sci. 2020, 21, 5

16 of 25

1

6

4

6

62.43, 153.65 (2C), 142.81, 139.64, 133.64, 129.39, 128.34, 124.57, 120.19, 112.16, 104.08 (2C), 100.29,

+

1.06, 56.42 (2C), 48.37, 48.00, 47.99, 43.83, 31.55, 26.92, 20.48, 18.42. ESI-MS (m/z): 391.1 [M-C H NO] ,

4

7

+

78.3 [M + H] , 500.2 [M + Na] , 539.1 [M+C H CONa] . Anal. Calcd for C H N O : C, 67.91; H,

2

8

27 31

3

5

.54; N, 8.80%. Found: C, 68.33; H, 6.69; N, 9.04%. The NMR and ESI-MS data match the previously

reported data [21].

0

5-((6 -methoxy-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-

phenylisoxazole (6h): 42% yield; Brown oil; IR (ATR): 2949, 1661, 1502, 1421, 1285, 1039 cm ¹. ¹H NMR

−

(

400 MHz, CDCl₃) δ (ppm): 7.74–7.72 (2H, m), 7.45–7.39 (3H, m), 6.69 (1H, dd, J = 2.9, 0.7 Hz), 6.61 (1H,

d, J = 9.0 Hz), 6.49 (1H, dd, J = 2.9, 0.7 Hz), 6.35 (1H, s), 5.39 (1H, dd, J = 9.3, 5.6 Hz), 4.57 (1H, d,

J = 17.4 Hz), 4.49 (1H, d, J = 17.4 Hz), 3.68 (3H, s), 3.51–3.32 (2H, m), 3.26–3.10 (2H, m), 2.50–2.45 (2H,

13

m), 2.19–2.06 (2H, m), 2.06–1.93 (2H, m). C NMR (100 MHz, CDCl ) δ (ppm): 175.69, 170.16, 162.46,

3

1

4

52.08, 139.43, 130.12, 128.95 (2C), 128.84, 126.85 (2C), 121.77, 114.12, 113.68, 113.48, 100.45, 55.78, 48.70,

8.35, 48.16, 43.68, 31.45, 26.85, 18.36. ESI-MS (m/z): 426.1 [M + Na] , 829.1 [2M + Na] . Anal. Calcd.

+

for C H N O (403.48 g/mol). Anal. Calcd for C H N O : C, 71.44; H, 6.25; N, 10.41%. Found: C,

24

25

3

24 25

3

7

1.08; H, 6.39; N, 10.13%. The NMR and ESI-MS data match the previously reported data [21].

0 0 0 0 0 0 0 0

-(4-methoxyphenyl)-5-((6 -methoxy-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-

3

−

1 1

yl)methyl)isoxazole (6i): 74% yield; Red oil; IR (ATR): 2937, 1662, 1427, 1249, 1174 cm . H NMR

400 MHz, CDCl₃) (ppm): 7.67 (2H, dd, J = 6.8, 2.1 Hz), 6.92 (2H, dd, J = 6.8, 1.9 Hz), 6.70 (1H, dd,

J = 8.9, 2.7 Hz) 6.61 (1H, d, J = 8.9 Hz), 6.49 (1H, d, J = 0.9 Hz), 6.29 (1H, s), 5.36 (1H, dd, J = 8.9, 6.5 Hz),

.56 (1H, d, J = 17.6 Hz), 4.48 (1H, d, J = 17.6 Hz), 3.82 (3H, s), 3.69 (3H, s), 3.51–3.32 (2H, m), 3.18–3.00

(

δ

4

13

(

2H, m), 2.50–2.45 (2H, m), 2.20–2.04 (2H, m), 2.09–1.91 (2H, m). C NMR (100 MHz, CDCl₃)

δ

(ppm):

1

8

75.72, 169.80, 161.65, 152.08, 138.32, 130.40, 128.98, 128.20 (2C), 121.23, 114.32 (2C), 114.14, 113.51,

+

11.72, 100.22, 55.77, 55.42, 48.17, 47.99, 47.00, 43.98, 31.23, 26.87, 18.38. ESI-MS (m/z): 456.1 [M + Na] ,

+

89.2 [2M + Na] . Anal. Calcd for C H N O : C, 69.27; H, 6.28; N, 9.69%. Found: C, 68.87; H, 6.15;

25

27

3

4

N, 9.44%.

0

5

-((8 -chloro-6 -methoxy-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-

(

3,4-dimethoxyphenyl)isoxazole (6j): 86% yield; Beige solid; IR (ATR): 2943, 2839, 1670, 1429, 1261

−1 1

cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.40 (1H, d, J = 2.0 Hz), 7.29 (1H, dd, J = 8.5, 2.0 Hz), 6.91

1H, d, J = 8.5 Hz), 6.90 (1H, dd, J = 2.8, 0.8 Hz), 6.62 (1H, s), 6.50 (1H, dd, J = 2.9, 0.9 Hz), 5.39 (1H,

t, J = 8.2 Hz), 4.34 (1H, d, J = 16.3 Hz), 4.28 (1H, d, J = 16.3 Hz), 3.93 (3H, s), 3.91 (3H, s), 3.72 (3H,

s), 3.40–3.19 (2H, m), 3.27–3.01 (2H, m), 2.49–2.45 (2H, m), 2.15–1.96 (2H, m), 1.95–1.83 (2H, m). ¹³

NMR (100 MHz, CDCl₃) (ppm): 175.71, 171.32, 162.42, 155.47, 150.63, 149.31, 138.32, 130.42, 128.98,

21.74, 120.03, 115.91, 111.73, 111.06, 109.25, 100.78, 56.09, 56.02, 55.77 49.86, 47.96, 46.95, 42.97, 31.23,

3

(

C

δ

1

2

+

1.58, 18.28. ESI-MS (m/z): 486.2 [[M-Cl]+Na] , 520.1 [M + Na] , 1017.2 [2M + Na] . Anal. Calcd for

C H ClN O : C, 62.71; H, 5.67; N, 8.44%. Found: C, 62.54; H, 5.57; N, 8.67%.

2

6

28

3

5

0 0 0 0 0 0

3-(3,4,5-trimethoxyphenyl)-5-((6 -methoxy-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-

0

−1

1

(2 H)-yl)methyl)isoxazole (6k): 74% yield; Red oil; IR (ATR): 2937, 1664, 1421, 1238, 1124 cm . H

NMR (400 MHz, CDCl₃) (ppm): 6.97 (2H, s), 6.71 (1H, dd, J = 9.0, 2.9 Hz), 6.61 (1H, d, J = 9.0 Hz),

.50 (1H, dd, J = 2.9, 0.8 Hz), 6.31 (1H, s), 5.43 (1H, dd, J = 9.6, 5.7 Hz), 4.62 (1H, d, J = 17.4 Hz), 4.48

1H, d, J = 17.4 Hz), 3.90 (6H, s), 3.87 (3H, s), 3.70 (3H, s), 3.56–3.35 (2H, m), 3.28–3.14 (2H, m), 2.53–2.48

2H, m), 2.21–2.08 (2H,m), 2.07–1.97 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 175.77, 170.34,

62.44, 153.68 (2C), 152.18, 139.67, 139.43, 124.38, 121.88, 114.12, 113.63, 113.44, 104.10 (2C), 100.38,

δ

6

(

1

6

1

δ

+

1.09, 56.44 (2C), 55.86, 48.57, 48.35, 48.19, 43.64, 31.51, 26.86, 18.44. ESI-MS (m/z): 516.1 [M + Na] ,

009.2 [2M + Na] . Anal. Calcd for C H N O : C, 65.71; H, 6.33; N, 8.51%. Found: C, 65.49; H, 6.21;

27 31 3 6

+

N, 8.30%. The NMR and ESI-MS data match the previously reported data [21].

0 0 0 0 0 0 0 0

-((6 -chloro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-

5

phenylisoxazole (6l): 55% yield; Red oil; IR (ATR): 2930, 1657, 1496, 1127 cm . ¹H NMR

−

1

(

400 MHz, CDCl₃) (ppm): 7.77–7.72 (2H, m), 7.44–7.41 (3H, m), 7.04 (1H, dd, J = 8.9, 2.5 Hz), 6.83 (1H,

δ

dd, J = 2.5, 1.0 Hz), 6.57 (1H, d, J = 8.9 Hz), 6.37 (1H, s), 5.37 (1H, dd, J = 9.6, 5.9 Hz), 4.60 (1H, d,

J = 17.5 Hz), 4.52 (1H, d, J = 17.5 Hz), 3.60–3.39 (2H, m), 3.28–3.11 (2H, m), 2.55–2.44 (2H, m), 2.21–2.03

Int. J. Mol. Sci. 2020, 21, 5

17 of 25

(

2H, m), 2.09–1.98 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

δ

(ppm): 175.77, 169.43, 162.55, 143.55, 130.25,

1

3

29.01 (2C), 128.69, 128.60, 127.28, 126.87 (2C), 122.56, 121.90, 113.23, 100.47, 48.24, 47.75, 47.69, 43.54,

+

1.15, 26.41, 18.34. ESI-MS (m/z): 430.0 [M + Na] , 837.1 [2M + Na] . Anal. Calcd for C H ClN O :

23

22

3

2

C, 67.73; H, 5.44; N, 10.30%. Found: C, 67.36; H, 5.58; N, 10.09%. The NMR and ESI-MS data match the

previously reported data [21].

0

5

-((6 -chloro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(4-

methoxyphenyl)isoxazole (6m): 51% yield; Orange oil; IR (ATR): 2976, 1678, 1431, 1253,

−

1 1

1

2

178 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.67 (2H, dd, J = 6.1, 2.2 Hz), 7.03 (1H, ddd, J = 8.9,

.6, 0.8 Hz), 6.93 (2H, dd, J = 6.1, 2.2 Hz), 6.83 (1H, dd, J = 2.6, 1.0 Hz), 6.57 (1H, d, J = 8.9 Hz), 6.31 (1H,

s), 5.37 (1H, dd, J = 9.6, 5.4 Hz), 4.58 (1H, d, J = 17.6 Hz), 4.50 (1H, d, J = 17.6 Hz), 3.82 (3H, s), 3.60–3.39

3

13

(

2H, m), 3.28–3.12 (2H, m), 2.56–2.46 (2H, m), 2.19–2.06 (2H, m), 2.09–2.01 (2H, m). C NMR (100 MHz,

CDCl₃) (ppm): 175.94, 169.14, 162.16, 161.14, 143.60, 128.64, 128.30 (2C), 127.30, 122.54, 121.82, 121.19,

14.37 (2C), 113.27, 100.25, 55.44, 48.21, 47.83, 47.70, 43.68, 31.36, 26.43, 18.16. ESI-MS (m/z): 460.1 [M +

δ

1

+

Na] , 897.1 [2M + Na] . Anal. Calcd for C H ClN O : C, 65.83; H, 5.52; N, 9.60%. Found: C, 67.47;

24

3

H, 5.66; N, 9.48%. The NMR and ESI-MS data match the previously reported data [20].

0 0 0 0 0 0 0 0

-((6 -chloro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4-

dimethoxyphenyl)isoxazole (6n): 68% yield; Orange oil; IR (ATR): 2935, 1665, 1420, 1257, 1022 cm .

H NMR (400 MHz, CDCl₃) δ (ppm): 7.34 (1H, d, J = 2.0 Hz), 7.22 (1H, dd, J = 8.3, 2.2 Hz), 7.02 (1H, dd,

5

−1

1

J = 8.8, 2.5 Hz), 6.87 (1H, d, J = 8.3 Hz), 6.82 (1H, dd, J = 2.5, 1.0 Hz), 6.56 (1H, d, J = 8.8 Hz), 6.31

1H, s), 5.36 (1H, dd, J = 9.7, 5.3 Hz), 4.59 (1H, d, J = 17.6 Hz), 4.49 (1H, d, J = 17.6 Hz), 3.91 (3H, s),

.89 (3H, s), 3.60–3.36 (2H, m), 3.27–3.11 (2H, m), 2.55–2.44 (2H, m), 2.21–2.10 (2H, m), 2.09–1.98 (2H,

(

3

13

m). C NMR (100 MHz, CDCl₃) δ (ppm): 175.76, 169.20, 162.24, 150.68, 149.27, 143.54, 128.57, 127.19,

1

2

22.50, 121.87, 121.36, 120.05, 113.20, 110.97, 109.16, 100.22, 56.07, 55.98, 48.25, 47.75, 47.68, 43.56, 31.32,

6.36, 18.34. ESI-MS (m/z): 456.1 [[M-Cl]+H] , 490.1 [M + Na] , 597.1 [2M + Na] . Anal. Calcd for

+

C H ClN O : C, 64.17; H, 5.60; N, 8.98%. Found: C, 63.91; H, 5.49; N, 8.81%.

2

5

26

3

4

0 0 0 0 0 0 0 0 0

5-((6 ,8 -dichloro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4-

−1

dimethoxyphenyl)isoxazole (6o): 88% yield; Orange solid; IR (ATR): 2954, 1662, 1427, 1249 cm .

H NMR (400 MHz, CDCl₃)

1

δ (ppm): 7.40 (1H, d, J = 2.0 Hz), 7.30 (1H, d, J = 0.8 Hz), 7.29 (1H, dd,

J = 8.3, 2.0 Hz), 6.92 (1H, d, J = 0.8 Hz), 6.91 (1H, d, J = 8.4 Hz), 6.61 (1H, s), 5.39 (1H, t, J = 8.3 Hz), 4.43

1H, d, J = 16.4 Hz), 4.36 (1H, d, J = 16.4 Hz), 3.94 (3H, s), 3.92 (3H, s), 3.32–3.24 (2H, m), 3.25–3.01 (2H,

m), 2.55–2.42 (2H, m), 2.06–1.97 (2H, m), 1.96–1.85 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm):

75.83, 170.79, 162.46, 150.73, 149.37, 143.62, 130.42, 129.95, 128.61, 128.25, 126.29, 121.63, 120.08, 111.08,

09.24, 100.93, 56.13, 56.06, 49.44, 47.80, 47.27, 42.96, 31.13, 21.41, 18.28. ESI-MS (m/z): 490.1 [[M-Cl] +

(

δ

1

+

Na] , 524.0 [M + Na] , 1027.0 [2M + Na] . Anal. Calcd for C H Cl N O : C, 59.77; H, 5.02; N,

25

2

3

4

8

.36%. Found: C, 59.52; H, 4.91; N, 8.46%.

0 0 0 0 0 0 0 0

-((6 -chloro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4,5-

5

−

1

trimethoxyphenyl)isoxazole (6p): 85% yield; Brown oil; IR (ATR): 2936, 1667, 1582, 1420, 1122 cm .

1

H NMR (400 MHz, CDCl₃)

.9 Hz), 6.57 (1H, d, J = 8.8 Hz), 6.31 (1H, s), 5.40 (1H, dd, J = 9.9, 5.4 Hz), 4.64 (1H, d, J = 17.6 Hz), 4.51

1H, d, J = 17.6 Hz), 3.90 (6H, s), 3.87 (3H, s), 3.64–3.40 (2H, m), 3.30–3.14 (2H, m), 2.59–2.44 (2H, m),

.23–2.05 (2H, m), 2.16–1.97 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 175.82, 169.55, 160.50,

δ (ppm): 7.05 (1H, dd, J = 8.8, 2.6 Hz), 6.96 (2H, s), 6.84 (1H, dd, J = 2.5,

0

(

2

δ

1

53.72 (2C), 143.55, 139.80, 128.67, 127.23, 124.20, 122.69, 122.02, 113.22, 104.14 (2C), 100.39, 61.08, 56.45

+

(

2C), 48.44, 47.86, 47.84, 43.54, 31.38, 26.40, 18.40. ESI-MS (m/z): 520.1 [M + Na] , 1017.2 [2M + Na]+,

+

1

513.0 [3M + Na] . Anal. Calcd for C H ClN O : C, 62.71; H, 5.67; N, 8.44%. Found: C, 63.12; H,

26 28 3 5

5

.53; N, 8.68%. The NMR and ESI-MS data match the previously reported data [21].

0 0 0 0 0 0 0 0

-((6 -ethyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-phenylisoxazole

5

−1

1

(

6q): 40% yield; Brown oil; IR (ATR): 2863, 2363, 1681, 1510, 1335, 1169 cm . H NMR (400 MHz,

CDCl₃) (ppm): 7.75 (2H, d, J = 4.8 Hz), 7.42 (3H, br), 6.95 (1H, d, J = 8.3 Hz), 6.74 (1H, s), 6.61 (1H,

d, J = 8.3 Hz), 6.38 (1H, s), 5.44–5.38 (1H, m), 4.63–4.52 (2H, m), 3.53 (1H, t, J = 10,1 Hz), 3.44–3.37

1H, m), 3.24 (1H, dd, J = 16.0, 8.0 Hz), 3.13 (1H, dd, J = 14.6, 8.0 Hz), 2.52 (4H, bs), 2.24–2.06 (2H, m),

δ

(

Int. J. Mol. Sci. 2020, 21, 5

18 of 25

2

.03–1.93 (2H, m), 1.15 (3H, t, J = 7.5 Hz). ¹³C NMR (100 MHz, CDCl₃)

δ

(ppm): 175.91, 170.61, 162.88,

1

4

43.43, 134.01, 130.47, 129.32 (2C), 128.99, 128.49, 127.79, 127.26 (2C), 120.56, 112.57, 100.75, 48.59, 48.37,

+

8.29, 44.33, 31.95, 28.24, 27.39, 18.87, 16.31. HR-ESI-MS (m/z): 402.2156 [M + H] , 420.2031 [M + Na] ,

+

40.1745 [M + K] . Anal. Calcd. for C H N O (401.2103 g/mol).

25

0

27

3

2

0

5

-((6 -ethyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(4-

methoxyphenyl)isoxazole (6r): 60% yield; Orange oil; IR (ATR): 2961, 2358, 1963, 1679, 1507,

−

436, 1255, 1027 cm . H NMR (400 MHz, CDCl ) δ (ppm): 7.79 (2H, d, J = 8.1 Hz), 7.36 (1H, s), 7.04

3

1 1

1

(

8

2H, d, J = 8.2 Hz), 6.84 (1H, s), 6.71 (1H, d, J = 8.2 Hz), 6.42 (1H, s), 5.54–5.48 (1H, m), 4.66 (2H, s), 3.94

3H, s), 3.63 (1H, t, J = 10.1 Hz), 3.53–3.46 (1H, m), 3.34 (1H, dd, J = 16.1, 8.0 Hz), 3.23 (1H, dd, J = 13.9,

.7 Hz), 2.64–2.54 (4H, m), 2.33–2.23 (2H, m), 2.13–2.03 (2H, m), 1.25 (3H, t, J = 7.5 Hz). ¹³C NMR

(100 MHz, CDCl₃)

δ

(ppm): 175.98, 170.32, 162.50, 161.47, 143.48, 133.98, 128.68 (2C), 128.51, 127.79,

1

21.85, 120.52, 114.73 (2C), 112.60, 100.52, 55.81, 48.57, 48.42, 48.30, 44.39, 31.97, 28.25, 27.40, 18.88,

+

6.32. HR-ESI-MS (m/z): 432.2271 [M + H] , 346.0085 [M-C H NO] . Anal. Calcd. for C H N O

3

4

7

26 29

3

(

431.2209 g/mol).

0

5-((8 -chloro-6 -ethyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4-

dimethoxyphenyl)isoxazole (6s): 73% yield; Beige solid; IR (ATR): 2966, 2360, 1679, 1470, 1267,

−

1

6

(

1

019 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.41 (1H, s), 7.30 (1H, d, J = 8.2 Hz), 7.15 (1H, s),

.92 (1H, d, J = 8.2 Hz), 6.77 (1H, s), 6.62 (1H, s), 5.42 (1H, m), 4.45–4.33 (2H, m), 3.95 (3H, s), 3.92

3H, s), 3.34–3.23 (2H, m), 3.22–2.99 (2H, m), 2.58–2.51 (2H, m), 2.50–2.45 (2H, m), 2.05–1.95 (2H, m),

3

13

.95–1.88 (2H, m), 1.18 (3H, t, J = 7.5 Hz). C NMR (100 MHz, CDCl₃) δ (ppm): 176.00, 171.73, 162.79,

1

5

51.07, 149.77, 142.88, 140.25, 129.93, 129.64, 128.21, 126.25, 122.23, 120.41, 111.53, 109.78, 101.14, 56.49,

+

6.41, 50.21, 48.21, 47.42, 43.47, 31.68, 28.39, 21.94, 18.74, 15.94. HR-ESI-MS (m/z): 496.2019 [M + H] ,

+

18.1739 [M + Na] , 534.1592 [M + K] . Anal. Calcd. for C H ClN O (495.1925 g/mol).

27

30

3

0

4

0

5

-((6 -ethyl-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4,5-

trimethoxyphenyl)isoxazole (6t): 72% yield; Orange oil; IR (ATR): 2961, 2929, 1679, 1581, 1230,

−

1 1

1

127 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.13 (2H, br), 7.10 (1H, d, J = 7.7 Hz), 6.88 (1H, s), 6.76

3

(1H, d, J = 8.4 Hz), 6.52 (1H, s), 5.59–5.53 (1H, m), 4.76 (1H, d, J = 17.4 Hz), 4.68 (1H, d, J = 17.5 Hz),

4

2

.05 (6H, s), 4.02 (3H, s), 3.75–3.52 (2H, m), 3.45–3.26 (2H, m), 2.70–2.62 (4H, m), 2.50–2.32 (2H, m),

13

.28–2.21 (2H, m), 1.30 (3H, t, J = 7.5 Hz). C NMR (100 MHz, CDCl₃)

δ (ppm): 176.16, 170.61, 162.61,

1

53.87 (2C), 143.27, 139.97, 133.85, 128.38, 127.45, 124.58, 120.28, 112.43, 104.45 (2C), 100.57, 61.22, 56.63

+

(

2C), 48.49, 48.38, 48.17, 44.22, 31.76, 28.08, 27.14, 18.68, 16.17. HR-ESI-MS (m/z): 492.2448 [M + H] .

Anal. Calcd. for C H N O (491.2420 g/mol).

28

33

3

5

0 0 0 0 0 0 0 0

5

-((6 -fluor-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-phenylisoxazole

−

1 1

(

6u): 89% yield; Orange oil; IR (ATR): 2941, 2870, 1665, 1499, 1281, 1155 cm . H NMR (400 MHz,

CDCl₃) (ppm): 7.75 (2H, d, J = 2.7 Hz), 7.43 (3H, br), 6.83 (1H, t, J = 8.4 Hz), 6.67–6.58 (2H, m), 6.37

1H, s), 5.45–5.39 (1H, m), 4.61 (1H, d, J = 17.4 Hz), 4.53 (1H, d, J = 17.4 Hz), 3.56 (1H, t, J = 10.7 Hz),

δ

(

3

2

.44–3.37 (1H, m), 3.27 (1H, dd, J = 16.2, 8.1 Hz), 3.15 (1H, dd, J = 14.6, 7.8 Hz), 2.55–2.46 (2H, m),

.23–2.07 (2H, m), 2.06–2.00 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

δ

(ppm): 175.56, 169.65, 162.46,

1

56.94, 154.59, 141.44, 130.11, 128.91 (2C), 126.81 (2C), 122.02, 115.64 (JC-F = 22.2), 114. 52 (JC-F = 22.2),

13.10 (JC-F = 7.2), 100.38, 48.38, 48.07, 47.86, 43.38, 31.26, 26.49, 18.27. HR-ESI-MS (m/z): 392.1917

+

[M + H] , 414.1691 [M + Na] , 430.1480 [M + K] . Anal. Calcd. for C H FN O (391.1696 g/mol).

2

3

22

3

2

0

5

-((6 -ﬂuoro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(4-

methoxyphenyl)isoxazole (6v): 77% yield; Orange oil; IR (ATR): 2954, 2836, 1669, 1608, 1429,

−

1 1

1

6

(

1

4

252, 1027 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.69 (d, J = 7.9 Hz, 1H), 6.94 (d, J = 7.9 Hz, 1H),

.82 (t, J = 8.4 Hz, 1H), 6.66–6.57 (m, 1H), 6.30 (s, 1H), 5.45–5.37 (m, 1H), 4.58 (d, J = 17.4 Hz, 1H), 4.51

d, J = 17.3 Hz, 1H), 3.84 (s, 1H), 3.59–3.36 (m, 1H), 3.31–3.11 (m, 1H), 2.58–2.44 (m, 1H), 2.23–2.06 (m,

3

13

H). C NMR (100 MHz, CDCl₃)

22.00, 121.25, 115.18 (JC-F = 19.3), 114.30 (2C), 114.05 (JC-F = 22.6), 113.12 (JC-F = 7.3), 100.13, 55.36,

8.35, 48.05, 47.87, 43.39, 31.26, 26.48, 18.27. HR-ESI-MS (m/z): 422.1874 [M + H] , 444.1710 [M + Na] .

δ (ppm): 175.57, 169.36, 162.06, 161.07, 156.92, 141.47, 128.21 (2C),

+

Anal. Calcd. for C H FN O (421.1802 g/mol).

24

3

Int. J. Mol. Sci. 2020, 21, 5

19 of 25

0

5

-((6 -ﬂuoro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4-

dimethoxyphenyl)isoxazole (6w): 75% yield; Orange oil; IR (ATR): 2934, 2834, 1679, 1502, 1264,

−

1

024 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.40 (1H, s), 7.29 (1H, d, J = 5.8 Hz), 6.93 (1H, d,

J = 8.2 Hz), 6.86 (1H, t, J = 8.4 Hz), 6.65 (2H, m), 6.35 (1H, s), 5.49–5.42 (1H, m), 4.63 (1H, d, J = 17.4 Hz),

.54 (1H, d, J = 17.4 Hz), 3.97 (3H, s), 3.95 (3H, s), 3.59 (1H, t, J = 10.7 Hz), 3.47–3.40 (1H, m), 3.30 (1H,

dd, J = 16.1, 8.1 Hz), 3.19 (1H, dd, J = 14.7, 7.6 Hz), 2.54 (2H, m), 2.27–2.10 (2H, m), 2.10–2.04 (2H,

3

4

13

m). C NMR (100 MHz, CDCl₃) δ (ppm): 176.05, 169.90, 162.63, 157.38, 155.02, 151.16, 149.77, 141.88,

1

5

21.91, 120.44, 115,62 (JC-F = 22.1), 114.46 (JC-F = 22.6), 113.55 (JC-F = 7.4), 111.48, 109.73, 100.61, 56.50,

+

6.40, 48.84, 48.52, 48.34, 43.81, 31.70, 26.90, 18.71. HR-ESI-MS (m/z): 452.1956 [M + H] , 474.1831

+

[M + Na] . Anal. Calcd. for C H FN O (451.1907 g/mol).

25 26 3 4

0

5

-((6 -ﬂuoro-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4,5-

trimethoxyphenyl)isoxazole (6x): 87% yield; Orange oil; IR (ATR): 2935, 2357, 1661, 1504, 1254,

−

126 cm . H NMR (400 MHz, CDCl ) δ (ppm): 6.97 (2H, s), 6.82 (1H, t, J = 8.4 Hz), 6.63 (1H, d,

3

1

J = 9.0 Hz), 6.59 (1H, dd, J = 8.8, 4.2 Hz), 6.31 (1H, s), 5.43 (1H, m), 4.63 (1H, d, J = 17.5 Hz), 4.50 (1H,

d, J = 17.4 Hz), 3.91 (6H, s), 3.88 (3H, s), 3.58 (1H, t, J = 10.8 Hz), 3.44–3.37 (1H, m), 3.28 (1H, dd,

J = 16.1, 8.1 Hz), 3.17 (1H, dd, J = 14.7, 7.8 Hz), 2.54–2.47 (2H, m), 2.25–2.08 (2H, m), 2.06–2.00 (2H,

m). ³C NMR (100 MHz, CDCl₃)

1

δ

(ppm): 176.07, 170.19, 162.80, 157.40, 155.05, 154.06, 141.82, 140.25,

1

5

24.59, 122.45, 115.62 (JC-F = 22.1), 114.40 (JC-F = 22.7), 113.49 (JC-F = 7.3), 104.60 (2C), 100.72, 61.40,

+

6.81 (2C), 48.93, 48.56, 48.38, 43.76, 31.69, 26.86, 18.71. HR-ESI-MS (m/z): 482.2097 [M + H] , 504.1880

+

[M + Na] , 520.1732 [M + K] . Anal. Calcd. for C H FN O (481.2013 g/mol).

26 28 3 5

0 0 0 0 0 0 0 0

-((6 -bromo-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-phenylisoxazole

5

−

1

(

6y): 95% yield; Beige solid; IR (ATR): 2949, 2877, 1676, 1497, 1277 cm . H NMR (400 MHz,

CDCl₃) (ppm): 7.74 (2H, d, J = 1.6 Hz), 7.42 (3H, br), 7.17 (1H, d, J = 8.7 Hz), 6.97 (1H, s), 6.53

1H, d, J = 8.7 Hz), 6.37 (1H, s), 5.42–5.33 (1H, m), 4.59 (1H, d, J = 17.4 Hz), 4.53 (1H, d, J = 17.4 Hz),

δ

(

13

3

.61–3.38 (2H, m), 3.28–3.11 (2H, m), 2.56–2.42 (2H, m), 2.23–2.05 (2H, m), 2.05–1.95 (2H, m). C NMR

(ppm): 175.29, 169.05, 162.24, 143.71, 131.18, 129.91 (2C), 128.69 (2C), 128.44, 126.57

2C), 122.13, 113.35, 109.36, 100.15, 47.88, 47.39, 47.33, 43.33, 31.04, 26.14, 18.08. HR-ESI-MS (m/z):

(

100 MHz, CDCl₃) δ

(

+

4

52.0906 [M + H] , 368.8798 [M-C H NO] . Anal. Calcd. for C H BrN O (451.0895 g/mol).

4

7

23 22

0

3

0

2

0

3

-(4-methoxyphenyl)-5-((6 -bromo-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-

−1 1

yl)methyl)isoxazole (6z): 67% yield; Beige solid; IR (ATR): 2928, 2039, 1680, 1252, 1023 cm . H NMR

400 MHz, CDCl₃) (ppm): 7.68 (2H, d, J = 7.4 Hz), 7.17 (1H, d, J = 8.8 Hz), 6.97 (1H, s), 6.94 (2H, d,

(

δ

J = 7.4 Hz), 6.53 (1H, d, J = 8.4 Hz), 6.30 (1H, s), 5.41–5.34 (1H, m), 4.57 (1H, d, J = 17.5 Hz), 4.51 (1H, d,

J = 17.5 Hz), 3.83 (3H, s), 3.60–3.38 (2H, m), 3.28–3.10 (2H, m), 2.57–2.41 (2H, m), 2.22–2.06 (2H, m),

2

.05–1.99 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

δ

(ppm): 175.33, 168.76, 161.86, 160.87, 143.76, 131.20,

1

29.91, 128.00, 122.10, 120.95, 114.09, 113.39, 109.34, 99.92, 55.14, 47.88, 47.42, 47.35, 43.37, 31.06, 26.17,

8.10. HR-ESI-MS (m/z): 482.1091 [M + H] , 504.0896 [M + Na] , 520.0751 [M + K] . Anal. Calcd. for

+

C H BrN O (481.1001 g/mol).

24

3

0 0 0 0 0 0 0 0

3-(3,4-dimethoxyphenyl)-5-((6 -bromo-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-

−1

yl)methyl)isoxazole (6aa): 70% yield; Orange oil; IR (ATR): 2936, 2832, 1677, 1497, 1264, 1019 cm .

1

H NMR (400 MHz, CDCl₃)

δ (ppm): 7.35 (1H, s), 7.23 (1H, d, J = 8.2 Hz), 7.17 (1H, d, J = 8.7 Hz), 6.97

(

1H, s), 6.88 (1H, d, J = 8.2 Hz), 6.53 (1H, d, J = 8.7 Hz), 6.31 (1H, s), 5.40–5.35 (1H, m), 4.59 (1H, d,

J = 17.4 Hz), 4.50 (1H, d, J = 17.4 Hz), 3.92 (3H, s), 3.90 (3H, s), 3.60–3.38 (2H, m), 3.28–3.11 (2H, m),

.55–2.44 (2H, m), 2.22–2.05 (2H, m), 2.04–2.00 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 175.38,

2

δ

1

9

5

68.84, 161.97, 150.50, 149.09, 143.71, 131.19, 129.85, 122.08, 121.15, 119.79, 113.36, 110.81, 109.34, 109.05,

+

9.94, 55.82, 55.72, 47.91, 47.44, 47.35, 43.34, 31.04, 26.12, 18.08. HR-ESI-MS (m/z): 512.1180 [M + H] ,

+

34.0969 [M + Na] , 550.0728 [M + K] . Anal. Calcd. for C H BrN O (511.1107 g/mol).

25

26

3

4

0

5

-((6 -bromo-4 -(2 ’-oxopyrrolidin-1 ’-yl)-3 ,4 -dihydroquinolin-1 (2 H)-yl)methyl)-3-(3,4,5-

trimethoxyphenyl)isoxazole (6ab): 43% yield; Orange oil; IR (ATR): 2939, 2830, 1680, 1419, 1125,

−

1 1

1

000 cm . H NMR (400 MHz, CDCl )

δ

(ppm): 7.18 (1H, d, J = 8.7 Hz), 6.99–6.94 (3H, br.s.), 6.52 (1H,

3

d, J = 8.8 Hz), 6.31 (1H, s), 5.42–5.36 (1H, m), 4.62 (1H, d, J = 17.5 Hz), 4.51 (1H, d, J = 17.5 Hz), 3.90

Int. J. Mol. Sci. 2020, 21, 5

20 of 25

(

1

2

6H, s), 3.87 (3H, s), 3.63–3.40 (2H, m), 3.29–3.12 (2H, m), 2.55–2.45 (2H, m), 2.24–2.07 (2H, m), 2.06–2.02

2H, m). ¹³C NMR (100 MHz, CDCl₃)

(ppm): 176.04, 169.78, 162.81, 154.05, 144.31, 140.24, 131.87,

30.45, 124.50, 122.79, 113.97, 110.06, 104.58, 100.70, 77.16, 61.38, 56.79, 48.67, 48.13, 48.07, 43.93, 31.69,

δ

+

6.74, 18.75. HR-ESI-MS (m/z): 542.1245 [M + H] . Anal. Calcd. for C H BrN O (541.1212 g/mol).

26

28

3

5

3

.2. Inhibitory Activity against Cholinesterases

The AChE and BChE inhibitory activities of the synthesized compounds were performed using

the methodology described by Ellman [26]. AChE (from Electrophorus electricus), BChE (from bovine

0

serum), 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine and butyrylthiocholine iodides

(

AChI/BChI) were purchased from Sigma-Aldrich. The alkaloid galantamine was used as the reference

compound, considering its competitive mode of action as described in the literature [27]. Synthesized

compounds were tested at ﬁve diﬀerent concentrations to obtain cholinesterase inhibitory activity

values between 5 to 80%. This assay was performed in 96-well plates, where 50

µL of sample were

dissolved in phosphate buﬀer (8 mM K HPO , 2.3 mM NaH PO , 150 mM NaCl, and 0.05% Tween

2

4

2

4

20 at pH 7.6) and a solution of 50 µL of AChE/BuChE (0.25 unit/mL) from Electroporus electricus and

bovine serum, respectively, in the same buﬀer, was added. The assay solutions, without substrate, were

incubated with the enzyme for 30 min at room temperature. After incubation, the substrate was added.

The substrate solution consisted of Na HPO (40 mM), acetylthiocholine/butyrylthiocholine (0.24 mM)

2

4

0

and 5,5 -dithio-bis-(2-nitrobenzoic acid) (0.2 mM, DTNB, Ellman’s reagent). Absorbance of the yellow

anion product, due to the spontaneous hydrolysis of substrate, was measured at 405 nm for 5 min on

a Microtiter plate reader (Multiskan EX, Thermo, Vanta, Finland). The AChE/BuChE inhibition was

determined for each compound. The enzyme activity was calculated as a percentage compared to a

control sample using only the buﬀer and enzyme solution. Each assay was run in triplicate and each

reaction was repeated at least three independent times. The IC₅₀values were calculated by means of

regression analysis.

3

.3. Enzymatic Kinetic Study

For enzymatic kinetic studies, the enzyme was pre-incubated with diﬀerent substrate

−

3

concentrations ranging from 3.75

×

10 to 0.48 mM. For the determination of type of inhibition, Vmax

m

and K (Michaelis constant), double reciprocal plots (1/V versus 1/[S] where V = reaction rate and

S = substrate concentration) were constructed, using Lineweaver–Burk methods. Determinations were

made in the absence and presence of test compounds. For 5n, the concentrations were set to 0, 2.12,

and 4.24

µM and for 6aa 0, 1.985, and 3.97

µM. The enzymatic reaction was extended to 5 min before

the determination of the absorption. The Vmax and K

m

values of the Michaelis–Menten kinetics were

calculated by nonlinear regression from substrate–velocity curves. Data analysis was carried out with

Sigmaplot v10.0 and Enzyme kinetic v1.3 add-on (Systat Software, Inc, Richmond, CA, USA) [28].

3

.4. Statistical Analysis

All biological assays were performed in triplicate by independent assays, and obtained values were

analyzed and expressed as mean standard error of the mean (SEM) using Statistical Product and Service

Solutions, 17th version (SPSS, Inc., Chicago, IL, USA)). IC₅₀values for hybrid compound derivatives

against AChE were calculated using regression analysis. Statistical analyses were performed by

one-way analysis of variance (ANOVA).

3

.5. Protein and Ligand Structure Preparation

The tridimensional X-ray structures of AChE were obtained from the Protein Data Bank (PDB).

As of May 2017, there were sixty-four structures from Tetronarce californica (Paciﬁc electric ray) with a

resolution less than or equal to 2.5 Å. PDB entries having non-drug or covalent ligands, non-standard

residues, and/or missing loops near the active site were excluded, leading to a total of thirty-one

structures. The selected crystal structures were further clustered down while keeping those having

Int. J. Mol. Sci. 2020, 21, 5

21 of 25

unique conformations of the amino acids at the active site, thus obtaining only ten crystals with PDB

codes: 1E66, 1EA5, 1GPK, 1ODC, 1ZGC, 2C5G, 2CEK, 2CMF, 2CKM, and 2XI4. These structures

were processed with the Protein Preparation Wizard module [29], which encompasses bond order

assignment, the addition of hydrogen atoms, and prediction of the protonation states of polar residues.

X-ray waters beyond 5.0 Å of the corresponding co-crystallized ligand were deleted. Non-conserved

X-ray water molecules were also deleted, where one water was considered as conserved if it was

present in more than 80% of the initial sixty-four structures. Residues with alternative positions (mostly

in the protein surface) were set to that of highest occupancy. Ionization and tautomeric states of the

co-crystallized ligands were predicted using Epik [29–31] at pH = 7 and set to those of lowest penalty.

Then, the hydrogen-bond network was optimized at neutral pH by sampling Asn and Gln rotamers,

hydroxyl and thiol terminal groups, and water orientations. The protonation states for residues Asp,

Glu, Lys and His were predicted using PropKa 3.7 [32,33]. The structures were then relaxed by means

of a restrained molecular minimization using the impact reﬁnement module [34] with the OPLS3 force

ﬁeld [35], with heavy atoms restrained to the initial coordinates within a root-mean-square deviation

(

RMSD) of 0.30 Å. The compound structures were sketched using Maestro software [36] and then

prepared with the LigPrep module [29], where ionization and tautomeric states were generated at pH

7.0 ± 2.0 using Epik. All possible enantiomers were included, summing up to 120 structures.

=

3

.6. Molecular Cross-Docking

All molecular docking calculations were performed using the Glide program [37] with the standard

precision (SP) algorithm. As the chemical structure of the synthesized compounds resemble none of the

co-crystallized ligands, we employed the ensemble docking approach to take into account the protein

ﬂexibility, where the compounds are docked against multiple rigid protein structures. Using multiple

protein conformations often increases the success rate of the pose prediction as some compounds

may score poorly against some protein conformations but they bind positively to another protein

conformation [38]. Such knowledge is diﬃcult to know a priori, therefore careful consideration of the

protein structures during docking experiments is required. We followed a similar approach based on a

previous work on several PPARγ agonist series [39]. Docking grids for the ten selected AChE crystal

structures were generated with the default settings using the corresponding cognate ligand as centroid

while ensuring that the grid box size was big enough to cover the entire active site. Default docking

parameters were used with the following options enabled: enhance planarity of conjugated

π groups,

include aromatic carbons as H-bond donors, include halogens as halogen bond donors, and add strain

correction in the post-docking score. For a comprehensive pose sampling, one-hundred poses were

retained per ligand with the root-mean-square deviation (RMSD) criteria for duplicate pose elimination

reduced from 0.5 to 0 Å. It was recently shown that these options can improve the identiﬁcation of the

correct pose by enhancing ligand sampling [40]. The obtained poses were clustered by RMSD with a

threshold of 2.0 Å with the protein structures aligned beforehand, where only compounds with the

same chirality were grouped together. Then, the ﬁve most populated clusters with at least ten poses

per ligand were selected for pose rescoring. For each cluster, the representative poses (one per ligand)

were selected according to the best ﬁt between the calculated energies and the experimental values.

2

Finally, the cluster with the highest coeﬃcient of determination (R ) was chosen out of the top ﬁve and

further analyzed.

3

.7. Pose Re-Scoring with MM/GBSA

Free binding energies ( Gbind) for the poses from selected clusters were calculated using the more

∆

computationally intensive MM/GBSA method with the OPLS3 force ﬁeld and the variable dielectric

implicit solvent model VSGB 2.0 [41] as implemented in the Prime program [29]. The MM/GBSA

method considers that the energy of a molecular system can be approximated by the sum of the energy

from molecular mechanics (MM) in gas phase, polar continuum solvation energy (GB), and nonpolar

Int. J. Mol. Sci. 2020, 21, 5

22 of 25

solvation energy obtained from the solvent accessible surface area (SASA). Several investigations have

used MM/GBSA to re-score docking poses in virtual screening experiments with great success [42 45].

–

3

.8. ADME Predictions

The pharmacokinetic properties of hybrid compounds were predicted through ADME descriptors

using QikProp [29]. Some of the descriptors predicted were molecular weight, van der Waals, surface

areas of polar nitrogen and oxygen atoms, H-bond acceptors, H-bond donors, log p (octanol/water)

based on the Lipinski´s rule of 5.

4

. Conclusions

In conclusion, a series of THQ-isoxazole/isoxazoline hybrids with diﬀerent patterns of substitution

were designed based on previous report and obtained using a friendly and economical synthetic

chemical protocol under mild conditions using the 1,3-dipolar cycloaddition reaction to obtain inhibitors

of cholinergic enzymes. Most of compounds showed signiﬁcant inhibitory eﬀects against AChE and

BChE. In general, the tendency of selectivity of the compounds of both synthesized series was moderate.

However, some compounds of the series 5 (5k, 5m, 5n, and 5o) showed signiﬁcant selectivity for

inhibiting AChE, while compounds of the series 6 (6a, 6h, 6l, 6u, 6aa) were more selective against

to BChE. Therefore, compound 6aa could be introduced as a candidate for further modiﬁcation

in the development of anti-AD drugs. The mode and free binding energies of the most active

compound against AChE were analyzed through dual computational protocol using the molecular

docking algorithm from the Glide module and the MM/GBSA method from Prime module. Similarly,

the structure–activity relationships of the molecular hybrids were established by comparing their

computational

correlation was obtained between the predicted values of

∆

Gpred values against the biological activities measured experimentally. A good

Gbind and experimental inhibitory activity

∆

of the compounds against the AChE enzyme. The analysis of obtained IC₅₀values, the binding modes

and calculated aﬃnities as well as the pharmacokinetic properties, all calculated in silico for this

series of compounds, let us infer that most active compounds reported in this work can be used as

new modulators of cholinesterases. Eﬀorts are ongoing to further optimize and provide a systematic

structure–activity analysis at the proper position. The results obtained in this work show that the

design of hybrid compounds from propargyl fragment as a versatile entity, allows the development of

new synthetic molecules with structural diversity, enhancing the biological activity of propargylated

tetrahydroquinolines previously described by our work group [19].

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com /1422-0067 /21 /1 /5 /s1 .

Author Contributions: Conceptualization, M.G. and Y.A.R.N.; methodology, Y.A.R.N. and A.R.R.B.; software,

J.A.-M. and F.A.-C.; validation, Y.A.R.N., A.R.R.B. and M.G.; formal analysis, F.M.G. and C.C.B.; investigation,

Y.A.R.N., F.M.G. and C.C.B.; writing—original draft preparation, Y.A.R.N.; writing—review and editing, Y.A.R.N.,

A.R.R.B., J.A.-M. and M.G. supervision, A.R.R.B. and M.G. All authors have read and agreed to the published

version of the manuscript.

Funding: This research was funded by Conicyt-Chile, Colciencias-Colombia.

Acknowledgments: This research was supported by “Patrimonio Aut

para la Ciencia Francisco Jos de Caldas” (Colciencias) contract code 110265842934. J.A-M. and F.A. acknowledge

Centro de Bioinform tica, Simulaci n y Modelado (CBSM) and CONICYT-FONDEQUIP-EQM160063 project for

ónomo, Fondo Nacional de Financiamiento

é

á

ó

the access to computational time for performing ensemble molecular docking and MM/GBSA calculations. Y.R.N.

◦

thanks CONICYT-Chile for a doctoral fellowship N 63130185.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Int. J. Mol. Sci. 2020, 21, 5

23 of 25

Abbreviations

THQ

Tetrahydroquinoline

AD

Alzheimer’s disease

AChE

BChE

ADME-Tox

MM/GBSA

PDB

Acetylcholinestrerase

Butirylcholinesterase

Absorption, Distribution, Metabolism, Excretion and Toxicity

Molecular Mechanics/Generalized Born Surface Area

Protein Data bank

RMSD

NMR

IR

Root-Mean-Square Deviation

Nuclear Magnetic Resonance

Infrared spectroscopy

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Article Doi

Tetrahydroquinoline-isoxazole/isoxazoline hybrid compounds as potential cholinesterases inhibitors: Synthesis, enzyme inhibition assays, and molecular modeling studies

DOI: 10.3390/ijms21010005

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