Indoloquinoxaline derivatives as promising multi-functional anti-Alzheimer agents

Journal of Biomolecular Structure and Dynamics

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20

Indoloquinoxaline derivatives as promising multi-

functional anti-Alzheimer agents

Ashish M. Kanhed , Dushyant V. Patel , Nirav R. Patel , Anshuman Sinha ,

Priyanka S. Thakor , Kishan B. Patel , Navnit K. Prajapati , Kirti V. Patel &

Mange Ram Yadav

To cite this article: Ashish M. Kanhed , Dushyant V. Patel , Nirav R. Patel , Anshuman Sinha ,

Priyanka S. Thakor , Kishan B. Patel , Navnit K. Prajapati , Kirti V. Patel & Mange Ram Yadav

(2020): Indoloquinoxaline derivatives as promising multi-functional anti-Alzheimer agents, Journal

of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1840441

To link to this article: https://doi.org/10.1080/07391102.2020.1840441

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

https://doi.org/10.1080/07391102.2020.1840441

Indoloquinoxaline derivatives as promising multi-functional

anti-Alzheimer agents

Ashish M. Kanhed^a,b, Dushyant V. Patel^a, Nirav R. Patel^a, Anshuman Sinha^a, Priyanka S. Thakor^a, Kishan

B. Patel^a, Navnit K. Prajapati^a, Kirti V. Patel^aand Mange Ram Yadav^a

b

^aFaculty of Pharmacy, Kalabhavan Campus, The Maharaja Sayajirao University of Baroda, Vadodara, India; Shobhaben Pratapbhai

Patel - School of Pharmacy & Technology Management, SVKMs NMIMS University, Mumbai, India

Communicated by Ramaswamy H. Sarma

ABSTRACT

ARTICLE HISTORY

Received 15 July 2020

Accepted 18 October 2020

To confront a disease like Alzheimer’s disease having complex pathogenesis, development of multitar-

get-directed ligands has emerged as a promising drug discovery approach. In our endeavor towards

the development of multitarget-directed ligands for Alzheimer’s disease, a series of indoloquinoxaline

derivatives were designed and synthesized. In vitro cholinesterase inhibition studies revealed that all

the synthesized compounds exhibited moderate to good cholinesterase inhibitory activity. 6-(6-

(Piperidin-1-yl)hexyl)-6H-indolo[2,3-b]quinoxaline 9f was identified as the most potent and selective

BuChE inhibitor (IC₅₀¼ 0.96 mM, selectivity index ¼ 0.17) that possessed 2 fold higher BuChE inhibitory

activity compared to the commercially approved reference drug donepezil (IC₅₀¼ 1.87 mM). Moreover,

compound 9f is also endowed with self-induced Ab_1-42aggregation inhibitory activity (51.24% inhib-

ition at 50 lM concentration). Some of the compounds of the series also displayed moderate anti-oxi-

dant activity. To perceive a putative binding mode of the compound 9f, molecular docking studies

were carried out, and the results pointed out significant interactions of compound 9f with the

enzymes in the binding sites of cholinesterases as well as Ab_1-42. Additionally, compound 9f exhibited

favorable in silico ADMET properties. Put together these findings project compound 9f as a potential

multitarget-directed ligand in the direction of developing novel anti-AD drugs.

KEYWORDS

Alzheimer’s disease; MTDL;

cholinesterase inhibitor;

anti-Ab aggregation;

indoloquinoxaline

Introduction

BuChE can also hydrolyze other molecules, for example, suc-

cinylcholine, adipoylcholine, benzoylcholine, and neurotoxic

peptides (Lane et al., 2006; Pohanka, 2011). In a disease free

brain, AChE mainly causes ACh hydrolysis but as the

Alzheimer’s advances, AChE level declines, and the BuChE

level augments up to 40 to 90% in the temporal cortex and

hippocampus areas of the brain (Hartmann et al., 2007).

BuChE performs both neural as well as non-neural function-

ing roles. Also the clinical data has suggested the various

roles of elevated levels of BuChE in AD, such as the aggrega-

tion of hyperphosphorylated tau protein and extracellular

deposition of the Ab (Greig et al., 2002). Several ChE inhibi-

tors like donepezil, tacrine, rivastigmine, and galantamine

have been approved for the treatment of AD. These inhibi-

tors, however, induce classical cholinergic side effects, such

as digestive tract reactions and hepatotoxicity that severely

affect their therapeutic goals. Therefore, the development of

safe, effective and moderately selective BuChE inhibitors may

present a novel approach for the treatment of AD patients of

primary as well as late stages (Dighe et al., 2016; Greig

et al., 2005).

Alzheimer’s disease (AD), the most prominent type of

dementia, is a progressive neurodegenerative disease preva-

lent amongst elderly people (Scheltens et al., 2016). Almost

50 million people are suffering from AD worldwide, and if no

cure or preventive measures are discovered soon, the num-

ber will grow up significantly to 150 million by 2050

(Patterson, 2018). The etiology of AD is very intricate, various

aspects like scarcity of acetylcholine (ACh) (Talesa, 2001),

abnormal amyloid-b (Ab) accumulation (Hardy & Higgins,

1992; Selkoe, 2003), tau hyperphosphorylation (Maccioni

et al., 2010), oxidative stress (Bonda et al., 2010) and dysho-

meostasis of biometals (Greenough et al., 2013) are hypothe-

sized to play significant roles in the etiology of AD.

According to the cholinergic hypothesis, low levels of ACh

in the brain is a crucial factor in the pathogenesis of AD

(Talesa, 2001). Two types of cholinesterase enzymes (ChEs),

namely, acetylcholinesterase (AChE) and butyrylcholinester-

ase (BuChE) are present in the central nervous system (CNS)

that can quickly hydrolyze acetylcholine (ACh). Therefore,

ChE inhibition remains an effective strategy to enhance ACh

levels within the brain (Bartus et al., 1982). Both ChEs hydro-

Another significant hypothesis is amyloid hypothesis,

lyze ACh, but BuChE is less substrate-specific than AChE, as which suggests the role of Ab plaques in the AD

CONTACT Mange Ram Yadav

mryadav11@yahoo.co.in

Faculty of Pharmacy, Kalabhavan Campus, The Maharaja Sayajirao University of Baroda, Vadodara,

Gujarat 390001, India

Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1840441.

ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2

A. M. KANHED ET AL.

pathogenesis (Hardy & Higgins, 1992; Selkoe, 2003). The 1c, catharanthine 1d, vallesiachotamine lactone 1e, vallesia-

amyloid precursor protein (APP) is hydrolyzed sequentially by chotamine 1f, fascaplysin 1g, infractopicrin 1h, angustine 1i,

a-, b- and c-secretase enzymes to produce Ab peptides that prunifoleine 1j and cryptolepine 1k are reported to possess

can aggregate into monomers, oligomers, and large Ab pla- cholinesterase inhibitory activity (Figure 1) (Brunhofer et al.,

ques, and get deposited in the regions of hippocampal and 2012). Recently, various ring-hybrids containing indole

basal ganglia of the patient’s brain. These aggregates initiate

pathogenic cascade of events and finally lead to neuronal

loss and dementia (O’Brien & Wong, 2011). The Ab plaques

formed from Ab_1-42are neurotoxic and continuously activate

inflammatory factors, such as IL-6 and TNF-a. Moreover,

because of self oxygen-free radical donation activity of Ab_1-

₄₂, it directly activates reactive oxygen species (ROS) which

subsequently affect the regular physiological functions of

neurocytes (Cheignon et al., 2018). Hence, the Ab_1-42aggre-

gation prevention could serve as a coherent approach for

the treatment of AD.

Recent research has emphasized that oxidative stress is

one of the earliest events in AD pathogenesis. Inequity

among the production and quenching of free radicals

formed from oxygen species generates oxidative stress (Lobo

et al., 2010). Through pathological redox steps, ROS and

reactive nitrogen species (RNS) can denature biomolecules

like lipids, proteins and nucleic acids and can cause serious

damage to tissue by apoptosis and necrosis (Uttara et al.,

2009). This suggests that oxidative stress also plays a crucial

role in the pathogenesis of AD, causing neuronal dysfunction

and cell death (Zhao & Zhao, 2013).

Considering the facts that ChE inhibition provides a symp-

tomatic treatment to AD, increased levels of BuChE are

observed in the AD patients’ brains, Ab aggregation and the

ROS system play vital roles in neurodegeneration, in this

report a series of indolo[2,3-b]quinoxaline derivatives were

designed, synthesized, and tested for their multifactorial anti-

AD activities, which includes cholinesterase and Ab aggrega-

tion inhibitory activity along with promising antioxidant

properties. The ADMET properties of the synthesized deriva-

tives were predicted in silico. Molecular modeling studies

were carried out to understand the binding modes of the

derivatives with the target proteins.

nucleus, e.g. triazinoindole hybrids (Patel et al., 2019, Patel

et al., 2020), donepezil-chromone-melatonin hybrids (Pachon-

ꢀ

Angona et al., 2019), tacrine ꢀ melatonin hybrids (Rodrıguez-

Franco et al., 2006), carbamate derivatives of indolines

(Yanovsky et al., 2012) and melatonin-N,N-dibenzyl(N-methyl)-

ꢀ

amine hybrids (Lopez-Iglesias et al., 2014) have been reported

as multifunctional agents for AD treatment suggesting indole

ring as an important privileged scaffold for CNS-active agents

which could augment the search for novel therapeutics for AD.

E Ramos et al reported new tacrine derivatives like quinoxa-

linetacrine (QT) hybrid QT78 for the treatment of AD (Figure 2).

QT78 is less toxic but less potent than tacrine and showed

selective BuChE inhibition (hAChE, IC₅₀¼ 22.0 lM; hBuChE,

IC₅₀¼ 6.79 lM) (Ramos et al., 2019). The toxicity associated

with tacrine and QT78 molecule might be due to their oxida-

tive hydroxylation by CYP1A2 followed by rearrangement to

the reactive quinonemethide intermediates. These metabolites

have the potential to bind irreversibly to liver cells and lead to

hepatic necrosis (McEneny-King et al., 2017). Replacing the

5,6,7,8-tetrahydroquinoline ring in QT78 with an indole ring

could retard the rearrangement step in metabolism and the

presence of indole ring additionally might provide a beneficial

effect against the oxidative stress. A combination of these two

privileged scaffolds, indole ring and the quinoxaline ring by

molecular hybridization approach resulted in a tetracyclic

indolo[2,3-b]quinoxaline scaffold (Figure 2). The planar aro-

matic structure of the indolo[2,3-b]quinoxaline scaffold has the

potential to block the pie stacking in amyloid fibril formation

and could inhibit self-induced Ab_1-42aggregation.

The molecular interactions of the designed indolo[2,3-

b]quinoxaline scaffold were assessed by performing the

molecular docking of the scaffold within the binding sites of

ChE enzymes. The indolo[2,3-b]quinoxaline scaffold demon-

strated promising and stable binding affinities with both the

AChE and BuChE enzymes (Figure 3). The indolo[2,3-b]qui-

noxaline scaffold showed very good stability within the

active sites of the enzymes. In AChE, the ligand-receptor

complex stability was observed mainly due to strong p–p

interactions between the scaffold and Trp84 and Phe330

(hAChE: Trp86 and Tyr337) of the active site. Further, this

complex was stabilized by hydrogen bonding with His440

(hAChE: His447), while the same molecule in the active bind-

ing site of BuChE showed promising p–p interactions with

Trp82 along with a hydrogen bond with His438. In the

in vitro enzyme inhibition assay, indolo[2,3-b]quinoxaline

offered IC₅₀values of 14.96 lM against AChE and 13.26 lM

against BuChE. Based on this moderately promising activity

of the designed indolo[2,3-b]quinoxaline scaffold, chemical

modifications were carried out in it by including a range of

Rationale of designing

The multifactorial nature of AD prompts treatment with multi-

target-directing ligands (MTDLs) to tackle the important

pathological hallmarks (Cavalli et al., 2008). Though ChEIs ren-

der only symptomatic and momentary benefits to the patients,

they still are the preferred medicines. The oxidative stress and

neurotoxic Ab plaques perform important functions in the AD

pathogenesis. However, Ab aggregation inhibitors or antioxi-

dants all alone might not be sufficient to resist a highly intri-

cate pathological condition of AD. Thus focusing inhibition of

multiple targets including cholinesterase and Ab aggregation

along with antioxidant activities might be a desirable pick to

offset the progress of this multifaceted disease.

Some nitrogen-containing heterocycles, especially alka- alkyl/benzyl groups to come up with some potential leads

loids, have been recognized as promising candidates in our and to frame a tangible SAR for the series. Accordingly, com-

quest for new drug leads for the treatment of AD. In particular, pounds of the two series (II, III), as shown in Figure 2, were

several indole alkaloids nitrarine 1a, hirsutine 1b, rauwolscine synthesized and discussed here in this report.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

3

Figure 1. Some natural indole alkaloids reported in the literature for treatment of AD.

Figure 2. Design of indoloquinoxaline derivatives by molecular hybridization approach.

different alkyl/benzyl halides. These isatin/N-substituted isatins

were further condensed with 1,2-diaminobenzene in the pres-

ence of acetic acid under microwave conditions to yield the

indolo[2,3-b]quinoxaline derivatives 3a-3l (Avula et al., 2012).

5-Substituted indolo[2,3-b]quinoxaline derivatives 7a-7k

Results and discussion

Chemistry

Compounds 3a-3l were synthesized as depicted in Scheme 1.

N-substituted isatins 2b-2l were prepared from commercially

available isatin 2a by nucleophilic substitution reaction with were synthesized as per the route depicted in the Scheme 2.

4

A. M. KANHED ET AL.

Figure 3. Molecular interaction of indolo[2,3-b]quinoxaline with (A) AChE and (B) BuChE.

Scheme 1. Synthesis of compounds 3a-3l. Reagents and conditions: (i) 1,2-Diaminobenzene, AcOH, microwave 450 W, 8–10 min.

1-Fluoro-2-nitrobenzene 4 was treated with substituted ben-

zyl/phenethylamines in the presence of potassium carbonate

as the base in DMF to obtain the substituted N-benzyl/phe-

nethyl-2-nitrophenylamines 5a-5k. These nitro derivatives

5a-5k were reduced by zinc/acetic acid to N₁-substituted 1,2-

diamine intermediates 6a-6k which were condensed with

isatin 2a as per the procedure adopted for compound 3a to

give the cyclized 5-substituted indolo[2,3-b]quinoxaline deriv-

atives 7a-7k.

6-(Aminoalkyl)-6H-indolo[2,3-b]quinoxaline derivatives (9a-

9f) and 1-(6-(6H-indolo[2,3-b]quinoxalin-6-yl)hexyl)alkylamides

(10a, 10b) were synthesized as per the route showed in the

Scheme 3. N-Bromoalkyl-6H-indolo[2,3-b]quinoxalines (8a-8c)

were obtained from compound 3a by reacting it with dibro-

moalkane in the presence of sodium hydroxide (Gu et al.,

2017). Aminodebromination of compounds 8a-8c by excess

secondary amines in THF leads to 6-substituted aminoalkyl-

6H-indolo[2,3-b]quinoxalines. To assess the importance of the

basic alicyclic amine function in the structure, alicyclic

amides were also prepared. Compounds 10a and 10b were

Biological evaluation

Inhibition studies on AChE and BuChE

The anti-cholinesterases (anti-ChE) activity of synthesized

compounds were assessed in vitro using the Ellman’s assay,

as previously reported by our group (Kanhed et al., 2015;

Patel et al., 2019, Patel et al., 2020; Shidore et al., 2016 Sinha

et al., 2015). Tacrine and donepezil were used as standard

reference drugs for this study. The IC₅₀values determined

for the compounds against both the enzymes along with

their selectivity indices (SI) are summarized in Tables 1 and

2. As shown in Table 1 compound 3a showed considerable

inhibitory activity (AChE, IC₅₀¼ 14.96 mM; BuChE, IC₅₀

¼

13.26 mM). This encouraging finding of the lead 3a provoked

us to look at different substituents on the indole and qui-

noxaline ring nitrogen to frame a considerable structure-

activity relationship.

The alkyl substituents introduction on indole as in com-

pounds 3b-3e showed decrease in activity. Among them,

compound 3b with the methyl group showed comparable

synthesized by the reaction of pyrrolidinone and piperidi- inhibition activity (AChE, IC₅₀¼ 13.37 mM; BuChE, IC₅₀

¼

none with 8c in the presence of sodium hydride. 15.80 mM) to the parent compound 3a. Incorporation of

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

5

Scheme 2. Synthesis of compounds 7a-7k. Reagents and conditions: (i) RNH₂, K₂CO₃, DMF, 60 ^ꢁC; (ii) Zn, AcOH, MeOH, rt, 6–8 hrs; (iii) Isatin (2a), AcOH, microwave

450 W, 8–10 min.

Scheme 3. Synthesis of compounds 9a-9f, 10a and 10b. Reagents and conditions: (i) Br(CH₂)_nBr, NaOH, THF, reflux; (ii) R¹R²NH, THF, reflux; (iii) R¹R²NH, NaH, THF.

benzyl and substituted benzyls at indole-NH exhibited compared to the lead molecule, whereas BuChE activity

reduced inhibitory activity. So as compared to compound 3a, remained the same as that of compound 3a.

the presence of simple alkyls/substituted benzyls on indole-

When the N-butyl moiety in compound 3e was replaced

NH ring did not show any significant improvement in activ- with 4-(1-pyrrolidinyl)butyl (compound 9a) and 4-(1-piperidi-

ity. Introducing alkyl substituents on the nitrogen of the qui- nyl)butyl (compound 9d) moieties, considerable raise in

noxaline ring as in compounds 7a-7k exhibited a mixed inhibitory activities against both the enzymes observed, par-

effect on the inhibitory activity of the compounds. As men- ticularly BuChE inhibitory activity got increased notably.

tioned in Table 1, all the compounds 7a-7k showed IC₅₀in Compounds 9a and 9d exhibited inhibitory activities (AChE;

the range of 9–17 mM for AChE and 13–30 mM for BuChE. IC₅₀¼ 14.39 mM, 11.53 mM, respectively) and (BuChE; IC₅₀

¼

Amongst them, compound 7h having 3,4-dimethoxybenzyl 4.48 mM, 2.56 mM, respectively). Joining of the tetracyclic

side chain showed better AChE inhibitory activity as indoloquinoxaline moiety with cyclic amines like pyrrolidine

6

A. M. KANHED ET AL.

Table 1. In Vitro Inhibition activity and Selectivity Index (SI) of Compounds 3a-3l, 7a-7k against AChE and BuChE.

IC₅₀SEM (mM)

IC₅₀SEM(mM)

Compd

R

AChE^a

BuChE^b

SI^c

Compd

R

AChE^a

BuChE^b

SI^c

3a

14.96 0.71

13.26 1.05

0.89

7a

14.78 0.19

16.79 0.22

1.14

3b

3c

3d

3e

13.37 1.18

17.47 0.78

19.14 1.51

19.98 0.35

15.80 1.22

22.29 1.19

18.56 0.45

21.01 0.53

1.18

1.28

0.97

1.05

7b

7c

7d

7e

12.07 0.15

21.66 0.18

24.95 0.26

30.01 0.22

15.01 0.25

1.79

1.82

1.78

0.92

13.70 0.18

16.88 0.26

16.39 0.21

3f

24.09 1.16

20.28 0.84

13.99 1.21

35.99 1.18

0.58

1.77

7f

15.81 0.14

12.18 0.15

32.21 0.28

25.27 0.17

2.04

2.07

3g

7g

3h

3i

20.92 1.09

26.63 1.23

16.15 0.56

93.48 2.53

0.77

3.51

7h

7i

9.42 0.54

13.50 0.61

20.47 0.89

1.43

1.57

13.07 0.78

3j

20.59 1.28

24.19 1.76

24.88 1.16

33.12 1.52

1.21

1.37

7j

17.38 1.22

13.75 0.71

21.74 1.74

18.90 0.24

1.25

1.37

3k

7k

3l

19.08 1.08

11.95 0.85

0.63

^aAChE from human erythrocytes; IC₅₀, 50% inhibitory concentration (means SEM of three experiments).

^bBuChE from equine serum.

^cSelectivity index ¼ IC₅₀(BuChE)/IC₅₀(AChE).

and piperidine through four carbon atom spacers enhanced

Change in the length of carbon chain showed alteration in

BuChE inhibition significantly in comparison to that of simple inhibitory activity. A comparative analysis of the inhibitory

alkyl/benzyl substituted indoloquinoxaline derivatives. On the potential of compounds 9d, 9e and 9f carrying a piperidino

basis of this finding, it was decided to evaluate the require- ring, revealed that compound 9f (n ¼ 6, IC₅₀value of 0.96 lM)

ment of the attached basic amine and effect of the linker showed the highest BuChE inhibitory activity whereas, the

length on the cholinesterase inhibition activity of resulting compound 9d (n ¼ 4, IC₅₀value of 2.56 lM) and compound 9e

compounds. As mentioned in Table 2, all the compounds 9a- (n ¼ 5, IC₅₀value of 1.40 lM) exhibited 2.6-fold and 1.5-fold

9f showed promising inhibitory activity against both ChE less BuChE inhibitory activities in comparison to compound 9f

enzymes, IC₅₀values ranging from 0.96 to 2.56 lM for BuChE (Table 2). Similar type of pattern was also found for the com-

and from 5.80 to 11.53 lM for AChE. These observations rec- pounds 9a, 9b and 9c. The inhibitory potential of compounds

ommended the presence of alicyclic amines is necessary 9a, 9b and 9c having a pyrrolidino ring, revealed that com-

requirement for the ChE inhibitory activity as all the com- pound 9c (n ¼ 6, IC₅₀value of 1.07 lM) exhibited the highest

pounds bearing pyrrolidino and piperidino moieties exhib- BuChE inhibitory activity while compound 9a (n ¼ 4, IC₅₀value

ited strong ChE inhibition.

of 4.48 lM) and compound 9b (n ¼ 5, IC₅₀value of 1.70 lM)

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

7

Table 2. In Vitro Inhibition activity and Selectivity Index (SI) of Compounds 9a-9f, 10a and 10b against AChE and BuChE.

IC₅₀SEM (mM)

Ab1-42 aggregation Inhibition (%)

RP of DPPH

IC₅₀SEM (mM) or

Compd

n

4

R¹R²N

AChE^a

BuChE^b

SI^c

25 mM

50 mM

(% inhibition at 100 mM)^d

9a

14.39 1.12

10.31 0.43

6.2 0.41

4.48 0.22

0.31

17.25 0.31

41.37 0.75

144.88 3.12

9b

9c

5

6

4

5

6

1.70 0.27

1.07 0.51

2.56 0.23

1.40 0.70

0.96 0.31

4.48 0.15

0.16

0.18

0.22

0.19

0.17

0.68

14.63 0.64

21.37 0.28

18.98 0.15

18.35 0.33

26.40 0.54

17.58 0.42

38.72 0.28

49.77 0.46

42.39 0.44

40.32 0.28

51.24 0.55

39.43 0.74

168.43 4.56

146.19 2.12

155.81 4.56

132.37 3.12

134.43 3.12

> 500 (3.59 %)

9d

9e

9f

11.53 0.76

7.27 0.58

5.80 0.70

6.63 0.54

10a

10b

6

5.99 0.37

5.01 1.02

0.84

21.55 0.62

46.32 0.82

> 500 (3.17 %)

Tacrine

0.056 0.01

0.023 0.01

nd

0.008 0.00

1.87 0.08

nd

0.14

81.3

–

nd

> 500 (17.2 %)

> 500 (5.41 %)

nd

Donepezil

Curcumin

Ascorbic acid

20.43 0.72 mM (IC₅₀

nd

)

nd

–

nd

13.91 1.33 (95.43 %)

^aAChE from human erythrocytes; IC₅₀, 50% inhibitory concentration (means SEM of three experiments).

^bBuChE from equine serum.

^cSelectivity index ¼ IC₅₀(BuChE)/IC₅₀(AChE).

^dRP of DPPH (%) ¼ reduction percentage of DPPH.

showed 4.2-fold and 1.6-fold less BuChE inhibitory activities in

comparison to compound 9c (Table 2).

When the amino functions in compounds 9c and 9f were

replaced with the corresponding cyclic amide moieties, the

BuChE inhibitory activity got notably diminished. Compound

10a having pyrrolidinone moiety (IC₅₀value of 4.48 lM) and

compound 10b having piperidinone moiety (IC₅₀value of

5.01 lM) showed 4.2-fold and 5.2-fold less BuChE inhibitory

activities compared to compounds 9c and 9f, respectively.

However, the AChE inhibitory activity of the compounds 10a

and 10b remained almost unaltered in comparison to the

corresponding amine derivatives 9c and 9f. Similar to tacrine

(SI value of 0.14), compounds 9c (SI value of 0.18) and 9f (SI

value of 0.17) have shown higher selectivity for BuChE over

AChE than donepezil (SI value of 81.3).

However, the inhibitory activity of these compounds

against AChE was slightly lesser than that against BuChE, this

could be because of structural and conformational variation

among both the enzymes. Compound 9f (n ¼ 6, IC₅₀value of

5.80 lM) showed an acceptable level of AChE inhibition

whereas compound 9d (n ¼ 4, IC₅₀value of 11.53 lM) and

compound 9e (n ¼ 5, IC₅₀value of 7.27 lM) exhibited 1.9-fold

and 1.2-fold less AChE inhibitory activities compared to com-

pound 9f. Compound 9c (n ¼ 6, IC₅₀value of 6.2 lM) exhib-

ited an acceptable level of AChE inhibitory activity while

compound 9a (n ¼ 4, IC₅₀value of 14.39 lM) and compound

9b (n ¼ 4, IC₅₀value of 10.31 lM) showed 2.3-fold and 1.7-

fold less AChE inhibitory activities compared to compound

9c. Piperidino moiety appeared to be a better choice over

pyrrolidino moiety, as compounds 9d-9f bearing piperidino

moiety exhibited higher activity than the compounds 9a-9c

bearing pyrrolidino moiety as attachments.

Self-mediated Ab_1–42aggregation inhibition study

The Ab peptides forming amyloid plaques are product of

APP due to cleavage mainly by b- and c-secretases. Ab

8

A. M. KANHED ET AL.

Table 3. Permeability (P_e10^ꢀ6cm s^ꢀ1) of selected commercial drugs for the

validation of the PAMPA-BBB permeation assay.

(P_e10^ꢀ6cm s^ꢀ1

)

Sr. No.

Commercial drugs

Reference value^a

Experimental value

1

2

3

4

5

6

7

Dopamine

Atenolol

Ofloxacin

Lomefloxacin

Corticosterone

Progesterone

Donepezil

0.2

0.8

1.1

5.1

9.3

12.0

0.3 0.1

1.1 0.3

1.7 0.5

1.9 0.3

4.3 0.6

11.5 1.2

14.3 1.7

^aTaken from reference (Di et al., 2003, Di et al., 2009). Data are expressed as

mean SEM of three independent experiments.

Figure 4. Linear correlation between experimental and reference permeabilities

of selected commercial drugs using PAMPA-BBB assay. P_e(exp.) ¼ 1.16 P_e(ref.)

þ 0.1668 (R²¼ 0.9781).

with 40 and 42 units are the main forms present in the pla-

ques. Though Ab_1-40is the predominant product in the pro-

teolytic cleavage, we chose Ab_1-42to study the inhibition of

compounds as it is more fibrillogenic in nature (Lane et al.,

2018). Thioflavin T (ThT) fluorescence assay was used to

understand the potency of the compounds to prevent self-

mediated Ab_1–42aggregation (Li et al., 2013). Compounds

having IC₅₀values (BuChE) less than 5 lM were considered

for this study. Here as positive control Curcumin was used

in the assay and the compounds were tested at 25 lM and

50 lM concentrations and listed in Table 2. All these com-

pounds exhibited moderate Ab_1–42aggregation inhibition

ranging from 14.63 to 26.40% at 25 lM concentration and

38.72–51.24% at 50 lM concentration. Compound 9f

showed 26.40% (at 25 lM concentration) and 51.24% (at

50 lM concentration) inhibition of Ab_1–42aggregation.

These anti-Ab aggregatory activities could be due to the

presence of planar aromatic tetracyclic indoloquinoxaline

ring in their structures.

Table 4. Permeability range (P_e10^ꢀ6cm s^ꢀ1) of PAMPA-BBB assay.

PBS:ethanol (70:30)

Compounds of low BBB permeation (CNS-)

Compounds of uncertain BBB permeation (CNSþ/ꢀ)

Compounds of high BBB permeation (CNSþ)

2.5 > P_e

4.8 > P_e> 2.5

P_e> 4.8

Table 5. Permeability (P_e10^ꢀ6cm s^ꢀ1) of compound 9f in the PAMPA-BBB

permeation assay with its predicted penetration into the CNS.

Compd.

(P_e10^ꢀ6cm s^ꢀ1

)

Prediction

9f

16.2 2.4

14.3 1.7

CNSþ

Donepezil

Data expressed as mean SEM of three independent experiments.

In vitro blood-brain barrier (BBB) permeation assay

The permeability through BBB is a key parameter for the

evaluation of novel CNS active molecules. The potential of

indoloquinoxaline derivatives to penetrate the BBB was

studied using a parallel artificial membrane permeation assay

(PAMPA) (Di et al., 2003, Di et al., 2009). This assay is used

for the prediction of passive diffusion of a molecule through

BBB. The BBB permeability (P_e) of the most active compound

9f was determined through a porcine brain lipid. The experi-

mental permeability values [P_e(exp)] of seven commercial

drugs with the reported permeability values [P_e(ref)] (Table

3) were compared for validation of the protocol, indicating a

Antioxidant activity [1,1-Diphenyl-2-picrylhydrazyl

(DPPH) radical scavenging activity]

To evaluate the antioxidant or free radical scavenging

potency of compounds, the DPPH radical scavenging assay is

common and reliable method. Being a stable free radical,

DPPH can accept an electron or hydrogen radical to become

a stable molecule. On the basis of the ability of compounds

under study to reduce DPPHꢂradical (purple color) to DPPHH

(yellow) and the corresponding radical-scavenging potential

were evaluated by a fall in the absorbance level at 517 nm

(Patel et al., 2019, Patel et al., 2020). Compounds with IC₅₀

(BuChE) less than 5 lM were considered here for this study.

Ascorbic acid was used as the positive control in this assay.

All the test compounds showed moderate free radical scav-

enging activity with their IC₅₀values ranging from 132 lM to

168 lM (Table 2). Compounds 9e and 9f also showed moder-

ate free radical scavenging activity (IC₅₀values 132.37 lM

and 134.43 lM, respectively) compared to ascorbic acid

(95.43% inhibition at 100 lM concentrations), whereas tacrine

and donepezil (IC₅₀values > 500 lM) were found to be

almost devoid of any free radical scavenging activity at these

concentrations.

linear relationship i.e. P_e(exp) ¼ 1.16 P_e(ref) þ 0.1668 (R²

¼

0.9781) (Figure 4). From this equation and considering the

limits for BBB permeation established by Di et al. (2003), it

was reckoned that the compounds with P_e(exp) greater than

4.8 ꢃ 10^ꢀ6cm s^ꢀ1(Table 4) would be able to cross the BBB.

Compound 9f exhibited permeability value above this limit

(Table 5). Therefore, P_e(exp) value propounded a high poten-

tial of the compound 9f to cross the BBB by pas-

sive diffusion.

Computational studies

Molecular docking studies of compound 9f and 9c

with ChEs

To comprehend the interactions and binding mode of the

most active compound 9f with the ChEs, docking studies

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

9

Figure 5. Docking interactions of 9f with (A) AChE and (B) BuChE.

were carried out with the active sites of Torpedo californica hydrophobic residues comprising of Ala328, Phe329, Trp430

AChE (TcAChE) (PDB code: 2CKM) and human BuChE

(hBuChE) (PDB code: 4BDS). The validation of the developed

grids for docking in Schrodinger suit was carried out by

knocking out the existing co-crystallized ligands and re-dock-

ing the same ligands drawn afresh and energy minimized,

using the prepared grids. In this re-performance study, very

identical interactions to that of original co-crystallized ligands

were observed between the enzymes and the re-docked

ligands. In this re-docking study the root-mean-square devi-

ation (RMSD) values of the redocked ligands in comparison

to the original cocrystallized forms in the active site of 2CKM

and 4BDS were found to be 0.40 and 0.26 Å, respectively.

To understand the molecular interactions of 9f with AChE,

TcAChE (PDB code: 2CKM) was retrieved from RCSB and

humanized with hAChE to identify the sequence of amino

acids from human interacting with compound 9f. In the

docking study of compound 9f with AChE (Figure 5), the aro-

matic indolo[2,3-b]quinoxaline ring was observed to be stabi-

lized comfortably in the active site of the enzyme by forming

p–p interactions with Trp84 and Phe330 (hAChE: Trp86 and

Tyr337). Due to the presence of six methylene linker, the

piperidine ring of compound 9f was observed to be oriented

well towards the PAS. At physiological pH, the nitrogen of

this ring could be protonated and showed highly stable pi-

cation interaction with Trp279 (hAChE: Trp284) which con-

ferred additional stability to the complex. In the docking

study of compound 9c with AChE, it was observed that the

nitrogen of the pyrrolidine ring formed a weak hydrogen

bond with Tyr121 (hAChE: Tyr124) and also part of the pyr-

rolidine ring was observed to be exposed to the solvent

front and oriented differently. Due to these conformational

differences, compound 9c having pyrrolidine moiety possibly

showed less AChE inhibitory activity than compound 9f.

In the docking study of compound 9f with BuChE (Figure

5), the terminal aromatic rings of indolo[2,3-b]quinoxaline

exhibited p–p interactions with Trp82 and Trp231. Further,

the nitrogen of the quinoxaline ring formed a stable hydro-

gen bond with His438. The pi-cation interaction between the

nitrogen of the piperidine ring and Phe329 imparted add-

itional stability to the ligand-receptor complex. Additionally,

and Met434. While in the docking study of compound 9c

with BuChE, it was observed that the pyrrolidine ring didn’t

cause any promising interactions within the active site of the

enzyme. Furthermore, part of the ring was observed to be

oriented in the polar region of Ser79 and nonpolar aliphatic

amino acid Gly78. These conformational differences could

have made compound 9f more active against BuChE than

compound 9c.

Molecular docking studies of compound 9f with Ab_1-42

To understand the binding interaction of compound 9f with

Ab_1-42,docking study was performed using the X-ray crystal

structure of human Ab_1-42(PDB code: 1IYT). As the active site

or specific binding site is not precisely known, a blind dock-

ing study was performed using AutoDock 4.2. In blind dock-

ing, the ligand under study is allowed to freely move over

the entire sequence of the protein and possible interactions

are checked. In this study, the most stable ligand-receptor

complex showed promising interactions (Figure 6). The aro-

matic indole ring exhibited p–p interaction with Phe19,

whereas the quinoxaline ring formed strong pi-cation inter-

action with Lys16. Further, the protonated piperidine ring

showed promising interaction by forming salt bridge as well

as hydrogen bond with Glu11. These stacking and other

non-covalent interactions could be responsible for the pre-

vention of aggregation and deposition of insoluble Ab_1-42

plaques and further events.

Molecular dynamics simulation studies

From the docking studies and biological evaluations, com-

pound 9f was observed as promising inhibitor. In order to

validate, and know the time-dependent stability of the iden-

tified most potent compound 9f with the AChE and BuChE

receptor, a molecular dynamics study was carried out for the

period of 10 ns. In order to understand the binding stability

of the ligand receptor complex during the period of simula-

tion time, statistical properties like RMSD-P, RMSF-P, and

the piperidine ring was surrounded by the nonpolar/ RMSD-L (P ¼ protein; L ¼ ligand), Van der Waals and

10

A. M. KANHED ET AL.

Figure 6. Docking interactions of compound 9f with Ab_{1 ꢀ 42}(PDB code 1IYT). The possible hydrogen bonding between compound 9f and Glu11 residue is shown

by the red line.

Figure 7. RMSD and RMSF plot for AChE with compound 9f.

electrostatic interaction energies were examined to cross- The average of ꢀ12.41 2.6 kJ/mol (Coul-SR) and ꢀ212.54

check and support the stability of interactions. To calculate 2.6 kJ/mol (LJ-SR) were observed. These observations sug-

all these parameters, the original pose of the ligand-receptor gested that the ligand interacted promisingly with the recep-

tor active site by the contribution of both hydrophobic and

complex was used as the reference frame.

The RMSD-P is fundamentally calculated to comprehend electrostatic interactions; wherein the role of hydrophobic

the extent of movements of different atoms or groups in the interactions was observed to be higher than that of electro-

receptor in presence of ligand in the binding site of the static interactions throughout the simulation period.

Similarly, the MD study of compound 9f with the BuChE

receptor was also carried out. Wherein the average protein

RMSD for BuChE was found to be 0.14 nm while for the lig-

and in contact with the protein it was 0.11 nm indicating

that was stable in the active site and did not diffuse out of it

during the complete simulation period. Here the RMSF for all

the residues was found below 0.2 nm (Figure 8).

The average of ꢀ41.88 0.65 kJ/mol (Coul-SR) and

ꢀ179.11 0.61 kJ/mol (LJ-SR) were observed suggesting that

the role of hydrophobic interaction is more than electrostatic

interaction in ligand-receptor stability.

receptor. Over the period of time, this explains the structural

deviations and conformations of the receptor. For AChE in

ligand-receptor complex the protein RMSD was observed in

the range of 0.08 to 0.18 nm, and the average RMSD was

0.15 nm. Whereas the RMSD for ligand in contact with the

residues of receptor active site was observed in the range of

0.04 to 0.18 nm with an average RMSD of 0.12 nm. Here, des-

pite of having more rotatable bonds in the ligand strucutre,

the RMSD value is found in the acceptable range, this

strongly suggests that the compound 9f is quite stable in

the active site of receptor and not diffused from the binding

site throughout the simulation period. The residual mobility

and structural integrity of the receptor were enumerated

with the help of RMSF. Including loop and terminal residues

of the receptor, all the residues while having the compound

9f in the active site, showed the RMSF below 0.4 nm (Figure

7). Further, the short-range electrostatic (Coul-SR) and van

In silico prediction of physicochemical and

pharmacokinetics parameters

Approximately 40% of drug candidates abort in the clinical

trials due to unacceptable ADME (absorption, distribution,

der Waal/hydrophobic (LJ-SR) interaction energies between metabolism, and excretion) profile. These late-stage dead

the ligand and the receptor were calculated within Gromacs. ducks impart a massive hike in the cost of development of a

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

11

Figure 8. RMSD and RMSF plot for BuChE with compound 9f.

Table 6. In-silico calculated ADME properties of 9c, 9f, Tacrine and Donepezil^a.

Properties

Limit

9c

9f

Donepezil

Tacrine

MW

HBD

HBA

130–725

0–6

2–20

372.521

386.539

379.498

0

5.5

198.267

1.5

0

4

0

4

2

QPlogP_o/w

Rule of Five violation

NRB

ꢀ2 to 6.5

5.64

1

7

5.947

1

7

4.242

0

6

2.536

0

1

0–1

0–8

PSA

SASA

7 to 200

300 to 1000

500–2000

32.268

749.37

1316.084

1387.038

100

779.49

0.247

1

31.315

769.868

1359.562

1487.168

100

840.486

0.279

1

46.234

681.675

1248.451

1070.771

100

589.289

0.223

1

33.825

425.06

701.299

2965.755

100

1602.036

0.047

1

Volume

QPPCaco

% HOA

QPPMDCK

QPlogBB

CNS

–

ꢀ3 to 1.2

–

QPlogKhSa

QPlogS

#rtvFG

ꢀ1.5 to 1.5

1.071

ꢀ5.964

0

1.198

ꢀ6.353

0

0.516

ꢀ4.059

0

0.049

ꢀ3.036

0

ꢀ6.5 to 0.5

–

#star

0

^aMW: molecular weight, HBD: hydrogen-bond donor atoms, HBA: hydrogen-bond acceptor atoms, QPlogP_o/w: Predicted octanol/water partition coefficient, NRB:

number of rotatable bonds, PSA: polar surface area, SASA: total solvent accessible surface area, QPPCaco: Caco-2 cell permeability in nm/s, % HOA: human oral

absorption on 0–100% scale, QPlogBB: brain/blood partition coefficient, QPPMDCK: Predicted apparent MDCK cell permeability in nm/s, CNS: predicted central

nervous system activity on a ꢀ2 (inactive) to þ2 (active) scale, QPlogKhsa: binding to human serum albumin, QPlogS: predicted aqueous solubility, #rtvFG:

number of reactive functional groups; #star: number of parameters with values that fall outside the 95% range of similar values for known drugs.

new drug. Hence, we planned to check virtual ADME param- and the topological Polar Surface Area (TPSA) (Veber et al.,

eters to affirm acceptable ADME performance during clinical 2002). NRB which explains the molecular flexibility, is also a

trials. The in silico calculation of the ADME properties very promising descriptor explaining the drug oral bioavail-

becomes comparatively simple and trustworthy because of ability. For the better oral bioavailability, a molecule could

significant advancements made in the computational science have 0 ꢀ 8 number of rotatable bonds or less than 7 linear

field. For the most active compounds 9c and 9f, the virtual chains outside the rings. TPSA is another important descrip-

physicochemical and pharmacokinetic parameters like tor that compared well with passive transport through the

Lipinski’s parameters, NRB, PSA, QPPCaco, QPlogBB, membranes and thus, enables prediction of bioavailability,

QPPMDCK, QPlogKhsa were calculated using QikProp module intestinal absorption, and blood-brain barrier (BBB) penetra-

(Table 6) (QikProp, 2018).

tion of drug (Kelder et al., 1999). The mean value of TPSA for

Lipinski’s rule of five suggests that most “drug-like” mole- the marketed drugs acting on CNS is 40.5 Å²(range of

cules contain logP ꢄ 5, hydrogen bond donors ꢄ 5, hydro- 4.63–108 Å²) (Pajouhesh & Lenz, 2005). Molecules 9c and 9f

gen bond acceptors ꢄ 10 and molecular weight ꢄ 500 have seven rotatable bonds each with TPSA respective values

(Lipinski et al., 1997). This rule explains the reason for various of 32.27 Å²and 31.32 Å². In In-silico the oral absorption of

physic-chemical behavior of drug-like molecules, such as drug is indicated by QPCaco-2 value. This QPCaco-2 explains

poor absorption or permeation occurs mainly when drug-like the permeability through gut-blood barrier. Values for com-

molecules deviate from more than one Lipinski’s rule. pound 9c and 9f are observed above 500 which forecast bet-

Compounds 9c and 9f violate only one limit of the Lipinski’s ter oral absorption. Similarly, The oral bioavailability of test

rule of five, making them promising leads for further drug compounds are also supported by the human oral absorp-

development. Veber and co-worker introduced two key tion percent (% HOA). The potential of compound to cross

parameters namely, the number of rotatable bonds (NRB) the blood-brain barrier is very well predicted by the Brain/

12

A. M. KANHED ET AL.

blood partition coefficient (QPlogBB), apparent MDCK cell

permeability (QPPMDCK) and n-octanol ꢀ water partition

coefficient (QPlogP_o/w). For the drugs which penetrate the

BBB passively have the QPlogP_o/wvalues around 3.76.

QPPMDCK value, which is considered as apparent MDCK cell

Experimental section

General

All the required chemicals, reagents and solvents were pro-

cured from S. d. fine chemicals, Sigma-Aldrich, Spectrochem

permeability in nm/s, is considered to be a good imitate for and Avra chemicals, and purified using general laboratory

techniques whenever required. Precoated silica gel thin-layer

chromatography (TLC), was used to monitor reaction pro-

gress and ultraviolet (UV) light (k ¼ 254 nm) or an iodine

chamber was used for visualization. Flash column chroma-

tography (Teledyne ISCO CombiFlash Rf system) with

RediSep Rf columns were used for purification of com-

pounds. All the reported yields are unoptimized. Melting

points were determined using melting point apparatus

the BBB. Any value above 25 for QPPMDCK is considered as

good, and the test compounds under study have qualified

this criterion. As the predicted CNS value is 1 for the test

and reference compounds, these can be predicted as CNS

active. The binding ability of test compound with human

serum albumin is predicted by QPlogKhsa value. The

QPlogKhsa values for the test molecules under study, 9c and

9f, are observed in the recommended range of values. #Star

indicates the number of parameters with values that fall out-

side the 95% range of similar values for known drugs. A

large #star value suggests that the molecule is less druglike

in comparison to a molecule with few #star value. Zero value

of #star for compounds 9c and 9f make them to be druglike.

Further, a compound having a tertiary nitrogen-containing

moiety in its structure is a common feature in many CNS

(Veego) or by differential scanning calorimetry (DSC

-

Shimadzu DSC-60 instrument), and the reported melting

points are uncorrected. Using Bruker ALPHA-T (Germany) FT-

IR spectrophotometer all IR spectra were recorded. NMR (¹H

and ¹³C) spectra were recorded on a Bruker Advance-II

400 MHz spectrometer either in CDCl₃or DMSO-d₆solvents

and corresponding chemical shifts (d) are expressed in parts

per million (ppm) relative to the standard TMS, and the peak

patterns are indicated as s (singlet), d (doublet), t (triplet), m

(multiplet), and br (broad signal). Thermo Fisher mass spec-

trometer with an EI ion source was used to record the mass

of compounds. For elemental analyses Thermo Fisher FLASH

2000 organic elemental analyzer was used. The elemental

compositions of the compounds were within 0.4% range of

the calculated values.

active drugs which confers

a good brain penetration

(Pajouhesh & Lenz, 2005) which both of these compounds

9c and 9f have. Thus, compounds 9c and 9f are predicted to

have a promising pharmacokinetics profile, which would aug-

ment their pharmacological potential.

Conclusion

Considering the heterogeneity and multifaceted nature of

AD, it was contemplated to merge two different biologically

active moieties, indole and quinoxaline in a single scaffold to

design a potential anti-AD drug. By molecular hybridization

approach, a combination of indole ring and the quinoxaline

ring resulted in indoloquinoxaline scaffold. Incorporation of

various substituents in the indoloquinoxaline scaffold

resulted in a novel series of anti-AD agents demonstrating

good in vitro cholinesterase inhibitory activity. Among the

three classes of compounds which were synthesized, 9f was

identified as the most potent and selective BuChE inhibitor

(IC₅₀¼ 0.96 mM, selectivity index ¼ 0.17) having a two-fold

higher BuChE inhibitory activity compared to the commer-

cially approved reference drug donepezil (IC₅₀¼ 1.87 mM).

Moreover, compound 9f is also endowed with self-induced

Ab_1-42aggregation inhibitory activity (51.24% inhibition at

100 lM concentration). This anti-Ab aggregatory activity is

likely to be due to the presence of a planar aromatic tetra-

cyclic indoloquinoxaline scaffold and a piperidine ring side

chain in its structure. These derivatives also possessed mod-

erate antioxidant activity. Molecular modeling studies indi-

cated significant interactions between compound 9f and

ChEs and Ab_1-42in their active sites. Compound 9f showed

good BBB permeation in PAMPA-BBB assay and favourable in

silico ADME properties. Taken together, all these findings

suggest compound 9f to be a potential candidate for further

development as a novel anti-AD drug.

Chemistry

6h-Indolo[2,3-b]quinoxaline (3a)

General procedure A

A mixture of isatin (2a) (1 g, 0.006 mol) and 1,2-diaminoben-

zene (0.734 g, 0.006 mol) in glacial acetic acid (10 mL) was

taken into RBF and irradiated in microwave for about 10 min

at 180 W. Progress of the reaction was monitored by TLC.

After completion of the reaction, the reaction mixture was

poured in ice-cold water and neutralized with a saturated

sodium bicarbonate solution to pH 7. The solid so obtained

was filtered, dried, and recrystallized to get compound 3a as

a yellow solid (0.37 g, 79%). m.p. 289.93 ^ꢁC (DSC); IR (KBr,

cm^ꢀ1): 3138, 2960, 2926, 1597, 1404, 744; H NMR (CDCl₃): d

1

11.94 (bs, 1H, –NH), 8.35–8.33 (d, 1H, ArH), 8.25–8.21 (m, 1H,

ArH), 8.07–8.05 (m, 1H, ArH), 7.79–7.75 (m, 1H, ArH),

7.71–7.65 (m, 2H, ArH), 7.58–7.56 (d, 1H, ArH), 7.37–7.33 (m,

1H, ArH); MS (m/z): 220.10 (M þ H)^þ.

Synthesis of 6-substituted 6H-indolo[2,3-b]quinoxaline

derivatives (3b-3l)

Following the General Procedure (A), 6-substituted 6H-

indolo[2,3-b]quinoxaline derivatives 3b-3l were synthesized

by condensation of N₁-substituted isatins with 1,2-diamino-

benzene. The obtained solids were recrystallized to yield the

titled compounds.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

13

6-Methyl-6H-indolo[2,3-b]quinoxaline (3b)

6-(3-Fluorobenzyl)-6H-indolo[2,3-b]quinoxaline (3h)

Pale yellow solid; yield 76%; m.p. 150–152 ^ꢁC; IR (KBr, cm^ꢀ1): Pale yellow solid; yield 76%; m.p. 137–140 ^ꢁC; IR (KBr, cm^ꢀ1):

3055, 2966, 1583, 1388, 1112, 748; ¹H NMR (CDCl₃) d

8.39–8.37 (d, 1H, ArH), 8.26–8.23 (m, 1H, ArH), 8.12–8.10 (m,

1H, ArH), 7.82–7.75 (m, 2H, ArH), 7.73–7.69 (m, 2H, ArH),

7.43–7.39 (m, 1H, ArH), 3.96 (s, 3H, –CH₃); MS (m/z):

234.20 (M þ H)^þ.

3062, 2980, 1587, 1408, 1253, 748; ¹H NMR (CDCl₃): d

8.42–8.40 (d, 1H, ArH), 8.30–8.28 (m, 1H, ArH), 8.14–8.12 (m,

1H, ArH), 7.84–7.59 (m, 4H, ArH), 7.43–7.39 (m, 1H, ArH),

7.32–7.28 (m, 1H, ArH), 7.21–7.16 (m, 2H, ArH), 7.05–7.00 (m,

1H, ArH), 5.59 (s, 2H, –CH₂); MS (m/z): 328.20 (M þ H)^þ.

6-Ethyl-6H-indolo[2,3-b]quinoxaline (3c)

6-(3-Trifluoromethylbenzyl)-6H-indolo[2,3-b]quinoxa-

line (3i)

Pale yellow solid; yield 75%; m.p. 136–138 ^ꢁC; IR (KBr, cm^ꢀ1):

3055, 2970, 1608, 1585, 1409, 1116, 742; ¹H NMR (CDCl₃): d

8.40–8.38 (d, 1H, ArH), 8.27–8.24 (m, 1H, ArH), 8.13–8.11 (m,

1H, ArH), 7.83–7.69 (m, 4H, ArH), 7.43–7.39 (m, 1H, ArH),

4.57–4.56 (m, 2H, –CH₂), 1.47–1.43 (t, 3H, –CH₃); ¹³C NMR

(DMSO-d₆): d 145.08, 144.30, 140.41, 140.07, 139.05, 131.94,

129.53, 127.97, 126.54, 122.80, 121.45, 119.06, 110.78, 36.31,

13.88; MS (m/z): 248.20 (M þ H)^þ.

Pale yellow solid; yield 68%; m.p. 183–185 ^ꢁC; IR (KBr, cm^ꢀ1):

3034, 2980, 1585, 1327, 1166, 750; ¹H NMR (CDCl₃): d

8.42–8.40 (d, 1H, ArH), 8.29–8.27 (m, 1H, ArH), 8.12–8.09 (m,

1H, ArH), 7.82–7.78 (m, 1H, ArH), 7.75–7.67 (m, 2H, ArH),

7.62–7.54 (m, 5H, ArH), 7.43–7.39 (m, 1H, ArH), 5.85 (s, 2H,

–CH₂); MS (m/z): 378.20 (M þ H)^þ.

6-(4-Cynobenzyl)-6H-indolo[2,3-b]quinoxaline (3j)

6-Isopropyl-6H-indolo[2,3-b]quinoxaline (3d)

Pale yellow solid; yield 74%; m.p. 148–150 ^ꢁC; IR (KBr, cm^ꢀ1):

2972, 2306, 1587, 1409, 1253, 750; ¹H NMR (CDCl₃): d

8.40–8.38 (d, 1H, ArH), 8.27–8.25 (m, 1H, ArH), 8.12–8.10 (m,

1H, ArH), 7.80–7.76 (m, 1H, ArH), 7.73–7.66 (m, 2H, ArH),

7.59–7.57 (d, 1H, ArH), 7.41–7.37 (m, 1H, ArH) 7.32–7.27 (m,

1H, ArH), 7.17–7.14 (m, 2H, ArH), 7.01–6.97 (m, 1H, ArH), 5.74

(s, 2H, –CH₂); ¹³C-NMR (CDCl₃): d 145.62, 144.40, 140.44,

140.07, 139.42, 132.01, 131.33, 131.24, 129.70, 129.62, 128.06,

126.85, 123.62, 122.86, 121.93, 119.33, 114.99, 114.78, 114.69,

114.47, 111.17, 44.22.

Pale yellow solid; yield 70%; m.p. 144–146 ^ꢁC; IR (KBr, cm^ꢀ1):

2985, 2968, 1608, 1579, 1382, 1234, 748; ¹H NMR (CDCl₃): d

8.41–8.39 (d, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 8.10–8.08 (m,

1H, ArH), 7.80–7.67 (m, 4H, ArH), 7.40–7.36 (m, 1H, ArH),

5.42–5.39 (m, 1H, –NCH), 1.78–1.77 (d, 6H, –CH₃); MS (m/z):

262.20 (M þ H)^þ.

6-Butyl-6H-indolo[2,3-b]quinoxaline (3e)

Pale yellow solid; yield 68%; m.p. 115–117 ^ꢁC; IR (KBr, cm^ꢀ1):

3055, 2960, 2887, 1606, 1581, 1371, 1112, 746; ¹H NMR

(CDCl₃): d 8.39–8.37 (d, 1H, ArH), 8.26–8.24 (m, 1H, ArH),

8.11–8.09 (m, 1H, ArH), 7.80–7.67 (m, 4H, ArH), 7.41–7.37 (m,

1H, ArH), 4.53–4.49 (m, 2H, –NCH₂), 1.94–1.86 (m, 2H, –CH₂),

1.42–1.37 (m, 2H, –CH₂), 0.97–0.94 (t, 3H, –CH₃); ¹³C-NMR

(CDCl₃): 145.51, 144.73, 140.44, 139.93, 139.05, 131.95, 129.54,

128.03, 126.57, 122.75, 121.45, 118.98, 110.96, 41.20, 30.54,

20.19, 14.15; MS (m/z): 276.20 (M þ H)^þ.

6-(4-Methylbenzyl)-6H-indolo[2,3-b]quinoxaline (3k)

Pale yellow solid; yield 72%; m.p. 206–208 ^ꢁC; IR (KBr, cm^ꢀ1):

3053, 2990, 1581, 1469, 1197, 742; ¹H NMR (CDCl₃): d

8.40–8.38 (d, 1H, ArH), 8.28–8.26 (m, 1H, ArH), 8.13–8.11 (m,

1H, ArH), 7.81–7.79 (m, 1H, ArH), 7.74–7.66 (m, 2H, ArH),

7.61–7.59 (d, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.26–7.24 (d,

2H, ArH), 7.09–7.07 (d, 2H, ArH), 5.70 (s, 2H, –CH₂), 2.24 (s,

3H, ArCH₃); MS (m/z): 324.15 (M þ H)^þ.

6-Benzyl-6H-indolo[2,3-b]quinoxaline (3f)

Pale yellow solid; yield 74%; m.p. 172–174 ^ꢁC; IR (KBr, cm^ꢀ1):

3084, 2980, 2960, 1581, 1408, 1197, 742; ¹H NMR (CDCl₃): d

8.41–8.39 (d, 1H, ArH), 8.28–8.26 (m, 1H, ArH), 8.13–8.11 (m,

1H, ArH), 7.81–7.77 (m, 1H, ArH), 7.73–7.65 (m, 2H, ArH),

7.58–7.56 (d, 1H, ArH), 7.41–7.35 (m, 3H, ArH), 7.30–7.21 (m,

3H, ArH), 5.75 (s, 2H, –CH₂–); ¹³C-NMR (CDCl₃): d 145.66,

144.53, 140.49, 140.02, 139.35, 137.36, 132.00, 129.76, 129.62,

129.24, 128.03, 127.64, 126.85, 122.85, 121.86, 119.27, 111.12,

44.72; MS (m/z): 310.14 (M þ H)^þ.

6-(4-Tert-Butylbenzyl)-6H-indolo[2,3-b]quinoxaline (3l)

Pale yellow solid; yield 77%; m.p. 164–166 ^ꢁC; IR (KBr, cm^ꢀ1):

3057, 2964, 1585, 1406, 1114, 746; ¹H NMR (CDCl₃): d

8.40–8.38 (d, 1H, ArH), 8.27–8.25 (d, 1H, ArH), 8.12–8.10 (d,

1H, ArH), 7.81–7.77 (m, 1H, ArH), 7.73–7.61 (m, 3H, ArH),

7.40–7.28 (m, 5H, ArH), 5.69 (s, 2H, –CH₂), 1.21 (s, 9H,

–C(CH₃)₃); MS (m/z): 366.20 (M þ H)^þ.

Synthesis of 5-substituted 5H-indolo[2,3-b]quinoxaline

derivatives (7a-7k)

6-(2-Bromobenzyl)-6H-indolo[2,3-b]quinoxaline (3g)

Pale yellow solid; yield 71%; m.p. 194–196 ^ꢁC; IR (KBr, cm^ꢀ1):

3057, 2980, 1587, 1404, 1154, 756; ¹H NMR (CDCl₃): d

8.45–8.43 (d, 1H, ArH), 8.29–8.27 (m, 1H, ArH), 8.08–8.06 (m,

1H, ArH), 7.80–7.66 (m, 4H, ArH), 7.46–7.41 (m, 2H, ArH),

7.22–7.18 (m, 1H, ArH), 7.15–7.11 (m, 1H, ArH), 6.73–6.71 (m,

1H, ArH), 5.76 (s, 2H, –CH₂); MS (m/z): 388.20 (M þ H)^þ.

Following the General Procedure (A), 5-substituted 5H-

indolo[2,3-b]quinoxaline derivatives 7a-7k were synthesized

by condensation of isatin (2a) with N₁-substituted 1,2-dia-

mines (6a-6k). The obtained solids were recrystallized to

yield the titled compounds.

14

A. M. KANHED ET AL.

1H, ArH), 7.67–7.57 (m, 2H, ArH), 7.35–7.28 (m, 3H, ArH),

6.86–6.84 (m, 3H, ArH), 6.06 (s, 2H, –CH₂), 3.69 (s, 3H, –OCH₃);

MS (m/z): 340.20 (M þ H)^þ.

5-Benzyl-5H-indolo[2,3-b]quinoxaline (7a)

Pale yellow solid; yield 67%; m.p. 215–218 ^ꢁC; IR (KBr, cm^ꢀ1):

3064, 2980, 2962, 1579, 1438, 1288, 746; ¹H NMR (CDCl₃): d

8.29–8.27 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 7.90–7.88 (m,

1H, ArH), 7.76–7.72 (m, 1H, ArH), 7.68–7.64 (m, 1H, ArH),

7.61–7.56 (m, 2H, ArH), 7.35–7.25 (m, 6H, ArH), 6.14 (s, 2H,

–CH₂); MS (m/z): 310.10 (M þ H)^þ.

5-(3,4-Dimethoxybenzyl)-5H-indolo[2,3-b]quinoxaline (7h)

Pale yellow solid; yield 59%; m.p. 224–226 ^ꢁC; IR (KBr, cm^ꢀ1):

2970, 2902, 1579, 1438, 1259, 752; ¹H NMR (CDCl₃): d

8.28–8.22 (m, 1H, ArH), 7.98–7.96 (m, 1H, ArH), 7.77–7.73 (m,

1H, ArH), 7.68–7.56 (m, 3H, ArH), 7.31–7.28 (m, 1H, ArH),

7.21–7.07 (m, 1H, ArH), 6.80–6.74 (m, 3H, ArH), 6.05 (s, 2H,

–CH₂), 3.78(s, 3H, –OCH₃), 3.76(s, 3H, –OCH₃); ¹³C-NMR

(CDCl₃): d 159.29, 153.45, 149.36, 147.04, 133.36, 130.95,

130.80, 128.30, 124.30, 123.08, 121.55, 119.60, 118.90, 116.13,

112.44, 112.07, 55.98, 48.56, 40.56; MS (m/z): 370.20 (M þ H)^þ.

5-(4-Methylbenzyl)-5H-indolo[2,3-b]quinoxaline (7b)

Pale yellow solid; yield 68%; m.p. 209–211 ^ꢁC; IR (KBr, cm^ꢀ1):

2980, 2887, 1579, 1566, 1438, 1290, 754; ¹H NMR (CDCl₃): d

8.28–8.26 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 7.90–7.88 (d,

1H, ArH), 7.75–7.71 (m, 1H, ArH), 7.68–7.64 (m, 1H, ArH),

7.61–7.55 (m, 2H, ArH), 7.32–7.28 (m, 1H, ArH), 7.25–7.23 (d,

2H, ArH), 7.11–7.09 (d, 2H, ArH), 6.08 (s, 2H, –CH₂), 2.24 (s,

3H, ArCH₃); MS (m/z): 324.20 (M þ H)^þ.

5-Phenylethyl-5H-indolo[2,3-b]quinoxaline (7i)

Pale yellow solid; yield 68%; m.p. 184–186 ^ꢁC; IR (KBr, cm^ꢀ1):

2980, 2885, 1564, 1454, 1244, 746; ¹H NMR (CDCl₃): d

8.28–8.26 (m, 1H, ArH), 8.21–8.19 (m, 1H, ArH), 8.07–8.05 (m,

1H, ArH), 7.83–7.79 (m, 1H, ArH), 7.66–7.58 (m, 3H, ArH),

7.41–7.39 (m, 2H, ArH), 7.33–7.22 (m, 4H, ArH), 5.09–5.05 (m,

2H, ArCH₂), 3.27–3.23 (m, 2H, –NCH₂); ¹³C-NMR (CDCl₃): d

159.19, 153.08, 146.14, 136.30, 134.77, 133.25, 130.93, 130.89,

129.45, 129.00, 127.20, 124.17, 123.15, 122.93, 121.41, 118.91,

115.64, 46.67, 33.32; MS (m/z): 324.120 (M þ H)^þ.

5-(2-Fluorobenzyl)-5H-indolo[2,3-b]quinoxaline (7c)

Pale yellow solid; yield 63%; m.p. 196–198 ^ꢁC; IR (KBr, cm^ꢀ1):

3032, 2960, 1579, 1438, 1138, 748; ¹H NMR (CDCl₃): d

8.28–8.21 (m, 2H, ArH), 7.92–7.90 (m, 1H, ArH), 7.76–7.72 (m,

1H, ArH), 7.68–7.64 (m, 1H, ArH), 7.61–7.56 (m, 2H, ArH),

7.45–7.42 (m, 2H, ArH), 7.32–7.28 (m, 1H, ArH), 7.10–7.06 (m,

2H, ArH), 6.11 (s, 2H, –CH₂); MS (m/z): 328.10 (M þ H)^þ.

5-(3-Fluorobenzyl)-5H-indolo[2,3-b]quinoxaline (7d)

Pale yellow solid; yield 59%; m.p. 202–204 ^ꢁC; IR (KBr, cm^ꢀ1):

2980, 2889, 1579, 1438, 1138, 748; ¹H NMR (CDCl₃): d

8.29–8.27 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 7.89–7.87 (m,

1H, ArH), 7.77–7.73 (m, 1H, ArH), 7.68–7.64 (m, 1H, ArH),

7.61–7.57 (m, 2H, ArH), 7.36–7.28 (m, 2H, ArH), 7.24–7.22 (d,

1H, ArH), 7.16–7.14 (d, 1H, ArH), 7.08–7.04 (m, 1H, ArH), 6.13

(s, 2H, –CH₂); MS (m/z): 328.10 (M þ H)^þ.

5-(4-Chlorophenylethyl)-5H-indolo[2,3-b]quinoxaline (7j)

Pale yellow solid; yield 63%; m.p. 185–187 ^ꢁC; IR (KBr, cm^ꢀ1):

2980, 2889, 1566, 1444, 1290, 752; ¹H NMR (CDCl₃): d

8.26–8.24 (m, 1H, ArH), 8.20–8.18 (m, 1H, ArH), 8.01–7.99 (m,

1H, ArH), 7.81–7.77 (m, 1H, ArH), 7.64–7.56 (m, 3H, ArH),

7.41–7.39 (m, 2H, ArH), 7.32–7.25 (m, 3H, ArH), 5.07–5.03 (m,

2H, ArCH₂), 3.28–3.24 (m, 2H, –NCH₂–); MS (m/z):

358.20 (M þ H)^þ.

5-(3-Chlorobenzyl)-5H-indolo[2,3-b]quinoxaline (7e)

Pale yellow solid; yield 66%; m.p. 198–200 ^ꢁC; IR (KBr, cm^ꢀ1):

3057, 2980, 1579, 1436, 1288, 746; ¹H NMR (CDCl₃): d

8.30–8.28 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 7.89–7.87 (m,

1H, ArH), 7.77–7.75 (m, 1H, ArH), 7.66–7.59 (m, 1H, ArH),

7.61–7.59 (m, 2H, ArH), 7.49 (s, 1H, ArH), 7.32–7.25 (m, 4H,

ArH), 6.13 (s, 2H, –CH₂); MS (m/z): 344.10 (M þ H)^þ.

5-(3,4-Dimethoxyphenylethyl)-5H-indolo[2,3-b]quinoxa-

line (7k)

Pale yellow solid; yield 57%; m.p. 159–160 ^ꢁC; IR (KBr, cm^ꢀ1):

2980, 2972, 1564, 1460, 1136, 746; ¹H NMR (CDCl₃): d

8.25–8.23 (m, 1H, ArH), 8.19–8.17 (m, 1H, ArH), 7.98–7.96 (m,

1H, ArH), 7.79–7.75 (m, 1H, ArH), 7.65–7.55 (m, 3H, ArH),

7.27–7.24 (m, 1H, ArH), 6.94 (s, 1H, ArH), 6.85–6.79 (m, 2H,

ArH), 5.07–5.03 (m, 2H, ArCH₂), 3.76 (s, 3H, –OCH₃), 3.74 (s,

3H, –OCH₃), 3.21–3.17 (m, 2H, –NCH₂); MS (m/z):

384.20 (M þ H)^þ.

5-(2-Methoxybenzyl)-5H-indolo[2,3-b]quinoxaline (7f)

Pale yellow solid; yield 53%; m.p. 218–220 ^ꢁC; IR (KBr, cm^ꢀ1):

2980, 2972, 1577, 1429, 1246, 752; ¹H NMR (CDCl₃): d

8.31–8.29 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 7.73–7.56 (m,

5H, ArH), 7.31–7.23 (m, 2H, ArH), 7.13–7.11 (m, 1H, ArH),

6.72–6.68 (m, 1H, ArH), 6.59–6.57 (m, 1H, ArH), 6.02 (s, 2H,

–CH₂), 4.10 (s, 3H, –OCH₃); MS (m/z): 340.10 (M þ H)^þ.

General procedure for the synthesis of 6-(bromoalkyl)-

6H-indolo[2,3-b]quinoxaline (8a-8c)

To a stirred suspension of 6H-indolo [2,3-b]quinoxaline (3a)

(2.39 g, 10 mmol) in THF (30 mL) was added sodium hydrox-

ide (2.81 g, 50 mmol). The obtained solution was stirred for

30 min at 45 ^ꢁC and dibromoalkane (50 mmol) was added.

The reaction mixture was heated to reflux for 6 h, the reac-

tion progress was monitored by TLC. After completion of the

5-(4-Methoxybenzyl)-5H-indolo[2,3-b]quinoxaline (7g)

Pale yellow solid; yield 58%; m.p. 216–218 ^ꢁC; IR (KBr, cm^ꢀ1):

3051, 2968, 1579, 1438, 1247, 742; ¹H NMR (CDCl₃): d

8.29–8.22 (m, 2H, ArH), 7.98–7.96 (m, 1H, ArH), 7.78–7.74 (m, reaction, the reaction mixture was evaporated under reduced

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

15

pressure. The residue was dissolved in chloroform and

6-(6-(Pyrrolidin-1-yl)hexyl)-6H-indolo[2,3-b]quinoxa-

washed with water. The collected organic layer was dried line (9c)

Pale yellow solid; yield 73%; m.p. 98–100 ^ꢁC; IR (KBr, cm^ꢀ1):

3053, 2926, 2856, 2778, 1606, 1579, 1464, 1112, 751; H NMR

over sodium sulfate, filtered and evaporated to give a crude

product which was purified by column chromatography.

1

(DMSO-d₆): d 8.49–8.46 (m, 1H, ArH), 8.30–8.28 (m, 1H, ArH),

8.14–8.12 (m, 1H, ArH), 7.76–7.66 (m, 3H, ArH), 7.46–7.35 (m,

2H, ArH), 4.48–4.45 (m, 2H, –NCH₂), 2.51–2.40 (m, 6H, –NCH₂),

1.97–1.95 (m, 2H, –NCH₂CH₂), 1.79–1.77 (m, 4H, –NCH₂CH₂),

1.54–1.52 (m, 2H, –NCH₂CH₂) 1.42–1.40 (m, 4H,

–NCH₂CH₂CH₂CH₂); ¹³C-NMR (DMSO-d₆): 145.05, 144.20,

139.97, 139.47, 138.62, 131.41, 129.10, 129.01, 127.54, 126.05,

122.25, 120.94, 118.53, 110.51, 55.39, 53.47, 53.45, 26.55,

26.19, 25.53, 25.05, 22.94; MS (m/z): 373.3 [M þ H]^þ.

6-(4-Bromobutyl)-6H-indolo[2,3-b]quinoxaline (8a)

Yellow solid; yield 85%; MS (m/z): 354.48 [M]^þ,

356.44 [M þ 2]^þ

6-(5-Bromopentyl)-6H-indolo[2,3-b]quinoxaline (8b)

Yellow solid; yield 79%; MS (m/z): 368.49 [M]^þ,

370.46 [M þ 2]^þ.

6-(4-(Piperidin-1-yl)butyl)-6H-indolo[2,3-b]quinoxaline (9d)

Pale yellow solid; yield 70%; m.p. 92–94 ^ꢁC; IR (KBr, cm^ꢀ1):

3056, 2926, 2860, 2782, 1607, 1580, 1467, 1237, 751; H NMR

6-(6-Bromohexyl)-6H-indolo[2,3-b]quinoxaline (8c)

Yellow solid; yield 79%; MS (m/z): 382.55 [M]^þ,

384.51 [M þ 2]^þ.

1

(DMSO-d₆): d 8.40 (d, 1H, ArH), 8.28 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H,

ArH), 8.13 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H, ArH), 7.85–7.72 (m, 4H,

ArH) 7.44–7.40 (m, 1H, ArH), 4.52–4.49 (m, 2H, –NCH₂),

2.52–2.34 (m, 6H, –NCH₂), 1.94–1.87 (m, 2H, –NCH₂CH₂),

1.63–1.60 (m, 4H, –NCH₂CH₂), 1.54–1.46 (m, 2H,

–NCH₂CH₂CH₂), 1.38–1.31 (m, 2H, –NCH₂CH₂CH₂); MS (m/z):

359.3 [M þ H]^þ.

General procedure for the synthesis of 6-(aminoalkyl)-

6H-indolo[2,3-b]quinoxaline (9a-9f)

To a solution of 6-(bromoalkyl)-6H-indolo[2,3-b]quinoxaline

(8a-8c) (1 mmol) in THF (15 mL), piperidine or pyrrolidine

(10 mmol) was added. The reaction mixture was refluxed

under a nitrogen atmosphere. Progress of the reaction was

monitored by TLC. After completion of the reaction, the reac-

tion mixture was evaporated under reduced pressure and

the residue was dissolved in 20 mL of water and extracted

with chloroform (3 ꢃ 20 mL). The collected organic layer was

again washed with water, dried over anhydrous magnesium

sulfate, filtered and evaporated to give a crude product

which was further purified by column chromatography.

6-(5-(Piperidin-1-yl)pentyl)-6H-indolo[2,3-b]quinoxa-

line (9e)

Pale yellow solid; yield 72%; m.p. 96–98 ^ꢁC; IR (KBr, cm^ꢀ1):

1

3058, 2930, 2851, 2793, 1610, 1579, 1467, 1114, 766; H NMR

(DMSO-d₆): d 8.50–8.45 (m, 1H, ArH), 8.34–8.27 (m, 1H, ArH),

8.14–8.12 (m, 1H, ArH), 7.75–7.66 (m,3 H, ArH), 7.44–7.36 (m,

2H, ArH), 4.45–4.48 (m, 2H, –NCH₂), 2.52–2.27 (m, 6H, –NCH₂),

1.94–1.87 (m, 2H, –NCH₂CH₂), 1.63–1.60 (m, 4H, –NCH₂CH₂),

1.42–1.39 (m, 4H, indole NCH₂CH₂CH₂); MS (m/z):

373.3 [M þ H]^þ.

6-(4-(Pyrrolidin-1-yl)butyl)-6H-indolo[2,3-b]quinoxa-

line (9a)

6-(6-(Piperidin-1-yl)hexyl)-6H-indolo[2,3-b]quinoxaline (9f)

Pale yellow solid; yield 70%; m.p. 71–73 ^ꢁC; IR (KBr, cm^ꢀ1):

Pale yellow solid; yield 69%; m.p. 99–101 ^ꢁC; IR (KBr, cm^ꢀ1):

1

3056, 2927, 2863, 2788, 1607, 1582, 1467, 1240, 751; H NMR

3053, 2927, 2852, 2762, 1607, 1579, 1463, 1112, 753; H NMR

(CDCl₃): d 8.48 (d, 1H, ArH), 8.29 (d, 1H, ArH) , 8.12 (d, 1H,

ArH), 7.77–7.65 (m, 3H, ArH), 7.50–7.48 (m, 1H, ArH),

7.39–7.36 (m, 1H, ArH), 4.54–4.51 (m, 2H, –NCH₂), 2.53–2.47

(m, 6H, –NCH₂), 2.03–1.99 (m, 2H, –NCH₂CH₂), 1.82–1.72 (m,

(DMSO-d₆): d 8.51–8.47 (m, 1H, ArH), 8.33–8.29 (m, 1H, ArH),

8.16–8.13 (m, 1H, ArH), 7.80–7.67 (m, 3H, ArH), 7.50–7.38 (m,

2H, ArH), 4.52–4.47 (m, 2H, –NCH₂), 2.40–2.24 (m, 6H, –NCH₂),

1.99–1.94 (m, 2H, –NCH₂CH₂), 1.70–1.42 (m, 6H, –NCH₂CH₂,

4H, –NCH₂CH₂), 1.68–1.64 (m, 2H, –NCH₂CH₂); MS (m/z): 4H, –NCH₂CH₂CH_2,2H, –NCH₂CH₂CH₂CH₂); ¹³C-NMR (DMSO-

345.3 [M þ H]^þ.

d₆): 145.05, 144.25, 139.98, 139.47, 138.62, 131.41, 129.10,

128.99, 127.55, 126.05, 122.25, 120.93, 118.53, 110.50, 58.35,

53.90, 27.74, 26.58, 26.19, 25.98, 25.53, 25.37, 24.00; MS (m/z):

387.3 [M þ H]^þ.

6-(5-(Pyrrolidin-1-yl)pentyl)-6H-indolo[2,3-b]quinoxa-

line (9b)

Pale yellow solid; yield 67%; m.p. 101–103 ^ꢁC; IR (KBr, cm^ꢀ1):

General procedure for the synthesis of 1-(6-(6H-

indolo[2,3-b]quinoxalin-6-yl)hexyl)alkylamide (10a, 10b)

1

3056, 2927, 2851, 2758, 1607, 1579, 1463, 1118, 747; H NMR

(DMSO-d₆): d 8.45 (d, 1H, ArH), 8.30–8.28 (m, 1H, ArH),

8.12–8.10 (m, 1H, ArH), 7.75–7.64 (m, 3H, ArH), 7.46–7.33 (m,

2H, ArH), 4.48–4.46 (m, 2H, –NCH₂), 2.41–2.37 (m, 6H, –NCH₂),

1.97–1.95 (m, 2H, –NCH₂CH₂), 1.65–1.57 (m, 6H, –NCH₂CH₂),

1.42–1.140 (m, 2H, –NCH₂CH₂CH₂); MS (m/z): 359.3 [M þ H]^þ.

To a stirred suspension of sodium hydride (1 mmol) in dry

THF (15 mL), 2-pyrrolidinone or 2-piperidinone (1 mmol) was

added at 0 ^ꢁC under a blanket of dry nitrogen. The reaction

mixture was stirred for 30 min. 6-(6-Bromohexyl)-6H-

16

A. M. KANHED ET AL.

indolo[2,3-b]quinoxaline (8c) dissolved in a minimum amount

of dry THF was added into the reaction mixture at 0 ^ꢁC, and

then brought slowly up to room temperature and left to stir

overnight. After completion of the reaction, the reaction mix-

ture was evaporated under reduced pressure and the residue

was dissolved in 20 mL of water and extracted with chloro-

form (3 ꢃ 20 mL). The collected organic layer was again

Self-induced Ab_1-42aggregation inhibition study

The potency, to inhibit the self-mediated Ab_1-42aggregation,

of indolo[2,3-b]quinoxaline derivatives was evaluated by a

thioflavin T (ThT)-based fluorescence assay reported previ-

ously (Li et al., 2013; Patel et al., 2020).

washed with water, dried over anhydrous magnesium sulfate, In vitro blood-brain barrier permeation assay

filtered and evaporated to give a crude product which was

The PAMPA assay was carried out to determine the BBB per-

further purified by column chromatography.

meation of the most active compound 9f as described previ-

ously (Patel et al., 2019, Patel et al., 2020).

1-(6-(6h-Indolo[2,3-b]quinoxalin-6-yl)hexyl)pyrrolidin-2-

Computational studies

one (10a)

Pale yellow solid; yield 52%; m.p. 102–104 ^ꢁC; IR (KBr, cm^ꢀ1):

Docking studies of compound 9f and 9c with ChEs

1

3053, 2927, 2852, 2762, 1607, 1579, 1463, 1112, 753; H NMR

Docking studies of the active compounds with ChEs were

(DMSO-d₆): d 8.50 (d, 1H, ArH), 8.31 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H,

carried out with the Glide (2018) module of Schrodinger

Suite. 3D structures of the ligand molecules were con-

structed by the Build module within Maestro, and low

energy conformation was searched for the ligand/compound

under consideration at physiological pH condition by using

the OPLS3e force field within the Ligprep (2018) module of

ArH), 8.15 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H, ArH) 7.80–7.68 (m, 3H,

ArH), 7.50–7.48 (m, 1H, ArH), 7.40–7.38 (m, 1H, ArH),

4.52–4.49 (m, 2H, –NCH₂), 3.33–3.30 (m, 2H, –CONCH₂),

3.27–3.23 (m, 2H, –CONCH₂) 1.99–1.94 (m, 4H, –COCH₂CH₂),

1.50–1.38 (m, 2H, –CONCH₂CH_2,4H, –NCH₂CH₂CH₂CH₂); MS

(m/z): 387.3 [M þ H]^þ.

€

Schrodinger. PDB structures of AChE (PDB code: 2CKM, 1B41)

and of BuChE (PDB code: 4BDS) were retrieved from the

Protein Data Bank (RCSB) (Protein Data Bank, 2019) and pre-

pared for docking using the protein preparation wizard.

Using extra-precision (XP) mode, molecular docking calcula-

tions were carried out within the active sites of the receptor

structures. The followed docking protocol was validated by

comparing the interactions of donepezil within the active

site (Cheung et al., 2012).

1-(6-(6h-Indolo[2,3-b]quinoxalin-6-yl)hexyl)piperidin-2-

one (10b)

Pale yellow solid; yield 58%; m.p. 63–65 ^ꢁC; IR (KBr, cm^ꢀ1):

3057, 2937, 2858, 1632, 1578, 1464, 1118, 761; ¹H NMR

(DMSO-d₆): d 8.40 (d, 1H, ArH), 8.28 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H,

ArH), 8.13 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H, ArH) 7.85–7.73 (m, 4H,

ArH), 7.44–7.42 (m, 1H, ArH), 4.52–4.49 (m, 2H, –NCH₂),

3.19–3.12 (m, 4H, –CONCH₂), 2.15–2.12 (m, 2H, –COCH₂),

1.91–1.87 (m, 2H, –COCH₂CH₂), 1.64–1.62 (m, 4H,

Docking studies of compound 9f with Ab_1-42

AutoDock4.2 (Morris et al., 2009; Sanner, 1999) was used to

perform this docking analysis. Ab_1-42peptide structure was

retrived from RCSB site (PDB Code: 1IYT). It was cleaned and

prepared for docking analysis within AutoDock tool. The

blind docking was performed for compound 9f. Grid was

generated over the entire protein structure, and compound

9f was allowed to dock with the entire amino acid sequence

to understand the most possible/stable interactions between

the 9f and the peptide sequence. For this study, 10 docking

experiments were run using the Lamarckian genetic algo-

rithm. The maximum number of energy evaluations of 25

million was applied for every docking experiment.

–CONCH₂CH₂),

1.40–1.28

(m,

2H,

–NCH₂CH₂,

4H,

–NCH₂CH₂CH₂CH₂); ¹³C-NMR (DMSO-d₆): 167.96, 145.03,

144.24, 139.97, 139.46, 138.61, 131.41, 129.09, 128.99, 127.55,

126.05, 122.25, 120.93, 118.53, 110.49, 60.39, 46.97, 45,97,

31.93, 27.76, 26.39, 26.13, 26.01, 22.73, 20.97; MS (m/z):

401.3 [M þ H]^þ.

Biology

Inhibition studies on AChE and BuChE

The ability of the test compounds to inhibit ChEs was

assessed using Ellman’s method as detailed in our earlier

report (Patel et al., 2019, Patel et al., 2020).

Molecular dynamics simulation studies

To determine the ligand receptor stability over a period of

time, molecular dynamics study was performed between the

most active compound 9f and AChE and BuChE protein

structures by using GROMACS 2018.1 software (Abraham

et al., 2018). The best docked pose of the ligand with the

respective receptor was taken as the starting point for simu-

lation. To determine the complex stability CHARMM36 all-

atom force field (Huang et al., 2017) was used and ligand-

Antioxidant activity [1,1-Diphenyl-2-picrylhydrazyl

(DPPH) radical scavenging activity]

The antioxidant potential of the compounds was assessed

using spectrophotometric DPPH assay as described earlier

(Patel et al., 2019, Patel et al., 2020).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

17

receptor parameters were derived in GROMACS. For this pur-

pose, the ligand topology was generated using the CGenFF

server (Vanommeslaeghe et al., 2010; Yu et al., 2012) and lig-

and-receptor complexes were built. These receptor-ligand

complex systems were solvated using SPC water model

(Mark & Nilsson, 2001). To neutralize the total charge on the

individual system, in AChE-9f complex 6 NA were added, in

BuChE-9f complex 7 CL ions were added and in Ab_1-42-9f

complex 2 NA were added to the system. All the complexes

were first energy minimized using steepest descent method

(Bixon & Lifson, 1967) followed by two sequential equilibra-

tion simulations using canonical (NVT) and isobaric-isother-

mic (NPT) ensemble for 100 picoseconds (ps) each. Using the

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Disclosure statement

There are no conflicts of interest to disclose.

Funding

Mange Ram Yadav acknowledges the University Grant Commission

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Article Doi

Indoloquinoxaline derivatives as promising multi-functional anti-Alzheimer agents

DOI: 10.1080/07391102.2020.1840441

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